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I generated my libraries and quantitated the purified pcr products by nanodrop, picogreen, and bioanalyzer. The bioanalyzer agreed with the picogreen although the absolute numbers differed.
As suggested, I blunt ligated my pcr products to a vector and sequenced 10 clones at random to validate my library. There were no duplicates. However, i did notice that homopolymer tracts reported in the annotated sequence of my organism were often deleted in my cloned sequence.
I know the aligned sequence i've gotten back from the SOLiD pipeline has reproducibly shown artifactual "deletions" of homopolymer tracts but I've never heard of a user of an Illumina-generated library of sequences complain about it.
I'm using iProof(TM) proofreading polymerase for my amplification and performing all reactions as described by Illumina (with the only difference that I'm shearing my DNA with Covaris techonology instead of the nebulizer). Is anyone else noticing this when validating your library or am i unique? Do most people actually look at the level of alignments when the check or do they just look for the hits specific to their genome?
Thanks,
der.
As suggested, I blunt ligated my pcr products to a vector and sequenced 10 clones at random to validate my library. There were no duplicates. However, i did notice that homopolymer tracts reported in the annotated sequence of my organism were often deleted in my cloned sequence.
I know the aligned sequence i've gotten back from the SOLiD pipeline has reproducibly shown artifactual "deletions" of homopolymer tracts but I've never heard of a user of an Illumina-generated library of sequences complain about it.
I'm using iProof(TM) proofreading polymerase for my amplification and performing all reactions as described by Illumina (with the only difference that I'm shearing my DNA with Covaris techonology instead of the nebulizer). Is anyone else noticing this when validating your library or am i unique? Do most people actually look at the level of alignments when the check or do they just look for the hits specific to their genome?
Thanks,
der.