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.