Earth Microbiome Locus Primers

[microgirl123]

Is anyone using 515F and 806R as locus primers for the Illumina two-step amplicon PCR library prep?

I have one investigator using them following the entire Earth Microbiome protocol (so adding primer pads and linkers) and sequencing with custom primers. This works great on the MiSeq and clusters properly.

Several other people have recently submitted samples prepared with the two-step PCR process (so no primer pads, etc.) and the sequencing just uses the Illumina primers in the MiSeq kit. These libraries are all over-clustering, even at loading concentrations of 2 pM.

Is anyone else seeing results like this? I'm starting to wonder if the locus-primers are somehow causing a problem with the sequencing or possibly the libraries are not working properly with the KAPA quantification.

I just started rerunning a library that was slightly over-clustered (4 pM loading concentration) at 2 pM, and the cluster density didn't decrease at all!

[nucacidhunter]

I have not used these exact primers but in my experience two step PCR libraries sequence just fine. Aim for cluster density at 550-750k with 20% PhiX. Read number and variation are similar with qPCR and dsDNA fluorophores quantifications. I would check the libraries to make sure that amplicons are clean with no primer dimers and double check calculations.

[ikripp]

We have run libraries containing amplicons from these primers, and I haven't seen any clustering issue related to them.

From my experience, the clustering of amplicons on the MiSeq is not linear, so when a run is severely over-clustered, I generally dilute the pool to half its concentration and then load the same volume of the diluted pool (I hope that makes sense).