NanoporeRNASeq contains RNA-Seq data from the K562 and MCF7 cell lines that were generated by the SG-NEx project (https://github.com/GoekeLab/sg-nex-data). Each of these cell line has three replicates, with 1 direct RNA sequencing data and 2 cDNA sequencing data. The files contains reads aligned to the human genome (Grch38) chromosome 22 (1:25500000).
data("SGNexSamples")
SGNexSamples
##> DataFrame with 6 rows and 6 columns
##> sample_id Platform cellLine protocol cancer_type
##> <character> <character> <character> <character> <character>
##> 1 K562_directcDNA_repl.. MinION K562 directcDNA Leukocyte
##> 2 K562_directcDNA_repl.. GridION K562 directcDNA Leukocyte
##> 3 K562_directRNA_repli.. GridION K562 directRNA Leukocyte
##> 4 MCF7_directcDNA_repl.. MinION MCF7 directcDNA Breast
##> 5 MCF7_directcDNA_repl.. GridION MCF7 directcDNA Breast
##> 6 MCF7_directRNA_repli.. GridION MCF7 directRNA Breast
##> fileNames
##> <character>
##> 1 NanoporeRNASeq/versi..
##> 2 NanoporeRNASeq/versi..
##> 3 NanoporeRNASeq/versi..
##> 4 NanoporeRNASeq/versi..
##> 5 NanoporeRNASeq/versi..
##> 6 NanoporeRNASeq/versi..
data("HsChr22BambuAnnotation")
HsChr22BambuAnnotation
##> GRangesList object of length 1500:
##> $ENST00000043402
##> GRanges object with 2 ranges and 2 metadata columns:
##> seqnames ranges strand | exon_rank exon_endRank
##> <Rle> <IRanges> <Rle> | <integer> <integer>
##> [1] 22 20241415-20243110 - | 2 1
##> [2] 22 20268071-20268531 - | 1 2
##> -------
##> seqinfo: 1 sequence from an unspecified genome; no seqlengths
##>
##> $ENST00000086933
##> GRanges object with 3 ranges and 2 metadata columns:
##> seqnames ranges strand | exon_rank exon_endRank
##> <Rle> <IRanges> <Rle> | <integer> <integer>
##> [1] 22 19148576-19149095 - | 3 1
##> [2] 22 19149663-19149916 - | 2 2
##> [3] 22 19150025-19150283 - | 1 3
##> -------
##> seqinfo: 1 sequence from an unspecified genome; no seqlengths
##>
##> $ENST00000155674
##> GRanges object with 8 ranges and 2 metadata columns:
##> seqnames ranges strand | exon_rank exon_endRank
##> <Rle> <IRanges> <Rle> | <integer> <integer>
##> [1] 22 17137511-17138357 - | 8 1
##> [2] 22 17138550-17138738 - | 7 2
##> [3] 22 17141059-17141233 - | 6 3
##> [4] 22 17143098-17143131 - | 5 4
##> [5] 22 17145024-17145117 - | 4 5
##> [6] 22 17148448-17148560 - | 3 6
##> [7] 22 17149542-17149745 - | 2 7
##> [8] 22 17165209-17165287 - | 1 8
##> -------
##> seqinfo: 1 sequence from an unspecified genome; no seqlengths
##>
##> ...
##> <1497 more elements>
We can visualize the one sample for a single gene ENST00000215832 (MAPK1)
library(ggbio)
range <- HsChr22BambuAnnotation$ENST00000215832
# plot mismatch track
library(BSgenome.Hsapiens.NCBI.GRCh38)
# plot annotation track
tx <- autoplot(range, aes(col = strand), group.selfish = TRUE)
# plot coverage track
coverage <- autoplot(bamFiles[[1]], aes(col = coverage), which = range)
# merge the tracks into one plot
tracks(annotation = tx, coverage = coverage, heights = c(1, 3)) + theme_minimal()
Applying bambu to bamFiles
bambu returns a SummarizedExperiment object
se
##> class: RangedSummarizedExperiment
##> dim: 1542 6
##> metadata(2): incompatibleCounts warnings
##> assays(4): counts CPM fullLengthCounts uniqueCounts
##> rownames(1542): BambuTx1 BambuTx2 ... ENST00000641933 ENST00000641967
##> rowData names(11): TXNAME GENEID ... txid eqClassById
##> colnames(6): 96f922b312965_3844 96f9262bf7c1a_3846 ...
