IGV shows bowtie2 mapped reads not corresponding to genomic sequence

[Sarah_Tulin]

Hi,
I have used Bowtie2 to map my reads to my genome. The reads are similar to ChIP seq reads. I filtered my reads for a MAPQ score > 10. When I look at the alignments using IGV, reads with a very high MAPQ score (say, 42 for example), seem to not match very well at all on the base-pair level for the genomic regions of interest I'm looking at (regulatory regions). How can this be? Doesn't a high MAPQ score from Bowtie2 mean it is certain that read maps there?

My Bowtie command:
bowtie2 --phred33 --fr -I 100 -X 500 -p 8 --seed 123 -q -t -x Sea_Urchin_Bowtie2_ref_index -1 clean_R1.fastq -2 clean_R2.fastq -S Sea_Urchin_sample_1_bowtie2.sam

[IMG][/IMG]

Any suggestions or comments or help?

Thanks!

[swbarnes2]

There's a discrepancy between what genome you aligned to, and what genome is in IGV, or the reference index is messed up.

So I'd start by removing that reference genome from IGV, close IGV, to make sure it's not remembering the old index, remaking the index with samtools faidx, and then reimporting it into IGV, and trying that. That's pretty fast.

If that doesn't work, reindex the genome with bowtie, and realign, to make 100% sure that the genome you are aligning to is consistent all the way through.

[Sarah_Tulin]

Thank you, that makes sense. Even though I thought they were both using the 3.0 version of the S. Purpuratus genome, I just used whatever IGV had rather than importing my own.