SpliceWiz: Quick Start
Alex CH Wong
10/29/2024
Abstract
This Quick-Start is a runnable example showing the functionalities of the SpliceWiz workflow. Version 1.8.0
Introduction
SpliceWiz is a graphical interface for differential alternative splicing and visualization in R. It differs from other alternative splicing tools as it is designed for users with basic bioinformatic skills to analyze datasets containing up to hundreds of samples! SpliceWiz contains a number of innovations including:
- Super-fast handling of alignment BAM files using ompBAM, our developer resource for multi-threaded BAM processing,
- Alternative splicing event (ASE) filters to remove problematic ASEs from analysis
- Group-averaged coverage plots: publication-ready figures to clearly visualize differential alternative splicing between biological / experimental conditions
- Interactive figures, including scatter and volcano plots, gene ontology (GO) analysis, heatmaps, and scrollable coverage plots, powered using the shinyDashboard interface
This vignette is a runnable working example of the SpliceWiz workflow. The purpose is to quickly demonstrate the basic functionalities of SpliceWiz.
We provide here a brief outline of the workflow for users to get started as quickly as possible. However, we also provide more details for those wishing to know more. Many sections will contain extra information that can be displayed when clicked on, such as these:
Click on me for more details
In most sections, we offer more details about each step of the workflow, that can be revealed in text segments like this one. Be sure to click on buttons like these, where available.
FAQ
What are the system memory requirements for running SpliceWiz
We recommend the following memory requirements (RAM) for running various steps of SpliceWiz:
buildRef()
- Building human / mouse SpliceWiz reference: 8 gigabytes
processBAM()
- Processing small alignment (BAM) files (~20 million paired end reads): 8 gigabytes
- Processing large BAM files (~100 million paired end reads): 16 gigabytes
collateData()
- Collating routine experiments (e.g. 3 replicates, 2 conditions): 8-16 gigabytes
- Collating large experiments (20+ samples, using
lowMemoryMode=TRUE): 32 gigabytes - Collating large experiments (20+ samples, using
lowMemoryMode=FALSE): 8 gigabytes per thread
Differential analysis
- Differential analysis (routine experiments): 8 gigabytes
- Differential analysis (large experiments - 20+ samples): 16 gigabytes
- DESeq2-based differential analysis of large experiments: 32 gigabytes
How does SpliceWiz measure alternative splicing?
SpliceWiz defines alternative splicing events (ASEs) as binary events between two possibilities, the included and excluded isoform. It detects and measures: skipped (casette) exons (SE), mutually-exclusive exons (MXE), alternative 5’/3’ splice site usage (A5SS / A3SS), alternate first / last exon usage (AFE / ALE), and retained introns (IR or RI).
SpliceWiz uses splice-specific read counts to measure ASEs. Namely, these are junction reads (reads that align across splice sites). The exception is intron retention (IR) whereby the (trimmed) mean read depth across the intron is measured (identical to the method used in IRFinder).
SpliceWiz provides two metrics:
- Percent spliced in (PSI): is the expression of the included isoform as a proportion of both included/excluded isoform. PSIs are measured for all types of alternative splicing, including annotated retained introns (RI)
- IR-ratio: For introns, we also measure IR-ratios, which is the expression of IR-transcript as a proportion of IR- and spliced-transcripts. Spliced transcript expression is measured using either
SpliceOverorSpliceMaxmethod (the latter is identical to that used in IRFinder)
Does SpliceWiz detect novel splicing events?
Novel splicing events are those in which at least one isoform is not an annotated transcript in the given gene annotation. SpliceWiz DOES detect novel splicing events.
It detects novel events by using novel junctions, using pairs of junctions that originate from or terminate at a common coordinate (novel alternate splice site usage).
Additionally, SpliceWiz detects “tandem junction reads”. These are reads that span across two or more splice junctions. The region between splice junctions can then be annotated as novel exons (if they are not identical to annotated exons). These novel exons can then be used to measure novel casette exon usage.
Workflow from a glance
The basic steps of SpliceWiz are as follows:
- Building the SpliceWiz reference
- Process BAM files using SpliceWiz
- Collate results of individual samples into an experiment
- Importing the collated experiment as an NxtSE object
- Alternative splicing event filtering
- Differential ASE analysis
- Visualization
Quick-Start
Installation
To install SpliceWiz, start R (devel version) and enter:
if (!requireNamespace("BiocManager", quietly = TRUE))
install.packages("BiocManager")
BiocManager::install("SpliceWiz")Setting up a Conda environment to use SpliceWiz
For those wishing to set up a self-contained environment with SpliceWiz installed (e.g. on a high performance cluster), we recommend using miniconda. For installation instructions, see the documentation on how to install miniconda
After installing miniconda, create a conda environment as follows:
After following the prompts, activate the environment:
Next, install R 4.2.1 as follows:
NB: We have not been able to successfuly use r-base=4.3, so we recommend using r-base=4.2.1 (until further notice).
Many of SpliceWiz’s dependencies are up-to-date from the conda-forge channel, so they are best installed via conda:
conda install -c conda-forge r-devtools r-essentials r-xml r-biocmanager \
r-fst r-plotly r-rsqlite r-rcurlAfter this is done, the remainder of the packages need to be installed from the R terminal. This is because most Bioconductor packages are from the bioconda channel and appear not to be routinely updated.
So, lets enter the R terminal from the command line:
Set up Bioconductor 3.16 (which is the latest version compatible with R 4.2):
Again, follow the prompts to update any necessary packages.
Once this is done, install SpliceWiz (devel) from github:
The last step will install any remaining dependencies, taking approximately 20-30 minutes depending on your system.
Enabling OpenMP (multi-threading) for MacOS users (Optional)
For MacOS users, make sure OpenMP libraries are installed correctly. We recommend users follow this guide, but the quickest way to get started is to install libomp via brew:
Installing statistical package dependencies (Optional)
SpliceWiz uses established statistical tools to perform alternative splicing differential analysis:
- limma: models included and excluded counts as log-normal distributions
- DESeq2: models included and excluded counts as negative binomial distributions
- edgeR: models included and excluded counts as negative binomial distributions. SpliceWiz uses the quasi-likelihood method which deals better with zero-counts.
- DoubleExpSeq: models included and excluded counts using beta binomial distributions
To install all of these packages:
Loading SpliceWiz
Details
The SpliceWiz package loads the
NxtIRFdata data package. This data package contains the example “chrZ” genome / annotations and 6 example BAM files that are used in this working example. Also, NxtIRFdata provides pre-generated mappability exclusion annotations for building human and mouse SpliceWiz references
The SpliceWiz Graphics User Interface (GUI)
SpliceWiz offers a graphical user interface (GUI) for interactive users, e.g. in the RStudio environment. To start using SpliceWiz GUI:
Navigating the GUI
The SpliceWiz GUI uses the shinyDashboard interface. Use the side menu on the left hand side of the interface to navigate across the various sub-panels in the SpliceWiz GUI.
SpliceWiz GUI - Menu Side Bar
Building the SpliceWiz reference
Why do we need the SpliceWiz reference?
SpliceWiz first needs to generate a set of reference files. The SpliceWiz reference is used to quantitate alternative splicing in BAM files, as well as in downstream collation, differential analysis and visualisation.
Using the example FASTA and GTF files, use the buildRef() function to build the SpliceWiz reference:
ref_path <- file.path(tempdir(), "Reference")
buildRef(
reference_path = ref_path,
fasta = chrZ_genome(),
gtf = chrZ_gtf(),
ontologySpecies = "Homo sapiens"
)The SpliceWiz reference can be viewed as data frames using various getter functions. For example, to view the annotated alternative splicing events (ASE):
See ?View-Reference-methods for a comprehensive list of getter functions
Using the GUI
After starting the SpliceWiz GUI in demo mode, click the Reference tab from the menu side bar. The following interface will be shown:
Building the SpliceWiz reference using the GUI
- The first step to building a SpliceWiz reference is to select a directory in which to create the reference.
- SpliceWiz provides an interface to retrieve the genome sequence (FASTA) and transcriptome annotation (GTF) files from the Ensembl FTP server, by first selecting the “Release” and then “Species” from the drop-down boxes.
- Alternatively, users can provide their own FASTA and GTF files.
- Human (hg38, hg19) and mouse genomes (mm10, mm9) have the option of further refining IR analysis using built-in mappability exclusion annotations, allowing SpliceWiz to ignore intronic regions of low mappability.
Load Demo FASTA/GTF (5), and then click Build Reference (6)
Where did the FASTA and GTF files come from?
The helper functions chrZ_genome() and chrZ_gtf() returns the paths to the example genome (FASTA) and transcriptome (GTF) file included with the NxtIRFdata package that contains the working example used by SpliceWiz:
What is the chrZ genome?
For the purpose of generating a running example to demonstrate SpliceWiz, we created an artificial genome / gene annotation. This was created using 7 human genes (SRSF1, SRSF2, SRSF3, TRA2A, TRA2B, TP53 and NSUN5). The SRSF and TRA family of genes all contain poison exons flanked by retained introns. Additionally, NSUN5 contains an annotated IR event in its terminal intron. Sequences from these 7 genes were aligned into one sequence to create an artificial chromosome Z (chrZ). The gene annotations were modified to only contain the 7 genes with the modified genomic coordinates.
What is the gene ontology species?
SpliceWiz supports gene ontology analysis. To enable this capability, we first need to generate the gene ontology annotations for the appropriate species.
To see a list of supported species:
getAvailableGO()
#> [1] "Anopheles gambiae"
#> [2] "Arabidopsis thaliana"
#> [3] "Bos taurus"
#> [4] "Canis familiaris"
#> [5] "Gallus gallus"
#> [6] "Pan troglodytes"
#> [7] "Escherichia coli"
#> [8] "Drosophila melanogaster"
#> [9] "Homo sapiens"
#> [10] "Mus musculus"
#> [11] "Sus scrofa"
#> [12] "Rattus norvegicus"
#> [13] "Macaca mulatta"
#> [14] "Caenorhabditis elegans"
#> [15] "Xenopus laevis"
#> [16] "Saccharomyces cerevisiae"
#> [17] "Danio rerio"
#> [18] "Triticum aestivum"
#> [19] "Triticum aestivum_subsp._aestivum"
#> [20] "Triticum vulgare"
#> [21] "Brassica napus"
#> [22] "Arachis hypogaea"
#> [23] "Hibiscus syriacus"
#> [24] "Acridium cancellatum"
#> [25] "Schistocerca cancellata"
#> [26] "Triticum dicoccoides"
#> [27] "Triticum turgidum_subsp._dicoccoides"
#> [28] "Triticum turgidum_var._dicoccoides"
#> [29] "Dendrohyas sarda"
#> [30] "Hyla arborea_sarda"
#> [31] "Hyla sarda"
#> [32] "Locusta gregaria"
#> [33] "Schistocerca gregaria"
#> [34] "Gossypium hirsutum"
#> [35] "Gossypium hirsutum_subsp._mexicanum"
#> [36] "Gossypium lanceolatum"
#> [37] "Gossypium purpurascens"
#> [38] "Camelina sativa"
#> [39] "Myagrum sativum"
#> [40] "Carassius auratus_gibelio"
#> [41] "Carassius gibelio_gibelio"
#> [42] "Carassius gibelio"
#> [43] "Carassius gibelio_subsp._gibelio"
#> [44] "Cyprinus gibelio"
#> [45] "Schistocerca piceifrons"
#> [46] "Papaver somniferum"
#> [47] "Zingiber officinale"
#> [48] "Trichomonas vaginalis_G3"
#> [49] "Trichomonas vaginalis_strain_G3"
#> [50] "Helianthus annuus"
#> [51] "Schistocerca americana"
#> [52] "Acipenser ruthenus"
#> [53] "Schistocerca serialis_cubense"
#> [54] "Panicum virgatum"
#> [55] "Nicotiana tabacum"
#> [56] "Oncorhynchus mykiss"
#> [57] "Oncorhynchus nerka_mykiss"
#> [58] "Parasalmo mykiss"
#> [59] "Salmo mykiss"
#> [60] "Schistocerca nitens"
#> [61] "Schistocerca vaga"
#> [62] "Salvia splendens"
#> [63] "Carassius carassius"
#> [64] "Cyprinus carassius"
#> [65] "Vicia villosa"
#> [66] "Camellia sinensis"
#> [67] "Thea sinensis"
#> [68] "Oncorhynchus keta"
#> [69] "Salmo keta"
#> [70] "Pisum sativum"
#> [71] "Salmo salar"
#> [72] "Raphanus sativus"
#> [73] "Oncorhynchus kisutch"
#> [74] "Oncorhyncus kisutch"
#> [75] "Salmo kisatch"
#> [76] "Lolium rigidum"
#> [77] "Aegilops squarrosa_subsp._squarrosa"
#> [78] "Aegilops squarrosa"
#> [79] "Aegilops tauschii"
#> [80] "Patropyrum tauschii_subsp._tauschii"
#> [81] "Patropyrum tauschii"
#> [82] "Triticum aegilops"
#> [83] "Triticum tauschii"
#> [84] "Salmo trutta"
#> [85] "Cryptomeria japonica"
#> [86] "Cupressus japonica"
#> [87] "Coregonus clupeaformis"
#> [88] "Salmo clupeaformis"
#> [89] "Oncorhynchus gorbuscha"
#> [90] "Salmo gorbuscha"
#> [91] "Cyprinus carpio"
#> [92] "Glycine max_subsp._soja"
#> [93] "Glycine soja"
#> [94] "Salmo fontinalis"
#> [95] "Salvelinus fontinalis"
#> [96] "Glycine max"
#> [97] "Phaseolus max"
#> [98] "Chenopodium quinoa"
#> [99] "Hordeum sativum"
#> [100] "Hordeum vulgare_subsp._vulgare"
#> [101] "Hordeum vulgare_var._nudum"
#> [102] "Hordeum vulgare_var._vulgare"
#> [103] "Festuca perennis_(L.)_Columbus_&_J.P.Sm.,_2010"
#> [104] "Festuca perennis"
#> [105] "Lolium perenne"
#> [106] "Lolium vulgare"
#> [107] "Coffea arabica"
#> [108] "Barbus grahami"
#> [109] "Sinocyclocheilus grahami"
#> [110] "Sinocyclocheilus rhinocerous"
#> [111] "Gossypium arboreum"
#> [112] "Brassica oleracea"
#> [113] "Malus sylvestris"
#> [114] "Pyrus malus_var._sylvestris"
#> [115] "Astyanax mexicanus"
#> [116] "Tetragonopterus mexicanus"
#> [117] "Arachis stenosperma"
#> [118] "Prosopis alba"
#> [119] "Sinocyclocheilus anshuiensis"
#> [120] "Brassica rapa"
#> [121] "Lactuca sativa"
#> [122] "Dreissena polymorpha"
#> [123] "Mytilus polymorphus"
#> [124] "Hydractinia symbiolongicarpus"
#> [125] "Triticum urartu"
#> [126] "Hevea brasiliensis"
#> [127] "Siphonia brasiliensis"
#> [128] "Oncorhynchus tschawytscha"
#> [129] "Oncorhynchus tshawytscha"
#> [130] "Salmo tshawytscha"
#> [131] "Arachis ipaensis"
#> [132] "Zea mays"
#> [133] "Zea mays_var._japonica"
#> [134] "Salmo namaycush"
#> [135] "Salvelinus namaycush"
#> [136] "Capsicum annuum"
#> [137] "Brienomyrus brachyistius"
#> [138] "Marcusenius brachyistius"
#> [139] "Convolvulus nil"
#> [140] "Ipomoea nil"
#> [141] "Pharbitis nil"
#> [142] "Olea europaea_subsp._europaea_var._sylvestris"
#> [143] "Olea europaea_var._oleaster"
#> [144] "Olea europaea_var._sylvestris"
#> [145] "Olea europea_subsp._sylvestris"
#> [146] "Alosa sapidissima"
#> [147] "Clupea sapidissima"
#> [148] "Carpiodes asiaticus"
#> [149] "Myxocyprinus asiaticus"
#> [150] "Actinidia eriantha"
#> [151] "Gossypium raimondii"
#> [152] "Salmo alpinus"
#> [153] "Salvelinus alpinus"
#> [154] "Catostomus texanus"
#> [155] "Xyrauchen texanus"
#> [156] "Doryrhamphus excisus"
#> [157] "Quercus lobata"
#> [158] "Malus communis"
#> [159] "Malus domestica"
#> [160] "Malus pumila_auct."
#> [161] "Malus pumila_var._domestica"
#> [162] "Malus sylvestris_var._domestica"
#> [163] "Malus x_domestica"
#> [164] "Pyrus malus"
#> [165] "Pyrus malus_var._domestica"
#> [166] "Quercus suber"
#> [167] "Oncorhynchus nerka"
#> [168] "Salmo nerka"
#> [169] "Nicotiana tomentosiformis"
#> [170] "Carya illinoensis"
#> [171] "Carya illinoinensis"
#> [172] "Mercenaria mercenaria"
#> [173] "Venus mercenaria"
#> [174] "Quercus robur"
#> [175] "Durio zibethinus"
#> [176] "Pongo abelii"
#> [177] "Pongo pygmaeus_abelii"
#> [178] "Pongo pygmaeus_abeli"
#> [179] "Mya arenaria"
#> [180] "Arachis duranensis"
#> [181] "Arachis spegazzinii"
#> [182] "Pyrus x_bretschneideri"
#> [183] "Trifolium pratense"
#> [184] "Gorilla gorilla_gorilla"
#> [185] "Cobitis anguillicaudata"
#> [186] "Misgurnus anguillicaudatus"
#> [187] "Scaphiopus bombifrons"
#> [188] "Spea bombifrons"
#> [189] "Haliotis rufenscens"
#> [190] "Haliotis rufescens"
#> [191] "Oreochromis nilotica"
#> [192] "Oreochromis niloticus"
#> [193] "Perca nilotica"
#> [194] "Tilapia nilotica"
#> [195] "Acropora convexa"
#> [196] "Acropora millepora"
#> [197] "Acropora singularis"
#> [198] "Cebus apella"
#> [199] "Sapajus apella"
#> [200] "Simia apella"
#> [201] "Eucalyptus grandis"
#> [202] "Dasypus fenestratus"
#> [203] "Dasypus novemcinctus"
#> [204] "Callithrix jacchus_jacchus"
#> [205] "Callithrix jacchus"
#> [206] "Simia jacchus"
#> [207] "Pistacia vera"
#> [208] "greater Indian_fruit_bat"
#> [209] "Pteropus giganteus"
#> [210] "Pteropus medius"
#> [211] "Salvia miltiorhiza"
#> [212] "Salvia miltiorrhiza"
#> [213] "Daphnia pulicaria"
#> [214] "Magnolia sinica"
#> [215] "Manglietia sinica"
#> [216] "Manglietiastrum sinicum"
#> [217] "Pachylarnax sinica_(Y.W.Law)_N.H.Xia_&_C.Y.Wu"
#> [218] "Rosa chinensis"
#> [219] "Rosa indica_auct.,_non_L."
#> [220] "Mytilus californianus"
#> [221] "Pteropus vampyrus"
#> [222] "Vespertilio vampyrus"
#> [223] "Chinemys reevesii"
#> [224] "Chinemys reevesi"
#> [225] "Emys reevesii"
#> [226] "Geoclemys reevesii"
#> [227] "Geoclemys reevessi"
#> [228] "Mauremys reevesii"
#> [229] "Mauremys reevesi"
#> [230] "Choloepus brasiliensis_Fitzinger_1871"
#> [231] "Choloepus brasiliensis"
#> [232] "Choloepus didactylus"
#> [233] "Macaca nemestrina"
#> [234] "Simia nemestrina"
#> [235] "Lotus corniculatus_var._japonicus"
#> [236] "Lotus japonicus"
#> [237] "Nicotiana sylvestris"
#> [238] "Tupaia belangeri_chinensis"
#> [239] "Tupaia chinensis"
#> [240] "Clarias gariepinus"
#> [241] "Clarias lazera"
#> [242] "Silurus gariepinus"
#> [243] "Barbus tetrazona"
#> [244] "Capoeta tetrazona"
#> [245] "Puntigrus tetrazona"
#> [246] "Puntius tetrazona"
#> [247] "Systomus tetrazona"
#> [248] "Lycium ferocissimum"
#> [249] "Nicotiana attenuata"
#> [250] "Octodon degus"
#> [251] "Haliotis rubra"
#> [252] "Aedes albopictus"
#> [253] "Stegomyia albopicta"
#> [254] "Spinacia oleracea"
#> [255] "Paramecium aurelia_syngen_4"
#> [256] "Paramecium tetraurelia"
#> [257] "Salvia hispanica"
#> [258] "Medicago truncatula"
#> [259] "Crassostrea virginica"
#> [260] "Ostrea virginica"
#> [261] "Felis catus"
#> [262] "Felis domesticus"
#> [263] "Felis silvestris_catus"
#> [264] "Anubis baboon"
#> [265] "Papio anubis"
#> [266] "Papio cynocephalus_anubis"
#> [267] "Papio doguera"
#> [268] "Papio hamadryas_anubis"
#> [269] "Papio hamadryas_doguera"
#> [270] "Simia anubis"
#> [271] "Pongo pygmaeus"
#> [272] "Simia pygmaeus"
#> [273] "Sorex etruscus"
#> [274] "Suncus etruscus"
#> [275] "Mimosa cineraria"
#> [276] "Prosopis cineraria"
#> [277] "Nycticebus coucang"
#> [278] "Tardigradus coucang"
#> [279] "Rhododendron vialii"
#> [280] "Pan paniscus"
#> [281] "Nematostella vectensis"
#> [282] "Ixodes dammini"
#> [283] "Ixodes scapularis"
#> [284] "Lupinus angustifolius"
#> [285] "Ipomoea triloba"
#> [286] "Equus asinus"
#> [287] "Emiliania huxleyi_CCMP1516"
#> [288] "Emiliania huxleyi_CCMP2090"
#> [289] "Mangifera indica"
#> [290] "Pteropus alecto"
#> [291] "Rana temporaria"
#> [292] "Crassostrea gigas"
#> [293] "Magallana gigas"
#> [294] "Ostrea gigas"
#> [295] "Etheostoma spectabile"
#> [296] "Poecilichthys spectabilis"
#> [297] "Macadamia integrifolia"
#> [298] "Megalobrama amblycephala"
#> [299] "Halichoerus grypus"
#> [300] "Phoca grypus"
#> [301] "Juglans regia"
#> [302] "Selaginella moellendorffii"
#> [303] "Selaginella moellendorfii"
#> [304] "Pleuronectes platessa"
#> [305] "Presbytis francoisi"
#> [306] "Trachypithecus francoisi"
#> [307] "Tripterygium wilfordii"
#> [308] "Argiope bruennichi"
#> [309] "Lepus cuniculus"
#> [310] "Oryctolagus cuniculus"
#> [311] "Huro salmoides"
#> [312] "Labrus salmoides"
#> [313] "Labrus salmonides"
#> [314] "Micropterus nigricans"
#> [315] "Micropterus salmoides"
#> [316] "Solanum stenotomum"
#> [317] "Heterocephalus glaber"
#> [318] "Neosciurus carolinensis"
#> [319] "Sciurus carolinensis"
#> [320] "Cervus elaphus"
#> [321] "Polyodon spathula"
#> [322] "Squalus spathula"
#> [323] "Gadus chalcogrammus"
#> [324] "Theragra chalcogramma"
#> [325] "Nothobranchius furzeri"
#> [326] "Bos bubalis"
#> [327] "Bubalus arnee_bubalis"
#> [328] "Bubalus bubalis"
#> [329] "Pleuronectes solea"
#> [330] "Solea solea"
#> [331] "Solea vulgaris"
#> [332] "Mastomys coucha"
#> [333] "Praomys coucha"
#> [334] "Impatiens glandulifera"
#> [335] "Dermacentor andersoni"
#> [336] "Felis nebulosa"
#> [337] "Neofelis nebulosa"
#> [338] "Pteropus egyptiacus"
#> [339] "Rousettus aegyptiacus"
#> [340] "Rousettus aegypticus"
#> [341] "Rousettus egyptiacus"
#> [342] "Phoenix dactylifera"
#> [343] "Pimephales promelas"
#> [344] "Ostrea edulis"
#> [345] "Peromyscus maniculatus_bairdii"
#> [346] "Gasterosteus pungitius"
#> [347] "Pungitius pungitius"
#> [348] "Populus alba"
#> [349] "Cricetus auratus"
#> [350] "Golden hamsters"
#> [351] "Mesocricetus auratus"
#> [352] "Syrian hamsters"
#> [353] "Chromis aureus"
#> [354] "Oreochromis aurea"
#> [355] "Oreochromis aureus"
#> [356] "Daucus carota_subsp._sativus"
#> [357] "Daucus carota_var._sativus"
#> [358] "Dermacentor silvarum"
#> [359] "Hylobates syndactylus"
#> [360] "Simia syndactyla"
#> [361] "Symphalangus syndactylus"
#> [362] "Saccharolobus solfataricus"
#> [363] "Sulfolobus solfataricus"
#> [364] "Felis geoffroyi"
#> [365] "Leopardus geoffroyi"
#> [366] "Oncifelis geoffroyi"
#> [367] "Felis yagouaroundi"
#> [368] "Herpailurus yagouaroundi"
#> [369] "Herpailurus yaguarondi"
#> [370] "Puma yagouaroundii"
#> [371] "Puma yagouaroundi"
#> [372] "Cervus canadensis"
#> [373] "Populus diversifolia"
#> [374] "Populus euphratica"
#> [375] "Cucurbita pepo_subsp._pepo"
#> [376] "Cucurbita pepo_var._medullosa"
#> [377] "Cucurbita pepo_var._pepo"
#> [378] "Macaca cynomolgus"
#> [379] "Macaca fascicularis"
#> [380] "Macaca irus"
#> [381] "Simia fascicularis"
#> [382] "Emys muticus"
#> [383] "Geoclemmys mutica"
#> [384] "Mauremys mutica"
#> [385] "Suricata suricatta"
#> [386] "Viverra suricatta"
#> [387] "Hylobates moloch"
#> [388] "Simia moloch"
#> [389] "Solanum dulcamara"
#> [390] "Cucurbita moschata"
#> [391] "Coffea eugeniodes"
#> [392] "Coffea eugenioides"
#> [393] "Cucurbita maxima"
#> [394] "Labrus bergylta"
#> [395] "Centropristis striata"
#> [396] "Labrus striatus"
#> [397] "Oryza sativa_(japonica_cultivar-group)"
#> [398] "Oryza sativa_Japonica_Group"
#> [399] "Oryza sativa_subsp._japonica"
#> [400] "Jaculus jaculus"
#> [401] "Mus jaculus"
#> [402] "Dioscorea cayenensis_subsp._rotundata_(Poir.)_J.Miege,_1968"
#> [403] "Dioscorea cayenensis_subsp._rotundata"
#> [404] "Dioscorea rotundata"
#> [405] "Cercopithecus aethiops_sabaeus"
#> [406] "Cercopithecus sabaeus"
#> [407] "Cercopithecus sabeus"
#> [408] "Chlorocebus aethiops_sabaeus"
#> [409] "Chlorocebus aethiops_sabeus"
#> [410] "Chlorocebus sabaeus"
#> [411] "Chlorocebus sabeus"
#> [412] "Simia sabaea"
#> [413] "Marmota monax"
#> [414] "Mus monax"
#> [415] "Pygathrix roxellana"
#> [416] "Rhinopithecus roxellana"
#> [417] "Semnopithecus roxellana"
#> [418] "Callorhinus ursinus"
#> [419] "Callorhynus ursius"
#> [420] "Phoca ursina"
#> [421] "Cricetulus barabensis_griseus"
#> [422] "Cricetulus griseus"
#> [423] "Elephantulus edwardii"
