homopolymer rich amplicons

[ilma]

Hi,

I have one direction amplicon sequencing (using Lib-L) data with 95% filtered out due to "too short quality". I tried both the amplicon and shotgut pipelines but the outcome is the same. The majority of passed filter reads are short. By checking them I can see that most of them get trimmed in the middle of the 6G homopolymer that is 30bp down the forward primer. My amplicons have a few more homopolymers. I checked a number of well flowgrams that failed to pass the filters and in all of them sequencing has been going on until the end of the amplicon. I am playing a bit with the filter parameters but would like to ask if there would be suggestions how to treat the homopolymer rich amplicons or how to adjust the filters?
Thanks in advance!

[Anthony.287]

Redesign your amplicon. Roche recommends no more than a 5mer, which is the largest homopolymer stretch in the Type-1 control beads. I've tried to sequence a few libraries with larger stretches, and they work inconsistently at best.

[ilma]

Thanks Anthony for response.