Best way to map paired-end, uniquely mapped reads with Bowtie2?

Question

[Ian]

This has come up before, but I have never been satisfied with, or completly understood, the answers. My issue is how can Bowtie2 be used to map uniquely mapping paired-end reads (Illumina) to a genome for the purpose of ChIP-seq analysis. Unlike the original Bowtie there is no magical -m1 flag to enforce the reporting of unqiely mapped reads.

My current strategy is to run Bowtie2 with -k 2, which reports up to two alignments per read. The resulting SAM file can then be filtered to remove reads containing the XS flag, which reports the mapping quality of a second best alignment.

I am fairly happy with this when using single-end reads, but with paired-end reads samtools flagstat on the filtered BAM file can show an odd number for properly paired reads.

I would be interest to get an opinion on this method and whether anyone else has tried an tested alternatives.

Thanks.

Answer 1

[Istvan Albert]

The reason that there is no direct solution for this is that there are several definitions of what uniquely mapped paired end reads could mean.

Even for single end mapping the entire concept of "uniquely mapping read" can be interpreted in different way. A discussion of this can be found on Heng Li&'s webpage, see http://lh3lh3.users.sourceforge.net/mapuniq.shtml

It states there that:

Uniqueness was initially introduced to measure the reliability of ungapped short read alignment with a read aligned in full length. It is not a proper concept for generic alignments.

Answer 2

[Ian]

I think the following answers my question:

How To Extract Reads-Pairs Aligned Concordantly Exactly 1 Time?