#> [424] "Macroscelides edwardii"
#> [425] "Cobitis heteroclita"
#> [426] "Fundulus heteroclitus"
#> [427] "Neothunnus macropterus"
#> [428] "Scomber albacares"
#> [429] "Thunnus albacares"
#> [430] "Telopea speciosissima"
#> [431] "Danio aesculapii"
#> [432] "Danio sp._'snakeskin'"
#> [433] "Danio sp._snakeskin"
#> [434] "Apodemus sylvaticus"
#> [435] "Mus sylvaticus"
#> [436] "Sylvaemus sylvaticus"
#> [437] "Populus balsamifera_subsp._trichocarpa"
#> [438] "Populus trichocarpa"
#> [439] "Mercurialis ambigua"
#> [440] "Mercurialis annua"
#> [441] "Eugenia oleosa"
#> [442] "Syzygium oleosum"
#> [443] "Citellus tridecemlineatus"
#> [444] "Ictidomys tridecemlineatus"
#> [445] "Spermophilus tridecemlineatus"
#> [446] "Ovis ammon_aries"
#> [447] "Ovis aries"
#> [448] "Ovis orientalis_aries"
#> [449] "Ovis ovis"
#> [450] "Solanum verrucosum"
#> [451] "Leo pardus"
#> [452] "Panthera pardus"
#> [453] "Microtus oregoni"
#> [454] "Arabidopsis lyrata_subsp._lyrata"
#> [455] "Arabis lyrata_subsp._lyrata"
#> [456] "Arabis lyrata"
#> [457] "Cardaminopsis lyrata"
#> [458] "Manihot esculenta"
#> [459] "Manihot utilissima"
#> [460] "Mustela erminea"
#> [461] "Dolichos unguiculatus"
#> [462] "Phaseolus unguiculatus"
#> [463] "Vigna unguiculata"
#> [464] "Lycopersicon pennellii_(Correll)_D'Arcy,_1982"
#> [465] "Solanum pennellii_Correll,_1958"
#> [466] "Solanum pennellii"
#> [467] "Panicum viride"
#> [468] "Setaria viridis"
#> [469] "Musa AA_Group"
#> [470] "Musa acuminata_AA_Group"
#> [471] "Musa acuminata"
#> [472] "Musa nana"
#> [473] "Gymnostomus macrolepis"
#> [474] "Onychostoma macrolepis"
#> [475] "Scaphesthes macrolepis"
#> [476] "Varicorhinus macrolepis"
#> [477] "Varicorhinus (Scaphesthes)_macrolepis"
#> [478] "Oryza glaberrima"
#> [479] "Pelteobagrus fulvidraco"
#> [480] "Pimelodus fulvidraco"
#> [481] "Pseudobagrus fulvidraco"
#> [482] "Tachysurus fulvidraco"
#> [483] "Hylobates concolor_leucogenys"
#> [484] "Hylobates concolor_leucogyneus"
#> [485] "Hylobates leucogenys_leucogenys"
#> [486] "Hylobates leucogenys"
#> [487] "Nomascus leucogenys_leucogenys"
#> [488] "Nomascus leucogenys"
#> [489] "Nomascus leukogenys"
#> [490] "Nannospalax ehrenbergi_galili"
#> [491] "Nannospalax galili"
#> [492] "Spalax galili"
#> [493] "Equus caballus"
#> [494] "Equus przewalskii_f._caballus"
#> [495] "Equus przewalskii_forma_caballus"
#> [496] "Thunnus maccoyii"
#> [497] "Thynnus maccoyii"
#> [498] "Chromis diagramma"
#> [499] "Simochromis diagramma"
#> [500] "Diplophysa dalaica"
#> [501] "Triplophysa dalaica"
#> [502] "Felis tigris"
#> [503] "Panthera tigris"
#> [504] "Echinus purpuratus"
#> [505] "Strongylocentrotus purpuratus"
#> [506] "Lucioperca lucioperca"
#> [507] "Perca lucioperca"
#> [508] "Sander lucioperca"
#> [509] "Stizostedion lucioperca"
#> [510] "Dipodomys spectabilis"
#> [511] "Acinonyx jubatus"
#> [512] "Felis jubata"
#> [513] "Conyza canadensis"
#> [514] "Erigeron canadensis"
#> [515] "Mustela lutreola"
#> [516] "Camelus bactrianus_ferus"
#> [517] "Camelus ferus"
#> [518] "Cajanus cajan"
#> [519] "Didelphys domestica"
#> [520] "Monodelphis domestica"
#> [521] "Pygathrix bieti"
#> [522] "Rhinopithecus bieti"
#> [523] "Saimiri boliviensis"
#> [524] "Hesperomys eremicus"
#> [525] "Peromyscus eremicus"
#> [526] "Arabidopsis salsuginea"
#> [527] "Eutrema salsugineum"
#> [528] "Hesperis salsuginea"
#> [529] "Sisymbrium salsugineum"
#> [530] "Stenophragma salsugineum"
#> [531] "Thellungiella salsuginea"
#> [532] "Thelypodium salsugineum"
#> [533] "Coetomys damarensis"
#> [534] "Cryptomys damarensis"
#> [535] "Fukomys damarensis"
#> [536] "Leptonychotes weddellii"
#> [537] "Leptonychotes weddelli"
#> [538] "Otaria weddellii"
#> [539] "Grammomys dolichurus_surdaster"
#> [540] "Grammomys surdaster"
#> [541] "Thamnomys surdaster"
#> [542] "Solanum aracc-papa"
#> [543] "Solanum tuberosum"
#> [544] "Andropogon sorghum"
#> [545] "Sorghum bicolor"
#> [546] "Sorghum bicolor_subsp._bicolor"
#> [547] "Sorghum nervosum"
#> [548] "Sorghum saccharatum"
#> [549] "Sorghum vulgare"
#> [550] "Holocentrus calcarifer"
#> [551] "Lates calcarifer"
#> [552] "Hippopotamus amphibius_kiboko"
#> [553] "Ixodes sanguineus"
#> [554] "Rhipicephalus sanguineus"
#> [555] "Clupea harengus_harengus"
#> [556] "Clupea harengus"
#> [557] "Bos indicus_x_Bos_taurus"
#> [558] "Bos primigenius_indicus_x_Bos_primigenius_taurus"
#> [559] "Bos taurus_indicus_x_Bos_taurus_taurus"
#> [560] "Bos taurus_x_Bos_indicus"
#> [561] "Chrysochloris asiatica"
#> [562] "Talpa asiatica"
#> [563] "Bufo bufo"
#> [564] "Rana bufo"
#> [565] "Maylandia zebra"
#> [566] "Metriaclima zebra"
#> [567] "Pseudotropheus sp._'Pseudotropheus_zebra_complex'"
#> [568] "Pseudotropheus zebra"
#> [569] "Ictalurus punctatus"
#> [570] "Silurus punctatus"
#> [571] "Oryx dammah"
#> [572] "Camelus dromedarius"
#> [573] "Asparagus litoralis"
#> [574] "Asparagus officinalis"
#> [575] "Amaranthus gangeticus"
#> [576] "Amaranthus mangostanus"
#> [577] "Amaranthus tricolor"
#> [578] "Pneumatophorus japonicus"
#> [579] "Scomber japonicus"
#> [580] "Lutra lutra"
#> [581] "Peromyscus leucopus"
#> [582] "Perca fluviatilis"
#> [583] "Pagothenia bernacchii"
#> [584] "Pseudotrematomus bernacchii"
#> [585] "Trematomus bernacchii"
#> [586] "Trematomus bernacchi"
#> [587] "Lipurus cinereus"
#> [588] "Phascolarctos cinereus"
#> [589] "Mustela furo"
#> [590] "Mustela putorius_furo"
#> [591] "Chaetochloa italica"
#> [592] "Panicum italicum"
#> [593] "Pennisetum macrochaetum"
#> [594] "Setaria italica"
#> [595] "Setaria viridis_subsp._italica"
#> [596] "Elaeis guineensis"
#> [597] "Mus rattus"
#> [598] "Rattus rattoides"
#> [599] "Rattus rattus"
#> [600] "Rattus wroughtoni"
#> [601] "Acropora digitifera"
#> [602] "Madrepora digitifera"
#> [603] "Echinops telfairii"
#> [604] "Echinops telfairi"
#> [605] "Madrepora verrucosa"
#> [606] "Pocillopora danae"
#> [607] "Pocillopora verrucosa"
#> [608] "Myotis daubentonii"
#> [609] "Myotis daubentoni"
#> [610] "Vespertilio daubentonii"
#> [611] "Limia formosa"
#> [612] "Mollienesia formosa"
#> [613] "Poecilia formosa"
#> [614] "Phyllostomus discolor"
#> [615] "Microcebus murinus"
#> [616] "Peromyscus californicus_insignis"
#> [617] "Peromyscus californicus_subsp._insignis"
#> [618] "Galago garnettii"
#> [619] "Galago garnetti"
#> [620] "Otolemur garnettii"
#> [621] "Arvicanthis niloticus"
#> [622] "Mus niloticus"
#> [623] "Didelphis ursina"
#> [624] "Vombatus ursinus"
#> [625] "Phaseolus angularis"
#> [626] "Vigna angularis"
#> [627] "Haitia acuta"
#> [628] "Physa acuta"
#> [629] "Physa heterostropha"
#> [630] "Physa integra"
#> [631] "Physella acuta"
#> [632] "Physella heterostropha"
#> [633] "Physella integra"
#> [634] "Ctenopharyngodon idella"
#> [635] "Ctenopharyngodon idellus"
#> [636] "Leuciscus idella"
#> [637] "Thalassophryne amazonica"
#> [638] "Cyprinus rohita"
#> [639] "Labeo rohita"
#> [640] "Talpa occidentalis"
#> [641] "Bombina bombina"
#> [642] "Rana bombina"
#> [643] "Cavia aperea_porcellus"
#> [644] "Cavia cobaya"
#> [645] "Cavia porcellus"
#> [646] "Mus porcellus"
#> [647] "Odocoileus virginianus"
#> [648] "Amphibalanus amphitrite"
#> [649] "Balanus amphitrite"
#> [650] "Panicum hallii"
#> [651] "Angill angill"
#> [652] "Anguilla anguilla_anguilla"
#> [653] "Anguilla anguilla"
#> [654] "Muraena anguilla"
#> [655] "Delphinus orca"
#> [656] "Orcinus orca"
#> [657] "Cannabis sativa"
#> [658] "Penaeus bubulus"
#> [659] "Penaeus carinatus"
#> [660] "Penaeus durbani"
#> [661] "Penaeus monodon"
#> [662] "Penaeus (Penaeus)_monodon"
#> [663] "Didelphis vulpecula"
#> [664] "Trichosurus vulpecula"
#> [665] "Myotis lucifugus"
#> [666] "Vespertilio lucifugus"
#> [667] "Brachypodium distachyon"
#> [668] "Bromus distachyos"
#> [669] "Aotus nancymaae"
#> [670] "Aotus nancymai"
#> [671] "Rhamnus zizyphus"
#> [672] "Ziziphus jujuba"
#> [673] "Ailuropoda melanoleuca"
#> [674] "Micropterus dolomieu"
#> [675] "Micropterus velox"
#> [676] "Lycopersicon esculentum"
#> [677] "Lycopersicon esculentum_var._esculentum"
#> [678] "Solanum esculentum"
#> [679] "Solanum lycopersicum"
#> [680] "Solanum lycopersicum_var._humboldtii"
#> [681] "Poecilia mexicana"
#> [682] "Manis pentadactyla"
#> [683] "Meles meles"
#> [684] "Ursus meles"
#> [685] "Ornithorhynchus anatinus"
#> [686] "Platypus anatinus"
#> [687] "Felis uncia"
#> [688] "Panthera uncia"
#> [689] "Uncia uncia"
#> [690] "Alligator mississippiensis"
#> [691] "Crocodilus mississipiensis"
#> [692] "Myrmecophaga aculeata"
#> [693] "Tachyglossus aculeatus"
#> [694] "Colossoma macropomum"
#> [695] "Myletes macropomus"
#> [696] "Cordylus capensis"
#> [697] "Cordylus (Hemicordylus)_capensis"
#> [698] "Hemicordylus capensis"
#> [699] "Pseudocordylus capensis"
#> [700] "Zonurus capensis"
#> [701] "Eptesicus fuscus"
#> [702] "Vespertilio fuscus"
#> [703] "Dromiciops australis"
#> [704] "Dromiciops gliroides"
#> [705] "Camelus pacos"
#> [706] "Lama guanicoe_pacos"
#> [707] "Lama pacos"
#> [708] "Vicugna pacos"
#> [709] "Mollienesia latipinna"
#> [710] "Poecilia latipinna"
#> [711] "Elephas maximus_indicus"
#> [712] "Corylus avellana"
#> [713] "Ostrea maxima"
#> [714] "Pecten maximus"
#> [715] "Felis viverrina"
#> [716] "Prionailurus viverrinus"
#> [717] "Gymnodraco acuticeps"
#> [718] "Thalarctos maritimus"
#> [719] "Ursus maritimus"
#> [720] "Lemur catta"
#> [721] "Myotis myotis"
#> [722] "Vespertilio myotis"
#> [723] "Lytechinus pictus"
#> [724] "Psammechinus pictus"
#> [725] "Litopenaeus vannamei"
#> [726] "Penaeus (Litopenaeus)_vannamei"
#> [727] "Penaeus vannamei"
#> [728] "Ursus arctos"
#> [729] "Vitis riparia"
#> [730] "Felis bengalensis"
#> [731] "Prionailurus bengalensis"
#> [732] "Clethrionomys glareolus"
#> [733] "Mus glareolus"
#> [734] "Myodes glareolus"
#> [735] "Mustela nigripes"
#> [736] "Putorius nigripes"
#> [737] "Alopex lagopus"
#> [738] "Canis lagopus"
#> [739] "Vulpes lagopus"
#> [740] "Cercocebus atys"
#> [741] "Cercocebus torquatus_atys"
#> [742] "Simia atys"
#> [743] "Lepidosiren annectens"
#> [744] "Protopterus annectens"
#> [745] "Rhinocryptis annectens"
#> [746] "Cerasus avium"
#> [747] "Prunus avium"
#> [748] "Prunus cerasus_var._avium"
#> [749] "Procambarus clarkii"
#> [750] "Sorex fumeus"
#> [751] "Macrorhinus angustirostris"
#> [752] "Mirounga angustirostris"
#> [753] "Beta vulgaris_subsp._vulgaris"
#> [754] "Beta vulgaris_subsp._vulgaris_var._altissima"
#> [755] "Beta vulgaris_Sugar_Beet_Group"
#> [756] "Beta vulgaris_var._altissima"
#> [757] "Eumetopias jubatus"
#> [758] "Phoca jubata"
#> [759] "Centruroides sculpturatus"
#> [760] "Diceros bicornis_minor"
#> [761] "Cicer arietinum"
#> [762] "Cleome hassleriana_Chodat,_1898"
#> [763] "Tarenaya hassleriana"
#> [764] "Sebastes umbrosus"
#> [765] "Sebastichthys umbrosus"
#> [766] "Eriocheir chinensis"
#> [767] "Eriocheir japonica_sinensis"
#> [768] "Eriocheir sinensis"
#> [769] "Dicentrarchus labrax"
#> [770] "Labrax labrax"
#> [771] "Morone labrax"
#> [772] "Perca labrax"
#> [773] "Roccus labrax"
#> [774] "Sciaena labrax"
#> [775] "Acanthopagrus latus"
#> [776] "Sparus latus"
#> [777] "Xiphophorus hellerii"
#> [778] "Xiphophorus helleri"
#> [779] "Acanthochromis polyacanthus"
#> [780] "Acanthochromis polyacathus"
#> [781] "Dascyllus polyacanthus"
#> [782] "Mustela vison"
#> [783] "Neogale vison"
#> [784] "Neovison vison"
#> [785] "Lingula anatina"
#> [786] "Lingula lingua"
#> [787] "Lingula nipponica"
#> [788] "Lingula unguis"
#> [789] "Madrepora faveolata"
#> [790] "Montastraea faveolata"
#> [791] "Montastrea faveolata"
#> [792] "Orbicella faveolata"
#> [793] "Esox lucius"
#> [794] "Chinchilla lanigera"
#> [795] "Chinchilla velligera"
#> [796] "Chinchilla villidera"
#> [797] "Mirounga leonina"
#> [798] "Phoca leonina"
#> [799] "Perognathus longimembris_pacificus"
#> [800] "Cynocephalus variegatus"
#> [801] "Galeopithecus variegatus"
#> [802] "Galeopterus variegatus"
#> [803] "Vigna radiata"
#> [804] "Vitis vinifera"
#> [805] "Vitis vinifera_subsp._vinifera"
#> [806] "Characodon multiradiatus"
#> [807] "Girardinichthys multiradiatus"
#> [808] "Marmota flaviventris"
#> [809] "Phaseolus calcaratus"
#> [810] "Phaseolus chrysanthos"
#> [811] "Phaseolus chrysanthus"
#> [812] "Vigna calcarata"
#> [813] "Vigna umbellata"
#> [814] "Balaenoptera acutorostrata"
#> [815] "Canis procyonoides"
#> [816] "Nyctereutes procyonoides"
#> [817] "Amphioxus floridae"
#> [818] "Branchiostoma floridae"
#> [819] "Moschus berezovskii"
#> [820] "Erythranthe guttata"
#> [821] "Mimulus guttatus_subsp._guttatus"
#> [822] "Mimulus guttatus"
#> [823] "Camelus bactrianus"
#> [824] "Desmodus rotundus"
#> [825] "Phyllostoma rotundum"
#> [826] "Octopus sinensis"
#> [827] "Physeter catodon"
#> [828] "Physeter macrocephalus"
#> [829] "Alexandromys fortis"
#> [830] "Microtus fortis"
#> [831] "Dendronephthya gigantea"
#> [832] "Canis hyaena"
#> [833] "Hyaena hyaena"
#> [834] "Helicophagus hypophthalmus"
#> [835] "Pangasianodon hypophthalmus"
#> [836] "Pangasius hypophthalmus"
#> [837] "Pangasius sutchi"
#> [838] "Pseudochaenichthys georgianus"
#> [839] "Capsella rubella"
#> [840] "Perkinsus marinus_ATCC_50983"
#> [841] "Holocentrus leopardus"
#> [842] "Plectropomus leopardus"
#> [843] "Hippocampus zosterae"
#> [844] "Artibeus jamaicensis"
#> [845] "Citrus sinensis"
#> [846] "Citrus x_sinensis"
#> [847] "Punica granatum"
#> [848] "Abrus cyaneus"
#> [849] "Abrus precatorius"
#> [850] "Polypterus senegalus"
#> [851] "Acomys russatus"
#> [852] "Mus russatus"
#> [853] "Hemibagrus wyckioides"
#> [854] "Macrones wyckioides"
#> [855] "Mystus wyckioides"
#> [856] "Melanotaenia boesemani"
#> [857] "Sturnira hondurensis"
#> [858] "Amphilophus centrarchus"
#> [859] "Archocentrus centrarchus"
#> [860] "Cichlasoma centrarchus"
#> [861] "Heros centrarchus"
#> [862] "Delphinus melas"
#> [863] "Globicephala melaena"
#> [864] "Globicephala melas"
#> [865] "Manis javanica"
#> [866] "Phyllostomus hastatus"
#> [867] "Vespertilio hastatus"
#> [868] "Scyliorhinus canicula"
#> [869] "Squalus canicula"
#> [870] "Silurana tropicalis"
#> [871] "Xenopus laevis_tropicalis"
#> [872] "Xenopus (Silurana)_tropicalis"
#> [873] "Xenopus tropicalis"
#> [874] "Pipistrellus kuhlii"
#> [875] "Pipistrellus kuhli"
#> [876] "Vespertilio kuhlii"
#> [877] "Solea senegalensis"
#> [878] "Mugil cephalotus"
#> [879] "Mugil cephalus"
#> [880] "Mugil galapagensis"
#> [881] "Mugil japonicus"
#> [882] "Siphostoma scovelli"
#> [883] "Syngnathus scovelli"
#> [884] "Capra aegagrus_hircus"
#> [885] "Capra hircus"
#> [886] "Poeciliopsis prolifica"
#> [887] "Gopherus flavomarginatus"
#> [888] "Lontra canadensis"
#> [889] "Lutra canadensis"
#> [890] "Hesperomys torridus"
#> [891] "Onychomys torridus"
#> [892] "Elephas africanus"
#> [893] "Loxodonta africana_africana"
#> [894] "Loxodonta africana"
#> [895] "Boophilus microplus"
#> [896] "Rhipicephalus (Boophilus)_microplus"
#> [897] "Rhipicephalus microplus"
#> [898] "Molossus molossus"
#> [899] "Vespertilio molossus"
#> [900] "Lagenorhynchus obliquidens"
#> [901] "Delphinus cymodoce"
#> [902] "Delphinus truncatus"
#> [903] "Tursiops cymodoce"
#> [904] "Tursiops truncatus"
#> [905] "Morone flavescens"
#> [906] "Perca flavescens"
#> [907] "Euarctos americanus"
#> [908] "Ursus americanus"
#> [909] "Arvicola nivalis"
#> [910] "Chionomys nivalis"
#> [911] "Microtus nivalis"
#> [912] "Felis rufus"
#> [913] "Lynx rufus"
#> [914] "Myotis brandtii"
#> [915] "Vespertilio brandtii"
#> [916] "Astatotilapia burtoni"
#> [917] "Chromis burtoni"
#> [918] "Haplochromis burtoni"
#> [919] "Silurus meridionalis"
#> [920] "Silurus soldatovi_meridionalis"
#> [921] "Cucumis melo"
#> [922] "Hydra attenuata"
#> [923] "Hydra carnea"
#> [924] "Hydra littoralis"
#> [925] "Hydra magnipapillata"
#> [926] "Hydra vulgaris"
#> [927] "Anoplopoma fimbria"
#> [928] "Gadus fimbria"
#> [929] "Alosa alosa"
#> [930] "Clupea alosa"
#> [931] "Chelonia mydas"
#> [932] "Testudo mydas"
#> [933] "Ctenocephalides felis"
#> [934] "Stylophora pistillata"
#> [935] "Cyrtodiopsis dalmanii"
#> [936] "Diopsis dalmanni"
#> [937] "Teleopsis dalmanni"
#> [938] "Rhagoletis zephyria"
#> [939] "Rhodamnia argentea"
#> [940] "Gasterosteus aculeatus"
#> [941] "Labrus celidotus"
#> [942] "Notolabrus celidotus"
#> [943] "Budorcas taxicolor"
#> [944] "Nelumbo nucifera"
#> [945] "Amphiprion ocellaris"
#> [946] "Arvicola amphibius"
#> [947] "Arvicola terrestris_(Linnaeus,_1758)"
#> [948] "Mus amphibius"
#> [949] "Daphnia magna"
#> [950] "Phaseolus vulgaris"
#> [951] "Psammomys obesus"
#> [952] "Carlito syrichta"
#> [953] "Simia syrichta"
#> [954] "Tarsius syrichta"
#> [955] "Cyprinodon tularosa"
#> [956] "Arvicola princeps"
#> [957] "Ochotona princeps"
#> [958] "Phytophthora sojae"
#> [959] "Equus caballus_przewalskii"
#> [960] "Equus ferus_przewalskii"
#> [961] "Equus przewalskii"
#> [962] "Phoca vitulina"
#> [963] "Coecilia bivitatum"
#> [964] "Rhinatrema bivitattum"
#> [965] "Rhinatrema bivittatum"
#> [966] "Lagomys curzoniae"
#> [967] "Ochotona curzonae"
#> [968] "Ochotona curzoniae"
#> [969] "Kogia breviceps"
#> [970] "Physeter breviceps"
#> [971] "Clupea cyprinoides"
#> [972] "Megalops cyprinoides"
#> [973] "Diospyros lotus"
#> [974] "Hippoglossus stenolepis"
#> [975] "Phacochoerus africanus"
#> [976] "Corythoichthys intestinalis"
#> [977] "Syngnatus intestinalis"
#> [978] "Mandrillus leucophaeus"
#> [979] "Papio leucophaeus"
#> [980] "Simia leucophaea"
#> [981] "Epinephelus fuscoguttatus"
#> [982] "Perca summana_fuscoguttata"
#> [983] "Asterias miniata"
#> [984] "Asterina miniata"
#> [985] "Patiria miniata"
#> [986] "Rhinolophus rouxii_sinicus"
#> [987] "Rhinolophus sinicus"
#> [988] "Lampris incognitus"
#> [989] "Monachus schauinslandi"
#> [990] "Neomonachus schauinslandi"
#> [991] "Hippoglossus hippoglossus"
#> [992] "Pleuronectes hippoglossus"
#> [993] "Andrographis paniculata"
#> [994] "Etheostoma cragini"
#> [995] "Perca chuatsi"
#> [996] "Siniperca chuatsi"
#> [997] "Meriones unguiculatus"
#> [998] "Colobus angolensis_palliatus"
#> [999] "Notothenia coriiceps"
#> [1000] "Hypomesus transpacificus"
#> [1001] "Dermochelys coriacea"
#> [1002] "Testudo coriacea"
#> [1003] "Bufo bufo_gargarizans"
#> [1004] "Bufo gargarizans"
#> [1005] "Bufo japonicus_gargarizans"
#> [1006] "Delphinapterus leucas"
#> [1007] "Delphinus leucas"
#> [1008] "Fugu flavidus"
#> [1009] "Takifugu flavidus"
#> [1010] "Pteronotus mesoamericanus"
#> [1011] "Pteronotus parnellii_mesoamericanus"
#> [1012] "Citrus clementina"
#> [1013] "Citrus deliciosa_x_Citrus_sinensis"
#> [1014] "Citrus x_clementina"
#> [1015] "Fugu rubripes"
#> [1016] "Sphaeroides rubripes"
#> [1017] "Takifugu rubripes"
#> [1018] "Tetraodon rubripes"
#> [1019] "Homarus americanus"
#> [1020] "Osteoglossum formosum"
#> [1021] "Scleropages formosus"
#> [1022] "Larimichthys crocea"
#> [1023] "Pseudosciaena amblyceps"
#> [1024] "Pseudosciaena crocea"
#> [1025] "Sciaena crocea"
#> [1026] "Fragaria vesca"
#> [1027] "Folsomia candida"
#> [1028] "Limulus polyphemus"
#> [1029] "Monoculus polyphemus"
#> [1030] "Doryrhamphus dactyliophorus"
#> [1031] "Dunckerocampus dactyliophorus"
#> [1032] "Syngnathus dactyliophorus"
#> [1033] "Epinephelus lanceolatus"
#> [1034] "Holocentrus lanceolatus"
#> [1035] "Promicrops lanceolatus"
#> [1036] "Mizuhopecten yessoensis"
#> [1037] "Patinopecten yessoensis"
#> [1038] "Patiopecten yessoensis"
#> [1039] "Pecten yessoensis"
#> [1040] "Platypoecilus maculatus"
#> [1041] "Xiphophorus maculatus"
#> [1042] "Triplophysa rosa"
#> [1043] "Antechinus flavipes"
#> [1044] "Phascogale flavipes"
#> [1045] "Balaena musculus"
#> [1046] "Balaenoptera musculus"
#> [1047] "Rhinolophus ferrumequinum"
#> [1048] "Vespertilio ferrumequinum"
#> [1049] "Oryza brachyantha"
#> [1050] "Chrysemys picta"
#> [1051] "Testudo picta"
#> [1052] "Trachemys picta"
#> [1053] "Tetrahymena thermophila_SB210"
#> [1054] "Amygdalus communis"
#> [1055] "Amygdalus dulcis"
#> [1056] "Prunus amygdalus"
#> [1057] "Prunus communis"
#> [1058] "Prunus dulcis"
#> [1059] "Prunus dulcis_var._sativa"
#> [1060] "Oryzias latipes"
#> [1061] "Poecilia latipes"
#> [1062] "Sarcophilus harrisii"
#> [1063] "Sarcophilus laniarius_(Owen,_1838)"
#> [1064] "Sarcophilus laniarius"
#> [1065] "Ursinus harrisii"
#> [1066] "Ictalurus furcatus"
#> [1067] "Pimelodus furcatus"
#> [1068] "Amphioxus belcheri"
#> [1069] "Branchiostoma belcheri"
#> [1070] "Gigantopelta aegis"
#> [1071] "Echinus variegatus"
#> [1072] "Lytechinus variegatus"
#> [1073] "Diaphorina citri"
#> [1074] "Epinephelus moara"
#> [1075] "Serranus moara"
#> [1076] "Stegodyphus dumicola"
#> [1077] "Boleophthalmus pectinirostris"
#> [1078] "Gobius pectinirostris"
#> [1079] "Austrofundulus limnaeus"
#> [1080] "Columba livia_domestica"
#> [1081] "Columba livia"
#> [1082] "Latimeria chalumnae"
#> [1083] "Pleuronectes maximus"
#> [1084] "Psetta maxima"
#> [1085] "Rhombus maximus"
#> [1086] "Scophthalmus maximus"
#> [1087] "Sesamum indicum"
#> [1088] "Sesamum orientale"
#> [1089] "Cyclopterus lumpus"
#> [1090] "Armeniaca mume"
#> [1091] "Prunus mume"
#> [1092] "Myotis davidii"
#> [1093] "Vespertilio Davidii"
#> [1094] "Didelphys agilis"
#> [1095] "Gracilinanus agilis"
#> [1096] "Acanthophacelus reticulata"
#> [1097] "Poecilia (Acanthophacelus)_reticulata"
#> [1098] "Poecilia latipinna_reticulata"
#> [1099] "Poecilia reticulata"
#> [1100] "Australorbis glabratus"
#> [1101] "Biomphalaria glabrata"
#> [1102] "Planorbis glabratus"
#> [1103] "Hypudaeus ochrogaster"
#> [1104] "Microtus ochrogaster"
#> [1105] "Amygdalus persica"
#> [1106] "Persica vulgaris"
#> [1107] "Prunus persica"
#> [1108] "Prunus persica_var._densa"
#> [1109] "Chiloscyllium plagiosum"
#> [1110] "Scyllium plagiosum"
#> [1111] "Cheilinus undulatus"
#> [1112] "Phodopus roborovskii"
#> [1113] "Caenorhabditis remanei"
#> [1114] "Caenorhabditis vulgaris"
#> [1115] "Lamprologus brichardi"
#> [1116] "Neolamprologus brichardi"
#> [1117] "Gymnopis unicolor"
#> [1118] "Microcaecilia unicolor"
#> [1119] "Rhinatrema unicolor"
#> [1120] "Sciaena jaculatrix"
#> [1121] "Toxotes jaculatrix"
#> [1122] "Bos indicus"
#> [1123] "Bos primigenius_indicus"
#> [1124] "Bos taurus_indicus"
#> [1125] "Lacerta sicula_raffonei"
#> [1126] "Podarcis raffoneae"
#> [1127] "Podarcis raffonei"
#> [1128] "Podarcis wagleriana_raffonei"
#> [1129] "Benincasa cerifera"
#> [1130] "Benincasa hispida"
#> [1131] "Benincasa pruriens"
#> [1132] "Cucurbita hispida"
#> [1133] "Lagenaria siceraria_var._hispida"
#> [1134] "Dendrobium catenatum"
#> [1135] "Marsupenaeus japonicus"
#> [1136] "Penaeus japonicus"
#> [1137] "Penaeus (Marsupenaeus)_japonicus"
#> [1138] "Penaeus (Melicertus)_japonicus"
#> [1139] "Chaetodon argus"
#> [1140] "Scatophagus argus"
#> [1141] "Chanos chanos"
#> [1142] "Mugil chanos"
#> [1143] "Bison bison_bison"
#> [1144] "Bos bison_bison"
#> [1145] "Amblyraja radiata"
#> [1146] "Raja radiata"
#> [1147] "Amphimedon queenslandica"
#> [1148] "Hippocampus comes"
#> [1149] "Hipposideros armiger"
#> [1150] "Rhinolophus armiger"
#> [1151] "Cynoglossus (Arelia)_semilaevis"
#> [1152] "Cynoglossus semilaevis"
#> [1153] "Alecto japonica"
#> [1154] "Anneissia japonica"
#> [1155] "Oxycomanthus japonicus"
#> [1156] "Ananas comosus"
#> [1157] "Ananas comosus_var._comosus"
#> [1158] "Ananas lucidus"
#> [1159] "Bromelia comosa"
#> [1160] "Callionymus splendidus"
#> [1161] "Pterosynchiropus splendidus"
#> [1162] "Synchiropus splendidus"
#> [1163] "Neophocaena asiaeorientalis_asiaeorientalis"
#> [1164] "Coluber guttatus"
#> [1165] "Elaphe guttata"
#> [1166] "Pantherophis guttatus"
#> [1167] "Pollicipes cornucopia"
#> [1168] "Pollicipes pollicipes"
#> [1169] "Pseudoliparis swirei"
#> [1170] "Chelonoidis abingdonii"
#> [1171] "Chelonoidis abingdoni"
#> [1172] "Chelonoidis nigra_abingdonii"
