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Old 11-15-2014, 11:53 PM   #9
bunce
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Location: Perth

Join Date: Sep 2012
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Quote:
Originally Posted by nucacidhunter View Post
I wonder if you have considered possibility of carry over from a previous run on MiSeq. We use two step PCR and it works well. In addition, it is convenient and we can pool and sequence other libraries as well without risk of unwanted interaction between custom and Illumina sequencing primers. Nano is the most expensive option for Illumina systems per Gb of data.
Hi, Sure - carry-over is always a possibility (we have been using the bleach wash protocol since february). I suspect (with no evidence) that people may 'blame' carry-over when it is on fact the lab workflow causing issues.

I don't doubt that a 2-step PCR protocol works well - it will always generate a truck-load of library. The question is what portion or reads are chimeric or have tags that you have not used on that run? I would be interested if anyone (using 2-step PCR and re-using indexes), has quantified this?
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