Output bam files become smaller after shiftGAlignmentsList

[Rui-Jing]

Hi, Thanks for developing this user-friendly tool for ATAC-seq. I found some problems after running shiftGAlignmentsList, and the output BAM file is about half the size of the input file. I don't know if this is a normal situation or if ATACseqQC does some filtering.

My code :

library(ATACseqQC)
bamfile <- '/home/zfzf/atac_ljh/alignment/neg2_701504_sorted.bam'
bamfile.labels <- gsub(".bam", "", basename(bamfile))
#bamQC(bamfile, outPath=NULL)
estimateLibComplexity(readsDupFreq(bamfile))
## generate fragement size distribution
fragSize <- fragSizeDist(bamfile, bamfile.labels)


## bamfile tags to be read in
possibleTag <- list("integer"=c("AM", "AS", "CM", "CP", "FI", "H0", "H1", "H2", 
                                "HI", "IH", "MQ", "NH", "NM", "OP", "PQ", "SM",
                                "TC", "UQ"), 
                    "character"=c("BC", "BQ", "BZ", "CB", "CC", "CO", "CQ", "CR",
                                  "CS", "CT", "CY", "E2", "FS", "LB", "MC", "MD",
                                  "MI", "OA", "OC", "OQ", "OX", "PG", "PT", "PU",
                                  "Q2", "QT", "QX", "R2", "RG", "RX", "SA", "TS",
                                  "U2"))

library(Rsamtools)

bamTop100 <- scanBam(BamFile(bamfile, yieldSize = 100),
                     param = ScanBamParam(tag=unlist(possibleTag)))[[1]]$tag
tags <- names(bamTop100)[lengths(bamTop100)>0]
tags

## files will be output into outPath
outPath <- "align_adjusted"
dir.create(outPath)

## shift the coordinates of 5'ends of alignments in the bam file
library(TxDb.Mmusculus.UCSC.mm10.knownGene)
gal <- readBamFile(bamfile, tag=tags, asMates=TRUE, bigFile=TRUE)
shiftedBamfile <- file.path(outPath, "shifted.bam")
gal1 <- shiftGAlignmentsList(gal, outbam=shiftedBamfile)

[jianhong]

Hi Rui-Jing,

Thank you for trying ATACseqQC to analyze your ATAC-seq data. Could you let me know the size of file by running the following code?

samtools view -b -f 2 -F 2816 neg2_701504_sorted.bam > clean.reads.bam

Thank you.

Jianhong.

From: Rui-Jing @.> Date: Wednesday, June 8, 2022 at 10:42 PM To: jianhong/ATACseqQC @.> Cc: Subscribed @.***> Subject: [jianhong/ATACseqQC] Output bam files become smaller after shiftGAlignmentsList (Issue #51)

Hi, Thanks for developing this user-friendly tool for ATAC-seq. I found some problems after running shiftGAlignmentsList, and the output BAM file is about half the size of the input file. I don't know if this is a normal situation or if ATACseqQC does some filtering.

My code :

library(ATACseqQC)

bamfile <- '/home/zfzf/atac_ljh/alignment/neg2_701504_sorted.bam'

bamfile.labels <- gsub(".bam", "", basename(bamfile))

#bamQC(bamfile, outPath=NULL)

estimateLibComplexity(readsDupFreq(bamfile))

generate fragement size distribution

fragSize <- fragSizeDist(bamfile, bamfile.labels)

bamfile tags to be read in

possibleTag <- list("integer"=c("AM", "AS", "CM", "CP", "FI", "H0", "H1", "H2",

                            "HI", "IH", "MQ", "NH", "NM", "OP", "PQ", "SM",

                            "TC", "UQ"),

                "character"=c("BC", "BQ", "BZ", "CB", "CC", "CO", "CQ", "CR",

                              "CS", "CT", "CY", "E2", "FS", "LB", "MC", "MD",

                              "MI", "OA", "OC", "OQ", "OX", "PG", "PT", "PU",

                              "Q2", "QT", "QX", "R2", "RG", "RX", "SA", "TS",

                              "U2"))

library(Rsamtools)

bamTop100 <- scanBam(BamFile(bamfile, yieldSize = 100),

                 param = ScanBamParam(tag=unlist(possibleTag)))[[1]]$tag

tags <- names(bamTop100)[lengths(bamTop100)>0]

tags

files will be output into outPath

outPath <- "align_adjusted"

dir.create(outPath)

shift the coordinates of 5'ends of alignments in the bam file

library(TxDb.Mmusculus.UCSC.mm10.knownGene)

gal <- readBamFile(bamfile, tag=tags, asMates=TRUE, bigFile=TRUE)

shiftedBamfile <- file.path(outPath, "shifted.bam")

gal1 <- shiftGAlignmentsList(gal, outbam=shiftedBamfile)

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[Rui-Jing]

Thanks for your quikly reply. The input bam size is 4810847KB, and the output bam size is 2120454KB. So, I wonder if ATACseqQC has done some filtering?

[jianhong]

You may want to ask help by ?readBamFile There is flag filters when you read bam file by ATACseqQC. You can change the flags to meet your design.

Jianhong.

From: Rui-Jing @.> Date: Thursday, June 9, 2022 at 9:26 AM To: jianhong/ATACseqQC @.> Cc: JIANHONG OU @.>, Comment @.> Subject: Re: [jianhong/ATACseqQC] Output bam files become smaller after shiftGAlignmentsList (Issue #51)

Thanks for your quikly reply. The input bam size is 4810847KB, and the output bam size is 2120454KB. So, I wonder if ATACseqQC has done some filtering?

— Reply to this email directly, view it on GitHubhttps://github.com/jianhong/ATACseqQC/issues/51#issuecomment-1151118336, or unsubscribehttps://github.com/notifications/unsubscribe-auth/ABLBEA2CLQ2EUJVEPYNOZT3VOHWJFANCNFSM5YIPMRXA. You are receiving this because you commented.Message ID: @.***>

[Rui-Jing]

Great, thanks for the feedback !