#> [1173] "Geochelone nigra_abigdonii"
#> [1174] "Geochelone nigra_abingdoni"
#> [1175] "Geochelone nigra_ephippium"
#> [1176] "Testudo abingdonii"
#> [1177] "Rhincodon typus"
#> [1178] "Ricinus communis"
#> [1179] "Ricinus sanguineus"
#> [1180] "Malania oleifera"
#> [1181] "Ceratotherium simum_simum"
#> [1182] "Kryptolebias marmoratus"
#> [1183] "Rivulus marmoratus"
#> [1184] "Patella vulgata"
#> [1185] "Rhagoletis pomonella"
#> [1186] "Trypanosoma cruzi"
#> [1187] "Squalus fasciatus"
#> [1188] "Squalus tigrinus"
#> [1189] "Stegostoma fasciatum"
#> [1190] "Stegostoma tigrinum"
#> [1191] "Cistudo triunguis"
#> [1192] "Terrapene mexicana_triunguis"
#> [1193] "Terrapene triunguis"
#> [1194] "Odobenus rosmarus_divergens"
#> [1195] "Manatus latirostris"
#> [1196] "Trichechus manatus_latirostris"
#> [1197] "Carcharodon carcharias"
#> [1198] "Squalus carcharias"
#> [1199] "Macrognathus armatus"
#> [1200] "Mastacembelus armatus"
#> [1201] "Anas boschas"
#> [1202] "Anas domesticus"
#> [1203] "Anas platyrhynchos_f._domestica"
#> [1204] "Anas platyrhynchos"
#> [1205] "Theobroma cacao"
#> [1206] "Diabrotica virgifera_virgifera"
#> [1207] "Actinia diaphana"
#> [1208] "Aiptasia pallida"
#> [1209] "Aiptasia pulchella"
#> [1210] "Dysactis pallida"
#> [1211] "Exaiptasia diaphana"
#> [1212] "Exaiptasia pallida"
#> [1213] "Syngnathus acus_rubescens"
#> [1214] "Syngnathus acus"
#> [1215] "Syngnathus rubescens"
#> [1216] "Caretta caretta"
#> [1217] "Testudo caretta"
#> [1218] "Guillardia theta_CCMP2712"
#> [1219] "Anarrhichthys ocellatus"
#> [1220] "Pelodiscus sinensis"
#> [1221] "Trionyx sinensis"
#> [1222] "Hippoglossus olivaceus"
#> [1223] "Paralichthys olivaceus"
#> [1224] "Xiphias gladius"
#> [1225] "Cyprinodon variegatus"
#> [1226] "Bos grunniens_mutus"
#> [1227] "Bos mutus"
#> [1228] "Poephagus mutus"
#> [1229] "Alligator sinensis"
#> [1230] "Morus notabilis"
#> [1231] "Nymphaea colorata"
#> [1232] "Photinus pyralis"
#> [1233] "Periophthalmus magnuspinnatus"
#> [1234] "Meleagris gallopavo"
#> [1235] "Pomacea canaliculata"
#> [1236] "Haplochromis nyererei"
#> [1237] "Pundamilia nyererei"
#> [1238] "Caranx dumerili"
#> [1239] "Seriola dumerili"
#> [1240] "Macrosteles (Macrosteles)_quadrilineatus"
#> [1241] "Macrosteles quadrilineatus"
#> [1242] "Enhydra lutris_kenyoni"
#> [1243] "Fluta alba"
#> [1244] "Monopterus albus"
#> [1245] "Muraena alba"
#> [1246] "Caecilia seraphini"
#> [1247] "Caecilia Seraphini"
#> [1248] "Geotrypetes seraphini"
#> [1249] "Hypogeophis seraphini"
#> [1250] "Chaetodon rostratus"
#> [1251] "Chelmon rostratus"
#> [1252] "Cucumis sativus"
#> [1253] "Cyrtodactylus macularius"
#> [1254] "Eublepharis macularius"
#> [1255] "Felis concolor"
#> [1256] "Panthera concolor"
#> [1257] "Puma concolor"
#> [1258] "Cancer chinensis"
#> [1259] "Fenneropenaeus chinensis"
#> [1260] "Penaeus chinensis"
#> [1261] "Pomacentrus partitus"
#> [1262] "Stegastes partitus"
#> [1263] "Phascum patens"
#> [1264] "Physcomitrella patens_subsp._patens"
#> [1265] "Physcomitrella patens"
#> [1266] "Physcomitrium patens"
#> [1267] "Anas jamaicensis"
#> [1268] "Oxyura jamaicensis"
#> [1269] "Drosophila miranda"
#> [1270] "Lottia gigantea"
#> [1271] "Eurytemora affinis"
#> [1272] "Temora affinis"
#> [1273] "Crotalus tigris"
#> [1274] "Argentina anserina"
#> [1275] "Potentilla anserina"
#> [1276] "Achaearanea tepidariorum"
#> [1277] "Parasteatoda tepidariorum"
#> [1278] "Theridion tepidariorum"
#> [1279] "Uranotaenia lowii"
#> [1280] "Cynolebias whitei"
#> [1281] "Nematolebias whitei"
#> [1282] "Simpsonichthys whitei"
#> [1283] "Sceloporus undulatus"
#> [1284] "Stellio undulatus"
#> [1285] "Helobdella robusta"
#> [1286] "Styela clava"
#> [1287] "Manis afer_afer"
#> [1288] "Orycteropus afer_afer"
#> [1289] "Leucoraja erinacea"
#> [1290] "Raja erinacea"
#> [1291] "Raja erinaceus"
#> [1292] "Raja erinacia"
#> [1293] "Phytophthora nicotianae_INRA-310"
#> [1294] "Anas olor"
#> [1295] "Cygnus olor"
#> [1296] "Lacerta agilis"
#> [1297] "Millepora damicornis"
#> [1298] "Pocillopora caespitosa_laysanensis"
#> [1299] "Pocillopora damicornis_laysanensis"
#> [1300] "Pocillopora damicornis"
#> [1301] "Morone saxatilis"
#> [1302] "Perca saxatilis"
#> [1303] "Miniopterus natalensis"
#> [1304] "Miniopterus schreibersii_natalensis"
#> [1305] "Vespertilio natalensis"
#> [1306] "Anas cygnoid"
#> [1307] "Anser cygnoides"
#> [1308] "Actinia tenebrosa"
#> [1309] "Neptunus trituberculatus"
#> [1310] "Portunus (Portunus)_trituberculatus"
#> [1311] "Portunus trituberculatus"
#> [1312] "Lacerta vivipara"
#> [1313] "Zootoca vivipara"
#> [1314] "Propithecus coquereli"
#> [1315] "Propithecus verreauxi_coquereli"
#> [1316] "Erinaceus europaeus"
#> [1317] "Jatropha curcas"
#> [1318] "Caenorhabditis briggsae"
#> [1319] "Rhabditis briggsae"
#> [1320] "Cherax quadricarinatus"
#> [1321] "Homalodisca coagulata"
#> [1322] "Homalodisca vitripennis"
#> [1323] "Tettigonia coagulata"
#> [1324] "Tettigonia vitripennis"
#> [1325] "Anolis carolinensis"
#> [1326] "Python bivittatus"
#> [1327] "Python molurus_bivittatus"
#> [1328] "Chrysemys scripta_elegans"
#> [1329] "Emys elegans"
#> [1330] "Pseudemys scripta_elegans"
#> [1331] "Trachemys scripta_elegans"
#> [1332] "Protobothrops mucrosquamatus"
#> [1333] "Trigonocephalus mucrosquamatus"
#> [1334] "Trimeresurus mucrosquamatus"
#> [1335] "Daphnia pulex"
#> [1336] "Paramacrobiotus metropolitanus"
#> [1337] "Lipotes vexillifer"
#> [1338] "Petromyzon marinus"
#> [1339] "Poephila guttata"
#> [1340] "Taeniopygia guttata"
#> [1341] "Taenopygia guttata"
#> [1342] "Aplysia californica"
#> [1343] "Phalaenopsis equestris"
#> [1344] "Stauroglottis equestris"
#> [1345] "Balanoglossus kowalevskii"
#> [1346] "Saccoglossus kowalevskii"
#> [1347] "Saccoglossus kowalevskyi"
#> [1348] "Numida meleagris"
#> [1349] "Phasianus meleagris"
#> [1350] "Momordica charantia"
#> [1351] "Callorhinchus milii"
#> [1352] "Sphaerodactylus townsendi"
#> [1353] "Eutainia elegans"
#> [1354] "Thamnophis elegans"
#> [1355] "Corvus hawaiiensis"
#> [1356] "Manacus candei"
#> [1357] "Pipra candei"
#> [1358] "Euleptes europaea"
#> [1359] "Euleptes europea"
#> [1360] "Phyllodactylus europaea"
#> [1361] "Phyllodactylus europaeus"
#> [1362] "Ptyodactylus caudivolvolus"
#> [1363] "Lepisosteus oculatus"
#> [1364] "Altirana parkeri"
#> [1365] "Nanorana parkeri"
#> [1366] "Ahaetulla prasina"
#> [1367] "Dryophis prasinus"
#> [1368] "Fusarium oxysporum_f._sp._lycopersici_4287"
#> [1369] "Heteropelma chrysocephalum"
#> [1370] "Neopelma chrysocephalum"
#> [1371] "Musca domestica"
#> [1372] "Pristis pectinata"
#> [1373] "Ischnura elegans"
#> [1374] "Fringilla chalybeata"
#> [1375] "Vidua chalybeata"
#> [1376] "Coturnix coturnix_japanica"
#> [1377] "Coturnix coturnix_japonica"
#> [1378] "Coturnix coturnix_Japonicus"
#> [1379] "Coturnix japonica_japonica"
#> [1380] "Coturnix japonica"
#> [1381] "Gekko japonicus"
#> [1382] "Platydactylus japonicus"
#> [1383] "Nilaparvata lugens"
#> [1384] "Ardea americana"
#> [1385] "Grus americana"
#> [1386] "Grus americanus"
#> [1387] "Harpia harpyja"
#> [1388] "Vultur harpyja"
#> [1389] "Pipra filicauda"
#> [1390] "Herrania umbratica"
#> [1391] "Ilyonectria robusta"
#> [1392] "Cupidonia cupido_pallidicincta"
#> [1393] "Tympanuchus pallidicinctus"
#> [1394] "Topomyia yanbarensis"
#> [1395] "Parus atricapillus"
#> [1396] "Poecile atricapilla"
#> [1397] "Poecile atricapillus"
#> [1398] "Corapipo altera"
#> [1399] "Acyrthosiphon pisum"
#> [1400] "Acyrthosiphum pisum"
#> [1401] "Varanus komodoensis"
#> [1402] "Saprolegnia parasitica_CBS_223.65"
#> [1403] "Fringilla macroura"
#> [1404] "Vidua macroura"
#> [1405] "Carica papaya"
#> [1406] "Chiroxiphia lanceolata"
#> [1407] "Pipra lanceolata"
#> [1408] "Octopus bimaculoides"
#> [1409] "Lagopus muta"
#> [1410] "Tetrao mutus"
#> [1411] "Bradysia coprophila"
#> [1412] "Sciara coprophila"
#> [1413] "Coluber sirtalis"
#> [1414] "Thamnophis sirtalis"
#> [1415] "Falco peregrinus"
#> [1416] "Falco cherrug"
#> [1417] "Asteracanthion distichum"
#> [1418] "Asterias attenuata"
#> [1419] "Asterias clathrata"
#> [1420] "Asterias disticha"
#> [1421] "Asterias gigantea"
#> [1422] "Asterias pallida"
#> [1423] "Asterias rubens"
#> [1424] "Asterias stimpsoni"
#> [1425] "Asterias vulgaris"
#> [1426] "Manduca sexta"
#> [1427] "Sphinx sexta"
#> [1428] "Condylura cristata"
#> [1429] "Sorex cristatus"
#> [1430] "Cuculus canorus"
#> [1431] "Pezoporus wallicus"
#> [1432] "Aedes aegypti"
#> [1433] "Aedes (Stegomyia)_aegypti"
#> [1434] "Culex aegypti"
#> [1435] "Stegomyia aegypti"
#> [1436] "Falco naumanni"
#> [1437] "Corvus kubaryi"
#> [1438] "Necator americanus"
#> [1439] "Larus tridactylus"
#> [1440] "Rissa tridactyla"
#> [1441] "Aphanomyces astaci"
#> [1442] "Culex (Culex)_pipiens_pallens"
#> [1443] "Culex pipiens_pallens"
#> [1444] "Catharus ustulatus"
#> [1445] "Turdus ustulatus"
#> [1446] "Accipiter gentilis"
#> [1447] "Accipiter gentillis"
#> [1448] "Falco gentilis"
#> [1449] "Crocodylus porosus"
#> [1450] "Amborella trichopoda"
#> [1451] "Falco biarmicus"
#> [1452] "Lagopus leucura"
#> [1453] "Lagopus leucurus"
#> [1454] "Tetrao leucurus"
#> [1455] "Falco rusticolus"
#> [1456] "Phasianus colchicus"
#> [1457] "Corvus brachyrhynchos"
#> [1458] "Uloborus diversus"
#> [1459] "Phytophthora infestans_strain_T30-4"
#> [1460] "Phytophthora infestans_T30-4"
#> [1461] "Empidonax traillii"
#> [1462] "Muscicapa traillii"
#> [1463] "Strix alba"
#> [1464] "Tyto alba"
#> [1465] "Parus major"
#> [1466] "Lepeophtheirus salmonis"
#> [1467] "Gavialis gangeticus"
#> [1468] "Lacerta gangetica"
#> [1469] "Daphnia carinata"
#> [1470] "Aphis gossypii"
#> [1471] "Ampithoe aztecus"
#> [1472] "Hyalella azteca"
#> [1473] "Hyalella knickerbockeri"
#> [1474] "Colletotrichum lupini"
#> [1475] "Gloeosporium lupini"
#> [1476] "Sphaeroforma arctica_JP610"
#> [1477] "Suillus fuscotomentosus"
#> [1478] "Mollisia scopiformis"
#> [1479] "Phialocephala scopiformis"
#> [1480] "Muscicapa cayanensis"
#> [1481] "Myiozetetes cayanensis"
#> [1482] "Hyaloscypha bicolor_E"
#> [1483] "Melopsittacus undulatus"
#> [1484] "Psittacus undulatus"
#> [1485] "Fringilla montana"
#> [1486] "Passer montanus"
#> [1487] "Coccinella axyridis"
#> [1488] "Harmonia axyridis"
#> [1489] "Aimophila crissalis"
#> [1490] "Kieneria crissalis"
#> [1491] "Kieneria crissalis_(Vigors,_1839)"
#> [1492] "Melozone crissalis"
#> [1493] "Pipilo crissalis"
#> [1494] "Pipilo fuscus_crissalis"
#> [1495] "Stomoxis calcitrans"
#> [1496] "Stomoxys calcitrans"
#> [1497] "Anas atrata"
#> [1498] "Cygnus atratus"
#> [1499] "Culex fatigans"
#> [1500] "Culex pipiens_fatigans"
#> [1501] "Culex pipiens_quinquefasciatus"
#> [1502] "Culex quinquefasciatus"
#> [1503] "Hirundo rustica"
#> [1504] "Acanthaster planci"
#> [1505] "Asterias planci"
#> [1506] "Molothrus ater"
#> [1507] "Oriolus ater"
#> [1508] "Laccaria bicolor_S238N-H82"
#> [1509] "Anastrepha obliqua"
#> [1510] "Tephritis obliqua"
#> [1511] "Grapholitha glycinivorella"
#> [1512] "Leguminivora glycinivorella"
#> [1513] "Ammodromus caudacutus_nelsoni"
#> [1514] "Ammospiza nelsoni"
#> [1515] "Nylanderia fulva"
#> [1516] "Paratrechina fulva"
#> [1517] "Agelaius phoeniceus"
#> [1518] "Agelaius phoniceus"
#> [1519] "Oriolus phoeniceus"
#> [1520] "Colletotrichum fructicola"
#> [1521] "Colletotrichum ignotum"
#> [1522] "Euthrips occidentalis"
#> [1523] "Frankliniella brunnescens"
#> [1524] "Frankliniella californica"
#> [1525] "Frankliniella occidentalis_brunnescens"
#> [1526] "Frankliniella occidentalis"
#> [1527] "Motacilla alba_alba"
#> [1528] "Fusarium solani"
#> [1529] "Fusisporium solani"
#> [1530] "Neocosmospora solani"
#> [1531] "Sitophilus oryzae"
#> [1532] "Corvus cornix_cornix"
#> [1533] "Fringilla canaria_Linnaeus,_1758"
#> [1534] "Serinus canaria"
#> [1535] "Serinus canarius"
#> [1536] "Drosophila subpulchrella"
#> [1537] "Chlamydomonas reinhardtii"
#> [1538] "Chlamydomonas smithii"
#> [1539] "Puccinia striiformis_f._sp._tritici"
#> [1540] "Bactrocera cucurbitae"
#> [1541] "Bactrocera (Zeugodacus)_cucurbitae"
#> [1542] "Zeugodacus cucurbitae"
#> [1543] "Zeugodacus (Zeugodacus)_cucurbitae"
#> [1544] "Antrodia serialis"
#> [1545] "Fomitopsis serialis"
#> [1546] "Neoantrodia serialis"
#> [1547] "Polyporus serialis"
#> [1548] "Drosophila suzukii"
#> [1549] "Leucophenga suzukii"
#> [1550] "Aedes smithii"
#> [1551] "Wyeomyia smithii"
#> [1552] "Montifringilla ruficollis"
#> [1553] "Pyrgilauda ruficollis"
#> [1554] "Gymnogyps californianus"
#> [1555] "Vultur californianus"
#> [1556] "Bactrocera (Bactrocera)_dorsalis"
#> [1557] "Bactrocera (Bactrocera)_invadens"
#> [1558] "Bactrocera dorsalis"
#> [1559] "Bactrocera invadens"
#> [1560] "Bactrocera papayae"
#> [1561] "Bactrocera philippinensis"
#> [1562] "Dacus dorsalis"
#> [1563] "Trichoplusia ni"
#> [1564] "Leptothorax curvispinosus"
#> [1565] "Temnothorax curvispinosus"
#> [1566] "Saprolegnia declina_VS20"
#> [1567] "Saprolegnia diclina_VS20"
#> [1568] "Fringilla albicollis"
#> [1569] "Zonotrichia albicollis"
#> [1570] "Bactrocera neohumeralis"
#> [1571] "Dacus tryoni_var._neohumeralis"
#> [1572] "Sphaeria pertusa"
#> [1573] "Trematosphaeria pertusa"
#> [1574] "Fusarium oxysporum_var._redolens"
#> [1575] "Fusarium redolens"
#> [1576] "Anastrepha ludens"
#> [1577] "Trypeta ludens"
#> [1578] "Cantharellus anzutake"
#> [1579] "Malurus melanocephalus"
#> [1580] "Muscicapa melanocephala"
#> [1581] "Melitaea cinxia"
#> [1582] "Papilio cinxia"
#> [1583] "Maniola jurtina"
#> [1584] "Papilio jurtina"
#> [1585] "Anas fuligula"
#> [1586] "Aythya fuligula"
#> [1587] "Bombyx mori"
#> [1588] "Phalaena mori"
#> [1589] "Botys furnacalis"
#> [1590] "Ostrinia furnacalis"
#> [1591] "Priapula caudata"
#> [1592] "Priapulus caudatus"
#> [1593] "Apanteles glomeratus"
#> [1594] "Cotesia glomerata"
#> [1595] "Ichneumon glomeratus"
#> [1596] "Centrocercus urophasianus"
#> [1597] "Centrocerus urophasianus"
#> [1598] "Tetrao urophasianus"
#> [1599] "Montifringilla taczanowskii_(Przewalski,_1876)"
#> [1600] "Onychostruthus taczanowskii"
#> [1601] "Monomorium pharaonis"
#> [1602] "Daktulosphaira vitifoliae"
#> [1603] "Pemphigus vitifoliae"
#> [1604] "Viteus vitifoliae"
#> [1605] "Helicoverpa armigera"
#> [1606] "Heliothis armigera"
#> [1607] "Heliothis (Helicoverpa)_armigera"
#> [1608] "Noctua armigera"
#> [1609] "Drosophila biarmipes"
#> [1610] "Myzus (Nectarosiphon)_persicae"
#> [1611] "Myzus persicae"
#> [1612] "Lucilia sericata"
#> [1613] "Phaenicia sericata"
#> [1614] "Tinamus guttatus"
#> [1615] "Solenopsis invicta"
#> [1616] "Fringilla georgiana"
#> [1617] "Melospiza georgiana"
#> [1618] "Helicoverpa zea"
#> [1619] "Heliothis zea"
#> [1620] "Phalaena zea"
#> [1621] "Drosophila ananassae"
#> [1622] "Drosophila annanassae"
#> [1623] "Fusarium odoratissimum_NRRL_54006"
#> [1624] "Coccinella 7-punctata"
#> [1625] "Coccinella septempunctata"
#> [1626] "Spodoptera frugiperda"
#> [1627] "Tigriopus californicus"
#> [1628] "Tisbe californica"
#> [1629] "Ficedula albicollis"
#> [1630] "Muscicapa albicollis"
#> [1631] "Drosophila pseudoobscura"
#> [1632] "Mytilidion resinicola"
#> [1633] "Mytilinidion resinicola"
#> [1634] "Halyomorpha halys"
#> [1635] "Phycomyces blakesleeanus_NRRL_1555(-)"
#> [1636] "Drosophila willistoni"
#> [1637] "Monoraphidium neglectum"
#> [1638] "Sturnus vulgaris"
#> [1639] "Bactrocera tryoni"
#> [1640] "Tephritis tryoni"
#> [1641] "Apus apus"
#> [1642] "Hirundo apus"
#> [1643] "Suillus paluster"
#> [1644] "Naegleria gruberi"
#> [1645] "Suillus discolor"
#> [1646] "Suillus tomentosus_var._discolor"
#> [1647] "Trichina spiralis"
#> [1648] "Trichinella spiralis"
#> [1649] "Onthophagus taurus"
#> [1650] "Epistrophe balteatus"
#> [1651] "Episyrphus balteatus"
#> [1652] "Episyrphus (Episyrphus)_balteatus"
#> [1653] "Musca balteata"
#> [1654] "Leptinotarsa decemlineata"
#> [1655] "Leptinotarsa decimlineata"
#> [1656] "Stilodes decemlineata"
#> [1657] "Boletus plorans"
#> [1658] "Suillus plorans"
#> [1659] "Dryobates pubescens"
#> [1660] "Picoides pubescens_(Linnaeus,_1766)"
#> [1661] "Picoides pubescens"
#> [1662] "Fusarium proliferatum_ET1"
#> [1663] "Fusarium oxysporum_Fo47"
#> [1664] "Drosophila sechellia"
#> [1665] "Schizophyllum commune_H4-8"
#> [1666] "Depressaria gossypiella"
#> [1667] "Pectinophora gossypiella"
#> [1668] "Parus humilis"
#> [1669] "Podoces humilis"
#> [1670] "Pseudopoces humilis"
#> [1671] "Pseudopodoces humilis"
#> [1672] "Ascidia intestinalis"
#> [1673] "Ciona intestinalis"
#> [1674] "Distomum viverrini"
#> [1675] "Opisthorchis viverrini"
#> [1676] "Puccinia graminis_f._sp._tritici_CRL_75-36-700-3"
#> [1677] "Plutella xylostella"
#> [1678] "Melampsora larici-populina_98AG31"
#> [1679] "Drosophila obscura"
#> [1680] "Fusarium verticillioides_7600"
#> [1681] "Anoplophora glabripennis"
#> [1682] "Anoplophora nobilis"
#> [1683] "Cerosterna glabripennis"
#> [1684] "Melanauster nobilis"
#> [1685] "Calypte anna"
#> [1686] "Ornismya anna"
#> [1687] "Microdochium trichocladiopsis"
#> [1688] "Anopheles merus"
#> [1689] "Bactrocera (Daculus)_oleae"
#> [1690] "Bactrocera (Dacus)_oleae"
#> [1691] "Bactrocera oleae"
#> [1692] "Dacus oleae"
#> [1693] "Musca oleae"
#> [1694] "Fusarium mangiferae"
#> [1695] "Drosophila yakuba"
#> [1696] "Contarinia nasturtii"
#> [1697] "Parastagonospora nodorum_SN15"
#> [1698] "Drosophila virilis"
#> [1699] "Zasmidium cellare_ATCC_36951"
#> [1700] "Drosophila mauritiana"
#> [1701] "Geospiza fortis"
#> [1702] "Eupeodes corollae"
#> [1703] "Eupeodes (Eupeodes)_corollae"
#> [1704] "Metasyrphus corollae"
#> [1705] "Spodoptera litura"
#> [1706] "Sitodiplosis mosellana"
#> [1707] "Microgaster mediator"
#> [1708] "Microplitis medianus"
#> [1709] "Microplitis mediator"
#> [1710] "Drosophila kikkawai"
#> [1711] "Diaporthe citri"
#> [1712] "Phomopsis citri"
#> [1713] "Mesites unicolor"
#> [1714] "Mesitornis unicolor"
#> [1715] "Suillus subaureus"
#> [1716] "Colletotrichum capsici"
#> [1717] "Colletotrichum dematium_f._truncatum_(Schwein.)_Arx,_1957"
#> [1718] "Colletotrichum truncatum"
#> [1719] "Glomerella glycines"
#> [1720] "Vermicularia capsici"
#> [1721] "Vermicularia truncata"
#> [1722] "Drosophila simulans"
#> [1723] "Anisochrysa carnea"
#> [1724] "Chrysopa carnea"
#> [1725] "Chrysoperla carnea"
#> [1726] "Drosophila takahashii"
#> [1727] "Lucilia cuprina"
#> [1728] "Drosophila persimilis"
#> [1729] "Falco albicilla"
#> [1730] "Haliaeetus albicilla"
#> [1731] "Antrostomus carolinensis"
#> [1732] "Caprimulgus carolinensis"
#> [1733] "Nasonia vitripennis"
#> [1734] "Colias crocea"
#> [1735] "Colias croceus"
#> [1736] "Papilio croceus"
#> [1737] "Leptidea sinapis"
#> [1738] "Papilio sinapis"
#> [1739] "Anopheles arabiensis"
#> [1740] "Drosophila ficusphila"
#> [1741] "Vollenhovia emeryi"
#> [1742] "Hermetia illucens"
#> [1743] "Fusarium vanettenii_77-13-4"
#> [1744] "Nectria haematococca_mpVI_77-13-4"
#> [1745] "Thrips palmi"
#> [1746] "Falco leucocephalus"
#> [1747] "Haliaeetus leucocephalus"
#> [1748] "Malaya genurostris"
#> [1749] "Colletotrichum gloeosporioides_(Penz.)_Penz._&_Sacc.,_1884"
#> [1750] "Colletotrichum gloeosporioides"
#> [1751] "Glomerella cingulata"
#> [1752] "Glomerella rufomaculans-vaccinii"
#> [1753] "Gnomoniopsis cingulata"
#> [1754] "Vermicularia gloeosporioides"
#> [1755] "Acanthamoeba castellanii_Neff_strain"
#> [1756] "Acanthamoeba castellanii_strain_Neff"
#> [1757] "Acanthamoeba castellanii_str._Neff"
#> [1758] "Drosophila albomicans"
#> [1759] "Drosophila nasuta_albomicans"
#> [1760] "Spinulophila albomicans"
#> [1761] "Diaporthe amygdali"
#> [1762] "Fusicoccum amygdali"
#> [1763] "Phomopsis amygdali"
#> [1764] "Pelecanus crispus"
#> [1765] "Pelecanus philippensis_crispus"
#> [1766] "Drosophila rhopaloa"
#> [1767] "Aphantopus hyperantus"
#> [1768] "Maniola hyperantus"
#> [1769] "Papilio hyperantus"
#> [1770] "Drosophila serrata"
#> [1771] "Leptopilina heterotoma"
#> [1772] "Peronospora halstedii"
#> [1773] "Plasmopara halstedii"
#> [1774] "Cuculus discolor"
#> [1775] "Leptosomus discolor"
#> [1776] "Aphanomyces invadans"
#> [1777] "Drosophila santomea"
#> [1778] "Sipha flava"
#> [1779] "Drosophila teissieri"
#> [1780] "Aptenodytes forsteri"
#> [1781] "Phaethon lepturus"
#> [1782] "Drosophila bipectinata"
#> [1783] "Fulmaris glacialis"
#> [1784] "Fulmarus glacialis"
#> [1785] "Procellaria glacialis"
#> [1786] "Ardea garzetta"
#> [1787] "Egretta garzetta"
#> [1788] "Anopheles mysorensis"
#> [1789] "Anopheles stephensi_mysorensis"
#> [1790] "Anopheles stephensi"
#> [1791] "Anopheles stephensi_var._mysorensis"
#> [1792] "Neocellia intermedia_Rothwell,_1907"
#> [1793] "Neocellia intermedia"
#> [1794] "Cryptotermes secundus"
#> [1795] "Pestalotiopsis fici_W106-1"
#> [1796] "Aricia agestis"
#> [1797] "Papilio agestis"
#> [1798] "Polyommatus agestis"
#> [1799] "Artogeia napi"
#> [1800] "Papilio napi"
#> [1801] "Pieris napi"
#> [1802] "Drosophila eugracilis"
#> [1803] "Wasmannia auropunctata"
#> [1804] "Oppia nitens"
#> [1805] "Adelges cooleyi"
#> [1806] "Chermes cooleyi"
#> [1807] "Gilletteella cooleyi"
#> [1808] "Acanthisitta chloris"
#> [1809] "Sitta chloris"
#> [1810] "Agrilus feretrius"
#> [1811] "Agrilus marcopoli"
#> [1812] "Agrilus planipennis"
#> [1813] "Drosophila elegans"
#> [1814] "Hyposmocoma kahamanoa"
#> [1815] "Cariama cristata"
#> [1816] "Palamedea cristata"
#> [1817] "Aleurodes tabaci"
#> [1818] "Aleyrodes tabaci"
#> [1819] "Bemisia tabaci"
#> [1820] "Ibis nippon"
#> [1821] "Nipponia nippon"
#> [1822] "Balearica gibbericeps"
#> [1823] "Balearica pavonina_gibbericeps"
#> [1824] "Balearica regulorum_gibbericepse"
#> [1825] "Balearica regulorum_gibbericeps"
#> [1826] "Bombus affinis"
#> [1827] "Varroa jacobsoni"
#> [1828] "Drosophila gunungcola"
#> [1829] "Colius striatus"
#> [1830] "Tauraco erythrolophus"
#> [1831] "Colletotrichum aenigma"
#> [1832] "Colletotrichum communis"
#> [1833] "Colletotrichum dianesei"
#> [1834] "Colletotrichum endomangiferae"
#> [1835] "Colletotrichum hymenocallidis"
#> [1836] "Colletotrichum jasmini-sambac"
#> [1837] "Colletotrichum melanocaulon"
#> [1838] "Colletotrichum siamense"
#> [1839] "Pterocles gutturalis"
#> [1840] "Aethina tumida"
#> [1841] "Galleria mellonella"
#> [1842] "Phalaena mellonella"
#> [1843] "Bicyclus anynana"
#> [1844] "Mycalesis anynana"
#> [1845] "Leptopilina boulardi"
#> [1846] "Zootermopsis nevadensis"
#> [1847] "Achroia grisella"
#> [1848] "Tinea grisella"
#> [1849] "Acremonium falciforme"
#> [1850] "Cephalosporium falciforme"
#> [1851] "Fusarium falciforme"
#> [1852] "Neocosmospora falciformis"
#> [1853] "Drosophila mohavensis"
#> [1854] "Drosophila mojavensis"
#> [1855] "Drosophila innubila"
#> [1856] "Bombus huntii"
#> [1857] "Cuculus indicator"
#> [1858] "Indicator indicator"
#> [1859] "Dendroctonus ponderosae"
#> [1860] "Ardea helias"
#> [1861] "Eurypyga helias"
#> [1862] "Dracunculus loa"
#> [1863] "Loa loa"
#> [1864] "Cadophora gregata"
#> [1865] "Cephalosporium gregatum"
#> [1866] "Phialophora gregata"
#> [1867] "Nestor notabilis_notabilis"
#> [1868] "Nestor notabilis"
#> [1869] "Colymbus stellatus"
#> [1870] "Gavia stellata"
#> [1871] "Cynthia cardui"
#> [1872] "Papilio cardui"
#> [1873] "Vanessa cardui"
#> [1874] "Cladosporium fulvum"
#> [1875] "Fulvia fulva"
#> [1876] "Mycovellosiella fulva"
#> [1877] "Passalora fulva"
#> [1878] "Plodia interpunctella"
#> [1879] "Tinea interpunctella"
#> [1880] "Stilbospora angustata"
#> [1881] "Truncatella angustata"
#> [1882] "Truncatella truncata"
#> [1883] "Chlamydotis macqueenii_macqueenii"
#> [1884] "Chlamydotis macqueenii_macqueeni"
#> [1885] "Chlamydotis macqueenii"
#> [1886] "Chlamydotis undulata_macqueenii"
#> [1887] "Otis macqueenii"
#> [1888] "Anopheles funestus"
#> [1889] "Fusarium fujikuroi_IMI_58289"
#> [1890] "Cephalosporium keratoplasticum_(nom._inval.)"
