View Single Post
Old 08-19-2012, 08:46 AM   #34
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,253
Default

Hi Carmen,
I think enrichment PCR is the "sawdust in the steering column" of next gen library construction protocols. That is, it is a bad idea (for the reasons you describe.) But it obscures a host of library issues that otherwise would have to be dealt with directly. Not the least of which is that some characteristic of the TruSeq adapters in TruSeq Prep kits makes them perform extremely poorly for clustering without at least 1 cycle of "enrichment" PCR.

Oh, let's tack on the low percentage conversion of library molecules to clusters. 12 pM is roughly 7.2 million molecules/ul. 120 ul required per HS cBot lane comes to 864 million amplicons. (Or is it twice that, because they began the day double-stranded?) 12 pM might get you 180 million clusters. But no one is going to bother to optimize on that factor because enrichment PCR gives you more amplicons than you are ever going to use anyway.

To be fair, it also works around the issues of dealing with very low concentration solutions of DNA. But the original protocol, that Illumina blessed with their official adoption of it, was a no-enrichment protocol.

--
Phillip
pmiguel is offline   Reply With Quote