Hi all,
first the TLDR:
Do you know of any factors that might cause PhiX denaturation to fail or reverse, outside of typical mistakes (NaOH concentration/pipetting mistakes/age)?
We have performed nearly one hundred MiSeq runs to date in our lab, and mostly they perform as expected. However, in some recent amplicon runs we have seen far less PhiX align than expected based on the % added, e.g. 1-2% aligned rather than 20% expected. This has happened both with an old and a fresh stock of PhiX. It cannot be explained by wrong library quantification, as the cluster densities were approximately as expected from the loading concentration. I quantified the new PhiX stock by qPCR, and it is very close to 10 nM. Also I verfied the insert size from what little did align. I checked the pH of the NaOH stock and dilution (made fresh every time) and it is OK. We have also done a system check of the instrument (optics alignment, volume test, thermal test) and everything passed.
This leads me to believe something sometimes goes wrong with the denaturation of the PhiX. Once we had some old 12.5 pM aliquots of denatured PhiX (several months at -20), and we saw that the % aligned gradually declined, as expected for old aliquots. But when I made fresh aliquots from the stock it got much worse; almost nothing aligned. At the time I though the stock had gone bad. But then the same thing happened to a completely new stock. I am at a loss to understand why this is happening. I did contact Illumina about this problem and other amplicon run problems we had and got some help, but nothing specifically about the PhiX problem.
Jon
Hellenic Centre for Marine Research
first the TLDR:
Do you know of any factors that might cause PhiX denaturation to fail or reverse, outside of typical mistakes (NaOH concentration/pipetting mistakes/age)?
We have performed nearly one hundred MiSeq runs to date in our lab, and mostly they perform as expected. However, in some recent amplicon runs we have seen far less PhiX align than expected based on the % added, e.g. 1-2% aligned rather than 20% expected. This has happened both with an old and a fresh stock of PhiX. It cannot be explained by wrong library quantification, as the cluster densities were approximately as expected from the loading concentration. I quantified the new PhiX stock by qPCR, and it is very close to 10 nM. Also I verfied the insert size from what little did align. I checked the pH of the NaOH stock and dilution (made fresh every time) and it is OK. We have also done a system check of the instrument (optics alignment, volume test, thermal test) and everything passed.
This leads me to believe something sometimes goes wrong with the denaturation of the PhiX. Once we had some old 12.5 pM aliquots of denatured PhiX (several months at -20), and we saw that the % aligned gradually declined, as expected for old aliquots. But when I made fresh aliquots from the stock it got much worse; almost nothing aligned. At the time I though the stock had gone bad. But then the same thing happened to a completely new stock. I am at a loss to understand why this is happening. I did contact Illumina about this problem and other amplicon run problems we had and got some help, but nothing specifically about the PhiX problem.
Jon
Hellenic Centre for Marine Research