#> [1891] "Fusarium keratoplasticum"
#> [1892] "Neocosmospora keratoplastica"
#> [1893] "Drosophila lebanonensis"
#> [1894] "Scaptodrosophila lebanonensis"
#> [1895] "Merops nubicus"
#> [1896] "Coniothyrium fuckelii_var._sporulosum"
#> [1897] "Coniothyrium sporulosum"
#> [1898] "Paraconiothyrium sporulosum"
#> [1899] "Paraconyotrichium sporulosum"
#> [1900] "Paraphaeosphaeria sporulosa"
#> [1901] "Fusarium venenatum"
#> [1902] "Fusarium venetum"
#> [1903] "Amyelois transitella"
#> [1904] "Nephopteryx transitella"
#> [1905] "Pelecanus carbo"
#> [1906] "Phalacrocorax carbo"
#> [1907] "Naegleria lovaniensis"
#> [1908] "Papilio machaon"
#> [1909] "Gaeumannomyces tritici_R3-111a-1"
#> [1910] "Papilio aegeria"
#> [1911] "Pararge aegeria"
#> [1912] "Lophyrus lecontei"
#> [1913] "Neodiprion lecontei"
#> [1914] "Sclerotinia sclerotiorum_1980_UF-70"
#> [1915] "Aegialitis vocifera"
#> [1916] "Charadrius vociferous"
#> [1917] "Charadrius vociferus"
#> [1918] "Oxyechus vociferus"
#> [1919] "Drosophila erecta"Note that, if genome_type is specified to a supported genome, the human / mouse gene ontology annotation will be automatically generated.
What is Mappability and why should I care about it?
For the most part, the SpliceWiz reference can be built with just the FASTA and GTF files. This is sufficient for assessment for most forms of alternative splicing events.
For intron retention, accurate assessment of intron depth is important. However, introns contain many repetitive regions that are difficult to map. We refer to these regions as “mappability exclusions”.
We adopt IRFinder’s algorithm to identify these mappability exclusions. This is determined empirically by generating synthetic reads systematically from the genome, then aligning these reads back to the same genome. Regions that contain less than the expected coverage depth of reads define “mappability exclusions”.
See the vignette: SpliceWiz cookbook for details on how to generate “mappability exclusions” for any genome.How do I use pre-built mappability exclusions to generate human and mouse references?
For human and mouse genomes, SpliceWiz provides pre-built mappability exclusion references that can be used to build the SpliceWiz reference. SpliceWiz provides these annotations via the NxtIRFdata package.
Simply specify the genome in the parameter genome_type in the buildRef() function (which accepts hg38, hg19, mm10 and mm9).
Additionally, a reference for non-polyadenylated transcripts is used. This has a minor role in QC of samples (to assess the adequacy of polyA capture).
For example, assuming your genome file "genome.fa" and a transcript annotation "transcripts.gtf" are in the working directory, a SpliceWiz reference can be built using the built-in hg38 low mappability regions and non-polyadenylated transcripts as follows:
Process BAM files using SpliceWiz
The function SpliceWiz_example_bams() retrieves 6 example BAM files from ExperimentHub and places a copy of these in the temporary directory.
What are these example BAM files and how were they generated?
In this vignette, we provide 6 example BAM files. These were generated based on aligned RNA-seq BAMs of 6 samples from the Leucegene AML dataset (GSE67039). Sequences aligned to hg38 were filtered to only include genes aligned to that used to create the chrZ chromosome. These sequences were then re-aligned to the chrZ reference using STAR.
How can I easily locate multiple BAM files?
Often, alignment pipelines process multiple samples. SpliceWiz provides convenience functions to recursively locate all the BAM files in a given folder, and tries to ascertain sample names. Often sample names can be gleaned when: * The BAM files are named by their sample names, e.g. “sample1.bam”, “sample2.bam”. In this case, level = 0 * The BAM files have generic names but are contained inside parent directories labeled by their sample names, e.g. “sample1/Unsorted.bam”, “sample2/Unsorted.bam”. In this case, level = 1
Process these BAM files using SpliceWiz:
pb_path <- file.path(tempdir(), "pb_output")
processBAM(
bamfiles = bams$path,
sample_names = bams$sample,
reference_path = ref_path,
output_path = pb_path
)Using the GUI
After building the demo reference as shown in the previous section, start SpliceWiz GUI in demo mode. Then, click the Experiment tab from the menu side bar. The following interface will be shown:
The Experiment Panel - GUI
The buttons on the left hand side are as follows:
- Set the folders containing the SpliceWiz reference, BAM files, and the output (NxtSE) folder
- Run
processBAM(process BAM files) - Import annotations from a tabular text file (such as a csv file)
- Settings for
collateData- collating the experiment - Run
collateData(collating the experiment) - Import/Export current sample annotations from/to the NxtSE folder, or export annotations as a csv file
- Set the number of threads used to run
processBAMand `collateData functions
Also, (8) is a row of tabs that toggle between different tables, showing details of the Reference, BAM files, processBAM output Files, and sample Annotations
To continue with our example, click on (1) Define Project Folders to bring up the following drop-down box:
Define Project Folders
We need to define the folders that contain our reference, BAM files, as well as the experiment (NxtSE) output folder for the final compiled experiment
- Click on
Choose Reference Folderand select theReferencedirectory (where the SpliceWiz reference was generated by the previous step. Then, - Click on
Choose BAM Folderand select thebamsdirectory (where the demo BAM files have been generated). - Click on
Choose / Create Experiment (NxtSE) Folderand select theNxtSEdirectory (which should currently be empty except for thepbOutputsubdirectory). Note that when an Experiment folder is chosen via this step, apbOutputsubdirectory will be created if it does not already exist
After our folders have been defined, on the right hand side, an interactive table should be displayed that looks like the following:
Running processBAM
To process the example BAM files, first make sure the BAM files you wish to process have been selected (BAM files can be unselected by removing the ticks in the selected column). Also, users have the option of renaming the samples (by setting the names in the sampleName column).
To continue with our example, lets leave the names as-is. Click the Process Selected BAMs button. A prompt should pop up asking for confirmation. Click OK to start running processBAM.
What is the
processBAM() function
SpliceWiz’s processBAM() function can process one or more BAM files. This function is ultra-fast, relying on an internal native C++ function that uses OpenMP multi-threading (via the ompBAM C++ API).
Input BAM files can be either read-name sorted or coordinate sorted (although SpliceWiz prefers the former). Indexing of coordinate-sorted BAMs are not necessary.
processBAM() loads the SpliceWiz reference. Then, it reads each BAM file in their entirety, and quantifies the following:
- Basic QC parameters including number of reads, directionality, etc
- Counts of gapped (junction) reads / fragments
- Intron coverage depths and other parameters (identical output to IRFinder)
- COV files (which are like BigWig files but record strand-specific coverage)
- Miscellaneous quants including coverage of chromosomes, intergenic regions, rRNAs, and non-polyadenylated regions
processBAM() generates two output files. The first is a gzipped text file containing all the quantitation data. The second is a COV file which contains the per-nucleotide RNA-seq coverage of the sample.More details on the
processBAM() function
At minimum, processBAM() requires four parameters:
bamfiles: The paths of the BAM filessample_names: The sample names corresponding to the given BAM filesreference_path: The directory containing the SpliceWiz referenceoutput_path: The directory where the output ofprocessBAM()should go
pb_path <- file.path(tempdir(), "pb_output")
processBAM(
bamfiles = bams$path,
sample_names = bams$sample,
reference_path = ref_path,
output_path = pb_path
)processBAM() also takes several optional, but useful, parameters:
n_threads: The number of threads for multi-threadingoverwrite: Whether existing files in the output directory should be overwrittenrun_featureCounts: (Requires the Rsubread package) runs featureCounts to obtain gene counts (which outputs results as an RDS file)
For example, to run processBAM() using 2 threads, disallow overwrite of existing processBAM() outputs, and run featureCounts afterwards, one would run the following:
# NOT RUN
# Re-run IRFinder without overwrite, and run featureCounts
require(Rsubread)
processBAM(
bamfiles = bams$path,
sample_names = bams$sample,
reference_path = ref_path,
output_path = pb_path,
n_threads = 2,
overwrite = FALSE,
run_featureCounts = TRUE
)
# Load gene counts
gene_counts <- readRDS(file.path(pb_path, "main.FC.Rds"))
# Access gene counts:
gene_counts$counts
Collate the experiment
The helper function findSpliceWizOutput() organises the output files of SpliceWiz’s processBAM() function. It identifies matching "txt.gz" and "cov" files for each sample, and organises these file paths conveniently into a 3-column data frame:
Using this data frame, collate the experiment using collateData(). We name the output directory as NxtSE_output as this folder will contain the data needed to import the NxtSE object:
nxtse_path <- file.path(tempdir(), "NxtSE_output")
collateData(
Experiment = expr,
reference_path = ref_path,
output_path = nxtse_path
)What is the
collateData() function
collateData() combines the processBAM() output files of multiple samples and builds a single database. collateData() creates a number of files in the chosen output directory. These outputs can then be imported into the R session as a NxtSE data object for downstream analysis.
At minimum, collateData() takes the following parameters:
Experiment: The 2- or 3- column data frame. The first column should contain (unique) sample names. The second and (optional) third columns contain the"txt.gz"and"cov"file pathsreference_path: The directory containing the SpliceWiz referenceoutput_path: The directory where the output ofprocessBAM()should go
collateData() can take some optional parameters:
IRMode: Whether to use SpliceWiz’sSpliceOvermethod, or IRFinder’sSpliceMaxmethod, to determine total spliced transcript abundance. Briefly,SpliceMaxconsiders junction reads that have either flanking splice site coordinate.SpliceOverconsiders additional junction reads that splices across exon clusters in common. Exon clusters are groups of mutually-overlapping exons.SpliceOveris the default option.overwrite: Whether files in the output directory should be overwrittenn_threads: Use multi-threaded operations where possiblelowMemoryMode: Minimise memory usage where possible. Note that most of the collateData pipeline will be single-threaded if this is set toTRUE.
collateData() is a memory-intensive operation when run using multiple threads. We estimate it can use up to 6-7 Gb per thread. lowMemoryMode will minimise RAM usage to ~ 8 Gb, but will be slower and run on a single thread.Enabling novel splicing detection
Novel splicing detection can be switched on by setting novelSplicing = TRUE from within the collateData() function:
# Modified pipeline - collateData with novel ASE discovery:
nxtse_path <- file.path(tempdir(), "NxtSE_output_novel")
collateData(
Experiment = expr,
reference_path = ref_path,
output_path = nxtse_path,
novelSplicing = TRUE ## NEW ##
)collateData() uses split reads that are not annotated introns to help construct hypothetical minimal transcripts. These are then injected into the original transcriptome annotation (GTF) file, whereby the SpliceWiz reference is rebuilt. The new SpliceWiz reference (which contains these novel transcripts) is then used to collate the samples.
To reduce false positives in novel splicing detection, SpliceWiz provides several filters to reduce the number of novel junctions fed into the analysis:
- Novel junctions that are lowly expressed (only in a small number of samples) are removed. The minimum number of samples required to retain a novel junction is set using
novelSplicing_minSamplesparameter - Alternately, junctions are retained if its expression exceeds a certain threshold (set using
novelSplicing_countThreshold) in a smaller number of samples (set usingnovelSplicing_minSamplesAboveThreshold) - Further, novel junctions can be filtered by requiring at least one end to be an annotated splice site (this is enabled using
novelSplicing_requireOneAnnotatedSJ = TRUE).
For example, if one wished to retain novel reads seen in 3 or more samples, or novel spliced reads with 10 or more counts in at least 1 sample, and requiring at least one end of a novel junction being an annotated splice site:
By default, tandem junction reads (reads that align across two or more splice junctions) are used to detect novel exons. This can be turned off by setting novelSplicing_useTJ = FALSE.
nxtse_path <- file.path(tempdir(), "NxtSE_output_novel")
collateData(
Experiment = expr,
reference_path = ref_path,
output_path = nxtse_path,
## NEW ##
novelSplicing = TRUE,
# switches on novel splice detection
novelSplicing_requireOneAnnotatedSJ = TRUE,
# novel junctions must share one annotated splice site
novelSplicing_minSamples = 3,
# retain junctions observed in 3+ samples (of any non-zero expression)
novelSplicing_minSamplesAboveThreshold = 1,
# only 1 sample required if its junction count exceeds a set threshold
novelSplicing_countThreshold = 10 ,
# threshold for previous parameter
novelSplicing_useTJ = TRUE
# whether tandem junction reads should be used to identify novel exons
)Using the GUI to annotate the experiment
After running the Reference and processBAM steps as indicated in the previous sections (of the GUI instructions), there is an option to assign annotations to the experiment prior to collation. Annotations can be assigned from existing tabular files (such as csv files). For this example, we will demonstrate how to use a csv file containing annotations to annotate our experiment. Note that the annotation table should contain matching sample names in its leftmost column.
To select a file containing annotations, click on Import Annotations from file, then select the demo_annotations.csv file that should be in the working directory. Press ok. Your interface should now look like this:
Importing annotations
Note that an extra button Add / Remove Annotation Columns (*) has appeared. Clicking on this button allows us to add/remove annotation columns
Adding annotation columns
Using the GUI to collate the experiment
After annotating the experiment in the step above, click on the Experiment Settings button. Then enable Look for Novel Splicing to bring up the following display:
Customizing the Experiment Settings
This drop-down dialog box contains several parameters related to novel splicing detection:
- Toggle on/off novel splice detection
- Restrict novel junction reads to having one annotated splice site
- Filter novel junction counts based on the number of samples in which the novel junction was observed (any non-zero amount)
- Filter novel junction counts based on expression threshold (min number of samples set here)
- Threshold junction read count (expression threshold based novel junction filter)
- Whether to utilize tandem junction to define novel exons
Also, there is an option (7) to overwrite a previously-compiled NxtSE in the same folder. There is also an option to clear all the Reference/BAM/NxtSE folders (8).
For this example, we will leave the settings as above. Proceed to run collateData() by clicking on Collate Experiment. After several moments, a pop-up message should be shown when the experiment has been successfully collated.
Importing the experiment
Before differential analysis can be performed, the collated experiment must be imported into the R session as an NxtSE data object.
After running collateData(), import the experiment using the makeSE() function:
Using the GUI
After running the steps in the previous GUI sections, navigate to Analysis and then click the Load Experiment on the menu bar. The display should look like this:
Analysis - Loading the Experiment - GUI
The buttons on the left hand side are as follows:
- Select the NxtSE folder containing the collated experiment
- Import annotations from file (tabular format such as csv file)
- Load NxtSE from folder (into current session for downstream analysis)
- After NxtSE has been loaded, it can be saved as an RDS file to send to collaborators (note that COV files will be disconnected once the file has been moved to a different location; it is best to give your collaborators the NxtSE folder instead, with the COV files inside the
pbOutputsubdirectory) - Load NxtSE from RDS file (saved via the prior step)
To continue with our example, click the Select Experiment (NxtSE) Folder, then select the NxtSE directory. The interface should now look like this:
Loading the NxtSE after reviewing the samples and annotations - GUI
To view any existing annotations, click the Annotations tab in the Experiment Display above the sample table. If you followed the prior steps in the Collate the experiment section, there should already be annotations here. If not, feel free to add annotations using the Import Annotations from File (and click on “demo_annotations.csv” file), or manually add and annotate columns by clicking on the Add / Remove Annotation Columns button to open the drop-down box.
To load the NxtSE object, click the Load NxtSE from Folder to which will load the NxtSE object into the current session. A pop-up will appear once the NxtSE object has been successfully completed.
What is the makeSE() function
The
makeSE() function imports the compiled data generated by the collateData() function. Data is imported as an NxtSE object. Downstream analysis, including differential analysis and visualization, is performed using the NxtSE object.More details about the
makeSE() function
By default, makeSE() uses delayed operations to avoid consuming memory until the data is actually needed. This is advantageous in analysis of hundreds of samples on a computer with limited resources. However, it will be slower. To load all the data into memory, we need to “realize” the NxtSE object, as follows:
Alternatively, makeSE() can realize the NxtSE object at construction:
By default, makeSE() constructs the NxtSE object using all the samples in the collated data. It is possible (and particularly useful in large data sets) to read only a subset of samples. In this case, construct a data frame object with the first column containing the desired sample names and parse this into the colData parameter as shown:
subset_samples <- colnames(se)[1:4]
df <- data.frame(sample = subset_samples)
se_small <- makeSE(nxtse_path, colData = df, RemoveOverlapping = TRUE)RemoveOverlapping = FALSE.
Differential analysis
Assigning annotations to samples
NB: to add annotations via the GUI workflow, see the Collate the experiment section.
Saving and reloading the NxtSE as an RDS file (GUI)
Once an NxtSE object has been loaded into memory, you can save it as an RDS object so it can be reloaded in a later session. To do this, click the Save NxtSE as RDS button. Choose a file name and location and press OK. This RDS file can be loaded as an NxtSE object in a later GUI session by clicking the Load NxtSE from RDS button.
What is the
NxtSE object
NxtSE is a data object which contains all the required data for downstream analysis after all the BAM alignment files have been process and the experiment is collated.
se
#> class: NxtSE
#> dim: 167 6
#> metadata(14): Up_Inc Down_Inc ... ref row_gr
#> assays(5): Included Excluded Depth Coverage minDepth
#> rownames(167): TRA2B/ENST00000453386_Intron8/clean
#> TRA2B/ENST00000453386_Intron7/clean ...
#> RI:SRSF2-203-exon2;SRSF2-202-intron2
#> RI:SRSF2-203-exon2;SRSF2-206-intron1
#> rowData names(20): EventName EventType ... is_annotated_IR
#> NMD_direction
#> colnames(6): 02H003 02H025 ... 02H043 02H046
#> colData names(2): condition batchThe NxtSE object inherits the SummarizedExperiment object. This means that the functions for SummarizedExperiment can be used on the NxtSE object. These include row and column annotations using the rowData() and colData() accessors.
Rows in the NxtSE object contain information about each alternate splicing event. For example:
head(rowData(se))
#> DataFrame with 6 rows and 20 columns
#> EventName EventType
#> <character> <character>
#> TRA2B/ENST00000453386_Intron8/clean TRA2B/ENST0000045338.. IR
#> TRA2B/ENST00000453386_Intron7/clean TRA2B/ENST0000045338.. IR
#> TRA2B/ENST00000453386_Intron6/clean TRA2B/ENST0000045338.. IR
#> TRA2B/ENST00000453386_Intron5/clean TRA2B/ENST0000045338.. IR
#> TRA2B/ENST00000453386_Intron4/clean TRA2B/ENST0000045338.. IR
#> TRA2B/ENST00000453386_Intron3/clean TRA2B/ENST0000045338.. IR
#> EventRegion gene_id
#> <character> <character>
#> TRA2B/ENST00000453386_Intron8/clean chrZ:1921-2559/- ENSG00000136527
#> TRA2B/ENST00000453386_Intron7/clean chrZ:2634-3631/- ENSG00000136527
#> TRA2B/ENST00000453386_Intron6/clean chrZ:3692-5298/- ENSG00000136527
#> TRA2B/ENST00000453386_Intron5/clean chrZ:5383-6205/- ENSG00000136527
#> TRA2B/ENST00000453386_Intron4/clean chrZ:6322-7990/- ENSG00000136527
#> TRA2B/ENST00000453386_Intron3/clean chrZ:8180-9658/- ENSG00000136527
#> gene_id_b intron_id
#> <character> <character>
#> TRA2B/ENST00000453386_Intron8/clean ENSG00000136527 ENST00000453386_Intr..
#> TRA2B/ENST00000453386_Intron7/clean ENSG00000136527 ENST00000453386_Intr..
#> TRA2B/ENST00000453386_Intron6/clean ENSG00000136527 ENST00000453386_Intr..
#> TRA2B/ENST00000453386_Intron5/clean ENSG00000136527 ENST00000453386_Intr..
#> TRA2B/ENST00000453386_Intron4/clean ENSG00000136527 ENST00000453386_Intr..
#> TRA2B/ENST00000453386_Intron3/clean ENSG00000136527 ENST00000453386_Intr..
#> Inc_Is_Protein_Coding Exc_Is_Protein_Coding
#> <logical> <logical>
#> TRA2B/ENST00000453386_Intron8/clean TRUE TRUE
#> TRA2B/ENST00000453386_Intron7/clean TRUE TRUE
#> TRA2B/ENST00000453386_Intron6/clean TRUE TRUE
#> TRA2B/ENST00000453386_Intron5/clean TRUE TRUE
#> TRA2B/ENST00000453386_Intron4/clean TRUE TRUE
#> TRA2B/ENST00000453386_Intron3/clean TRUE TRUE
#> Exc_Is_NMD Inc_Is_NMD Inc_TSL
#> <logical> <logical> <character>
#> TRA2B/ENST00000453386_Intron8/clean FALSE TRUE 1
#> TRA2B/ENST00000453386_Intron7/clean FALSE TRUE 1
#> TRA2B/ENST00000453386_Intron6/clean FALSE TRUE 1
#> TRA2B/ENST00000453386_Intron5/clean FALSE TRUE 1
#> TRA2B/ENST00000453386_Intron4/clean FALSE TRUE 1
#> TRA2B/ENST00000453386_Intron3/clean FALSE TRUE 1
#> Exc_TSL Event1a Event2a
#> <character> <character> <character>
#> TRA2B/ENST00000453386_Intron8/clean 1 chrZ:1921-2559/- NA
#> TRA2B/ENST00000453386_Intron7/clean 1 chrZ:2634-3631/- NA
#> TRA2B/ENST00000453386_Intron6/clean 1 chrZ:3692-5298/- NA
#> TRA2B/ENST00000453386_Intron5/clean 1 chrZ:5383-6205/- NA
#> TRA2B/ENST00000453386_Intron4/clean 1 chrZ:6322-7990/- NA
#> TRA2B/ENST00000453386_Intron3/clean 1 chrZ:8180-9658/- NA
#> Event1b Event2b
#> <character> <character>
#> TRA2B/ENST00000453386_Intron8/clean NA NA
#> TRA2B/ENST00000453386_Intron7/clean NA NA
#> TRA2B/ENST00000453386_Intron6/clean NA NA
#> TRA2B/ENST00000453386_Intron5/clean NA NA
#> TRA2B/ENST00000453386_Intron4/clean NA NA
#> TRA2B/ENST00000453386_Intron3/clean NA NA
#> is_always_first_intron
#> <logical>
#> TRA2B/ENST00000453386_Intron8/clean FALSE
#> TRA2B/ENST00000453386_Intron7/clean FALSE
#> TRA2B/ENST00000453386_Intron6/clean FALSE
#> TRA2B/ENST00000453386_Intron5/clean FALSE
#> TRA2B/ENST00000453386_Intron4/clean FALSE
#> TRA2B/ENST00000453386_Intron3/clean FALSE
#> is_always_last_intron is_annotated_IR
#> <logical> <logical>
#> TRA2B/ENST00000453386_Intron8/clean FALSE FALSE
#> TRA2B/ENST00000453386_Intron7/clean FALSE FALSE
#> TRA2B/ENST00000453386_Intron6/clean FALSE FALSE
#> TRA2B/ENST00000453386_Intron5/clean FALSE FALSE
#> TRA2B/ENST00000453386_Intron4/clean FALSE TRUE
#> TRA2B/ENST00000453386_Intron3/clean FALSE FALSE
#> NMD_direction
#> <numeric>
#> TRA2B/ENST00000453386_Intron8/clean 1
#> TRA2B/ENST00000453386_Intron7/clean 1
#> TRA2B/ENST00000453386_Intron6/clean 1
#> TRA2B/ENST00000453386_Intron5/clean 1
#> TRA2B/ENST00000453386_Intron4/clean 1
#> TRA2B/ENST00000453386_Intron3/clean 1Columns contain information about each sample. By default, no annotations are assigned to each sample. These can be assigned as shown above.
