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Old 03-19-2017, 02:04 PM   #2
Location: Nottingham, England

Join Date: Nov 2009
Posts: 26

Originally Posted by seb42 View Post

We are using custom sequencing primers containing multiple degenerate bases for MiSeq sequencing of the V4 SSU region. We have included these degenerate bases to hit more taxa during the PCR steps. Since the degenerate bases are in the V4 primer region they are pretty close to the start of the sequenced region so might have a disproportional affect on sequencing.

We have been getting poor quality scores for multiple libraries (40-60% reads pass) and only getting between 3-5 million reads back. We were wondering if anyone else has had any trouble with this and if so how they overcame the problem.

We were thinking that due to the mix of primers the poor results could be due to low sequencing primer density. Has anyone seen results similar to ours due to low primer densities or mixing of multiple sequencing primers?

Thank you for any advice you can give.
You should make sure the sequencing run is been carried out with about 20% PhiX in order to increase the base diversity during the run. Sounds like the low diversity is leading to a number of the clusters being removed during filtering.
barrmur is offline   Reply With Quote