We just started using a new MiSeq and are discovering that the devil can be in the details. We have done two runs, the first with four indexed yeast genomes and the second with three indexed yeast genomes. The libraries were prepared using the Nextera kit and quantified by picogreen. The reads in these runs contained a high number of Ns (no base call) that increased as read length got longer. Illumina says this should not happen. The cluster densities for these runs were 437 and 312 K/mm2, respectively. The distribution of reads between the pooled genomes was skewed away from the desired 25/25/25/25 and 33/33/33 for the two runs as well.
I have very carefully quantified the yeast genomic DNA preps with picogreen and then run 200 ng next to 200 ng of lambda DNA on a gel. There is certainly some variability in prep quality from the gel image. In particular, yeast DNA preps always have a significant fraction of the DNA that is trapped at the well. I don't know if this is a problem or not.
Is there anyone out there with tips for maximizing quantity and quality of sequence data from a MiSeq run with three indexed yeast genomes?
I have very carefully quantified the yeast genomic DNA preps with picogreen and then run 200 ng next to 200 ng of lambda DNA on a gel. There is certainly some variability in prep quality from the gel image. In particular, yeast DNA preps always have a significant fraction of the DNA that is trapped at the well. I don't know if this is a problem or not.
Is there anyone out there with tips for maximizing quantity and quality of sequence data from a MiSeq run with three indexed yeast genomes?
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