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): 3558ad441f2c9f_3844 3558ad54743faf_3846 ...
##> 3558ad50564daa_3852 3558ad417a4eca_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.248]
sessionInfo()
##> R Under development (unstable) (2023-10-22 r85388)
##> Platform: x86_64-pc-linux-gnu
##> Running under: Ubuntu 22.04.3 LTS
##>
##> Matrix products: default
##> BLAS: /home/biocbuild/bbs-3.19-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_3.5.0
##> [2] SummarizedExperiment_1.33.0
##> [3] Biobase_2.63.0
##> [4] MatrixGenerics_1.15.0
##> [5] matrixStats_1.0.0
##> [6] BSgenome.Hsapiens.NCBI.GRCh38_1.3.1000
##> [7] BSgenome_1.71.0
##> [8] rtracklayer_1.63.0
##> [9] BiocIO_1.13.0
##> [10] ggbio_1.51.0
##> [11] ggplot2_3.4.4
##> [12] Rsamtools_2.19.0
##> [13] Biostrings_2.71.1
##> [14] XVector_0.43.0
##> [15] GenomicRanges_1.55.1
##> [16] GenomeInfoDb_1.39.0
##> [17] IRanges_2.37.0
##> [18] S4Vectors_0.41.1
##> [19] NanoporeRNASeq_1.13.0
##> [20] ExperimentHub_2.11.0
##> [21] AnnotationHub_3.11.0
##> [22] BiocFileCache_2.11.1
##> [23] dbplyr_2.4.0
##> [24] BiocGenerics_0.49.0
##>
##> loaded via a namespace (and not attached):
##> [1] RColorBrewer_1.1-3 rstudioapi_0.15.0
##> [3] jsonlite_1.8.7 magrittr_2.0.3
##> [5] GenomicFeatures_1.55.1 farver_2.1.1
##> [7] rmarkdown_2.25 zlibbioc_1.49.0
##> [9] vctrs_0.6.4 memoise_2.0.1
##> [11] RCurl_1.98-1.12 base64enc_0.1-3
##> [13] htmltools_0.5.6.1 S4Arrays_1.3.0
##> [15] progress_1.2.2 curl_5.1.0
##> [17] xgboost_1.7.5.1 SparseArray_1.3.0
##> [19] Formula_1.2-5 sass_0.4.7
##> [21] bslib_0.5.1 htmlwidgets_1.6.2
##> [23] plyr_1.8.9 cachem_1.0.8
##> [25] GenomicAlignments_1.39.0 mime_0.12
##> [27] lifecycle_1.0.3 pkgconfig_2.0.3
##> [29] Matrix_1.6-1.1 R6_2.5.1
##> [31] fastmap_1.1.1 GenomeInfoDbData_1.2.11
##> [33] shiny_1.7.5.1 digest_0.6.33
##> [35] colorspace_2.1-0 GGally_2.1.2
##> [37] reshape_0.8.9 OrganismDbi_1.45.0
##> [39] AnnotationDbi_1.65.0 Hmisc_5.1-1
##> [41] RSQLite_2.3.2 labeling_0.4.3
##> [43] filelock_1.0.2 fansi_1.0.5
##> [45] httr_1.4.7 abind_1.4-5
##> [47] compiler_4.4.0 bit64_4.0.5
##> [49] withr_2.5.2 htmlTable_2.4.2
##> [51] backports_1.4.1 BiocParallel_1.37.0
##> [53] DBI_1.1.3 highr_0.10
##> [55] biomaRt_2.59.0 rappdirs_0.3.3
##> [57] DelayedArray_0.29.0 rjson_0.2.21
##> [59] tools_4.4.0 foreign_0.8-85
##> [61] interactiveDisplayBase_1.41.0 httpuv_1.6.12
##> [63] nnet_7.3-19 glue_1.6.2
##> [65] restfulr_0.0.15 promises_1.2.1
##> [67] grid_4.4.0 checkmate_2.3.0
##> [69] cluster_2.1.4 reshape2_1.4.4
##> [71] generics_0.1.3 gtable_0.3.4
##> [73] tidyr_1.3.0 ensembldb_2.27.0
##> [75] data.table_1.14.8 hms_1.1.3
##> [77] xml2_1.3.5 utf8_1.2.4
##> [79] BiocVersion_3.19.1 pillar_1.9.0
##> [81] stringr_1.5.0 later_1.3.1
##> [83] dplyr_1.1.3 lattice_0.22-5
##> [85] bit_4.0.5 biovizBase_1.51.0
##> [87] RBGL_1.79.0 tidyselect_1.2.0
##> [89] knitr_1.45 gridExtra_2.3
##> [91] ProtGenerics_1.35.0 xfun_0.40
##> [93] stringi_1.7.12 lazyeval_0.2.2
##> [95] yaml_2.3.7 evaluate_0.22
##> [97] codetools_0.2-19 tibble_3.2.1
##> [99] graph_1.81.0 BiocManager_1.30.22
##> [101] cli_3.6.1 rpart_4.1.21
##> [103] xtable_1.8-4 munsell_0.5.0
##> [105] jquerylib_0.1.4 dichromat_2.0-0.1
##> [107] Rcpp_1.0.11 png_0.1-8
##> [109] XML_3.99-0.14 parallel_4.4.0
##> [111] ellipsis_0.3.2 blob_1.2.4
##> [113] prettyunits_1.2.0 AnnotationFilter_1.27.0
##> [115] bitops_1.0-7 VariantAnnotation_1.49.0
##> [117] scales_1.2.1 purrr_1.0.2
##> [119] crayon_1.5.2 rlang_1.1.1
##> [121] KEGGREST_1.43.0 formatR_1.14