This is not directly a Mothur problem, but I’m posting here in the off chance someone else has run across this problem and might have some insight.
My lab uses the V4 custom amplicon and sequencing primers detailed in the MiSeq wetlab SOP on the Schloss github. We have used these primers for ~4yrs and have always had good results with them. Recently, however, we have been getting really low cluster densities on the MiSeq machine (we would spike in to another run with 30% of the DNA pool and would only get back 1% of the total reads). If we use an entire flow cell for our 16S data, we will get reads but the run is still markedly under-clustered. The reads we do get are of good quality, however. This started a year ago when our MiSeq stage temperature was re-calibrated (it was too low before so the technician reset it higher).
The Illumina reps believe this is due to the custom primers we use for 16S library prep and/or sequencing (despite having used these primers ~20 times in the last 4 years without a problem). We did Sanger sequence a library through the amplicon primer and do see a small region in the adapter sequence that Sanger always has trouble with (perhaps due to secondary structure). But, again, these primers have always worked for us in the past and we can’t change the adapter sequence regardless.
Has anyone else run into this issue with the Schloss custom primers?! I’m guessing at least some of the people on this forum use these same primers…Any help would be greatly appreciated.