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Old 06-24-2019, 12:38 PM   #4
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Location: Eugene, OR

Join Date: May 2013
Posts: 496

If they loaded PhiX at 5% but the library was mis-estimated and actually much higher, then the actual PhiX could be lower, leading to poor quality scores.

Are you sequencing just one of the cut sites in read 1?

It may be that you are still sequencing ddRAD fragments and not random genomic but the poor quality of the cut site (because of overloading) is changing the cut site sequence. If you take 30 bp from the middle of "bad" read, can you find it in other reads with some of those reads starting from a good sequence and has a cut site?

It can be tough to amplify a very tight size selection and that does give artifacts a chance to become significant. But I think you should characterize the "off site" reads and determine if they are random and scattered or real ddRAD loci that just have bad quality cut sites that have changed the cut site sequence.

Overall though the core problem is the overloading. You aren't going to get a good number of reads. But you do want to figure this out to see if you can just load at a better concentration and you'll be fine or if other issues are present.
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