[bradfish]

As a follow up, the PI decided to not try the 0.083 cpb ratio, and we went with 2 cpb instead. I don't know if the ratio is the reason or not, but we always get a large number of reads filtered out by the numFilteredTooShortQuality filter (about 60% are filtered out). The enrichments are a little high (20% or a little above). But the thing is, in one of the two control bead sets, the same thing happens. The CATG key control beads also lose 60% to the filter; the ATGC key control beads are fine. Not sure how to interpret this.

We have been having the exact same issue, even using 1 CPB for a 400 bp amplicon. Everything you describe, from the control beads to the 60% short quality. We are going to try to change the valley filter to less flows so that maybe some reads won't be discarded as short. In the meantime, underloading the PTP by 100k beads seams to help. I suspect it is an issue with the DNA fragments getting out of sync at the end of the run and the signal intensities then throwing the valley filter off.

[Anthony.287]

Getting the enrichment down to 5-10% will probably help dramatically with the passing filter. I know of one lab that won't sequence amplicons that enrich over 7%! High enrichment leads to high numbers of mixed beads which leads to high percentage of too short quality, and several of the filters are included in the too short quality category, which makes it even more confusing!
Also, are you reducing the amplification primer? I had one set of amplicons that I was seeing the same problem with, except more extreme. There were no CATG control beads showing up at all. Turns out that the samples were amplifying so well that the signal drowned out the CATG control beads.

[bradfish]

Getting the enrichment down to 5-10% will probably help dramatically with the passing filter. I know of one lab that won't sequence amplicons that enrich over 7%! High enrichment leads to high numbers of mixed beads which leads to high percentage of too short quality, and several of the filters are included in the too short quality category, which makes it even more confusing!
Also, are you reducing the amplification primer? I had one set of amplicons that I was seeing the same problem with, except more extreme. There were no CATG control beads showing up at all. Turns out that the samples were amplifying so well that the signal drowned out the CATG control beads.

Yes we reduce amp primer to 1/4 of recommended volume. I know that getting the enrichment down will help, just am confused by why it is all of a sudden 200% higher enrichment that I used to get. These are the same size ampliconn I always use so I am perplexed as to why everything is changing all of a sudden. I am working on trying a few new things, will keep members posted...

Thanks!
J