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Filtering fastq vs BAM for fragment size range

Wondering if you would get equivalent results in limiting fragment size to a range by doing it at the bowtie2 level (mapping only that fragment range indicated by the -I and -X flags) or filtering of the resulting BAM file after alignment of all fragments?

alignment bowtie2 bam fastq
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written by rbronste rbronste

Could you include a little more information about the type of sequencing you're doing and why you're only interested in a specific size range? If you're interested in multiple size ranges in something like ATAC-seq, I'd recommend mapping once then subsetting the bam file based on what sizes you want, that way you don't need to remap your fastq file. But I can't be sure that's what you're asking.

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written by shawn.w.foley shawn.w.foley

1 answer

Simply try it. Given that you asked how to subset a BAM by TLEN in a previous thread, here is a possible solution:

function TLEN {
  sambamba view -f sam -h $1 | \
    mawk -v LEN=$2 '{if ($9 <= LEN && $9 >= -(LEN) && $9 != 0 || $1 ~ /^@/) print $0}' | \
    sambamba view -S -f bam -h -o ${1%.bam}_isize${2}.bam /dev/stdin
}; export -f TLEN

## Subset bam for fragments with TLEN <= 100bp
TLEN in.bam 100
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written by ATpoint ATpoint

This is a nice approach thanks, I guess my question was moreso if the two approaches (before and after alignment) would be equivalent in their final resulting bam file?

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written by rbronste rbronste

Yeah, but I cannot answer this, as I never tried. I would always go for the after-alignment step because that way you have all data available and can do multiple filtering approaches without having to re-align everything in case the filtering options during alignment were not optimal.

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written by ATpoint ATpoint

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