We want to try making PEIn Illumina libraries and then mixing them followed by Sure Select whole exon cap and PEIn sequencing. Has anyone tried this and what did you use for blocking during capture?
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Hi Bruce,
We've done many of these types of experiments using custom-made indexed adapters (different to the new Illumina indexed adapters in their TrueSeq kits) and SR or PE sequencing. We didn't add any extra blockers for the capture process against the index and tested the efficiency of the capture process in indexed vs non-indexed samples and found that the extra index had no effect on the capture process. That study was published at the end of last year if you're interested in reading it: Multiplex Target Enrichment Using DNA Indexing for Ultra-High Throughput SNP Detection. Kenny EM et al. DNA Res. 2010 Dec 16.
If you're thinking of going with the Illumina multiplexing protocols as far as I know the newer TrueSeq mulitplexing method (use of indexed adapters) is more amenable to pre-capture indexing and pooling than the original multiplexing protocol, where the index was introduced during PCR using a 3rd primer. In order to get that protocol to work for pre-capture pooling you would have to modify your PCR enrichment primers for the post capture enrichment PCR.
Elaine
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