My last MiSeq 16S amplicon run (2x250) and a rerun with a lower cluster density both showed large spikes in corrected intensity (>1000) during the second sequencing read cycles (see attached). This resulted in poor read quality (see attached). The lower cluster density for the re-run produced one big spike in intensity (>2000) at cycle ~410 (spike also present in original run). We used custom 16S rRNA primers that closely reflect the Kozich et al., 2013 dual-indexed primers (our forward read has a couple more degeneracies). This is our first time using these primers on a MiSeq run. We multiplexed ~90 samples, and the rerun used the same library prep, but just a different cluster density.
Here's some stats on each MiSeq run:
Original run:
* cluster density = 963
* cluster PF = 49.38
* %>=Q30 = 52.53 (read1), 56.57 (read2)
* % aligned = 8.9 (read1), 6.69 (read2)
re-run:
* cluster density=752
* cluster PF=52.23
* %>=Q30 = 61.68 (read1), 67.92 (read2)
* % aligned = 29.8 (read1), 28.63 (read2)
Can anyone help me troubleshoot what may be causing these large spikes in corrected intensity (which I hope will improve read quality)?
Thanks.
Here's some stats on each MiSeq run:
Original run:
* cluster density = 963
* cluster PF = 49.38
* %>=Q30 = 52.53 (read1), 56.57 (read2)
* % aligned = 8.9 (read1), 6.69 (read2)
re-run:
* cluster density=752
* cluster PF=52.23
* %>=Q30 = 61.68 (read1), 67.92 (read2)
* % aligned = 29.8 (read1), 28.63 (read2)
Can anyone help me troubleshoot what may be causing these large spikes in corrected intensity (which I hope will improve read quality)?
Thanks.
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