correct mark duplicated error using python and pysam to filter out secondary alignments
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Entering edit mode
2.5 years ago
bioguy24 ▴ 220

I am new to python and trying to learn. The below is an attempt to filter out secondary reads in a all bam files in a directory using pysam. I ran mark duplicates and got an error on several bam files due to secondary alignments. I added comments to each line to illustrate my thought process. I think that when I algin using BWA-MEM with the -M option and there are readpairs with the same valid primary alignments and by removing the lower score alignments I will be ok. Is that correct or is there a better approach? Thank you :)

The specific mark duplicates error:

Value was put into PairInfoMap more than once

Attempt 1

#! /usr/bin/python  ## call python script
import sys ## import python system functions
import pysam  ## import module

bam = pysam.AlignmentFile(.bam, "rb")  ## open bam and read
click.echo("Reading BAM file")  ## output message
for read in bam.fetch():
    if read.is_secondary=true  ## not the primary alignment
        continue
    if read.has_tag('AS') and read.has_tag('XS') ## look for Primary and Secondary Alignment Score tag
        AS = read.get_tag('AS')  ## read and store AS value
        XS = read.get_tag('XS')  ## read and store XS value
             if AS >= XS  ## Alignment score greater then or equal to XS (these will be printed)
        bam.write(read)  ## open bam and only print primary alignments
             continue  ## process next line
bam.close()  ## end processing

Attempt 2

#! /usr/bin/python  ## call python script
import sys ## import python system functions
import pysam  ## import module

bam = pysam.AlignmentFile(bam, "rb")  ## open bam and read
click.echo("Reading BAM file")  ## output message
for read in bam.fetch():  ## start loop and iterate over each bam
    if read.is_secondary:  ## not the primary alignment
        continue
    if read.has_tag('AS') and read.has_tag('XS') ## look for Primary and Secondary Alignment Score tag
        AS = read.get_tag('AS')  ## read and store AS value
        XS = read.get_tag('XS')  ## read and store XS value
            if AS >= XS  ## Alignment score greater then or equal to XS (these will be printed)
    read.write(read)  ## only print primary alignments
    continue
bam.close()  ## end processing
python pysam • 927 views
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Entering edit mode

I have re-mapped and markduplicates seems ok but I am curious if the python script is close? Thank you :).

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