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Old 01-31-2012, 05:26 AM   #1
End User
Location: Heidelberg

Join Date: Jul 2011
Posts: 8
Default Pre-Capture Pooling with Nimblegen SeqCap EZ v3: SNP detection quality?

Dear all,

Does anyone have already some experience with the new Nimblegen SeqCap EZ v3 targeted Exome Enrichment kit, concerning pre-capture pooling?
The kit explicitly supports pre-capture pooling of samples (barcoded) and a subsequent pooled targeted enrichment.
(In a test at my institution, The v3 kit itself performed satisfactorily concerning on-target rate and target coverage).

Now I am especially looking for information concerning the specificity of SNP-calls when performing such a pre-capture pooling approach.

The enrichment involves some PCR cycles when the samples are already pooled (as far as I gathered), and I wondered if or to what degree cross-hybridization between the captured fragments of different samples occurs or can occur at this step. However, my knowledge (and so far also my understanding) about this technology is limited.

If such cross-hybridization occurs, wouldnít the specificity of the SNP calls become much worse compared to a single sample enrichment? I would expect that there is a considerable fraction of reads from a specific sample (assigned via the sample-specific barcodes) which bear SNPs or InDels stemming from cross-hybridization events with the fragments from another sample.

Or do I maybe misunderstand something in general, and cross-hybridization cannot happen? Or can those events easily be identified and filtered? Or does that only play a very minor role and can be neglected?

I asked Nimblegen about it... They seem to have a hard time to find someone in the company who can provide any informaton on that topic (I am asking repeatedly and waiting for weeks now). I also haunted the competitors from Agilent, but they just stated (of course) that the SNP detection specificity with the Nimblegen pre-capture pooling enrichment is worse than with their enrichment with the Human All Exon 50 MB or v4 kits, and that they would advise against pre-capture pooling with their kits, but they did not provide any arguments or data.

I studied the publications on in-solution targeted exome enrichment kit comparisons (see this thread:, and they largely agree that the Nimblegen capture probe design (DNA probes, shorter, but many) is in the end slightly more efficient than Agilentís design (RNA probes and longer, meaning higher binding specificity, but fewer of them) for SNP calling (Agilent won for the overall detection counts because of the larger target region compared to the older Nimblegen kits).
Although also the older Nimblegen version (v2) apparently also supports pre-capture enrichment, the two studies that compared that kit with Agilentís SureSelect Human All Exon 50 MB both used single sample enrichment as far as I can tell.

I should mention that I am comparably new to NGS, and I am an end user, but I tried getting myself read into field as good as possible during the past few months (btw, SeqAnswers was a great help). I am not directly involved in the practical steps concerning enrichment and sequencing, which is done by our NGS core facility. However, they also cannot answer the pre-capture enrichment question.

I thoroughly searched for available information on the topic in the web and on SeqAnswers, but I couldnít find any. If I missed or completely misunderstood something, I would be glad if you could point me towards it.

Any information or opinion is very welcome!

Thanks a lot!
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