Also, NxtSE objects can be subsetted by rows (ASEs) or columns (samples). This is useful if one wishes to perform analysis on a subset of the dataset, or only on a subset of ASEs (say for example, only skipped exon events). Subsetting is performed just like for SummarizedExperiment objects:
# Subset by columns: select the first 2 samples
se_sample_subset <- se[,1:2]
# Subset by rows: select the first 10 ASE events
se_ASE_subset <- se[1:10,]
Filtering high-confidence events
SpliceWiz offers default filters to identify and remove low confidence alternative splice events (ASEs). Run the default filter using the following:
se.filtered <- se[applyFilters(se),]
#> Running Depth filter
#> Running Participation filter
#> Running Participation filter
#> Running Consistency filter
#> Running Terminus filter
#> Running ExclusiveMXE filter
#> Running StrictAltSS filterUsing the GUI
After following the GUI tutorials in the prior sections, click on Analysis and then Filters from the menu bar. It should look like this:
Analysis - Filters - GUI
To load SpliceWiz’s default filters, click the top right button Load Default Filters. Then to apply these filters to the NxtSE, click Apply Filters. After the filters have been run, your session should now look like this:
SpliceWiz default filters - GUI
Why do we need to filter alternative splicing events?
Often, the gene annotations contain isoforms for all discovered splicing events. Most annotated transcripts are not expressed, and their inclusion in differential analysis complicates results including adjusting for multiple testing. It is prudent to filter these out using various approaches, akin to removing genes with low gene counts in differential gene analysis. We suggest using the default filters which work well for small experiments with sequencing depths at 100-million paired-end reads.
?ASEFilters
Performing differential analysis
Using the edgeR wrapper ASE_edgeR(), perform differential ASE analysis between conditions “A” and “B”:
# Requires edgeR to be installed:
require("edgeR")
res_edgeR <- ASE_edgeR(
se = se.filtered,
test_factor = "condition",
test_nom = "B",
test_denom = "A"
)Using the GUI
After running the previous sections (of the GUI instructions), click Analysis and then Differential Expression Analysis on the menu side bar. It should look something like this:
Analysis - Differential Expression Analysis - GUI
To perform edgeR-based differential analysis, first ensure Method is set to edgeR. Using the Variable drop-down box, select condition. Then, select the Nominator and Denominator fields to B and A, respectively. Leave the batch factor fields as (none). Then, click Perform DE.
Once differential expression analysis has finished, your session should look like below. The output is a DT-based data table equivalent to the ASE_edgeR() function.
Example Differential Analysis using edgeR - GUI
NB: The interface allows users to choose to sort the results either by nominal or (multiple-testing) adjusted P values
NB2: There are 3 different ways Intron Retention events can be quantified and analysed - see “What are the different ways intron retention is measured?” below for further details.
NB3: Analyses can be saved or loaded to/from RDS files using the corresponding buttons.
What are the options for differential ASE analysis?
SpliceWiz provides wrappers to three established algorithms:
ASE_limmauseslimmato model isoform counts as log-normal distributions. Limma is probably the fastest method and is ideal for large datasets. Time series analysis is available for this mode.ASE_DESequsesDESeq2to model isoform counts as negative binomial distribution. This method is the most computationally expensive, but gives robust results. Time series analysis is also available for this modeASE_edgeRusesedgeRto model isoform counts as negative binomial distributions. SpliceWiz uses the quasi-likelihood method that deals better with variance at near-zero junction counts, resulting in reduced false positives.ASE_DoubleExpSequses the lesser-known CRAN packageDoubleExpSeq. This package uses the beta-binomial distribution to model isoform counts. The method is at least as fast aslimma, but for now it is restricted to analysis between two groups (i.e. batch correction is not implemented)
We recommend the following for differential analysis:
- For quick comparisons between two groups, where no batch factors are involved, we recommend DoubleExpSeq
- For large complex experiments where quick results are required for a preliminary or exploratory analysis, we recommend limma
- For final analysis where accuracy is paramount, we recommend edgeR or DESeq2
# Requires limma to be installed:
require("limma")
res_limma <- ASE_limma(
se = se.filtered,
test_factor = "condition",
test_nom = "B",
test_denom = "A"
)
# Requires DoubleExpSeq to be installed:
require("DoubleExpSeq")
res_DES <- ASE_DoubleExpSeq(
se = se.filtered,
test_factor = "condition",
test_nom = "B",
test_denom = "A"
)
# Requires DESeq2 to be installed:
require("DESeq2")
res_deseq <- ASE_DESeq(
se = se.filtered,
test_factor = "condition",
test_nom = "B",
test_denom = "A",
n_threads = 1
)
# Requires edgeR to be installed:
require("edgeR")
res_edgeR <- ASE_edgeR(
se = se.filtered,
test_factor = "condition",
test_nom = "B",
test_denom = "A"
)What are the different ways intron retention is measured?
Intron retention can be measured via two approaches.
The first (and preferred) approach is using IR-ratio. We presume that every intron is potentially retained (thus ignoring annotation). Given this results in many overlapping introns, SpliceWiz adjusts for this via the following:
- Where there are mutually-overlapping introns, the less abundant intron is removed from the analysis. Abundance is estimated across the entire dataset, and less-abundant overlapping introns are removed at the
makeSE()step. - IR-ratio measures included (intronic) abundance using an identical approach to IRFinder, i.e., it calculates the trimmed mean of sequencing depth across the intron, excluding annotated outliers (other exons, intronic elements). Given we cannot assume whether the exact intron corresponds to the major isoform, we estimate splicing abundance by summing junction reads that share either exon cluster as the intron of interest (
SpliceOvermethod in SpliceWiz). Alternately, users can choose to use IRFinder’sSpliceMaxmethod, summing junction reads that share either splice junction with the intron of interest. This choice is also made by the user at themakeSE()step.
At the differential analysis step, users can choose the following:
IRmode = "all"- all introns are potentially retained, use IR-ratio to quantify IR (EventType = "IR")IRmode = "annotated"- only annotated retained intron events are considered, but use IR-ratio to quantify IR (EventType = "IR")IRmode = "annotated_binary"- only annotated retained intron events are considered, use PSI to quantify IR - which considers the IR-transcript and the transcript isoform with the exactly-spliced intron as binary alternatives. Splicing of overlapping introns are not considered in PSI quantitation.
res_edgeR_allIntrons <- ASE_edgeR(
se = se.filtered,
test_factor = "condition",
test_nom = "B",
test_denom = "A",
IRmode = "all"
)
res_edgeR_annotatedIR <- ASE_edgeR(
se = se.filtered,
test_factor = "condition",
test_nom = "B",
test_denom = "A",
IRmode = "annotated"
)
res_edgeR_annotated_binaryIR <- ASE_edgeR(
se = se.filtered,
test_factor = "condition",
test_nom = "B",
test_denom = "A",
IRmode = "annotated_binary"
)Can I account for batch factors?
ASE_limma, ASE_edgeR, and ASE_DESeq can accept up to 2 categories of batches from which to normalize. For example, to normalize the analysis by the batch category, one would run:
require("edgeR")
res_edgeR_batchnorm <- ASE_edgeR(
se = se.filtered,
test_factor = "condition",
test_nom = "B",
test_denom = "A",
batch1 = "batch"
)Can I do time series analysis?
Time series analysis can be performed using limma, edgeR, and DESeq2.
For limma and edgeR, time series analysis is done using the ASE_limma_timeseries() and ASE_edgeR_timeseries() function. test_factor, despite its name, should be a column in colData(se) containing numerical values that represent time series data.
Note that these time series wrappers function requires the splines package.
colData(se.filtered)$timevar <- rep(c(0,1,2), 2)
require("splines")
require("limma")
res_limma_cont <- ASE_limma_timeseries(
se = se.filtered,
test_factor = "timevar"
)
require("splines")
require("edgeR")
res_edgeR_cont <- ASE_edgeR_timeseries(
se = se.filtered,
test_factor = "timevar"
)For DESeq2, time series analysis is performed using the ASE_DESeq() funcction. The key difference is that, for time series analysis, simply do not specify the test_nom and test_denom parameters. As long as the test_factor contains numeric values, ASE_DESeq will treat it as a continuous variable. See the following example:
colData(se.filtered)$timevar <- rep(c(0,1,2), 2)
require("DESeq2")
res_deseq_cont <- ASE_DESeq(
se = se.filtered,
test_factor = "timevar"
)Advanced GLM-based differential ASE analysis with edgeR
We have implemented wrapper functions enabling advanced users to perform differential ASE analysis by constructing their own design matrices. This allows users to evaluate effects of covariates in complex experimental models.
We will be building a separate vignette to illustrate the full functionality of these edgeR-based functions, but for now a quick example can be found in the relevant documentation, which can be viewed via:
Visualization
Volcano plots
Volcano plots show changes in PSI levels (log fold change, x axis) against statistical significance (-log10 p values, y axis):
library(ggplot2)
ggplot(res_edgeR,
aes(x = logFC, y = -log10(FDR))) +
geom_point() +
labs(title = "Differential analysis - B vs A",
x = "Log2-fold change", y = "FDR (-log10)")
Can I visualize significant events for each modality of alternative splicing events?
Yes. We can use ggplot2’s facet_wrap function to separately plot volcanos for each modality of ASE. The type of ASE is contained in the EventType column of the differential results data frame.
ggplot(res_edgeR,
aes(x = logFC, y = -log10(FDR))) +
geom_point() + facet_wrap(vars(EventType)) +
labs(title = "Differential analysis - B vs A",
x = "Log2-fold change", y = "FDR (-log10)")
Using the GUI
After following the previous sections including differential analysis, navigate to Display and then Volcano Plot. Notice that there will be a message that says “No events found. Consider relaxing some filters”.
This message occurs because our example dataset has no differential events that surpass an adjusted P value of less than 0.05 (which is the default filter setting). The SpliceWiz GUI avoids plotting all ASEs as this will crowd the visualization. In this example, change the Filter Events by to Nominal P value, and move the P-value/FDR threshold all the way to the right. There should now be a volcano plot but most events have near-zero significance because the default y-axis setting is to Plot adjusted P values. Switch this off to show the following:
Volcano Plot - GUI
You can customize this volcano plot using the following controls:
- Use the filtering panel to adjust the number of events to show. Here, events can be filtered by Nominal or adjusted P values, or by top events. We can also filter by “highlighted events” which is useful for gene ontology analysis and heatmaps later. Events can also be filtered by the type of alternative splicing
- The volcano plot can be faceted by the type of alternative splicing.
- The y axis can show either nominal or adjusted P values (Benjamini Hochberg).
- If
NMD ModeisON, the horizontal axis represents whether splicing is shifted towards (positive values) or away from (negative values) a NMD substrate transcript - The plot can be exported as a pdf file (ggplot object)
- Clear settings
Also, on the (right hand side) main panel:
- This clears any selected events (selected events are highlighted in red)
- Toggle between using tools to select or deselect events
- Plotly interactive toolbox. Users can use the box-select (9a) or lasso- select (9b) to select (or de-select) one or more events of interest. Selected events are highlighted in other plots and non-selected points can be removed by selecting “highlighted events” in the event filter panel (1).
Scatter plots
Scatter plots are useful for showing splicing levels (percent-spliced-in, PSI) between two conditions. The results from differential analysis contains these values and can be plotted:
library(ggplot2)
ggplot(res_edgeR, aes(x = 100 * AvgPSI_B, y = 100 * AvgPSI_A)) +
geom_point() + xlim(0, 100) + ylim(0, 100) +
labs(title = "PSI values across conditions",
x = "PSI of condition B", y = "PSI of condition A")
#> Warning: Removed 1 row containing missing values or values outside the scale range
#> (`geom_point()`).
Using the GUI
After following the previous sections including differential analysis, navigate to Display and then Scatter Plot. After relaxing the event filters similar to the previous section, change the Variable to condition, X-axis condition to A and Y-axis condition to B. A scatter plot should be automatically generated as follows:
Scatter plot - GUI
You can customize the scatter plot using the following controls:
- Again, the filtering panel is available to filter the number of events by significance threshold, rank, or by highlighted events, as explained in the previous section (volcano plots - GUI)
Variablerefers to the annotation table column name- and (4) -
X-axisandY-axisdropdowns refer to the condition categories (that are used to contrast between two experimental conditions) - If
NMD ModeisON, the PSI values are altered such that they represent the inclusion values of the NMD substrate (instead of that of the “included” isoform) - The plot can be exported as a pdf file (ggplot object)
- Clear settings
As in the volcano plot, the scatter plot is interactive and points highlighted on this plot will stay highlighted in other plots.
- Clears all highlighted events
- Toggles between tools that select or de-select events
- Plotly interactive plot where users can use box or lasso select tools to select / de-select events.
Selecting ASEs of interest using the interactive plots (GUI)
SpliceWiz GUI generates plotly interactive figures. For volcano and scatter plots, points of interest can be selected using the lasso or box select tools. For example, we can select the top hits from the faceted volcano plot as shown:
Volcano plot - selecting ASEs of interest via the GUI
These ASEs of interest will then be highlighted in other plots, for example scatter plot:
Scatter plot - highlighted ASE events
How do I generate average PSI values for many conditions?
SpliceWiz provides the makeMeanPSI() function that can generate mean PSI values for each condition of a condition category. For example, the below code will calculate the mean PSIs of each “batch” of this example experiment:
Gene ontology (GO) analysis
Gene ontology analysis using the GUI
Our working example does not have enough genes to demonstrate a workable gene ontology analysis. Instead, the following explains the controls found in the Gene Ontology panel:
Gene ontology analysis
The controls are as follows:
- Again, the filtering panel is available to filter the number of events by significance threshold, rank, or by highlighted events, as explained in the previous sections. Filtered events are considered “enriched events” in the analysis.
- Type of gene ontology category - “Biological Function”, “Molecular Function” or “Cellular Compartment”
- Enrich for upregulated, downregulated events, or both
- Which set of genes to consider as background. Default is genes belonging to All ASE Events (as they contain introns). Alternatives are to either test for genes of ASEs of the specified modality (a popup select input box will appear, allowing users to select which modalities of ASE to derive background genes), or by all genes in the genome (NB this option will contain intronless genes which may bias the result).
- Gene ontology plots can be saved to PDF
- Gene ID’s of enriched genes (6a) or background genes (6b) can be exported to file, allowing users to repeat GO analysis using their own gene ontology enrichment tool of choice.
SpliceWiz has built-in gene ontology analysis. For now, only a limited number of species are supported for Ensembl gene ID’s. To see a list of supported organisms:
getAvailableGO()
#> [1] "Anopheles gambiae"
#> [2] "Arabidopsis thaliana"
#> [3] "Bos taurus"
#> [4] "Canis familiaris"
#> [5] "Gallus gallus"
#> [6] "Pan troglodytes"
#> [7] "Escherichia coli"
#> [8] "Drosophila melanogaster"
#> [9] "Homo sapiens"
#> [10] "Mus musculus"
#> [11] "Sus scrofa"
#> [12] "Rattus norvegicus"
#> [13] "Macaca mulatta"
#> [14] "Caenorhabditis elegans"
#> [15] "Xenopus laevis"
#> [16] "Saccharomyces cerevisiae"
#> [17] "Danio rerio"
#> [18] "Triticum aestivum"
#> [19] "Triticum aestivum_subsp._aestivum"
#> [20] "Triticum vulgare"
#> [21] "Brassica napus"
#> [22] "Arachis hypogaea"
#> [23] "Hibiscus syriacus"
#> [24] "Acridium cancellatum"
#> [25] "Schistocerca cancellata"
#> [26] "Triticum dicoccoides"
#> [27] "Triticum turgidum_subsp._dicoccoides"
#> [28] "Triticum turgidum_var._dicoccoides"
#> [29] "Dendrohyas sarda"
#> [30] "Hyla arborea_sarda"
#> [31] "Hyla sarda"
#> [32] "Locusta gregaria"
#> [33] "Schistocerca gregaria"
#> [34] "Gossypium hirsutum"
#> [35] "Gossypium hirsutum_subsp._mexicanum"
#> [36] "Gossypium lanceolatum"
#> [37] "Gossypium purpurascens"
#> [38] "Camelina sativa"
#> [39] "Myagrum sativum"
#> [40] "Carassius auratus_gibelio"
#> [41] "Carassius gibelio_gibelio"
#> [42] "Carassius gibelio"
#> [43] "Carassius gibelio_subsp._gibelio"
#> [44] "Cyprinus gibelio"
#> [45] "Schistocerca piceifrons"
#> [46] "Papaver somniferum"
#> [47] "Zingiber officinale"
#> [48] "Trichomonas vaginalis_G3"
#> [49] "Trichomonas vaginalis_strain_G3"
#> [50] "Helianthus annuus"
#> [51] "Schistocerca americana"
#> [52] "Acipenser ruthenus"
#> [53] "Schistocerca serialis_cubense"
#> [54] "Panicum virgatum"
#> [55] "Nicotiana tabacum"
#> [56] "Oncorhynchus mykiss"
#> [57] "Oncorhynchus nerka_mykiss"
#> [58] "Parasalmo mykiss"
#> [59] "Salmo mykiss"
#> [60] "Schistocerca nitens"
#> [61] "Schistocerca vaga"
#> [62] "Salvia splendens"
#> [63] "Carassius carassius"
#> [64] "Cyprinus carassius"
#> [65] "Vicia villosa"
#> [66] "Camellia sinensis"
#> [67] "Thea sinensis"
#> [68] "Oncorhynchus keta"
#> [69] "Salmo keta"
#> [70] "Pisum sativum"
#> [71] "Salmo salar"
#> [72] "Raphanus sativus"
#> [73] "Oncorhynchus kisutch"
#> [74] "Oncorhyncus kisutch"
#> [75] "Salmo kisatch"
#> [76] "Lolium rigidum"
#> [77] "Aegilops squarrosa_subsp._squarrosa"
#> [78] "Aegilops squarrosa"
#> [79] "Aegilops tauschii"
#> [80] "Patropyrum tauschii_subsp._tauschii"
#> [81] "Patropyrum tauschii"
#> [82] "Triticum aegilops"
#> [83] "Triticum tauschii"
#> [84] "Salmo trutta"
#> [85] "Cryptomeria japonica"
#> [86] "Cupressus japonica"
#> [87] "Coregonus clupeaformis"
#> [88] "Salmo clupeaformis"
#> [89] "Oncorhynchus gorbuscha"
#> [90] "Salmo gorbuscha"
#> [91] "Cyprinus carpio"
#> [92] "Glycine max_subsp._soja"
#> [93] "Glycine soja"
#> [94] "Salmo fontinalis"
#> [95] "Salvelinus fontinalis"
#> [96] "Glycine max"
#> [97] "Phaseolus max"
#> [98] "Chenopodium quinoa"
#> [99] "Hordeum sativum"
#> [100] "Hordeum vulgare_subsp._vulgare"
#> [101] "Hordeum vulgare_var._nudum"
#> [102] "Hordeum vulgare_var._vulgare"
#> [103] "Festuca perennis_(L.)_Columbus_&_J.P.Sm.,_2010"
#> [104] "Festuca perennis"
#> [105] "Lolium perenne"
#> [106] "Lolium vulgare"
#> [107] "Coffea arabica"
#> [108] "Barbus grahami"
#> [109] "Sinocyclocheilus grahami"
#> [110] "Sinocyclocheilus rhinocerous"
#> [111] "Gossypium arboreum"
#> [112] "Brassica oleracea"
#> [113] "Malus sylvestris"
#> [114] "Pyrus malus_var._sylvestris"
#> [115] "Astyanax mexicanus"
#> [116] "Tetragonopterus mexicanus"
#> [117] "Arachis stenosperma"
#> [118] "Prosopis alba"
#> [119] "Sinocyclocheilus anshuiensis"
#> [120] "Brassica rapa"
#> [121] "Lactuca sativa"
#> [122] "Dreissena polymorpha"
#> [123] "Mytilus polymorphus"
#> [124] "Hydractinia symbiolongicarpus"
#> [125] "Triticum urartu"
#> [126] "Hevea brasiliensis"
#> [127] "Siphonia brasiliensis"
#> [128] "Oncorhynchus tschawytscha"
#> [129] "Oncorhynchus tshawytscha"
#> [130] "Salmo tshawytscha"
#> [131] "Arachis ipaensis"
#> [132] "Zea mays"
#> [133] "Zea mays_var._japonica"
#> [134] "Salmo namaycush"
#> [135] "Salvelinus namaycush"
#> [136] "Capsicum annuum"
#> [137] "Brienomyrus brachyistius"
#> [138] "Marcusenius brachyistius"
#> [139] "Convolvulus nil"
#> [140] "Ipomoea nil"
#> [141] "Pharbitis nil"
#> [142] "Olea europaea_subsp._europaea_var._sylvestris"
#> [143] "Olea europaea_var._oleaster"
#> [144] "Olea europaea_var._sylvestris"
#> [145] "Olea europea_subsp._sylvestris"
#> [146] "Alosa sapidissima"
#> [147] "Clupea sapidissima"
#> [148] "Carpiodes asiaticus"
#> [149] "Myxocyprinus asiaticus"
#> [150] "Actinidia eriantha"
#> [151] "Gossypium raimondii"
#> [152] "Salmo alpinus"
#> [153] "Salvelinus alpinus"
#> [154] "Catostomus texanus"
#> [155] "Xyrauchen texanus"
#> [156] "Doryrhamphus excisus"
#> [157] "Quercus lobata"
#> [158] "Malus communis"
#> [159] "Malus domestica"
#> [160] "Malus pumila_auct."
#> [161] "Malus pumila_var._domestica"
#> [162] "Malus sylvestris_var._domestica"
#> [163] "Malus x_domestica"
#> [164] "Pyrus malus"
#> [165] "Pyrus malus_var._domestica"
#> [166] "Quercus suber"
#> [167] "Oncorhynchus nerka"
#> [168] "Salmo nerka"
#> [169] "Nicotiana tomentosiformis"
#> [170] "Carya illinoensis"
#> [171] "Carya illinoinensis"
#> [172] "Mercenaria mercenaria"
#> [173] "Venus mercenaria"
#> [174] "Quercus robur"
#> [175] "Durio zibethinus"
#> [176] "Pongo abelii"
#> [177] "Pongo pygmaeus_abelii"
#> [178] "Pongo pygmaeus_abeli"
#> [179] "Mya arenaria"
#> [180] "Arachis duranensis"
#> [181] "Arachis spegazzinii"
#> [182] "Pyrus x_bretschneideri"
#> [183] "Trifolium pratense"
#> [184] "Gorilla gorilla_gorilla"
#> [185] "Cobitis anguillicaudata"
#> [186] "Misgurnus anguillicaudatus"
#> [187] "Scaphiopus bombifrons"
#> [188] "Spea bombifrons"
#> [189] "Haliotis rufenscens"
#> [190] "Haliotis rufescens"
#> [191] "Oreochromis nilotica"
#> [192] "Oreochromis niloticus"
#> [193] "Perca nilotica"
#> [194] "Tilapia nilotica"
#> [195] "Acropora convexa"
#> [196] "Acropora millepora"
#> [197] "Acropora singularis"
#> [198] "Cebus apella"
#> [199] "Sapajus apella"
#> [200] "Simia apella"
#> [201] "Eucalyptus grandis"
#> [202] "Dasypus fenestratus"
#> [203] "Dasypus novemcinctus"
#> [204] "Callithrix jacchus_jacchus"
#> [205] "Callithrix jacchus"
#> [206] "Simia jacchus"
#> [207] "Pistacia vera"
#> [208] "greater Indian_fruit_bat"
#> [209] "Pteropus giganteus"
#> [210] "Pteropus medius"
#> [211] "Salvia miltiorhiza"
#> [212] "Salvia miltiorrhiza"
#> [213] "Daphnia pulicaria"
#> [214] "Magnolia sinica"
#> [215] "Manglietia sinica"
#> [216] "Manglietiastrum sinicum"
#> [217] "Pachylarnax sinica_(Y.W.Law)_N.H.Xia_&_C.Y.Wu"
#> [218] "Rosa chinensis"
#> [219] "Rosa indica_auct.,_non_L."