##> 96f921d913cf8_3852 96f923b8683e8_3854
##> colData names(1): name
We can visualize the annotated and novel isoforms identified in this gene example using plot functions from bambu
##> [[1]]
##> TableGrob (3 x 1) "arrange": 3 grobs
##> z cells name grob
##> 1 1 (2-2,1-1) arrange gtable[layout]
##> 2 2 (3-3,1-1) arrange gtable[layout]
##> 3 3 (1-1,1-1) arrange text[GRID.text.245]
sessionInfo()
##> R version 4.4.0 RC (2024-04-16 r86468)
##> Platform: x86_64-pc-linux-gnu
##> Running under: Ubuntu 22.04.4 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.3.10.0
##>
##> locale:
##> [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
##> [3] LC_TIME=en_GB LC_COLLATE=C
##> [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
##> [7] LC_PAPER=en_US.UTF-8 LC_NAME=C
##> [9] LC_ADDRESS=C LC_TELEPHONE=C
##> [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
##>
##> time zone: America/New_York
##> tzcode source: system (glibc)
##>
##> attached base packages:
##> [1] stats4 stats graphics grDevices utils datasets methods
##> [8] base
##>
##> other attached packages:
##> [1] bambu_3.7.0
##> [2] SummarizedExperiment_1.35.0
##> [3] Biobase_2.65.0
##> [4] MatrixGenerics_1.17.0
##> [5] matrixStats_1.3.0
##> [6] BSgenome.Hsapiens.NCBI.GRCh38_1.3.1000
##> [7] BSgenome_1.73.0
##> [8] rtracklayer_1.65.0
##> [9] BiocIO_1.15.0
##> [10] ggbio_1.53.0
##> [11] ggplot2_3.5.1
##> [12] Rsamtools_2.21.0
##> [13] Biostrings_2.73.0
##> [14] XVector_0.45.0
##> [15] GenomicRanges_1.57.0
##> [16] GenomeInfoDb_1.41.0
##> [17] IRanges_2.39.0
##> [18] S4Vectors_0.43.0
##> [19] NanoporeRNASeq_1.15.0
##> [20] ExperimentHub_2.13.0
##> [21] AnnotationHub_3.13.0
##> [22] BiocFileCache_2.13.0
##> [23] dbplyr_2.5.0
##> [24] BiocGenerics_0.51.0
##>
##> loaded via a namespace (and not attached):
##> [1] RColorBrewer_1.1-3 rstudioapi_0.16.0 jsonlite_1.8.8
##> [4] magrittr_2.0.3 GenomicFeatures_1.57.0 farver_2.1.1
##> [7] rmarkdown_2.26 zlibbioc_1.51.0 vctrs_0.6.5
##> [10] memoise_2.0.1 RCurl_1.98-1.14 base64enc_0.1-3
##> [13] progress_1.2.3 htmltools_0.5.8.1 S4Arrays_1.5.0
##> [16] curl_5.2.1 xgboost_1.7.7.1 SparseArray_1.5.0
##> [19] Formula_1.2-5 sass_0.4.9 bslib_0.7.0
##> [22] htmlwidgets_1.6.4 httr2_1.0.1 plyr_1.8.9
##> [25] cachem_1.0.8 GenomicAlignments_1.41.0 mime_0.12
##> [28] lifecycle_1.0.4 pkgconfig_2.0.3 Matrix_1.7-0
##> [31] R6_2.5.1 fastmap_1.1.1 GenomeInfoDbData_1.2.12
##> [34] digest_0.6.35 colorspace_2.1-0 GGally_2.2.1
##> [37] AnnotationDbi_1.67.0 OrganismDbi_1.47.0 Hmisc_5.1-2
##> [40] RSQLite_2.3.6 labeling_0.4.3 filelock_1.0.3
##> [43] fansi_1.0.6 httr_1.4.7 abind_1.4-5
##> [46] compiler_4.4.0 bit64_4.0.5 withr_3.0.0
##> [49] htmlTable_2.4.2 backports_1.4.1 BiocParallel_1.39.0
##> [52] DBI_1.2.2 ggstats_0.6.0 highr_0.10
##> [55] biomaRt_2.61.0 rappdirs_0.3.3 DelayedArray_0.31.0
##> [58] rjson_0.2.21 tools_4.4.0 foreign_0.8-86
##> [61] nnet_7.3-19 glue_1.7.0 restfulr_0.0.15
##> [64] grid_4.4.0 checkmate_2.3.1 cluster_2.1.6
##> [67] reshape2_1.4.4 generics_0.1.3 gtable_0.3.5
##> [70] tidyr_1.3.1 ensembldb_2.29.0 hms_1.1.3
##> [73] data.table_1.15.4 xml2_1.3.6 utf8_1.2.4
##> [76] BiocVersion_3.20.0 pillar_1.9.0 stringr_1.5.1
##> [79] dplyr_1.1.4 lattice_0.22-6 bit_4.0.5
##> [82] biovizBase_1.53.0 RBGL_1.81.0 tidyselect_1.2.1
##> [85] knitr_1.46 gridExtra_2.3 ProtGenerics_1.37.0
##> [88] xfun_0.43 stringi_1.8.3 UCSC.utils_1.1.0
##> [91] lazyeval_0.2.2 yaml_2.3.8 evaluate_0.23
##> [94] codetools_0.2-20 tibble_3.2.1 graph_1.83.0
##> [97] BiocManager_1.30.22 cli_3.6.2 rpart_4.1.23
##> [100] munsell_0.5.1 jquerylib_0.1.4 dichromat_2.0-0.1
##> [103] Rcpp_1.0.12 png_0.1-8 XML_3.99-0.16.1
##> [106] parallel_4.4.0 blob_1.2.4 prettyunits_1.2.0
##> [109] AnnotationFilter_1.29.0 bitops_1.0-7 txdbmaker_1.1.0
##> [112] VariantAnnotation_1.51.0 scales_1.3.0 purrr_1.0.2
##> [115] crayon_1.5.2 rlang_1.1.3 KEGGREST_1.45.0
##> [118] formatR_1.14