low quality 16s v3-v4

[sleeper]

i have tried 3 times to sequence 16S v3-V4 region, primer design is same like in earth microbiome project, reverse primer is exactly same , but forward is 338 f instead of 515f,
quality of reverse read is very poor, even we have included phix 10%

Earlier we did v4 region sequencing and it was perfect,..

totally confused, how same primer is perfect for v4 region but not for v3-v4, i have changed forward primer not reverse.

best

[kcchan]

What is the final fragment size of your library? If your inserts are too long you may run into problems getting good data with high cluster densities.

[snetmcom]

What do you mean by low quality? Are the quality values low, or are you seeing low cluster density?

[sleeper]

Hello
amlpcon size is about 550 (including adaptor/index and primers). we are using custom primers to sequence 430 bases (16S rRNA V3-V4 region).
we are getting more than 10Million reads but about 50 % reads have low q score (less than 20), specially on the ends of R1 and R2 reads (from 250-250 bases).
Problem is much more in reverse reads. would be obliged if someone could specify the reason and some solution.