#> [220] "Mytilus californianus"
#> [221] "Pteropus vampyrus"
#> [222] "Vespertilio vampyrus"
#> [223] "Chinemys reevesii"
#> [224] "Chinemys reevesi"
#> [225] "Emys reevesii"
#> [226] "Geoclemys reevesii"
#> [227] "Geoclemys reevessi"
#> [228] "Mauremys reevesii"
#> [229] "Mauremys reevesi"
#> [230] "Choloepus brasiliensis_Fitzinger_1871"
#> [231] "Choloepus brasiliensis"
#> [232] "Choloepus didactylus"
#> [233] "Macaca nemestrina"
#> [234] "Simia nemestrina"
#> [235] "Lotus corniculatus_var._japonicus"
#> [236] "Lotus japonicus"
#> [237] "Nicotiana sylvestris"
#> [238] "Tupaia belangeri_chinensis"
#> [239] "Tupaia chinensis"
#> [240] "Clarias gariepinus"
#> [241] "Clarias lazera"
#> [242] "Silurus gariepinus"
#> [243] "Barbus tetrazona"
#> [244] "Capoeta tetrazona"
#> [245] "Puntigrus tetrazona"
#> [246] "Puntius tetrazona"
#> [247] "Systomus tetrazona"
#> [248] "Lycium ferocissimum"
#> [249] "Nicotiana attenuata"
#> [250] "Octodon degus"
#> [251] "Haliotis rubra"
#> [252] "Aedes albopictus"
#> [253] "Stegomyia albopicta"
#> [254] "Spinacia oleracea"
#> [255] "Paramecium aurelia_syngen_4"
#> [256] "Paramecium tetraurelia"
#> [257] "Salvia hispanica"
#> [258] "Medicago truncatula"
#> [259] "Crassostrea virginica"
#> [260] "Ostrea virginica"
#> [261] "Felis catus"
#> [262] "Felis domesticus"
#> [263] "Felis silvestris_catus"
#> [264] "Anubis baboon"
#> [265] "Papio anubis"
#> [266] "Papio cynocephalus_anubis"
#> [267] "Papio doguera"
#> [268] "Papio hamadryas_anubis"
#> [269] "Papio hamadryas_doguera"
#> [270] "Simia anubis"
#> [271] "Pongo pygmaeus"
#> [272] "Simia pygmaeus"
#> [273] "Sorex etruscus"
#> [274] "Suncus etruscus"
#> [275] "Mimosa cineraria"
#> [276] "Prosopis cineraria"
#> [277] "Nycticebus coucang"
#> [278] "Tardigradus coucang"
#> [279] "Rhododendron vialii"
#> [280] "Pan paniscus"
#> [281] "Nematostella vectensis"
#> [282] "Ixodes dammini"
#> [283] "Ixodes scapularis"
#> [284] "Lupinus angustifolius"
#> [285] "Ipomoea triloba"
#> [286] "Equus asinus"
#> [287] "Emiliania huxleyi_CCMP1516"
#> [288] "Emiliania huxleyi_CCMP2090"
#> [289] "Mangifera indica"
#> [290] "Pteropus alecto"
#> [291] "Rana temporaria"
#> [292] "Crassostrea gigas"
#> [293] "Magallana gigas"
#> [294] "Ostrea gigas"
#> [295] "Etheostoma spectabile"
#> [296] "Poecilichthys spectabilis"
#> [297] "Macadamia integrifolia"
#> [298] "Megalobrama amblycephala"
#> [299] "Halichoerus grypus"
#> [300] "Phoca grypus"
#> [301] "Juglans regia"
#> [302] "Selaginella moellendorffii"
#> [303] "Selaginella moellendorfii"
#> [304] "Pleuronectes platessa"
#> [305] "Presbytis francoisi"
#> [306] "Trachypithecus francoisi"
#> [307] "Tripterygium wilfordii"
#> [308] "Argiope bruennichi"
#> [309] "Lepus cuniculus"
#> [310] "Oryctolagus cuniculus"
#> [311] "Huro salmoides"
#> [312] "Labrus salmoides"
#> [313] "Labrus salmonides"
#> [314] "Micropterus nigricans"
#> [315] "Micropterus salmoides"
#> [316] "Solanum stenotomum"
#> [317] "Heterocephalus glaber"
#> [318] "Neosciurus carolinensis"
#> [319] "Sciurus carolinensis"
#> [320] "Cervus elaphus"
#> [321] "Polyodon spathula"
#> [322] "Squalus spathula"
#> [323] "Gadus chalcogrammus"
#> [324] "Theragra chalcogramma"
#> [325] "Nothobranchius furzeri"
#> [326] "Bos bubalis"
#> [327] "Bubalus arnee_bubalis"
#> [328] "Bubalus bubalis"
#> [329] "Pleuronectes solea"
#> [330] "Solea solea"
#> [331] "Solea vulgaris"
#> [332] "Mastomys coucha"
#> [333] "Praomys coucha"
#> [334] "Impatiens glandulifera"
#> [335] "Dermacentor andersoni"
#> [336] "Felis nebulosa"
#> [337] "Neofelis nebulosa"
#> [338] "Pteropus egyptiacus"
#> [339] "Rousettus aegyptiacus"
#> [340] "Rousettus aegypticus"
#> [341] "Rousettus egyptiacus"
#> [342] "Phoenix dactylifera"
#> [343] "Pimephales promelas"
#> [344] "Ostrea edulis"
#> [345] "Peromyscus maniculatus_bairdii"
#> [346] "Gasterosteus pungitius"
#> [347] "Pungitius pungitius"
#> [348] "Populus alba"
#> [349] "Cricetus auratus"
#> [350] "Golden hamsters"
#> [351] "Mesocricetus auratus"
#> [352] "Syrian hamsters"
#> [353] "Chromis aureus"
#> [354] "Oreochromis aurea"
#> [355] "Oreochromis aureus"
#> [356] "Daucus carota_subsp._sativus"
#> [357] "Daucus carota_var._sativus"
#> [358] "Dermacentor silvarum"
#> [359] "Hylobates syndactylus"
#> [360] "Simia syndactyla"
#> [361] "Symphalangus syndactylus"
#> [362] "Saccharolobus solfataricus"
#> [363] "Sulfolobus solfataricus"
#> [364] "Felis geoffroyi"
#> [365] "Leopardus geoffroyi"
#> [366] "Oncifelis geoffroyi"
#> [367] "Felis yagouaroundi"
#> [368] "Herpailurus yagouaroundi"
#> [369] "Herpailurus yaguarondi"
#> [370] "Puma yagouaroundii"
#> [371] "Puma yagouaroundi"
#> [372] "Cervus canadensis"
#> [373] "Populus diversifolia"
#> [374] "Populus euphratica"
#> [375] "Cucurbita pepo_subsp._pepo"
#> [376] "Cucurbita pepo_var._medullosa"
#> [377] "Cucurbita pepo_var._pepo"
#> [378] "Macaca cynomolgus"
#> [379] "Macaca fascicularis"
#> [380] "Macaca irus"
#> [381] "Simia fascicularis"
#> [382] "Emys muticus"
#> [383] "Geoclemmys mutica"
#> [384] "Mauremys mutica"
#> [385] "Suricata suricatta"
#> [386] "Viverra suricatta"
#> [387] "Hylobates moloch"
#> [388] "Simia moloch"
#> [389] "Solanum dulcamara"
#> [390] "Cucurbita moschata"
#> [391] "Coffea eugeniodes"
#> [392] "Coffea eugenioides"
#> [393] "Cucurbita maxima"
#> [394] "Labrus bergylta"
#> [395] "Centropristis striata"
#> [396] "Labrus striatus"
#> [397] "Oryza sativa_(japonica_cultivar-group)"
#> [398] "Oryza sativa_Japonica_Group"
#> [399] "Oryza sativa_subsp._japonica"
#> [400] "Jaculus jaculus"
#> [401] "Mus jaculus"
#> [402] "Dioscorea cayenensis_subsp._rotundata_(Poir.)_J.Miege,_1968"
#> [403] "Dioscorea cayenensis_subsp._rotundata"
#> [404] "Dioscorea rotundata"
#> [405] "Cercopithecus aethiops_sabaeus"
#> [406] "Cercopithecus sabaeus"
#> [407] "Cercopithecus sabeus"
#> [408] "Chlorocebus aethiops_sabaeus"
#> [409] "Chlorocebus aethiops_sabeus"
#> [410] "Chlorocebus sabaeus"
#> [411] "Chlorocebus sabeus"
#> [412] "Simia sabaea"
#> [413] "Marmota monax"
#> [414] "Mus monax"
#> [415] "Pygathrix roxellana"
#> [416] "Rhinopithecus roxellana"
#> [417] "Semnopithecus roxellana"
#> [418] "Callorhinus ursinus"
#> [419] "Callorhynus ursius"
#> [420] "Phoca ursina"
#> [421] "Cricetulus barabensis_griseus"
#> [422] "Cricetulus griseus"
#> [423] "Elephantulus edwardii"
#> [424] "Macroscelides edwardii"
#> [425] "Cobitis heteroclita"
#> [426] "Fundulus heteroclitus"
#> [427] "Neothunnus macropterus"
#> [428] "Scomber albacares"
#> [429] "Thunnus albacares"
#> [430] "Telopea speciosissima"
#> [431] "Danio aesculapii"
#> [432] "Danio sp._'snakeskin'"
#> [433] "Danio sp._snakeskin"
#> [434] "Apodemus sylvaticus"
#> [435] "Mus sylvaticus"
#> [436] "Sylvaemus sylvaticus"
#> [437] "Populus balsamifera_subsp._trichocarpa"
#> [438] "Populus trichocarpa"
#> [439] "Mercurialis ambigua"
#> [440] "Mercurialis annua"
#> [441] "Eugenia oleosa"
#> [442] "Syzygium oleosum"
#> [443] "Citellus tridecemlineatus"
#> [444] "Ictidomys tridecemlineatus"
#> [445] "Spermophilus tridecemlineatus"
#> [446] "Ovis ammon_aries"
#> [447] "Ovis aries"
#> [448] "Ovis orientalis_aries"
#> [449] "Ovis ovis"
#> [450] "Solanum verrucosum"
#> [451] "Leo pardus"
#> [452] "Panthera pardus"
#> [453] "Microtus oregoni"
#> [454] "Arabidopsis lyrata_subsp._lyrata"
#> [455] "Arabis lyrata_subsp._lyrata"
#> [456] "Arabis lyrata"
#> [457] "Cardaminopsis lyrata"
#> [458] "Manihot esculenta"
#> [459] "Manihot utilissima"
#> [460] "Mustela erminea"
#> [461] "Dolichos unguiculatus"
#> [462] "Phaseolus unguiculatus"
#> [463] "Vigna unguiculata"
#> [464] "Lycopersicon pennellii_(Correll)_D'Arcy,_1982"
#> [465] "Solanum pennellii_Correll,_1958"
#> [466] "Solanum pennellii"
#> [467] "Panicum viride"
#> [468] "Setaria viridis"
#> [469] "Musa AA_Group"
#> [470] "Musa acuminata_AA_Group"
#> [471] "Musa acuminata"
#> [472] "Musa nana"
#> [473] "Gymnostomus macrolepis"
#> [474] "Onychostoma macrolepis"
#> [475] "Scaphesthes macrolepis"
#> [476] "Varicorhinus macrolepis"
#> [477] "Varicorhinus (Scaphesthes)_macrolepis"
#> [478] "Oryza glaberrima"
#> [479] "Pelteobagrus fulvidraco"
#> [480] "Pimelodus fulvidraco"
#> [481] "Pseudobagrus fulvidraco"
#> [482] "Tachysurus fulvidraco"
#> [483] "Hylobates concolor_leucogenys"
#> [484] "Hylobates concolor_leucogyneus"
#> [485] "Hylobates leucogenys_leucogenys"
#> [486] "Hylobates leucogenys"
#> [487] "Nomascus leucogenys_leucogenys"
#> [488] "Nomascus leucogenys"
#> [489] "Nomascus leukogenys"
#> [490] "Nannospalax ehrenbergi_galili"
#> [491] "Nannospalax galili"
#> [492] "Spalax galili"
#> [493] "Equus caballus"
#> [494] "Equus przewalskii_f._caballus"
#> [495] "Equus przewalskii_forma_caballus"
#> [496] "Thunnus maccoyii"
#> [497] "Thynnus maccoyii"
#> [498] "Chromis diagramma"
#> [499] "Simochromis diagramma"
#> [500] "Diplophysa dalaica"
#> [501] "Triplophysa dalaica"
#> [502] "Felis tigris"
#> [503] "Panthera tigris"
#> [504] "Echinus purpuratus"
#> [505] "Strongylocentrotus purpuratus"
#> [506] "Lucioperca lucioperca"
#> [507] "Perca lucioperca"
#> [508] "Sander lucioperca"
#> [509] "Stizostedion lucioperca"
#> [510] "Dipodomys spectabilis"
#> [511] "Acinonyx jubatus"
#> [512] "Felis jubata"
#> [513] "Conyza canadensis"
#> [514] "Erigeron canadensis"
#> [515] "Mustela lutreola"
#> [516] "Camelus bactrianus_ferus"
#> [517] "Camelus ferus"
#> [518] "Cajanus cajan"
#> [519] "Didelphys domestica"
#> [520] "Monodelphis domestica"
#> [521] "Pygathrix bieti"
#> [522] "Rhinopithecus bieti"
#> [523] "Saimiri boliviensis"
#> [524] "Hesperomys eremicus"
#> [525] "Peromyscus eremicus"
#> [526] "Arabidopsis salsuginea"
#> [527] "Eutrema salsugineum"
#> [528] "Hesperis salsuginea"
#> [529] "Sisymbrium salsugineum"
#> [530] "Stenophragma salsugineum"
#> [531] "Thellungiella salsuginea"
#> [532] "Thelypodium salsugineum"
#> [533] "Coetomys damarensis"
#> [534] "Cryptomys damarensis"
#> [535] "Fukomys damarensis"
#> [536] "Leptonychotes weddellii"
#> [537] "Leptonychotes weddelli"
#> [538] "Otaria weddellii"
#> [539] "Grammomys dolichurus_surdaster"
#> [540] "Grammomys surdaster"
#> [541] "Thamnomys surdaster"
#> [542] "Solanum aracc-papa"
#> [543] "Solanum tuberosum"
#> [544] "Andropogon sorghum"
#> [545] "Sorghum bicolor"
#> [546] "Sorghum bicolor_subsp._bicolor"
#> [547] "Sorghum nervosum"
#> [548] "Sorghum saccharatum"
#> [549] "Sorghum vulgare"
#> [550] "Holocentrus calcarifer"
#> [551] "Lates calcarifer"
#> [552] "Hippopotamus amphibius_kiboko"
#> [553] "Ixodes sanguineus"
#> [554] "Rhipicephalus sanguineus"
#> [555] "Clupea harengus_harengus"
#> [556] "Clupea harengus"
#> [557] "Bos indicus_x_Bos_taurus"
#> [558] "Bos primigenius_indicus_x_Bos_primigenius_taurus"
#> [559] "Bos taurus_indicus_x_Bos_taurus_taurus"
#> [560] "Bos taurus_x_Bos_indicus"
#> [561] "Chrysochloris asiatica"
#> [562] "Talpa asiatica"
#> [563] "Bufo bufo"
#> [564] "Rana bufo"
#> [565] "Maylandia zebra"
#> [566] "Metriaclima zebra"
#> [567] "Pseudotropheus sp._'Pseudotropheus_zebra_complex'"
#> [568] "Pseudotropheus zebra"
#> [569] "Ictalurus punctatus"
#> [570] "Silurus punctatus"
#> [571] "Oryx dammah"
#> [572] "Camelus dromedarius"
#> [573] "Asparagus litoralis"
#> [574] "Asparagus officinalis"
#> [575] "Amaranthus gangeticus"
#> [576] "Amaranthus mangostanus"
#> [577] "Amaranthus tricolor"
#> [578] "Pneumatophorus japonicus"
#> [579] "Scomber japonicus"
#> [580] "Lutra lutra"
#> [581] "Peromyscus leucopus"
#> [582] "Perca fluviatilis"
#> [583] "Pagothenia bernacchii"
#> [584] "Pseudotrematomus bernacchii"
#> [585] "Trematomus bernacchii"
#> [586] "Trematomus bernacchi"
#> [587] "Lipurus cinereus"
#> [588] "Phascolarctos cinereus"
#> [589] "Mustela furo"
#> [590] "Mustela putorius_furo"
#> [591] "Chaetochloa italica"
#> [592] "Panicum italicum"
#> [593] "Pennisetum macrochaetum"
#> [594] "Setaria italica"
#> [595] "Setaria viridis_subsp._italica"
#> [596] "Elaeis guineensis"
#> [597] "Mus rattus"
#> [598] "Rattus rattoides"
#> [599] "Rattus rattus"
#> [600] "Rattus wroughtoni"
#> [601] "Acropora digitifera"
#> [602] "Madrepora digitifera"
#> [603] "Echinops telfairii"
#> [604] "Echinops telfairi"
#> [605] "Madrepora verrucosa"
#> [606] "Pocillopora danae"
#> [607] "Pocillopora verrucosa"
#> [608] "Myotis daubentonii"
#> [609] "Myotis daubentoni"
#> [610] "Vespertilio daubentonii"
#> [611] "Limia formosa"
#> [612] "Mollienesia formosa"
#> [613] "Poecilia formosa"
#> [614] "Phyllostomus discolor"
#> [615] "Microcebus murinus"
#> [616] "Peromyscus californicus_insignis"
#> [617] "Peromyscus californicus_subsp._insignis"
#> [618] "Galago garnettii"
#> [619] "Galago garnetti"
#> [620] "Otolemur garnettii"
#> [621] "Arvicanthis niloticus"
#> [622] "Mus niloticus"
#> [623] "Didelphis ursina"
#> [624] "Vombatus ursinus"
#> [625] "Phaseolus angularis"
#> [626] "Vigna angularis"
#> [627] "Haitia acuta"
#> [628] "Physa acuta"
#> [629] "Physa heterostropha"
#> [630] "Physa integra"
#> [631] "Physella acuta"
#> [632] "Physella heterostropha"
#> [633] "Physella integra"
#> [634] "Ctenopharyngodon idella"
#> [635] "Ctenopharyngodon idellus"
#> [636] "Leuciscus idella"
#> [637] "Thalassophryne amazonica"
#> [638] "Cyprinus rohita"
#> [639] "Labeo rohita"
#> [640] "Talpa occidentalis"
#> [641] "Bombina bombina"
#> [642] "Rana bombina"
#> [643] "Cavia aperea_porcellus"
#> [644] "Cavia cobaya"
#> [645] "Cavia porcellus"
#> [646] "Mus porcellus"
#> [647] "Odocoileus virginianus"
#> [648] "Amphibalanus amphitrite"
#> [649] "Balanus amphitrite"
#> [650] "Panicum hallii"
#> [651] "Angill angill"
#> [652] "Anguilla anguilla_anguilla"
#> [653] "Anguilla anguilla"
#> [654] "Muraena anguilla"
#> [655] "Delphinus orca"
#> [656] "Orcinus orca"
#> [657] "Cannabis sativa"
#> [658] "Penaeus bubulus"
#> [659] "Penaeus carinatus"
#> [660] "Penaeus durbani"
#> [661] "Penaeus monodon"
#> [662] "Penaeus (Penaeus)_monodon"
#> [663] "Didelphis vulpecula"
#> [664] "Trichosurus vulpecula"
#> [665] "Myotis lucifugus"
#> [666] "Vespertilio lucifugus"
#> [667] "Brachypodium distachyon"
#> [668] "Bromus distachyos"
#> [669] "Aotus nancymaae"
#> [670] "Aotus nancymai"
#> [671] "Rhamnus zizyphus"
#> [672] "Ziziphus jujuba"
#> [673] "Ailuropoda melanoleuca"
#> [674] "Micropterus dolomieu"
#> [675] "Micropterus velox"
#> [676] "Lycopersicon esculentum"
#> [677] "Lycopersicon esculentum_var._esculentum"
#> [678] "Solanum esculentum"
#> [679] "Solanum lycopersicum"
#> [680] "Solanum lycopersicum_var._humboldtii"
#> [681] "Poecilia mexicana"
#> [682] "Manis pentadactyla"
#> [683] "Meles meles"
#> [684] "Ursus meles"
#> [685] "Ornithorhynchus anatinus"
#> [686] "Platypus anatinus"
#> [687] "Felis uncia"
#> [688] "Panthera uncia"
#> [689] "Uncia uncia"
#> [690] "Alligator mississippiensis"
#> [691] "Crocodilus mississipiensis"
#> [692] "Myrmecophaga aculeata"
#> [693] "Tachyglossus aculeatus"
#> [694] "Colossoma macropomum"
#> [695] "Myletes macropomus"
#> [696] "Cordylus capensis"
#> [697] "Cordylus (Hemicordylus)_capensis"
#> [698] "Hemicordylus capensis"
#> [699] "Pseudocordylus capensis"
#> [700] "Zonurus capensis"
#> [701] "Eptesicus fuscus"
#> [702] "Vespertilio fuscus"
#> [703] "Dromiciops australis"
#> [704] "Dromiciops gliroides"
#> [705] "Camelus pacos"
#> [706] "Lama guanicoe_pacos"
#> [707] "Lama pacos"
#> [708] "Vicugna pacos"
#> [709] "Mollienesia latipinna"
#> [710] "Poecilia latipinna"
#> [711] "Elephas maximus_indicus"
#> [712] "Corylus avellana"
#> [713] "Ostrea maxima"
#> [714] "Pecten maximus"
#> [715] "Felis viverrina"
#> [716] "Prionailurus viverrinus"
#> [717] "Gymnodraco acuticeps"
#> [718] "Thalarctos maritimus"
#> [719] "Ursus maritimus"
#> [720] "Lemur catta"
#> [721] "Myotis myotis"
#> [722] "Vespertilio myotis"
#> [723] "Lytechinus pictus"
#> [724] "Psammechinus pictus"
#> [725] "Litopenaeus vannamei"
#> [726] "Penaeus (Litopenaeus)_vannamei"
#> [727] "Penaeus vannamei"
#> [728] "Ursus arctos"
#> [729] "Vitis riparia"
#> [730] "Felis bengalensis"
#> [731] "Prionailurus bengalensis"
#> [732] "Clethrionomys glareolus"
#> [733] "Mus glareolus"
#> [734] "Myodes glareolus"
#> [735] "Mustela nigripes"
#> [736] "Putorius nigripes"
#> [737] "Alopex lagopus"
#> [738] "Canis lagopus"
#> [739] "Vulpes lagopus"
#> [740] "Cercocebus atys"
#> [741] "Cercocebus torquatus_atys"
#> [742] "Simia atys"
#> [743] "Lepidosiren annectens"
#> [744] "Protopterus annectens"
#> [745] "Rhinocryptis annectens"
#> [746] "Cerasus avium"
#> [747] "Prunus avium"
#> [748] "Prunus cerasus_var._avium"
#> [749] "Procambarus clarkii"
#> [750] "Sorex fumeus"
#> [751] "Macrorhinus angustirostris"
#> [752] "Mirounga angustirostris"
#> [753] "Beta vulgaris_subsp._vulgaris"
#> [754] "Beta vulgaris_subsp._vulgaris_var._altissima"
#> [755] "Beta vulgaris_Sugar_Beet_Group"
#> [756] "Beta vulgaris_var._altissima"
#> [757] "Eumetopias jubatus"
#> [758] "Phoca jubata"
#> [759] "Centruroides sculpturatus"
#> [760] "Diceros bicornis_minor"
#> [761] "Cicer arietinum"
#> [762] "Cleome hassleriana_Chodat,_1898"
#> [763] "Tarenaya hassleriana"
#> [764] "Sebastes umbrosus"
#> [765] "Sebastichthys umbrosus"
#> [766] "Eriocheir chinensis"
#> [767] "Eriocheir japonica_sinensis"
#> [768] "Eriocheir sinensis"
#> [769] "Dicentrarchus labrax"
#> [770] "Labrax labrax"
#> [771] "Morone labrax"
#> [772] "Perca labrax"
#> [773] "Roccus labrax"
#> [774] "Sciaena labrax"
#> [775] "Acanthopagrus latus"
#> [776] "Sparus latus"
#> [777] "Xiphophorus hellerii"
#> [778] "Xiphophorus helleri"
#> [779] "Acanthochromis polyacanthus"
#> [780] "Acanthochromis polyacathus"
#> [781] "Dascyllus polyacanthus"
#> [782] "Mustela vison"
#> [783] "Neogale vison"
#> [784] "Neovison vison"
#> [785] "Lingula anatina"
#> [786] "Lingula lingua"
#> [787] "Lingula nipponica"
#> [788] "Lingula unguis"
#> [789] "Madrepora faveolata"
#> [790] "Montastraea faveolata"
#> [791] "Montastrea faveolata"
#> [792] "Orbicella faveolata"
#> [793] "Esox lucius"
#> [794] "Chinchilla lanigera"
#> [795] "Chinchilla velligera"
#> [796] "Chinchilla villidera"
#> [797] "Mirounga leonina"
#> [798] "Phoca leonina"
#> [799] "Perognathus longimembris_pacificus"
#> [800] "Cynocephalus variegatus"
#> [801] "Galeopithecus variegatus"
#> [802] "Galeopterus variegatus"
#> [803] "Vigna radiata"
#> [804] "Vitis vinifera"
#> [805] "Vitis vinifera_subsp._vinifera"
#> [806] "Characodon multiradiatus"
#> [807] "Girardinichthys multiradiatus"
#> [808] "Marmota flaviventris"
#> [809] "Phaseolus calcaratus"
#> [810] "Phaseolus chrysanthos"
#> [811] "Phaseolus chrysanthus"
#> [812] "Vigna calcarata"
#> [813] "Vigna umbellata"
#> [814] "Balaenoptera acutorostrata"
#> [815] "Canis procyonoides"
#> [816] "Nyctereutes procyonoides"
#> [817] "Amphioxus floridae"
#> [818] "Branchiostoma floridae"
#> [819] "Moschus berezovskii"
#> [820] "Erythranthe guttata"
#> [821] "Mimulus guttatus_subsp._guttatus"
#> [822] "Mimulus guttatus"
#> [823] "Camelus bactrianus"
#> [824] "Desmodus rotundus"
#> [825] "Phyllostoma rotundum"
#> [826] "Octopus sinensis"
#> [827] "Physeter catodon"
#> [828] "Physeter macrocephalus"
#> [829] "Alexandromys fortis"
#> [830] "Microtus fortis"
#> [831] "Dendronephthya gigantea"
#> [832] "Canis hyaena"
#> [833] "Hyaena hyaena"
#> [834] "Helicophagus hypophthalmus"
#> [835] "Pangasianodon hypophthalmus"
#> [836] "Pangasius hypophthalmus"
#> [837] "Pangasius sutchi"
#> [838] "Pseudochaenichthys georgianus"
#> [839] "Capsella rubella"
#> [840] "Perkinsus marinus_ATCC_50983"
#> [841] "Holocentrus leopardus"
#> [842] "Plectropomus leopardus"
#> [843] "Hippocampus zosterae"
#> [844] "Artibeus jamaicensis"
#> [845] "Citrus sinensis"
#> [846] "Citrus x_sinensis"
#> [847] "Punica granatum"
#> [848] "Abrus cyaneus"
#> [849] "Abrus precatorius"
#> [850] "Polypterus senegalus"
#> [851] "Acomys russatus"
#> [852] "Mus russatus"
#> [853] "Hemibagrus wyckioides"
#> [854] "Macrones wyckioides"
#> [855] "Mystus wyckioides"
#> [856] "Melanotaenia boesemani"
#> [857] "Sturnira hondurensis"
#> [858] "Amphilophus centrarchus"
#> [859] "Archocentrus centrarchus"
#> [860] "Cichlasoma centrarchus"
#> [861] "Heros centrarchus"
#> [862] "Delphinus melas"
#> [863] "Globicephala melaena"
#> [864] "Globicephala melas"
#> [865] "Manis javanica"
#> [866] "Phyllostomus hastatus"
#> [867] "Vespertilio hastatus"
#> [868] "Scyliorhinus canicula"
#> [869] "Squalus canicula"
#> [870] "Silurana tropicalis"
#> [871] "Xenopus laevis_tropicalis"
#> [872] "Xenopus (Silurana)_tropicalis"
#> [873] "Xenopus tropicalis"
#> [874] "Pipistrellus kuhlii"
#> [875] "Pipistrellus kuhli"
#> [876] "Vespertilio kuhlii"
#> [877] "Solea senegalensis"
#> [878] "Mugil cephalotus"
#> [879] "Mugil cephalus"
#> [880] "Mugil galapagensis"
#> [881] "Mugil japonicus"
#> [882] "Siphostoma scovelli"
#> [883] "Syngnathus scovelli"
#> [884] "Capra aegagrus_hircus"
#> [885] "Capra hircus"
#> [886] "Poeciliopsis prolifica"
#> [887] "Gopherus flavomarginatus"
#> [888] "Lontra canadensis"
#> [889] "Lutra canadensis"
#> [890] "Hesperomys torridus"
#> [891] "Onychomys torridus"
#> [892] "Elephas africanus"
#> [893] "Loxodonta africana_africana"
#> [894] "Loxodonta africana"
#> [895] "Boophilus microplus"
#> [896] "Rhipicephalus (Boophilus)_microplus"
#> [897] "Rhipicephalus microplus"
#> [898] "Molossus molossus"
#> [899] "Vespertilio molossus"
#> [900] "Lagenorhynchus obliquidens"
#> [901] "Delphinus cymodoce"
#> [902] "Delphinus truncatus"
#> [903] "Tursiops cymodoce"
#> [904] "Tursiops truncatus"
#> [905] "Morone flavescens"
#> [906] "Perca flavescens"
#> [907] "Euarctos americanus"
#> [908] "Ursus americanus"
#> [909] "Arvicola nivalis"
#> [910] "Chionomys nivalis"
#> [911] "Microtus nivalis"
#> [912] "Felis rufus"
#> [913] "Lynx rufus"
#> [914] "Myotis brandtii"
#> [915] "Vespertilio brandtii"
#> [916] "Astatotilapia burtoni"
#> [917] "Chromis burtoni"
#> [918] "Haplochromis burtoni"
#> [919] "Silurus meridionalis"
#> [920] "Silurus soldatovi_meridionalis"
#> [921] "Cucumis melo"
#> [922] "Hydra attenuata"
#> [923] "Hydra carnea"
#> [924] "Hydra littoralis"
#> [925] "Hydra magnipapillata"
#> [926] "Hydra vulgaris"
#> [927] "Anoplopoma fimbria"
#> [928] "Gadus fimbria"
#> [929] "Alosa alosa"
#> [930] "Clupea alosa"
#> [931] "Chelonia mydas"
#> [932] "Testudo mydas"
#> [933] "Ctenocephalides felis"
#> [934] "Stylophora pistillata"
#> [935] "Cyrtodiopsis dalmanii"
#> [936] "Diopsis dalmanni"
#> [937] "Teleopsis dalmanni"
#> [938] "Rhagoletis zephyria"
#> [939] "Rhodamnia argentea"
#> [940] "Gasterosteus aculeatus"
#> [941] "Labrus celidotus"
#> [942] "Notolabrus celidotus"
#> [943] "Budorcas taxicolor"
#> [944] "Nelumbo nucifera"
#> [945] "Amphiprion ocellaris"
#> [946] "Arvicola amphibius"
#> [947] "Arvicola terrestris_(Linnaeus,_1758)"
#> [948] "Mus amphibius"
#> [949] "Daphnia magna"
#> [950] "Phaseolus vulgaris"
#> [951] "Psammomys obesus"
#> [952] "Carlito syrichta"
#> [953] "Simia syrichta"
#> [954] "Tarsius syrichta"
#> [955] "Cyprinodon tularosa"
#> [956] "Arvicola princeps"
#> [957] "Ochotona princeps"
#> [958] "Phytophthora sojae"
#> [959] "Equus caballus_przewalskii"
#> [960] "Equus ferus_przewalskii"
#> [961] "Equus przewalskii"
#> [962] "Phoca vitulina"
#> [963] "Coecilia bivitatum"
#> [964] "Rhinatrema bivitattum"
#> [965] "Rhinatrema bivittatum"
#> [966] "Lagomys curzoniae"
#> [967] "Ochotona curzonae"
#> [968] "Ochotona curzoniae"
#> [969] "Kogia breviceps"
#> [970] "Physeter breviceps"
#> [971] "Clupea cyprinoides"
#> [972] "Megalops cyprinoides"
#> [973] "Diospyros lotus"
#> [974] "Hippoglossus stenolepis"
#> [975] "Phacochoerus africanus"
#> [976] "Corythoichthys intestinalis"
#> [977] "Syngnatus intestinalis"
