Question: Multiplex In Exome Seq And "Customized/Capture" Seq Kits
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gravatar for CrazyB
6.4 years ago by
CrazyB210
United States
CrazyB210 wrote:

Does anyone have the experience of using Nimblegen exome seq kit or customized seq kit to enrich their samples before sequencing the DNA?

My question is how many samples could we multiplex per capture reaction? Clearly the answer depends on the desired coverage depth, and I am also aware that capture is not homogeneous so some regions may get more covered/captured than others. Moreover, the coverage depth will certainly increase if we sequence the capture product multiple times/lanes. But I was wondering what people's general experiences are with these kits when they multiplex the samples.

e.g. When we capture one sample per capture reaction, what's the expected/estimated coverage depth on average for one run sequencing in one sequencing lane (HiSeq)? When we multiplex 2 samples per capture reaction, what's the expected/estimated coverage depth on average? When we multiplex 8 samples per capture reaction, what's the expected/estimated coverage depth on average?

Any info sharing will be greatly appreciated !!

To clarify - The kit comes in different sizes - 4 rxn, 12 rxn, 24 rxn ..etc. Ideally, each rxn is for one sample capture. However, I assume the oligos in the rxn are in EXCESS, so in principle, we should be able to pool 2-3 (or more) samples per capture and still get enough read depth of sequence for each sample. I was wondering if people have done it and if so, what's recommended and not-recommended for multiplexing in the capture reaction.

ngs • 2.9k views
ADD COMMENTlink modified 6.4 years ago by Dan Gaston7.1k • written 6.4 years ago by CrazyB210

Use the Illumina coverage calculator: http://support.illumina.com/downloads/sequencing_coverage_calculator.ilmn

ADD REPLYlink written 6.4 years ago by Daniel Swan13k
0
gravatar for Dan Gaston
6.4 years ago by
Dan Gaston7.1k
Canada
Dan Gaston7.1k wrote:

I'm not familiar with the Nimblegen kit, as we have used either Illumina TruSeq or Agilent kits for our exome sequencing reactions. For TruSeq that have a recommended max multi-plex per reaction (6-plex), which I believe is what most people actually do. You would need to consult the product sheet for your particular kit for that info.

For our samples we then multiplex 3 exomes per lane of sequencing as we are targeting a mean of 100x coverage.

ADD COMMENTlink written 6.4 years ago by Dan Gaston7.1k

If I understand you correctly, this means each Agilent reaction has the capacity to enrich ~ 30 GB sequence ?? (each exons = 50 MB, 6-plex, average 100x read-depth). Nimblegen does not provide any number (or I couldn't find it), so I can only hope their capture capacity per reaction is comparable to that of Agilent.

ADD REPLYlink modified 6.4 years ago • written 6.4 years ago by CrazyB210

Sorry the figures I gave where for Illumina TruSeq, not the Agilent kit.

ADD REPLYlink written 6.4 years ago by Dan Gaston7.1k
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