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

Load the NanoporeRNASeq package

List the samples

List the available BamFile

Get the annotation GRangesList

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(type = model, 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

Visualizing gene examples

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.262]
sessionInfo()
##> R version 4.1.1 (2021-08-10)
##> Platform: x86_64-pc-linux-gnu (64-bit)
##> Running under: Ubuntu 20.04.3 LTS
##> 
##> Matrix products: default
##> BLAS:   /home/biocbuild/bbs-3.14-bioc/R/lib/libRblas.so
##> LAPACK: /home/biocbuild/bbs-3.14-bioc/R/lib/libRlapack.so
##> 
##> 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       
##> 
##> attached base packages:
##> [1] stats4    stats     graphics  grDevices utils     datasets  methods  
##> [8] base     
##> 
##> other attached packages:
##>  [1] bambu_1.99.0                          
##>  [2] SummarizedExperiment_1.23.5           
##>  [3] Biobase_2.53.0                        
##>  [4] MatrixGenerics_1.5.4                  
##>  [5] matrixStats_0.61.0                    
##>  [6] BSgenome.Hsapiens.NCBI.GRCh38_1.3.1000
##>  [7] BSgenome_1.61.0                       
##>  [8] rtracklayer_1.53.1                    
##>  [9] ggbio_1.41.0                          
##> [10] ggplot2_3.3.5                         
##> [11] Rsamtools_2.9.1                       
##> [12] Biostrings_2.61.2                     
##> [13] XVector_0.33.0                        
##> [14] GenomicRanges_1.45.0                  
##> [15] GenomeInfoDb_1.29.10                  
##> [16] IRanges_2.27.2                        
##> [17] S4Vectors_0.31.5                      
##> [18] NanoporeRNASeq_1.3.6                  
##> [19] ExperimentHub_2.1.4                   
##> [20] AnnotationHub_3.1.6                   
##> [21] BiocFileCache_2.1.1                   
##> [22] dbplyr_2.1.1                          
##> [23] BiocGenerics_0.39.2                   
##> 
##> loaded via a namespace (and not attached):
##>   [1] backports_1.2.1               Hmisc_4.6-0                  
##>   [3] plyr_1.8.6                    lazyeval_0.2.2               
##>   [5] splines_4.1.1                 BiocParallel_1.27.17         
##>   [7] digest_0.6.28                 ensembldb_2.17.4             
##>   [9] htmltools_0.5.2               fansi_0.5.0                  
##>  [11] magrittr_2.0.1                checkmate_2.0.0              
##>  [13] memoise_2.0.0                 cluster_2.1.2                
##>  [15] prettyunits_1.1.1             jpeg_0.1-9                   
##>  [17] colorspace_2.0-2              blob_1.2.2                   
##>  [19] rappdirs_0.3.3                xfun_0.26                    
##>  [21] dplyr_1.0.7                   crayon_1.4.1                 
##>  [23] RCurl_1.98-1.5                jsonlite_1.7.2               
##>  [25] graph_1.71.2                  survival_3.2-13              
##>  [27] VariantAnnotation_1.39.0      glue_1.4.2                   
##>  [29] gtable_0.3.0                  zlibbioc_1.39.0              
##>  [31] DelayedArray_0.19.4           scales_1.1.1                 
##>  [33] DBI_1.1.1                     GGally_2.1.2                 
##>  [35] Rcpp_1.0.7                    xtable_1.8-4                 
##>  [37] progress_1.2.2                htmlTable_2.3.0              
##>  [39] foreign_0.8-81                bit_4.0.4                    
##>  [41] OrganismDbi_1.35.0            Formula_1.2-4                
##>  [43] htmlwidgets_1.5.4             httr_1.4.2                   
##>  [45] RColorBrewer_1.1-2            ellipsis_0.3.2               
##>  [47] farver_2.1.0                  pkgconfig_2.0.3              
##>  [49] reshape_0.8.8                 XML_3.99-0.8                 
##>  [51] nnet_7.3-16                   sass_0.4.0                   
##>  [53] utf8_1.2.2                    labeling_0.4.2               
##>  [55] tidyselect_1.1.1              rlang_0.4.11                 
##>  [57] reshape2_1.4.4                later_1.3.0                  
##>  [59] AnnotationDbi_1.55.1          munsell_0.5.0                
##>  [61] BiocVersion_3.14.0            tools_4.1.1                  
##>  [63] cachem_1.0.6                  xgboost_1.4.1.1              
##>  [65] generics_0.1.0                RSQLite_2.2.8                
##>  [67] evaluate_0.14                 stringr_1.4.0                
##>  [69] fastmap_1.1.0                 yaml_2.2.1                   
##>  [71] knitr_1.36                    bit64_4.0.5                  
##>  [73] purrr_0.3.4                   KEGGREST_1.33.0              
##>  [75] AnnotationFilter_1.17.1       RBGL_1.69.0                  
##>  [77] mime_0.12                     formatR_1.11                 
##>  [79] xml2_1.3.2                    biomaRt_2.49.6               
##>  [81] compiler_4.1.1                rstudioapi_0.13              
##>  [83] filelock_1.0.2                curl_4.3.2                   
##>  [85] png_0.1-7                     interactiveDisplayBase_1.31.2
##>  [87] tibble_3.1.5                  bslib_0.3.1                  
##>  [89] stringi_1.7.5                 highr_0.9                    
##>  [91] GenomicFeatures_1.45.2        lattice_0.20-45              
##>  [93] ProtGenerics_1.25.1           Matrix_1.3-4                 
##>  [95] vctrs_0.3.8                   pillar_1.6.4                 
##>  [97] lifecycle_1.0.1               BiocManager_1.30.16          
##>  [99] jquerylib_0.1.4               data.table_1.14.2            
##> [101] bitops_1.0-7                  httpuv_1.6.3                 
##> [103] R6_2.5.1                      BiocIO_1.3.0                 
##> [105] latticeExtra_0.6-29           promises_1.2.0.1             
##> [107] gridExtra_2.3                 dichromat_2.0-0              
##> [109] assertthat_0.2.1              rjson_0.2.20                 
##> [111] withr_2.4.2                   GenomicAlignments_1.29.0     
##> [113] GenomeInfoDbData_1.2.7        parallel_4.1.1               
##> [115] hms_1.1.1                     grid_4.1.1                   
##> [117] rpart_4.1-15                  tidyr_1.1.4                  
##> [119] rmarkdown_2.11                biovizBase_1.41.0            
##> [121] shiny_1.7.1                   base64enc_0.1-3              
##> [123] restfulr_0.0.13