#> [978] "Mandrillus leucophaeus"
#> [979] "Papio leucophaeus"
#> [980] "Simia leucophaea"
#> [981] "Epinephelus fuscoguttatus"
#> [982] "Perca summana_fuscoguttata"
#> [983] "Asterias miniata"
#> [984] "Asterina miniata"
#> [985] "Patiria miniata"
#> [986] "Rhinolophus rouxii_sinicus"
#> [987] "Rhinolophus sinicus"
#> [988] "Lampris incognitus"
#> [989] "Monachus schauinslandi"
#> [990] "Neomonachus schauinslandi"
#> [991] "Hippoglossus hippoglossus"
#> [992] "Pleuronectes hippoglossus"
#> [993] "Andrographis paniculata"
#> [994] "Etheostoma cragini"
#> [995] "Perca chuatsi"
#> [996] "Siniperca chuatsi"
#> [997] "Meriones unguiculatus"
#> [998] "Colobus angolensis_palliatus"
#> [999] "Notothenia coriiceps"
#> [1000] "Hypomesus transpacificus"
#> [1001] "Dermochelys coriacea"
#> [1002] "Testudo coriacea"
#> [1003] "Bufo bufo_gargarizans"
#> [1004] "Bufo gargarizans"
#> [1005] "Bufo japonicus_gargarizans"
#> [1006] "Delphinapterus leucas"
#> [1007] "Delphinus leucas"
#> [1008] "Fugu flavidus"
#> [1009] "Takifugu flavidus"
#> [1010] "Pteronotus mesoamericanus"
#> [1011] "Pteronotus parnellii_mesoamericanus"
#> [1012] "Citrus clementina"
#> [1013] "Citrus deliciosa_x_Citrus_sinensis"
#> [1014] "Citrus x_clementina"
#> [1015] "Fugu rubripes"
#> [1016] "Sphaeroides rubripes"
#> [1017] "Takifugu rubripes"
#> [1018] "Tetraodon rubripes"
#> [1019] "Homarus americanus"
#> [1020] "Osteoglossum formosum"
#> [1021] "Scleropages formosus"
#> [1022] "Larimichthys crocea"
#> [1023] "Pseudosciaena amblyceps"
#> [1024] "Pseudosciaena crocea"
#> [1025] "Sciaena crocea"
#> [1026] "Fragaria vesca"
#> [1027] "Folsomia candida"
#> [1028] "Limulus polyphemus"
#> [1029] "Monoculus polyphemus"
#> [1030] "Doryrhamphus dactyliophorus"
#> [1031] "Dunckerocampus dactyliophorus"
#> [1032] "Syngnathus dactyliophorus"
#> [1033] "Epinephelus lanceolatus"
#> [1034] "Holocentrus lanceolatus"
#> [1035] "Promicrops lanceolatus"
#> [1036] "Mizuhopecten yessoensis"
#> [1037] "Patinopecten yessoensis"
#> [1038] "Patiopecten yessoensis"
#> [1039] "Pecten yessoensis"
#> [1040] "Platypoecilus maculatus"
#> [1041] "Xiphophorus maculatus"
#> [1042] "Triplophysa rosa"
#> [1043] "Antechinus flavipes"
#> [1044] "Phascogale flavipes"
#> [1045] "Balaena musculus"
#> [1046] "Balaenoptera musculus"
#> [1047] "Rhinolophus ferrumequinum"
#> [1048] "Vespertilio ferrumequinum"
#> [1049] "Oryza brachyantha"
#> [1050] "Chrysemys picta"
#> [1051] "Testudo picta"
#> [1052] "Trachemys picta"
#> [1053] "Tetrahymena thermophila_SB210"
#> [1054] "Amygdalus communis"
#> [1055] "Amygdalus dulcis"
#> [1056] "Prunus amygdalus"
#> [1057] "Prunus communis"
#> [1058] "Prunus dulcis"
#> [1059] "Prunus dulcis_var._sativa"
#> [1060] "Oryzias latipes"
#> [1061] "Poecilia latipes"
#> [1062] "Sarcophilus harrisii"
#> [1063] "Sarcophilus laniarius_(Owen,_1838)"
#> [1064] "Sarcophilus laniarius"
#> [1065] "Ursinus harrisii"
#> [1066] "Ictalurus furcatus"
#> [1067] "Pimelodus furcatus"
#> [1068] "Amphioxus belcheri"
#> [1069] "Branchiostoma belcheri"
#> [1070] "Gigantopelta aegis"
#> [1071] "Echinus variegatus"
#> [1072] "Lytechinus variegatus"
#> [1073] "Diaphorina citri"
#> [1074] "Epinephelus moara"
#> [1075] "Serranus moara"
#> [1076] "Stegodyphus dumicola"
#> [1077] "Boleophthalmus pectinirostris"
#> [1078] "Gobius pectinirostris"
#> [1079] "Austrofundulus limnaeus"
#> [1080] "Columba livia_domestica"
#> [1081] "Columba livia"
#> [1082] "Latimeria chalumnae"
#> [1083] "Pleuronectes maximus"
#> [1084] "Psetta maxima"
#> [1085] "Rhombus maximus"
#> [1086] "Scophthalmus maximus"
#> [1087] "Sesamum indicum"
#> [1088] "Sesamum orientale"
#> [1089] "Cyclopterus lumpus"
#> [1090] "Armeniaca mume"
#> [1091] "Prunus mume"
#> [1092] "Myotis davidii"
#> [1093] "Vespertilio Davidii"
#> [1094] "Didelphys agilis"
#> [1095] "Gracilinanus agilis"
#> [1096] "Acanthophacelus reticulata"
#> [1097] "Poecilia (Acanthophacelus)_reticulata"
#> [1098] "Poecilia latipinna_reticulata"
#> [1099] "Poecilia reticulata"
#> [1100] "Australorbis glabratus"
#> [1101] "Biomphalaria glabrata"
#> [1102] "Planorbis glabratus"
#> [1103] "Hypudaeus ochrogaster"
#> [1104] "Microtus ochrogaster"
#> [1105] "Amygdalus persica"
#> [1106] "Persica vulgaris"
#> [1107] "Prunus persica"
#> [1108] "Prunus persica_var._densa"
#> [1109] "Chiloscyllium plagiosum"
#> [1110] "Scyllium plagiosum"
#> [1111] "Cheilinus undulatus"
#> [1112] "Phodopus roborovskii"
#> [1113] "Caenorhabditis remanei"
#> [1114] "Caenorhabditis vulgaris"
#> [1115] "Lamprologus brichardi"
#> [1116] "Neolamprologus brichardi"
#> [1117] "Gymnopis unicolor"
#> [1118] "Microcaecilia unicolor"
#> [1119] "Rhinatrema unicolor"
#> [1120] "Sciaena jaculatrix"
#> [1121] "Toxotes jaculatrix"
#> [1122] "Bos indicus"
#> [1123] "Bos primigenius_indicus"
#> [1124] "Bos taurus_indicus"
#> [1125] "Lacerta sicula_raffonei"
#> [1126] "Podarcis raffoneae"
#> [1127] "Podarcis raffonei"
#> [1128] "Podarcis wagleriana_raffonei"
#> [1129] "Benincasa cerifera"
#> [1130] "Benincasa hispida"
#> [1131] "Benincasa pruriens"
#> [1132] "Cucurbita hispida"
#> [1133] "Lagenaria siceraria_var._hispida"
#> [1134] "Dendrobium catenatum"
#> [1135] "Marsupenaeus japonicus"
#> [1136] "Penaeus japonicus"
#> [1137] "Penaeus (Marsupenaeus)_japonicus"
#> [1138] "Penaeus (Melicertus)_japonicus"
#> [1139] "Chaetodon argus"
#> [1140] "Scatophagus argus"
#> [1141] "Chanos chanos"
#> [1142] "Mugil chanos"
#> [1143] "Bison bison_bison"
#> [1144] "Bos bison_bison"
#> [1145] "Amblyraja radiata"
#> [1146] "Raja radiata"
#> [1147] "Amphimedon queenslandica"
#> [1148] "Hippocampus comes"
#> [1149] "Hipposideros armiger"
#> [1150] "Rhinolophus armiger"
#> [1151] "Cynoglossus (Arelia)_semilaevis"
#> [1152] "Cynoglossus semilaevis"
#> [1153] "Alecto japonica"
#> [1154] "Anneissia japonica"
#> [1155] "Oxycomanthus japonicus"
#> [1156] "Ananas comosus"
#> [1157] "Ananas comosus_var._comosus"
#> [1158] "Ananas lucidus"
#> [1159] "Bromelia comosa"
#> [1160] "Callionymus splendidus"
#> [1161] "Pterosynchiropus splendidus"
#> [1162] "Synchiropus splendidus"
#> [1163] "Neophocaena asiaeorientalis_asiaeorientalis"
#> [1164] "Coluber guttatus"
#> [1165] "Elaphe guttata"
#> [1166] "Pantherophis guttatus"
#> [1167] "Pollicipes cornucopia"
#> [1168] "Pollicipes pollicipes"
#> [1169] "Pseudoliparis swirei"
#> [1170] "Chelonoidis abingdonii"
#> [1171] "Chelonoidis abingdoni"
#> [1172] "Chelonoidis nigra_abingdonii"
#> [1173] "Geochelone nigra_abigdonii"
#> [1174] "Geochelone nigra_abingdoni"
#> [1175] "Geochelone nigra_ephippium"
#> [1176] "Testudo abingdonii"
#> [1177] "Rhincodon typus"
#> [1178] "Ricinus communis"
#> [1179] "Ricinus sanguineus"
#> [1180] "Malania oleifera"
#> [1181] "Ceratotherium simum_simum"
#> [1182] "Kryptolebias marmoratus"
#> [1183] "Rivulus marmoratus"
#> [1184] "Patella vulgata"
#> [1185] "Rhagoletis pomonella"
#> [1186] "Trypanosoma cruzi"
#> [1187] "Squalus fasciatus"
#> [1188] "Squalus tigrinus"
#> [1189] "Stegostoma fasciatum"
#> [1190] "Stegostoma tigrinum"
#> [1191] "Cistudo triunguis"
#> [1192] "Terrapene mexicana_triunguis"
#> [1193] "Terrapene triunguis"
#> [1194] "Odobenus rosmarus_divergens"
#> [1195] "Manatus latirostris"
#> [1196] "Trichechus manatus_latirostris"
#> [1197] "Carcharodon carcharias"
#> [1198] "Squalus carcharias"
#> [1199] "Macrognathus armatus"
#> [1200] "Mastacembelus armatus"
#> [1201] "Anas boschas"
#> [1202] "Anas domesticus"
#> [1203] "Anas platyrhynchos_f._domestica"
#> [1204] "Anas platyrhynchos"
#> [1205] "Theobroma cacao"
#> [1206] "Diabrotica virgifera_virgifera"
#> [1207] "Actinia diaphana"
#> [1208] "Aiptasia pallida"
#> [1209] "Aiptasia pulchella"
#> [1210] "Dysactis pallida"
#> [1211] "Exaiptasia diaphana"
#> [1212] "Exaiptasia pallida"
#> [1213] "Syngnathus acus_rubescens"
#> [1214] "Syngnathus acus"
#> [1215] "Syngnathus rubescens"
#> [1216] "Caretta caretta"
#> [1217] "Testudo caretta"
#> [1218] "Guillardia theta_CCMP2712"
#> [1219] "Anarrhichthys ocellatus"
#> [1220] "Pelodiscus sinensis"
#> [1221] "Trionyx sinensis"
#> [1222] "Hippoglossus olivaceus"
#> [1223] "Paralichthys olivaceus"
#> [1224] "Xiphias gladius"
#> [1225] "Cyprinodon variegatus"
#> [1226] "Bos grunniens_mutus"
#> [1227] "Bos mutus"
#> [1228] "Poephagus mutus"
#> [1229] "Alligator sinensis"
#> [1230] "Morus notabilis"
#> [1231] "Nymphaea colorata"
#> [1232] "Photinus pyralis"
#> [1233] "Periophthalmus magnuspinnatus"
#> [1234] "Meleagris gallopavo"
#> [1235] "Pomacea canaliculata"
#> [1236] "Haplochromis nyererei"
#> [1237] "Pundamilia nyererei"
#> [1238] "Caranx dumerili"
#> [1239] "Seriola dumerili"
#> [1240] "Macrosteles (Macrosteles)_quadrilineatus"
#> [1241] "Macrosteles quadrilineatus"
#> [1242] "Enhydra lutris_kenyoni"
#> [1243] "Fluta alba"
#> [1244] "Monopterus albus"
#> [1245] "Muraena alba"
#> [1246] "Caecilia seraphini"
#> [1247] "Caecilia Seraphini"
#> [1248] "Geotrypetes seraphini"
#> [1249] "Hypogeophis seraphini"
#> [1250] "Chaetodon rostratus"
#> [1251] "Chelmon rostratus"
#> [1252] "Cucumis sativus"
#> [1253] "Cyrtodactylus macularius"
#> [1254] "Eublepharis macularius"
#> [1255] "Felis concolor"
#> [1256] "Panthera concolor"
#> [1257] "Puma concolor"
#> [1258] "Cancer chinensis"
#> [1259] "Fenneropenaeus chinensis"
#> [1260] "Penaeus chinensis"
#> [1261] "Pomacentrus partitus"
#> [1262] "Stegastes partitus"
#> [1263] "Phascum patens"
#> [1264] "Physcomitrella patens_subsp._patens"
#> [1265] "Physcomitrella patens"
#> [1266] "Physcomitrium patens"
#> [1267] "Anas jamaicensis"
#> [1268] "Oxyura jamaicensis"
#> [1269] "Drosophila miranda"
#> [1270] "Lottia gigantea"
#> [1271] "Eurytemora affinis"
#> [1272] "Temora affinis"
#> [1273] "Crotalus tigris"
#> [1274] "Argentina anserina"
#> [1275] "Potentilla anserina"
#> [1276] "Achaearanea tepidariorum"
#> [1277] "Parasteatoda tepidariorum"
#> [1278] "Theridion tepidariorum"
#> [1279] "Uranotaenia lowii"
#> [1280] "Cynolebias whitei"
#> [1281] "Nematolebias whitei"
#> [1282] "Simpsonichthys whitei"
#> [1283] "Sceloporus undulatus"
#> [1284] "Stellio undulatus"
#> [1285] "Helobdella robusta"
#> [1286] "Styela clava"
#> [1287] "Manis afer_afer"
#> [1288] "Orycteropus afer_afer"
#> [1289] "Leucoraja erinacea"
#> [1290] "Raja erinacea"
#> [1291] "Raja erinaceus"
#> [1292] "Raja erinacia"
#> [1293] "Phytophthora nicotianae_INRA-310"
#> [1294] "Anas olor"
#> [1295] "Cygnus olor"
#> [1296] "Lacerta agilis"
#> [1297] "Millepora damicornis"
#> [1298] "Pocillopora caespitosa_laysanensis"
#> [1299] "Pocillopora damicornis_laysanensis"
#> [1300] "Pocillopora damicornis"
#> [1301] "Morone saxatilis"
#> [1302] "Perca saxatilis"
#> [1303] "Miniopterus natalensis"
#> [1304] "Miniopterus schreibersii_natalensis"
#> [1305] "Vespertilio natalensis"
#> [1306] "Anas cygnoid"
#> [1307] "Anser cygnoides"
#> [1308] "Actinia tenebrosa"
#> [1309] "Neptunus trituberculatus"
#> [1310] "Portunus (Portunus)_trituberculatus"
#> [1311] "Portunus trituberculatus"
#> [1312] "Lacerta vivipara"
#> [1313] "Zootoca vivipara"
#> [1314] "Propithecus coquereli"
#> [1315] "Propithecus verreauxi_coquereli"
#> [1316] "Erinaceus europaeus"
#> [1317] "Jatropha curcas"
#> [1318] "Caenorhabditis briggsae"
#> [1319] "Rhabditis briggsae"
#> [1320] "Cherax quadricarinatus"
#> [1321] "Homalodisca coagulata"
#> [1322] "Homalodisca vitripennis"
#> [1323] "Tettigonia coagulata"
#> [1324] "Tettigonia vitripennis"
#> [1325] "Anolis carolinensis"
#> [1326] "Python bivittatus"
#> [1327] "Python molurus_bivittatus"
#> [1328] "Chrysemys scripta_elegans"
#> [1329] "Emys elegans"
#> [1330] "Pseudemys scripta_elegans"
#> [1331] "Trachemys scripta_elegans"
#> [1332] "Protobothrops mucrosquamatus"
#> [1333] "Trigonocephalus mucrosquamatus"
#> [1334] "Trimeresurus mucrosquamatus"
#> [1335] "Daphnia pulex"
#> [1336] "Paramacrobiotus metropolitanus"
#> [1337] "Lipotes vexillifer"
#> [1338] "Petromyzon marinus"
#> [1339] "Poephila guttata"
#> [1340] "Taeniopygia guttata"
#> [1341] "Taenopygia guttata"
#> [1342] "Aplysia californica"
#> [1343] "Phalaenopsis equestris"
#> [1344] "Stauroglottis equestris"
#> [1345] "Balanoglossus kowalevskii"
#> [1346] "Saccoglossus kowalevskii"
#> [1347] "Saccoglossus kowalevskyi"
#> [1348] "Numida meleagris"
#> [1349] "Phasianus meleagris"
#> [1350] "Momordica charantia"
#> [1351] "Callorhinchus milii"
#> [1352] "Sphaerodactylus townsendi"
#> [1353] "Eutainia elegans"
#> [1354] "Thamnophis elegans"
#> [1355] "Corvus hawaiiensis"
#> [1356] "Manacus candei"
#> [1357] "Pipra candei"
#> [1358] "Euleptes europaea"
#> [1359] "Euleptes europea"
#> [1360] "Phyllodactylus europaea"
#> [1361] "Phyllodactylus europaeus"
#> [1362] "Ptyodactylus caudivolvolus"
#> [1363] "Lepisosteus oculatus"
#> [1364] "Altirana parkeri"
#> [1365] "Nanorana parkeri"
#> [1366] "Ahaetulla prasina"
#> [1367] "Dryophis prasinus"
#> [1368] "Fusarium oxysporum_f._sp._lycopersici_4287"
#> [1369] "Heteropelma chrysocephalum"
#> [1370] "Neopelma chrysocephalum"
#> [1371] "Musca domestica"
#> [1372] "Pristis pectinata"
#> [1373] "Ischnura elegans"
#> [1374] "Fringilla chalybeata"
#> [1375] "Vidua chalybeata"
#> [1376] "Coturnix coturnix_japanica"
#> [1377] "Coturnix coturnix_japonica"
#> [1378] "Coturnix coturnix_Japonicus"
#> [1379] "Coturnix japonica_japonica"
#> [1380] "Coturnix japonica"
#> [1381] "Gekko japonicus"
#> [1382] "Platydactylus japonicus"
#> [1383] "Nilaparvata lugens"
#> [1384] "Ardea americana"
#> [1385] "Grus americana"
#> [1386] "Grus americanus"
#> [1387] "Harpia harpyja"
#> [1388] "Vultur harpyja"
#> [1389] "Pipra filicauda"
#> [1390] "Herrania umbratica"
#> [1391] "Ilyonectria robusta"
#> [1392] "Cupidonia cupido_pallidicincta"
#> [1393] "Tympanuchus pallidicinctus"
#> [1394] "Topomyia yanbarensis"
#> [1395] "Parus atricapillus"
#> [1396] "Poecile atricapilla"
#> [1397] "Poecile atricapillus"
#> [1398] "Corapipo altera"
#> [1399] "Acyrthosiphon pisum"
#> [1400] "Acyrthosiphum pisum"
#> [1401] "Varanus komodoensis"
#> [1402] "Saprolegnia parasitica_CBS_223.65"
#> [1403] "Fringilla macroura"
#> [1404] "Vidua macroura"
#> [1405] "Carica papaya"
#> [1406] "Chiroxiphia lanceolata"
#> [1407] "Pipra lanceolata"
#> [1408] "Octopus bimaculoides"
#> [1409] "Lagopus muta"
#> [1410] "Tetrao mutus"
#> [1411] "Bradysia coprophila"
#> [1412] "Sciara coprophila"
#> [1413] "Coluber sirtalis"
#> [1414] "Thamnophis sirtalis"
#> [1415] "Falco peregrinus"
#> [1416] "Falco cherrug"
#> [1417] "Asteracanthion distichum"
#> [1418] "Asterias attenuata"
#> [1419] "Asterias clathrata"
#> [1420] "Asterias disticha"
#> [1421] "Asterias gigantea"
#> [1422] "Asterias pallida"
#> [1423] "Asterias rubens"
#> [1424] "Asterias stimpsoni"
#> [1425] "Asterias vulgaris"
#> [1426] "Manduca sexta"
#> [1427] "Sphinx sexta"
#> [1428] "Condylura cristata"
#> [1429] "Sorex cristatus"
#> [1430] "Cuculus canorus"
#> [1431] "Pezoporus wallicus"
#> [1432] "Aedes aegypti"
#> [1433] "Aedes (Stegomyia)_aegypti"
#> [1434] "Culex aegypti"
#> [1435] "Stegomyia aegypti"
#> [1436] "Falco naumanni"
#> [1437] "Corvus kubaryi"
#> [1438] "Necator americanus"
#> [1439] "Larus tridactylus"
#> [1440] "Rissa tridactyla"
#> [1441] "Aphanomyces astaci"
#> [1442] "Culex (Culex)_pipiens_pallens"
#> [1443] "Culex pipiens_pallens"
#> [1444] "Catharus ustulatus"
#> [1445] "Turdus ustulatus"
#> [1446] "Accipiter gentilis"
#> [1447] "Accipiter gentillis"
#> [1448] "Falco gentilis"
#> [1449] "Crocodylus porosus"
#> [1450] "Amborella trichopoda"
#> [1451] "Falco biarmicus"
#> [1452] "Lagopus leucura"
#> [1453] "Lagopus leucurus"
#> [1454] "Tetrao leucurus"
#> [1455] "Falco rusticolus"
#> [1456] "Phasianus colchicus"
#> [1457] "Corvus brachyrhynchos"
#> [1458] "Uloborus diversus"
#> [1459] "Phytophthora infestans_strain_T30-4"
#> [1460] "Phytophthora infestans_T30-4"
#> [1461] "Empidonax traillii"
#> [1462] "Muscicapa traillii"
#> [1463] "Strix alba"
#> [1464] "Tyto alba"
#> [1465] "Parus major"
#> [1466] "Lepeophtheirus salmonis"
#> [1467] "Gavialis gangeticus"
#> [1468] "Lacerta gangetica"
#> [1469] "Daphnia carinata"
#> [1470] "Aphis gossypii"
#> [1471] "Ampithoe aztecus"
#> [1472] "Hyalella azteca"
#> [1473] "Hyalella knickerbockeri"
#> [1474] "Colletotrichum lupini"
#> [1475] "Gloeosporium lupini"
#> [1476] "Sphaeroforma arctica_JP610"
#> [1477] "Suillus fuscotomentosus"
#> [1478] "Mollisia scopiformis"
#> [1479] "Phialocephala scopiformis"
#> [1480] "Muscicapa cayanensis"
#> [1481] "Myiozetetes cayanensis"
#> [1482] "Hyaloscypha bicolor_E"
#> [1483] "Melopsittacus undulatus"
#> [1484] "Psittacus undulatus"
#> [1485] "Fringilla montana"
#> [1486] "Passer montanus"
#> [1487] "Coccinella axyridis"
#> [1488] "Harmonia axyridis"
#> [1489] "Aimophila crissalis"
#> [1490] "Kieneria crissalis"
#> [1491] "Kieneria crissalis_(Vigors,_1839)"
#> [1492] "Melozone crissalis"
#> [1493] "Pipilo crissalis"
#> [1494] "Pipilo fuscus_crissalis"
#> [1495] "Stomoxis calcitrans"
#> [1496] "Stomoxys calcitrans"
#> [1497] "Anas atrata"
#> [1498] "Cygnus atratus"
#> [1499] "Culex fatigans"
#> [1500] "Culex pipiens_fatigans"
#> [1501] "Culex pipiens_quinquefasciatus"
#> [1502] "Culex quinquefasciatus"
#> [1503] "Hirundo rustica"
#> [1504] "Acanthaster planci"
#> [1505] "Asterias planci"
#> [1506] "Molothrus ater"
#> [1507] "Oriolus ater"
#> [1508] "Laccaria bicolor_S238N-H82"
#> [1509] "Anastrepha obliqua"
#> [1510] "Tephritis obliqua"
#> [1511] "Grapholitha glycinivorella"
#> [1512] "Leguminivora glycinivorella"
#> [1513] "Ammodromus caudacutus_nelsoni"
#> [1514] "Ammospiza nelsoni"
#> [1515] "Nylanderia fulva"
#> [1516] "Paratrechina fulva"
#> [1517] "Agelaius phoeniceus"
#> [1518] "Agelaius phoniceus"
#> [1519] "Oriolus phoeniceus"
#> [1520] "Colletotrichum fructicola"
#> [1521] "Colletotrichum ignotum"
#> [1522] "Euthrips occidentalis"
#> [1523] "Frankliniella brunnescens"
#> [1524] "Frankliniella californica"
#> [1525] "Frankliniella occidentalis_brunnescens"
#> [1526] "Frankliniella occidentalis"
#> [1527] "Motacilla alba_alba"
#> [1528] "Fusarium solani"
#> [1529] "Fusisporium solani"
#> [1530] "Neocosmospora solani"
#> [1531] "Sitophilus oryzae"
#> [1532] "Corvus cornix_cornix"
#> [1533] "Fringilla canaria_Linnaeus,_1758"
#> [1534] "Serinus canaria"
#> [1535] "Serinus canarius"
#> [1536] "Drosophila subpulchrella"
#> [1537] "Chlamydomonas reinhardtii"
#> [1538] "Chlamydomonas smithii"
#> [1539] "Puccinia striiformis_f._sp._tritici"
#> [1540] "Bactrocera cucurbitae"
#> [1541] "Bactrocera (Zeugodacus)_cucurbitae"
#> [1542] "Zeugodacus cucurbitae"
#> [1543] "Zeugodacus (Zeugodacus)_cucurbitae"
#> [1544] "Antrodia serialis"
#> [1545] "Fomitopsis serialis"
#> [1546] "Neoantrodia serialis"
#> [1547] "Polyporus serialis"
#> [1548] "Drosophila suzukii"
#> [1549] "Leucophenga suzukii"
#> [1550] "Aedes smithii"
#> [1551] "Wyeomyia smithii"
#> [1552] "Montifringilla ruficollis"
#> [1553] "Pyrgilauda ruficollis"
#> [1554] "Gymnogyps californianus"
#> [1555] "Vultur californianus"
#> [1556] "Bactrocera (Bactrocera)_dorsalis"
#> [1557] "Bactrocera (Bactrocera)_invadens"
#> [1558] "Bactrocera dorsalis"
#> [1559] "Bactrocera invadens"
#> [1560] "Bactrocera papayae"
#> [1561] "Bactrocera philippinensis"
#> [1562] "Dacus dorsalis"
#> [1563] "Trichoplusia ni"
#> [1564] "Leptothorax curvispinosus"
#> [1565] "Temnothorax curvispinosus"
#> [1566] "Saprolegnia declina_VS20"
#> [1567] "Saprolegnia diclina_VS20"
#> [1568] "Fringilla albicollis"
#> [1569] "Zonotrichia albicollis"
#> [1570] "Bactrocera neohumeralis"
#> [1571] "Dacus tryoni_var._neohumeralis"
#> [1572] "Sphaeria pertusa"
#> [1573] "Trematosphaeria pertusa"
#> [1574] "Fusarium oxysporum_var._redolens"
#> [1575] "Fusarium redolens"
#> [1576] "Anastrepha ludens"
#> [1577] "Trypeta ludens"
#> [1578] "Cantharellus anzutake"
#> [1579] "Malurus melanocephalus"
#> [1580] "Muscicapa melanocephala"
#> [1581] "Melitaea cinxia"
#> [1582] "Papilio cinxia"
#> [1583] "Maniola jurtina"
#> [1584] "Papilio jurtina"
#> [1585] "Anas fuligula"
#> [1586] "Aythya fuligula"
#> [1587] "Bombyx mori"
#> [1588] "Phalaena mori"
#> [1589] "Botys furnacalis"
#> [1590] "Ostrinia furnacalis"
#> [1591] "Priapula caudata"
#> [1592] "Priapulus caudatus"
#> [1593] "Apanteles glomeratus"
#> [1594] "Cotesia glomerata"
#> [1595] "Ichneumon glomeratus"
#> [1596] "Centrocercus urophasianus"
#> [1597] "Centrocerus urophasianus"
#> [1598] "Tetrao urophasianus"
#> [1599] "Montifringilla taczanowskii_(Przewalski,_1876)"
#> [1600] "Onychostruthus taczanowskii"
#> [1601] "Monomorium pharaonis"
#> [1602] "Daktulosphaira vitifoliae"
#> [1603] "Pemphigus vitifoliae"
#> [1604] "Viteus vitifoliae"
#> [1605] "Helicoverpa armigera"
#> [1606] "Heliothis armigera"
#> [1607] "Heliothis (Helicoverpa)_armigera"
#> [1608] "Noctua armigera"
#> [1609] "Drosophila biarmipes"
#> [1610] "Myzus (Nectarosiphon)_persicae"
#> [1611] "Myzus persicae"
#> [1612] "Lucilia sericata"
#> [1613] "Phaenicia sericata"
#> [1614] "Tinamus guttatus"
#> [1615] "Solenopsis invicta"
#> [1616] "Fringilla georgiana"
#> [1617] "Melospiza georgiana"
#> [1618] "Helicoverpa zea"
#> [1619] "Heliothis zea"
#> [1620] "Phalaena zea"
#> [1621] "Drosophila ananassae"
#> [1622] "Drosophila annanassae"
#> [1623] "Fusarium odoratissimum_NRRL_54006"
#> [1624] "Coccinella 7-punctata"
#> [1625] "Coccinella septempunctata"
#> [1626] "Spodoptera frugiperda"
#> [1627] "Tigriopus californicus"
#> [1628] "Tisbe californica"
#> [1629] "Ficedula albicollis"
#> [1630] "Muscicapa albicollis"
#> [1631] "Drosophila pseudoobscura"
#> [1632] "Mytilidion resinicola"
#> [1633] "Mytilinidion resinicola"
#> [1634] "Halyomorpha halys"
#> [1635] "Phycomyces blakesleeanus_NRRL_1555(-)"
#> [1636] "Drosophila willistoni"
#> [1637] "Monoraphidium neglectum"
#> [1638] "Sturnus vulgaris"
#> [1639] "Bactrocera tryoni"
#> [1640] "Tephritis tryoni"
#> [1641] "Apus apus"
#> [1642] "Hirundo apus"
#> [1643] "Suillus paluster"
#> [1644] "Naegleria gruberi"
#> [1645] "Suillus discolor"
#> [1646] "Suillus tomentosus_var._discolor"
#> [1647] "Trichina spiralis"
#> [1648] "Trichinella spiralis"
#> [1649] "Onthophagus taurus"
#> [1650] "Epistrophe balteatus"
#> [1651] "Episyrphus balteatus"
#> [1652] "Episyrphus (Episyrphus)_balteatus"
#> [1653] "Musca balteata"
#> [1654] "Leptinotarsa decemlineata"
#> [1655] "Leptinotarsa decimlineata"
#> [1656] "Stilodes decemlineata"
#> [1657] "Boletus plorans"
#> [1658] "Suillus plorans"
#> [1659] "Dryobates pubescens"
#> [1660] "Picoides pubescens_(Linnaeus,_1766)"
#> [1661] "Picoides pubescens"
#> [1662] "Fusarium proliferatum_ET1"
#> [1663] "Fusarium oxysporum_Fo47"
#> [1664] "Drosophila sechellia"
#> [1665] "Schizophyllum commune_H4-8"
#> [1666] "Depressaria gossypiella"
#> [1667] "Pectinophora gossypiella"
#> [1668] "Parus humilis"
#> [1669] "Podoces humilis"
#> [1670] "Pseudopoces humilis"
#> [1671] "Pseudopodoces humilis"
#> [1672] "Ascidia intestinalis"
#> [1673] "Ciona intestinalis"
#> [1674] "Distomum viverrini"
#> [1675] "Opisthorchis viverrini"
#> [1676] "Puccinia graminis_f._sp._tritici_CRL_75-36-700-3"
#> [1677] "Plutella xylostella"
#> [1678] "Melampsora larici-populina_98AG31"
#> [1679] "Drosophila obscura"
#> [1680] "Fusarium verticillioides_7600"
#> [1681] "Anoplophora glabripennis"
#> [1682] "Anoplophora nobilis"
#> [1683] "Cerosterna glabripennis"
#> [1684] "Melanauster nobilis"
#> [1685] "Calypte anna"
#> [1686] "Ornismya anna"
#> [1687] "Microdochium trichocladiopsis"
#> [1688] "Anopheles merus"
#> [1689] "Bactrocera (Daculus)_oleae"
#> [1690] "Bactrocera (Dacus)_oleae"
#> [1691] "Bactrocera oleae"
#> [1692] "Dacus oleae"
#> [1693] "Musca oleae"
#> [1694] "Fusarium mangiferae"
#> [1695] "Drosophila yakuba"
