Water blanks

[AStretton]

Some colleagues of mine are running an Illumina TSCA panel on FFPE samples using the MiSeq, they have just starting using a water blank and putting it through the whole process including sequencing on the MiSeq. As a diagnostic lab using a water blank is common practise for every other technique we use.

They were surprised at how many reads were assigned to the blank. I was just wondering if anyone else had tried running this sort of blank on a MiSeq or any other NGS machine for that matter. Or if anyone has any thoughts on using water blanks on NGS?

Thanks in advance!!

[dpryan]

What are they hoping to achieve with a water blank? The amount of noise in such an experiment would likely be over-inflated.

[GenoMax]

Is the water blank part of the pool that is being run? If so you would be interested in looking at JackieBadger's post (#3) in this thread: http://seqanswers.com/forums/showthread.php?t=38697

[AStretton]

Hi dpryan - with the TSCA you run a gel after the PCR step, some of the samples on the gel, look as if there is no band but they are still run on the miseq and they still some times get good results from these and these results are then reported back. Sometimes they get quite low coverage less than 150x but are reporting if a mutation is found (we are looking at somatic mutations in FFPE DNA). So when they ran a water blank which is also showing as negative on a gel, to check that the mutations they are reporting are genuine and not an artefact of the assay and this water blank then had quite good coverage up in 100x coverage they are now worried that they are reporting false positives. We have a few things we are going to look at to try to address this but I was just wondering what other people thought about running water blanks.

Hi GenoMax - Yes it is part of the pooled samples, thanks I'll have a read of that paper

[GenoMax]

There are approved assays for CF diagnostics for MiSeqDX so you should ask your local FAS about what kind of controls that assay is using.

This kit for MiSeq is referring to a master negative control (which may be the water blank you are referring to).

[AStretton]

The TSCA kit doesn't come with a negative control, so we run a negative of our own, running through the entire prep but we haven't actually put any sample in. I will have a chat with our FAS about what the negative control is in that kit though, thanks for the suggestion.