Rstudio crashes at summarizeOverlaps()

[chuddy-ibk]

Hi there,

I am trying to use my 42 BAM files (aligned with Subjunc from RSubread package) to prepare a DEXSeqDataSet according to point 2 in the manual (https://bioconductor.org/packages/release/bioc/vignettes/DEXSeq/inst/doc/DEXSeq.html#2_Preparations)

Following would be my script I copied and adjusted from the mentioned link:

txdb = makeTxDbFromGFF("/home/chuddy/bioinformatics/ref_genomes/mouse_38/annotation/ensembl/Mus_musculus.GRCm38.102.gtf.gz")

flattenedAnnotation = exonicParts(txdb, linked.to.single.gene.only = TRUE)
names(flattenedAnnotation) = sprintf("%s:E%0.3d", flattenedAnnotation$gene_id, flattenedAnnotation$exonic_part)

bamFiles <- list.files("../BAM/subjunc/", full.names = T, include.dirs = F, pattern = "\\.BAM$")
bamFiles <- normalizePath(bamFiles) 

bamFiles = BamFileList(bamFiles)

se = summarizeOverlaps(flattenedAnnotation, 
                       bamFiles, 
                       singleEnd=T,
                       ignore.strand=TRUE)

I changed BamFileList(bamFiles) to bamFiles, because we used this function already on the bam files. But also with the original code snippet Rstudio crashed. Since I have seven condition with each six replicates, I would love to use all BAM files for more complex analyses. Are there any recommendations how to approach without the need of too much RAM (I have approx 60GB left over)?

Best Thomas

[NanoCoreUSA]

You can divide the BAMs into chunks by setting the yieldSize parameter when you create the BamFileList:

bamFiles = BamFileList(bamFiles, yieldSize = 1000000)

Setting it to 1000000 worked for me with 6 files and 32Gb of RAM, but might have to reduce it even further for 42 BAM files.