An introduction to the bambu package using NanoporeRNASeq data
Introduction
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).
Accessing NanoporeRNASeq data
List the samples
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..List the available BamFile
Get the annotation GRangesList
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
##>
##> $ENST00000155674
##> GRanges object with 8 ranges and 2 metadata columns:
##> seqnames ranges strand | exon_rank exon_endRank
##> <Rle> <IRanges> <Rle> | <integer> <integer>
##> [1] 22 17137511-17138357 - | 8 1
##> [2] 22 17138550-17138738 - | 7 2
##> [3] 22 17141059-17141233 - | 6 3
##> [4] 22 17143098-17143131 - | 5 4
##> [5] 22 17145024-17145117 - | 4 5
##> [6] 22 17148448-17148560 - | 3 6
##> [7] 22 17149542-17149745 - | 2 7
##> [8] 22 17165209-17165287 - | 1 8
##> -------
##> seqinfo: 1 sequence from an unspecified genome; no seqlengths
##>
##> ...
##> <1497 more elements>Visualizing gene of interest from a single bam file
We can visualize the one sample for a single gene ENST00000215832 (MAPK1)
library(ggbio)
range <- HsChr22BambuAnnotation$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()
Running Bambu with NanoporeRNASeq data
Load the bambu package
Run bambu
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): 1a0332368209_3844 1a034803f231_3846 ... 1a03a01b1fd_3852
##> 1a031829bbf2_3854
##> colData names(1): nameVisualizing gene examples
We can visualize the annotated and novel isoforms identified in this gene example using plot functions from bambu
sessionInfo()
##> R version 4.6.1 (2026-06-24)
##> Platform: x86_64-pc-linux-gnu
##> Running under: Ubuntu 26.04.1 LTS
##>
##> Matrix products: default
##> BLAS: /usr/lib/x86_64-linux-gnu/openblas-pthread/libblas.so.3
##> LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.32.so; LAPACK version 3.12.0
##>
##> locale:
##> [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
##> [3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8
##> [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: Etc/UTC
##> tzcode source: system (glibc)
##>
##> attached base packages:
##> [1] stats4 stats graphics grDevices utils datasets methods
##> [8] base
##>
##> other attached packages:
##> [1] bambu_3.15.1
##> [2] SummarizedExperiment_1.43.0
##> [3] Biobase_2.73.2
##> [4] MatrixGenerics_1.25.0
##> [5] matrixStats_1.5.0
##> [6] BSgenome.Hsapiens.NCBI.GRCh38_1.3.1000
##> [7] BSgenome_1.81.1
##> [8] rtracklayer_1.73.0
##> [9] BiocIO_1.23.3
##> [10] ggbio_1.61.0
##> [11] ggplot2_4.0.3
##> [12] Rsamtools_2.29.0
##> [13] Biostrings_2.81.9
##> [14] XVector_0.53.0
##> [15] GenomicRanges_1.65.4
##> [16] IRanges_2.47.5
##> [17] S4Vectors_0.51.10
##> [18] Seqinfo_1.3.2
##> [19] NanoporeRNASeq_1.23.1
##> [20] ExperimentHub_3.3.2
##> [21] AnnotationHub_4.3.2
##> [22] BiocFileCache_3.3.0
##> [23] dbplyr_2.6.0
##> [24] BiocGenerics_0.59.12
##> [25] generics_0.1.4
##> [26] rmarkdown_2.32
##>
##> loaded via a namespace (and not attached):
##> [1] RColorBrewer_1.1-3 sys_3.4.3 rstudioapi_0.19.0
##> [4] jsonlite_2.0.0 magrittr_2.0.5 GenomicFeatures_1.65.0
##> [7] farver_2.1.2 vctrs_0.7.3 memoise_2.0.1
##> [10] RCurl_1.98-1.20 base64enc_0.1-6 htmltools_0.5.9
##> [13] S4Arrays_1.13.2 BiocBaseUtils_1.15.1 curl_8.0.0
##> [16] xgboost_3.2.1.1 SparseArray_1.13.4 Formula_1.2-6
##> [19] sass_0.4.10 bslib_0.12.0 htmlwidgets_1.6.4
##> [22] plyr_1.8.9 httr2_1.3.0 cachem_1.1.0
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##> [46] S7_0.2.2 backports_1.5.1 BiocParallel_1.47.0
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##> [79] xfun_0.61 stringi_1.8.9 UCSC.utils_1.9.0
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##> [85] codetools_0.2-20 cigarillo_1.3.1 tibble_3.3.1
##> [88] BiocManager_1.30.27 graph_1.91.0 cli_3.6.6
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##> [97] XML_3.99-0.25 parallel_4.6.1 blob_1.3.0
##> [100] AnnotationFilter_1.37.0 bitops_1.1-0 VariantAnnotation_1.59.4
##> [103] scales_1.4.0 purrr_1.2.2 crayon_1.5.3
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