Mapped reads with many mismatched reads

Leinal Sejour

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Jun 4, 2018, 5:59:26 PM6/4/18

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My name is Leinal and I work with Genomic Expression, a small start up in Beverly, Massachusetts. Currently, I'm mapping reads, from a recent sequencing experiment, to the 6th chromosome of the human reference. We were hoping to learn more about where our reads mapped to, in respect to the AFG1L gene. When I performed the mapping, I saw good coverage over a specific region on the reference but I also saw a high number of mismatches. How should I interpret this? We were expecting the reads to be represented as grey bars. Why would these reads map to this location if there was such disagreement and mismatching with the reference?

I'm attaching a picture of our reads in IGV to give you a better picture. The version of IGV we are using is 2.4.6. The coordinates we are focused on is chr6: 108,292,874-108,527,784, the AFG1L gene.

Thank you for your time! I appreciate it!

IGV mapping photo.PNG

James Robinson

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Jun 4, 2018, 8:02:33 PM6/4/18

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This usually means you are viewing the alignments against a different reference other than the one they are aligned to. Are you certain the reads were aligned to hg38?

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Tgrandis

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Jun 5, 2018, 1:05:10 AM6/5/18

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Hello Leinal: 

If you are mapping reads from a human whole genome sequencing experiment to the Chromosome 6 ONLY, then reads that might map to a (somewhat) similar genome sequence on another chromosome might
be erroneously mapped to a region on Chr 6. Whole genome reads should always be mapped back to whole genome reference, and then you can use
IGV to view the reads that map to your genome region of interest. 

Your alignment seems quite odd, but it is not a problem in IGV.. it is a problem during the read mapping step. If you have paired end reads, then
definitely check the mapping / alignment of the paired read too.


Cheers, 

Hugo

gne...@broadinstitute.org

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Nov 1, 2018, 4:48:46 PM11/1/18

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I am experiencing a similar problem to Leinal. My colleague loads my .bam into IGV and it maps perfectly to hg38. When I do the same, also to hg38 on my computer, I see totally mismatched amplicons like Leinal. I still cannot figure out why I have this issue and it persists after re-installing IGV. Any tips would be appreciated!

-Greg

James Robinson

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Nov 1, 2018, 5:15:12 PM11/1/18

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You can see clearly from the sequence that those bases marked mismatch really do mismatch. Could you post a screen shot from your colleagues computer of the same bam file at exactly the same location? Actually zoomed in a bit would be more helpful so we can see the sequence, but post both screenshots for the same BAM and same location.

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Gregory Newby

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Nov 1, 2018, 5:47:59 PM11/1/18

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Hi James,

Thank you for your reply - actually I cannot post the side-by-side comparison anymore. After finding this forum, I noticed the "GenomeSpace" feature on the IGV website. I launched IGV from GenomeSpace, and it seemed to fix whatever was wrong with my original download. I had downloaded it twice over this last week and continually ran into the same problem of reads mapping incorrectly to hg38. However, now when I open IGV either the old way or through GenomeSpace, my reads are mapping correctly as they were on my colleagues' computer.

Thanks again,

-Greg

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Gregory A. Newby

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Laboratory of David Liu

Broad Institute of MIT and Harvard

75 Ames Street, Room 3010

Cambridge, MA 02142

James Robinson

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Nov 1, 2018, 5:56:14 PM11/1/18

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Hi Greg, could you verify the IGV version that is now, correctly, displaying the reads? Just select Help > About, it will show you the version. I'm not sure what GenomeSpace installs.

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Gregory A. Newby
Post-doctoral fellow
Laboratory of David Liu
Broad Institute of MIT and Harvard
75 Ames Street, Room 3010
Cambridge, MA 02142

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Gregory Newby

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Nov 1, 2018, 5:57:36 PM11/1/18

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Sure, James - it installed Version 2.4.14 08/14/2018

-Greg

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Gregory A. Newby
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Laboratory of David Liu
Broad Institute of MIT and Harvard
75 Ames Street, Room 3010
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James Robinson

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Nov 1, 2018, 5:59:21 PM11/1/18

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That's the current version. Oh well, I guess this will remain a mystery.

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Gregory A. Newby
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Laboratory of David Liu
Broad Institute of MIT and Harvard
75 Ames Street, Room 3010
Cambridge, MA 02142

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Gregory A. Newby
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Laboratory of David Liu
Broad Institute of MIT and Harvard
75 Ames Street, Room 3010
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