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Hi all,
I want to share my recent experience with Nimblegen enrichment and get some useful feedback. In general, enrichment did what it supposed to do with libraries from DNA of cancer xenografts. There were two complicated issues however, which I am still quite puzzled about.
First, with accurate TaqMan library quantitation, it is possible to sequence captured libraries directly, without subsequent LMPCR that is recommended in the manual. However, this produces a lot of short reads, sometimes up to 25-30%, wasting the GS Jr bandwith, so to say. Read distribution uniformly shows two peaks, one around 50-70 bp and another at expected 450 bp.
if library is used ater postcap LMPCR, which also enables fragment distribution analysis with BioAnalyser and quality of capture assesment by qPCR, another problem adds up - the number of reads without the key raises substantially, up to 10-20 fold. This looks like quite specific to the postcap LMPCR step, since keyless reads also quite low when libraries are sequenced without Nimblegen enrichment.
The first issue, with short reads, is also present, although BioAnalyser shows nothing in the low range, but I guess if one considers molar ratios, short fragments can contribute a lot of molecules without being visible on electrophoregrams. The problem can be taken care of by resizing the amplified captured library using 454 protocol from Rapid Library kit.
I wonder if blocking oligos used in the hybridization step contribute to these problems, if carried over to the isolated postcap library. The earllier protocol used 3'-blocked oligos, which somewhat more expensive and were eliminated from the later version.
I want to share my recent experience with Nimblegen enrichment and get some useful feedback. In general, enrichment did what it supposed to do with libraries from DNA of cancer xenografts. There were two complicated issues however, which I am still quite puzzled about.
First, with accurate TaqMan library quantitation, it is possible to sequence captured libraries directly, without subsequent LMPCR that is recommended in the manual. However, this produces a lot of short reads, sometimes up to 25-30%, wasting the GS Jr bandwith, so to say. Read distribution uniformly shows two peaks, one around 50-70 bp and another at expected 450 bp.
if library is used ater postcap LMPCR, which also enables fragment distribution analysis with BioAnalyser and quality of capture assesment by qPCR, another problem adds up - the number of reads without the key raises substantially, up to 10-20 fold. This looks like quite specific to the postcap LMPCR step, since keyless reads also quite low when libraries are sequenced without Nimblegen enrichment.
The first issue, with short reads, is also present, although BioAnalyser shows nothing in the low range, but I guess if one considers molar ratios, short fragments can contribute a lot of molecules without being visible on electrophoregrams. The problem can be taken care of by resizing the amplified captured library using 454 protocol from Rapid Library kit.
I wonder if blocking oligos used in the hybridization step contribute to these problems, if carried over to the isolated postcap library. The earllier protocol used 3'-blocked oligos, which somewhat more expensive and were eliminated from the later version.