Hi everyone,
We have been sequencing bacterial 16S rRNA gene libraires for about 3 years, prepared with custom dual-index primers (not staggered), followed Kozich paper (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753973/). But recently we have experienced some failures in sequencing these libraries, all showing very low cluster density and PF%. We generally load 4 pM (optimal based on Kozich paper), and spike in 15% PhiX. And our libraries were purified with AMPure beads twice and checked by running an agarose gel.
We have already re-ordered sequencing primers, re-made fresh dilutions of PCR primers and increased loading concentration to 8 pM, but cluster density was still not improved.
Has anybody experienced similar problems? Or can you please provide some advice? Thanks a lot!
We have been sequencing bacterial 16S rRNA gene libraires for about 3 years, prepared with custom dual-index primers (not staggered), followed Kozich paper (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753973/). But recently we have experienced some failures in sequencing these libraries, all showing very low cluster density and PF%. We generally load 4 pM (optimal based on Kozich paper), and spike in 15% PhiX. And our libraries were purified with AMPure beads twice and checked by running an agarose gel.
We have already re-ordered sequencing primers, re-made fresh dilutions of PCR primers and increased loading concentration to 8 pM, but cluster density was still not improved.
Has anybody experienced similar problems? Or can you please provide some advice? Thanks a lot!
Comment