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Old 03-14-2018, 10:13 AM   #4
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,317

(1) Yes, we accept libraries from customers and pool them with other libraries. But the libraries have to be unique dual indexed and we do some QC to make sure they would not be a major source of index hops.

(2) MiSeq titration run. We can tell from that if the customer was on the wrong index pair.

(3) Yes. Not a full run, just enough to allow titration. Alas the MiSeq run is not as good a predictive tool for relative cluster densities for the NovaSeq as it was for the HiSeq. Our Illumina FAS mentioned there was some hope that the iSeq might be better as it also has a patterned flowcell. But we don't have an iSeq, so I guess that is a moot point for us...

Also, we heat denature libraries and run them on a pico RNA chip for QC, rather than running them undenatured on a DNA high sensitivity chip. This catches libraries with large amounts of primers/adapter dimers even if they are annealed to full length library molecules. In most cases Ampure fixes these. But we have had one strange case recently that always produced a trail, or even a peak of lower molecular weight entities even with extra Ampure. The Ampure seemed to make no difference.

Okay, this is a tangent, but possibly of interest:
These libraries were constructed by a customer who cut them out of a gel after amplification. They were using a UV transluminator and "Gel Red" stain for this. We loaned them a Dark Reader hand-held lamp to use for this purpose and the issue disappeared.

We never use a UV transluminator. I always advise those starting a new lab not to buy one and to buy a Dark Reader instead. I was shocked to learn that a large percentage of them had never even heard of a Dark Reader!

But to tell you the truth I'm still not sure what about the UV exposure caused the issue described above. Almost like it made the DNA "brittle", or caused cross-strand linkage formation. The lore is that UV causes "pyrimidine dimers". Which I would not expect to produce the issue that we saw.

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