Good day,
We recently performed a 16S metagenomics sequencing run (288 samples) on the Illumina MiSeq using a v3 (600 cycle) kit.
We quantified the library using qPCR and calculated the size-adjusted concentration using the average fragment size from the Bioanalyzer.
As we have typically done for previous runs, we spiked-in our PhiX at 15% at the same loading concentration as our pooled libraries (~5.5 pM).
However, compared to previous runs, we achieved a much lower cluster density (~350 K/mm2) and data output (4.8 Gb) than normal (~700 K/mm2 and 7.5 GB, respectively). Also, the previous runs contained 384 samples.
We are highly confident that our qPCR and Bioanalyzer results are accurate. Obviously, one would question the manual steps involved in denaturation and dilution of the libraries/PhiX. We also ensured the 0.2 N NaOH was made up fresh!
The number of reads that aligned to PhiX was 23.94% rather than the expected 15%. I know that % of PhiX increases when the concentration of the library is low causing PhiX to take over.
One concern I have is that the 20 pM PhiX library (prepared the week before and used for a sequencing run that worked) was stored as suggested in the Illumina guide: 'You can store the denatured 20 pM PhiX library up to 3 weeks at -15°C to -25°C'.
I am wondering whether a more diluted form of PhiX was stored (rather than the 20 pM) after the previous run, and that perhaps the Phix was spiked into the present library at a lower concentration than the library. Would this influence the QC metrics of the run?
Any suggestions/guidance would be greatly appreciated.
Thank you!
Kind regards,
Justin
We recently performed a 16S metagenomics sequencing run (288 samples) on the Illumina MiSeq using a v3 (600 cycle) kit.
We quantified the library using qPCR and calculated the size-adjusted concentration using the average fragment size from the Bioanalyzer.
As we have typically done for previous runs, we spiked-in our PhiX at 15% at the same loading concentration as our pooled libraries (~5.5 pM).
However, compared to previous runs, we achieved a much lower cluster density (~350 K/mm2) and data output (4.8 Gb) than normal (~700 K/mm2 and 7.5 GB, respectively). Also, the previous runs contained 384 samples.
We are highly confident that our qPCR and Bioanalyzer results are accurate. Obviously, one would question the manual steps involved in denaturation and dilution of the libraries/PhiX. We also ensured the 0.2 N NaOH was made up fresh!
The number of reads that aligned to PhiX was 23.94% rather than the expected 15%. I know that % of PhiX increases when the concentration of the library is low causing PhiX to take over.
One concern I have is that the 20 pM PhiX library (prepared the week before and used for a sequencing run that worked) was stored as suggested in the Illumina guide: 'You can store the denatured 20 pM PhiX library up to 3 weeks at -15°C to -25°C'.
I am wondering whether a more diluted form of PhiX was stored (rather than the 20 pM) after the previous run, and that perhaps the Phix was spiked into the present library at a lower concentration than the library. Would this influence the QC metrics of the run?
Any suggestions/guidance would be greatly appreciated.
Thank you!
Kind regards,
Justin
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