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Old 12-04-2014, 06:22 PM   #16
Brian Bushnell
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Location: Walnut Creek, CA

Join Date: Jan 2014
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Originally Posted by GenoMax View Post
@nucacidhunder: All those appear to be "standard" (gold?) samples.

Brian: If PhiX standard does not look good then that is worrisome.
I agree, which is why it's surprising that Illumina has made it publicly available - and indeed, that's what they pointed me to when I first started questioning them about NextSeq's quality. Currently they are claiming that a NextSeq machine is 'in spec' as long as at least 70% of the bases are labeled by the machine as at least Q30, regardless of whether those bases are actually correct! I don't know whether this is a group of employees misinterpreting the specification, or if really is Illumina's official policy, but it's worrisome either way.

Our machine self-reports 87% of bases as having quality above 30, and therefore Illumina claims it is in-spec, but the true quality as measured by mapping for the highest-rated bases (claimed Q37) is only Q28, and the majority are much lower. In other words, bases the machine assigns Q37 are wrong 0.16% of the time rather than the claimed 0.02%, so their quality values are inflated by a factor of 8. In reality, 0% of the output is at least Q30, either from our machine or from Illumina's official PhiX data, which I used because they calibrate their machines on PhiX so it should represent the best case scenario.

Does anyone have a different experience?

Last edited by Brian Bushnell; 12-04-2014 at 08:30 PM.
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