Over the weekend I set up a V2 2x250 16S sequencing run on our Illumina MiSeq (I work in a Core lab). I came back on Monday and the results were terrible. Overall Q30 was 58.91, it overclustered at 1031 (we aim for 700-800ish) and Cluster PF was 49.72.
Anyway, I repeated the sequencing run and actually gel extracted the library pools just to be safe, quantification was good and I used a subnanomolar protocol to load the cartridge. I loaded 4pm of library with a 10% spike of PhiX.
So I checked the results this morning and they are actually worse! Cluster density is 931 with cluster PF at 20% and Q30 for read 1 is 85%
Typically we have excellent results: cluster PF should be between 80-90%, Q 30 used to be really good, though lately we've had some trouble with our read 2 results dipping below 80% (has anyone else seen this in the past 6 months?)
But this situation is really terrible and I wondered if anyone has any advice/has experienced it before?
Anyway, I repeated the sequencing run and actually gel extracted the library pools just to be safe, quantification was good and I used a subnanomolar protocol to load the cartridge. I loaded 4pm of library with a 10% spike of PhiX.
So I checked the results this morning and they are actually worse! Cluster density is 931 with cluster PF at 20% and Q30 for read 1 is 85%
Typically we have excellent results: cluster PF should be between 80-90%, Q 30 used to be really good, though lately we've had some trouble with our read 2 results dipping below 80% (has anyone else seen this in the past 6 months?)
But this situation is really terrible and I wondered if anyone has any advice/has experienced it before?
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