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Old 02-12-2018, 01:39 AM   #1
Justin87
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Default 16S Sequencing Run - Under-Clustered; Higher PhiX Alignment than expected

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 -15C to -25C'.

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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Old 02-12-2018, 06:58 AM   #2
thermophile
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Your phix isn't too low, it could be too high []. But if you used exactly that tube on a previous run, then I think it's fine and your library quant is off.
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Old 02-14-2018, 01:12 AM   #3
Justin87
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Default PhiX conc.

Thank you, thermophile!

Sorry for the ignorant question, but just out of interest, could I ask what would happen IF the phix was not spiked into the library at an equimolar concentration and was much lower than expected? Other than the % alignment, which other stats would be affected (cluster density, percentage of clusters passing filter, etc.)? Are any of these measurements based on PhiX conc.?

Regards,
Justin
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Old 02-14-2018, 06:07 AM   #4
thermophile
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% base and clusters PF are the biggest ones. A run of just 16S will likely fail because many of the first 20 cycles are only one base and the machine won't be able to focus for the other bases.

One thing to check, how old is your 2N NaOH that you use to make fresh 0.2N? If it's over 6months you might want to throw it out and make fresh 2N from 10N stock
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