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Old 06-19-2019, 12:00 PM   #2
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Location: Eugene, OR

Join Date: May 2013
Posts: 496

Did the facility add extra PhiX to help the basecalling in the low-complexity region of the cut site? What is the cut site?

It does seem like two issues. If you are getting lots of off-target sites then a common problem is your adapters having the overhang chewed back and blunt-ligating to random sheared ends. But then the restriction region should not be very low complexity and should have normal quality!

Are the off-target sites all at 1X read depth or is it a larger set of sites with most around 5X? If the latter, then is the size-selection different this time and you just ended up sequencing more ddRAD sites than hoped for? It doesn't sound like it if the fragments truly aren't near cut sites, but just checking.
Providing nextRAD genotyping and PacBio sequencing services.
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