#> [1696] "Contarinia nasturtii"
#> [1697] "Parastagonospora nodorum_SN15"
#> [1698] "Drosophila virilis"
#> [1699] "Zasmidium cellare_ATCC_36951"
#> [1700] "Drosophila mauritiana"
#> [1701] "Geospiza fortis"
#> [1702] "Eupeodes corollae"
#> [1703] "Eupeodes (Eupeodes)_corollae"
#> [1704] "Metasyrphus corollae"
#> [1705] "Spodoptera litura"
#> [1706] "Sitodiplosis mosellana"
#> [1707] "Microgaster mediator"
#> [1708] "Microplitis medianus"
#> [1709] "Microplitis mediator"
#> [1710] "Drosophila kikkawai"
#> [1711] "Diaporthe citri"
#> [1712] "Phomopsis citri"
#> [1713] "Mesites unicolor"
#> [1714] "Mesitornis unicolor"
#> [1715] "Suillus subaureus"
#> [1716] "Colletotrichum capsici"
#> [1717] "Colletotrichum dematium_f._truncatum_(Schwein.)_Arx,_1957"
#> [1718] "Colletotrichum truncatum"
#> [1719] "Glomerella glycines"
#> [1720] "Vermicularia capsici"
#> [1721] "Vermicularia truncata"
#> [1722] "Drosophila simulans"
#> [1723] "Anisochrysa carnea"
#> [1724] "Chrysopa carnea"
#> [1725] "Chrysoperla carnea"
#> [1726] "Drosophila takahashii"
#> [1727] "Lucilia cuprina"
#> [1728] "Drosophila persimilis"
#> [1729] "Falco albicilla"
#> [1730] "Haliaeetus albicilla"
#> [1731] "Antrostomus carolinensis"
#> [1732] "Caprimulgus carolinensis"
#> [1733] "Nasonia vitripennis"
#> [1734] "Colias crocea"
#> [1735] "Colias croceus"
#> [1736] "Papilio croceus"
#> [1737] "Leptidea sinapis"
#> [1738] "Papilio sinapis"
#> [1739] "Anopheles arabiensis"
#> [1740] "Drosophila ficusphila"
#> [1741] "Vollenhovia emeryi"
#> [1742] "Hermetia illucens"
#> [1743] "Fusarium vanettenii_77-13-4"
#> [1744] "Nectria haematococca_mpVI_77-13-4"
#> [1745] "Thrips palmi"
#> [1746] "Falco leucocephalus"
#> [1747] "Haliaeetus leucocephalus"
#> [1748] "Malaya genurostris"
#> [1749] "Colletotrichum gloeosporioides_(Penz.)_Penz._&_Sacc.,_1884"
#> [1750] "Colletotrichum gloeosporioides"
#> [1751] "Glomerella cingulata"
#> [1752] "Glomerella rufomaculans-vaccinii"
#> [1753] "Gnomoniopsis cingulata"
#> [1754] "Vermicularia gloeosporioides"
#> [1755] "Acanthamoeba castellanii_Neff_strain"
#> [1756] "Acanthamoeba castellanii_strain_Neff"
#> [1757] "Acanthamoeba castellanii_str._Neff"
#> [1758] "Drosophila albomicans"
#> [1759] "Drosophila nasuta_albomicans"
#> [1760] "Spinulophila albomicans"
#> [1761] "Diaporthe amygdali"
#> [1762] "Fusicoccum amygdali"
#> [1763] "Phomopsis amygdali"
#> [1764] "Pelecanus crispus"
#> [1765] "Pelecanus philippensis_crispus"
#> [1766] "Drosophila rhopaloa"
#> [1767] "Aphantopus hyperantus"
#> [1768] "Maniola hyperantus"
#> [1769] "Papilio hyperantus"
#> [1770] "Drosophila serrata"
#> [1771] "Leptopilina heterotoma"
#> [1772] "Peronospora halstedii"
#> [1773] "Plasmopara halstedii"
#> [1774] "Cuculus discolor"
#> [1775] "Leptosomus discolor"
#> [1776] "Aphanomyces invadans"
#> [1777] "Drosophila santomea"
#> [1778] "Sipha flava"
#> [1779] "Drosophila teissieri"
#> [1780] "Aptenodytes forsteri"
#> [1781] "Phaethon lepturus"
#> [1782] "Drosophila bipectinata"
#> [1783] "Fulmaris glacialis"
#> [1784] "Fulmarus glacialis"
#> [1785] "Procellaria glacialis"
#> [1786] "Ardea garzetta"
#> [1787] "Egretta garzetta"
#> [1788] "Anopheles mysorensis"
#> [1789] "Anopheles stephensi_mysorensis"
#> [1790] "Anopheles stephensi"
#> [1791] "Anopheles stephensi_var._mysorensis"
#> [1792] "Neocellia intermedia_Rothwell,_1907"
#> [1793] "Neocellia intermedia"
#> [1794] "Cryptotermes secundus"
#> [1795] "Pestalotiopsis fici_W106-1"
#> [1796] "Aricia agestis"
#> [1797] "Papilio agestis"
#> [1798] "Polyommatus agestis"
#> [1799] "Artogeia napi"
#> [1800] "Papilio napi"
#> [1801] "Pieris napi"
#> [1802] "Drosophila eugracilis"
#> [1803] "Wasmannia auropunctata"
#> [1804] "Oppia nitens"
#> [1805] "Adelges cooleyi"
#> [1806] "Chermes cooleyi"
#> [1807] "Gilletteella cooleyi"
#> [1808] "Acanthisitta chloris"
#> [1809] "Sitta chloris"
#> [1810] "Agrilus feretrius"
#> [1811] "Agrilus marcopoli"
#> [1812] "Agrilus planipennis"
#> [1813] "Drosophila elegans"
#> [1814] "Hyposmocoma kahamanoa"
#> [1815] "Cariama cristata"
#> [1816] "Palamedea cristata"
#> [1817] "Aleurodes tabaci"
#> [1818] "Aleyrodes tabaci"
#> [1819] "Bemisia tabaci"
#> [1820] "Ibis nippon"
#> [1821] "Nipponia nippon"
#> [1822] "Balearica gibbericeps"
#> [1823] "Balearica pavonina_gibbericeps"
#> [1824] "Balearica regulorum_gibbericepse"
#> [1825] "Balearica regulorum_gibbericeps"
#> [1826] "Bombus affinis"
#> [1827] "Varroa jacobsoni"
#> [1828] "Drosophila gunungcola"
#> [1829] "Colius striatus"
#> [1830] "Tauraco erythrolophus"
#> [1831] "Colletotrichum aenigma"
#> [1832] "Colletotrichum communis"
#> [1833] "Colletotrichum dianesei"
#> [1834] "Colletotrichum endomangiferae"
#> [1835] "Colletotrichum hymenocallidis"
#> [1836] "Colletotrichum jasmini-sambac"
#> [1837] "Colletotrichum melanocaulon"
#> [1838] "Colletotrichum siamense"
#> [1839] "Pterocles gutturalis"
#> [1840] "Aethina tumida"
#> [1841] "Galleria mellonella"
#> [1842] "Phalaena mellonella"
#> [1843] "Bicyclus anynana"
#> [1844] "Mycalesis anynana"
#> [1845] "Leptopilina boulardi"
#> [1846] "Zootermopsis nevadensis"
#> [1847] "Achroia grisella"
#> [1848] "Tinea grisella"
#> [1849] "Acremonium falciforme"
#> [1850] "Cephalosporium falciforme"
#> [1851] "Fusarium falciforme"
#> [1852] "Neocosmospora falciformis"
#> [1853] "Drosophila mohavensis"
#> [1854] "Drosophila mojavensis"
#> [1855] "Drosophila innubila"
#> [1856] "Bombus huntii"
#> [1857] "Cuculus indicator"
#> [1858] "Indicator indicator"
#> [1859] "Dendroctonus ponderosae"
#> [1860] "Ardea helias"
#> [1861] "Eurypyga helias"
#> [1862] "Dracunculus loa"
#> [1863] "Loa loa"
#> [1864] "Cadophora gregata"
#> [1865] "Cephalosporium gregatum"
#> [1866] "Phialophora gregata"
#> [1867] "Nestor notabilis_notabilis"
#> [1868] "Nestor notabilis"
#> [1869] "Colymbus stellatus"
#> [1870] "Gavia stellata"
#> [1871] "Cynthia cardui"
#> [1872] "Papilio cardui"
#> [1873] "Vanessa cardui"
#> [1874] "Cladosporium fulvum"
#> [1875] "Fulvia fulva"
#> [1876] "Mycovellosiella fulva"
#> [1877] "Passalora fulva"
#> [1878] "Plodia interpunctella"
#> [1879] "Tinea interpunctella"
#> [1880] "Stilbospora angustata"
#> [1881] "Truncatella angustata"
#> [1882] "Truncatella truncata"
#> [1883] "Chlamydotis macqueenii_macqueenii"
#> [1884] "Chlamydotis macqueenii_macqueeni"
#> [1885] "Chlamydotis macqueenii"
#> [1886] "Chlamydotis undulata_macqueenii"
#> [1887] "Otis macqueenii"
#> [1888] "Anopheles funestus"
#> [1889] "Fusarium fujikuroi_IMI_58289"
#> [1890] "Cephalosporium keratoplasticum_(nom._inval.)"
#> [1891] "Fusarium keratoplasticum"
#> [1892] "Neocosmospora keratoplastica"
#> [1893] "Drosophila lebanonensis"
#> [1894] "Scaptodrosophila lebanonensis"
#> [1895] "Merops nubicus"
#> [1896] "Coniothyrium fuckelii_var._sporulosum"
#> [1897] "Coniothyrium sporulosum"
#> [1898] "Paraconiothyrium sporulosum"
#> [1899] "Paraconyotrichium sporulosum"
#> [1900] "Paraphaeosphaeria sporulosa"
#> [1901] "Fusarium venenatum"
#> [1902] "Fusarium venetum"
#> [1903] "Amyelois transitella"
#> [1904] "Nephopteryx transitella"
#> [1905] "Pelecanus carbo"
#> [1906] "Phalacrocorax carbo"
#> [1907] "Naegleria lovaniensis"
#> [1908] "Papilio machaon"
#> [1909] "Gaeumannomyces tritici_R3-111a-1"
#> [1910] "Papilio aegeria"
#> [1911] "Pararge aegeria"
#> [1912] "Lophyrus lecontei"
#> [1913] "Neodiprion lecontei"
#> [1914] "Sclerotinia sclerotiorum_1980_UF-70"
#> [1915] "Aegialitis vocifera"
#> [1916] "Charadrius vociferous"
#> [1917] "Charadrius vociferus"
#> [1918] "Oxyechus vociferus"
#> [1919] "Drosophila erecta"To enable gene ontology analysis, one must use a SpliceWiz reference with prepared GO annotations for the specified organism. To view the gene ontology annotation for a given SpliceWiz reference:
ref_path <- file.path(tempdir(), "Reference")
ontology <- viewGO(ref_path)
head(ontology)
#> gene_id go_id evidence
#> 1 ENSG00000121410 GO:0002764 IBA
#> 2 ENSG00000121410 GO:0003674 ND
#> 3 ENSG00000121410 GO:0005576 HDA
#> 4 ENSG00000121410 GO:0005576 IDA
#> 5 ENSG00000121410 GO:0005576 TAS
#> 6 ENSG00000121410 GO:0005615 HDA
#> go_term ontology gene_name
#> 1 immune response-regulating signaling pathway BP <NA>
#> 2 molecular_function MF <NA>
#> 3 extracellular region CC <NA>
#> 4 extracellular region CC <NA>
#> 5 extracellular region CC <NA>
#> 6 extracellular space CC <NA>Note that gene_names are not available for our example reference because there are only 7 genes in the example reference. Nevertheless, the GO annotation is complete and relies on Ensembl gene_ids for matching.
For simple gene over-representation analysis, we use the goGenes function. As an example, to analyse for enriched biological functions of the first 1000 genes in the reference:
allGenes <- sort(unique(ontology$gene_id))
exampleGeneID <- allGenes[1:1000]
exampleBkgdID <- allGenes
go_byGenes <- goGenes(
enrichedGenes = exampleGeneID,
universeGenes = exampleBkgdID,
ontologyRef = ontology
)To visualize the top gene ontology categories of the above analysis:
Of course, we wish to perform GO analysis on the top differential events in our analysis:
res_edgeR <- ASE_edgeR(
se = se.filtered,
test_factor = "condition",
test_nom = "B",
test_denom = "A"
)
#> Oct 29 23:05:52 Performing edgeR contrast for included / excluded counts separately
#> Oct 29 23:05:54 Performing edgeR contrast for included / excluded counts together
go_byASE <- goASE(
enrichedEventNames = res_edgeR$EventName[1:10],
universeEventNames = NULL,
se = se
)
head(go_byASE)
#> go_id go_term
#> <char> <char>
#> 1: GO:0000381 regulation of alternative mRNA splicing, via spliceosome
#> 2: GO:0045892 negative regulation of DNA-templated transcription
#> 3: GO:0048026 positive regulation of mRNA splicing, via spliceosome
#> 4: GO:0000122 negative regulation of transcription by RNA polymerase II
#> 5: GO:0000375 RNA splicing, via transesterification reactions
#> 6: GO:0000423 mitophagy
#> pval padj foldEnrichment overlap size overlapGenes expected
#> <num> <num> <num> <int> <int> <list> <num>
#> 1: 0.4761905 0.7867495 1 2 2 ENSG0000.... 2
#> 2: 0.4761905 0.7867495 1 2 2 ENSG0000.... 2
#> 3: 0.4761905 0.7867495 1 2 2 ENSG0000.... 2
#> 4: 0.7142857 0.7867495 1 1 1 ENSG0000.... 1
#> 5: 0.7142857 0.7867495 1 1 1 ENSG0000.... 1
#> 6: 0.7142857 0.7867495 1 1 1 ENSG0000.... 1In most cases, users will wish to use the set of genes represented in the background ASEs as the background genes. This is because some genes do not have alternative splicing events, most often because they are intronless genes!
To perform GO analysis using background genes from analysed ASEs:
Heatmaps
Heatmaps are useful for visualizing differential expression of individual samples, as well as potential patterns of expression.
First, obtain a matrix of PSI values:
# Create a matrix of values of the top 10 differentially expressed events:
mat <- makeMatrix(
se.filtered,
event_list = res_edgeR$EventName[1:10],
method = "PSI"
)
How does
makeMatrix() work?
makeMatrix() provides a matrix of PSI values from the given NxtSE object. The parameters event_list and sample_list allows subsetting for ASEs and/or samples, respectively.
The parameter method accepts 3 options:
"PSI": outputs raw PSI values"logit": outputs logit PSI values"Z-score": outputs Z-score transformed PSI values
Also, makeMatrix() facilitates exclusion of low confidence PSI values. These can occur when counts of both isoforms are too low. Setting the depth_threshold (default 10) will set samples with total isoform count below this value to be converted to NA.
NA values are filtered out. Setting the parameter na.percent.max (default 0.1) means any ASE with the proportion of NA above this threshold will be removed from the final matrix.
Plot this matrix of values in a heatmap:
library(pheatmap)
anno_col_df <- as.data.frame(colData(se.filtered))
anno_col_df <- anno_col_df[, 1, drop=FALSE]
pheatmap(mat, annotation_col = anno_col_df)
Using the GUI
Navigate to Display, then Heatmap in the menu side bar. After relaxing the event filters as per the prior sections, a heatmap will be automatically generated:
Heatmap - GUI
The heatmap can be customized as follows:
- Again, the filtering panel is available to filter the number of events by significance threshold, rank, or by highlighted events, as explained in the previous sections. Highlighted events are particularly useful for heatmaps as users can (if they want) cherry-pick events of interest
- After above filtering, an additional filter by gene ontology category can be performed. GO analysis must first be performed, and the top enriched categories are listed here
- Samples can be annotated by one or more annotation categories
- Samples can be sorted by the chosen annotation category
- If (4) is selected, whether the sort order should be ascendeng or descending
- The maximum number of rows of the heatmap to display
- Whether the heatmap values are raw PSI, logit-transformed PSI, or Z-score transformed values
- Users can customize their heatmap color palettes from this list
- A static plot (using the pheatmap R package) will be saved to PDF
SpliceWiz Coverage Plots
Coverage plots visualize RNA-seq coverage in individual samples. SpliceWiz uses its coverage normalization algorithm to visualize group differences in PSIs.
What are SpliceWiz coverage plots and how are they generated?
SpliceWiz produces RNA-seq coverage plots of analysed samples. Coverage data is compiled simultaneous to the IR and junction quantitation performed by processBAM(). This data is saved in “COV” files, which is a BGZF compressed and indexed file. COV files show compression and performance gains over BigWig files.
Coverage plots of individual samples
First, lets obtain a list of differential events with delta PSI > 5%:
res_edgeR <- ASE_edgeR(
se = se.filtered,
test_factor = "condition",
test_nom = "B",
test_denom = "A"
)
#> Oct 29 23:05:56 Performing edgeR contrast for included / excluded counts separately
#> Oct 29 23:05:57 Performing edgeR contrast for included / excluded counts together
res_edgeR.filtered <- res_edgeR[res_edgeR$abs_deltaPSI > 0.05,]
res_edgeR.filtered$EventName[1]
#> [1] "NSUN5/ENST00000252594_Intron2/clean"We can see here the top differential event belongs to NSUN5. The first step to plotting this event is to create a data object that contains the requisite data for the gene NSUN5:
This step retrieves all coverage and junction data for NSUN5 (and surrounding genes).
Next, we need to create event-specific data for the IR event in NSUN5 intron 2.
This step normalizes the coverage and junction data from the viewpoint of NSUN5 intron 2.
The final step is to generate the plot:
plotView(
plotObj,
centerByEvent = TRUE, # whether the plot should be centered at the `Event`
trackList = list(1,2,3,4,5,6),
plotJunctions = TRUE
)
This plots all 6 individual coverage plots, each in its own track.
Why are the tracks referred to using numbers
In the covPlotObject which is returned by the above function, each “track” is a sample. These can be viewed using the tracks() function:
For convenience, users have the option of referring to tracks by their actual names, or by numbers. So, in the above example, the two parameters below are equivalent:
trackList = list(1,2)- `trackList = list(“02H003”, “02H025”)
Normalized Coverage plots of individual samples
To plot “normalized” coverage plots, where coverage is normalized to transcript depth (i.e. sum of all transcripts = 1), set normalizeCoverage = TRUE. We can also include multiple samples in each trace, so lets stack all samples in the same condition in the same trace:
plotView(
plotObj,
centerByEvent = TRUE,
trackList = list(c(1,2,3), c(4,5,6)),
# Each list element contains a vector of track id's
normalizeCoverage = TRUE
)
NB: junction (sashimi) arcs are not plotted in tracks with more than 1 trace (to avoid cluttering)
Group Coverage plots
To plot group coverage plots, first we need to generate a new covPlotObject. This is because the previous covPlotObject was generated on the basis of each track being an individual sample. To generate a plot object where each track represents a condition:
plotObj_group <- getPlotObject(
dataObj,
Event = res_edgeR.filtered$EventName[1],
condition = "condition",
tracks = c("A", "B")
)
# NB:
# when `condition` is not specified, tracks are assumed to be the same samples
# as that of the covDataObject
# when `condition` is specified, tracks must refer to the condition categories
# that are desired for the final plotNote that there are several scenarios where a new covPlotObject is required:
- When plotting normalized coverages using a different normalization ASE,
- When plotting by a different condition or different tracks,
- When plotting on a different strand (default is
*- unstranded)
To generate the group coverage plot, with the two conditions on the same track:
Coverage plots using exon windows
Sometimes, we are interested in the differential coverage of exons but not of the intervening introns. Given introns are often much longer than exons, it is useful to plot by the exons of interest.
For example, to plot the skipped casette exon in TRA2B:
dataObj <- getCoverageData(se, Gene = "TRA2B", tracks = colnames(se))
plotObj <- getPlotObject(
dataObj,
Event = "SE:TRA2B-206-exon2;TRA2B-205-int1",
condition = "condition", tracks = c("A", "B")
)
plotView(
plotObj,
centerByEvent = TRUE,
trackList = list(c(1,2)),
filterByEventTranscripts = TRUE
)
NB: setting filterByEventTranscripts = TRUE means only transcripts involved in the specified splicing event are plotted in the annotation
Note that the involved exons are in only a small area of the coverage plot. To zoom in on the exons, we first have to plot an “exon view” so the exons are labelled, and at the same time return their coordinates:
gr <- plotView(
plotObj,
centerByEvent = TRUE,
trackList = list(c(1,2)),
filterByEventTranscripts = TRUE,
showExonRanges = TRUE
)
By setting the parameter showExonRanges = TRUE, the plotView function shows a plot with exons and their names in the annotation track, and returns a GRanges object, with ranges named by their corresponding exon names:
names(gr)
#> [1] "TRA2B-205-E3" "TRA2B-206-E4" "TRA2B-206-E3" "TRA2B-206-E2" "TRA2B-206-E1"
#> [6] "TRA2B-205-E2" "TRA2B-213-E2" "TRA2B-213-E1" "TRA2B-205-E1"We can see in the above figure that he exons of interest are c("TRA2B-206-E1", "TRA2B-206-E2", "TRA2B-206-E3"). To plot these in an “exons view” coverage plot:
plotView(
plotObj,
centerByEvent = TRUE,
trackList = list(c(1,2)),
filterByEventTranscripts = TRUE,
plotRanges = gr[c("TRA2B-206-E1", "TRA2B-206-E2", "TRA2B-206-E3")]
)
Using the GUI
The main coverage plot interface is as follows
Coverage Plots Main Panel - GUI
The top bar contains controls to locate the genomic loci of interest:
- Genes - locate by the specified gene
- Events - locate by the specified alternate splicing event. The list of events can be modified by the right-hand controls in the top bar
- Coordinates - locate by the specified chromosome and start/end coordinate
- Zoom in or out (each step by a factor of 3)
- Strand - view coverage by strand-specific (+/-) or non-specific (*) modes
- Select events either by all events in differential analysis (which can be filtered using the html-table column filters), user-highlighted events (using volcano/scatter plots), or by gene ontology category. If using gene-ontology category, an extra drop-down box will appear allowing users to specify from the top gene ontology categories from prior GO analysis
- Limit the number of events displayed in the Event drop-down box (2)
In the plot control panel on the left hand side of the main plot area:
- Select which alternative splicing event that should be normalized against. This is critical when plotting normalized coverages (14) or plotting by condition (9)
- Select whether to plot by individual samples, or specify the condition to plot group-plots. See the next section for condition-specific controls.
- The track list table allows users to specify track ID’s for one or more of the specified controls. The left hand column displays available sample names (if plotting by individual samples), or condition categories (if a condition is specified)
- Interactive plots can be slow to render because of too many data points. Resolution (the number of data points across the main display) can be controlled using this slider.
- For group plots, variance of each group can be displayed either as standard deviation (SD), standard error of the mean (SEM), 95% confidence interval (95% CI) or none.
- Whether junction (sashimi) arcs should be plotted. Note that junction arcs will be disabled if there are multiple traces on the same track
- When plotting individual samples, whether raw or normalized coverages should be displayed. Junction counts will also be normalized (they are automatically normalized in group coverage plots)
- Whether to display isoforms belonging to the included / excluded isoform described in the selected normalizing alternative splicing event
- Whether transcripts should be condensed by gene. This is useful if there are too many transcripts cluttering the display.
- If selected, brings up a transcript table where users can select 1 or more transcripts to display in the annotation track.
- If selected, converts the annotation track into an exons display track with named exons. A table will pop up allowing users to select 2 or more exon ranges. Selecting these exons will generate a static exons-only coverage plot.
The main plot (19) is a plotly-based interactive plot. Users can zoom in to a genomic loci of interest using the zoom tool.
There are several options that appear if a specific condition is set:
Group Coverage Plots - GUI
- Note that the track list table now shows condition categories instead of names of samples
- Difference of normalized coverage between conditions can be objectively measured. Users have the option to use the Student’s T-test to measure difference in normalized coverage between replicates of a pair of conditions
- Two drop-down boxes allowing users to select a pair of conditions to assess difference in normalized coverage
As mentioned, when the “Exon Plot mode” is selected, an exons table is displayed where users can select one or more exon ranges can be selected, as shown below:
Generating Exon Coverage (Static) Plots - GUI
Selecting 2 or more exon ranges will trigger (after a 3 second delay) a static exon-window coverage plot to be generated.
Note that at the bottom of the left hand panel, users can save the interactive (top) plot, or the static exon-window coverage (bottom) plot to PDF as ggplot- based figures.
SessionInfo
sessionInfo()
#> R version 4.4.1 (2024-06-14)
#> Platform: x86_64-pc-linux-gnu
#> Running under: Ubuntu 24.04.1 LTS
#>
#> Matrix products: default
#> BLAS: /home/biocbuild/bbs-3.20-bioc/R/lib/libRblas.so
#> LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.12.0
#>
#> locale:
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#> [3] LC_TIME=en_GB LC_COLLATE=C
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#>
#> time zone: America/New_York
#> tzcode source: system (glibc)
#>
#> attached base packages:
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#> [8] methods base
#>
#> other attached packages:
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#> [5] Biobase_2.66.0 MatrixGenerics_1.18.0
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#>
#> loaded via a namespace (and not attached):
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