Exclude Reads With Xa Tag

Question

[fo3c]

Hello there,

Does anyone know of a tool to remove reads that have alternative alignments? Similarly to filtering for specific flags, I would like to filter out all reads that have more than one alignment.

I am aware that depending on the aligner the XA tag might not be set. I used bwa, which by default does not set an XA tag if there are more than 3 alternative alignments. However, this should not be a problem since multiple alignments would result in a lower mapping quality (and can thus be removed on this basis).

A simple grep would work, but that could leave me with broken flags (filter out one read in a pair and not the other without adjusting the flag).

Any suggestions?

[JC]

I don't remember if BWA reports it, but the NH tag reports the total hits, so NH:i:1 means unique hits.

Answer 1

[matted]

It's a bit ugly as well, but for these types of tasks I usually identify the read names I want to filter via grep, then use grep again to remove both read pairs that match the name. This assumes that paired reads have the same name, which I think is usually the case.

For example:

samtools view in.bam | fgrep XA | cut -f 1 > bad_names.txt
samtools view -h in.bam | fgrep -vf bad_names.txt | samtools view -Sb - > out.bam

This would filter out a complete pair if either (or both) read has an XA tag, and leave the BAM consistent. If you'd prefer to keep a read whose mate has an XA tag but fix the flags, I think you'd have to do it yourself with something like pysam or Bio-Samtools (or just hack the flags and text on your own).

Answer 2

[Pierre Lindenbaum]

The following java program will filter a BAM using javascript (rhino) and the picard library. It reads a BAM file/stdin and applies the javascript script to accept/reject each SamRecord. The samRecord is injected as "record" and the SamFileHeader is injected as "header" in the js context. To answer your question you can use the script:

record.getAttribute("XA")==null;

The java program, how to compile and a test file :