Our core facility just did a long tag paired end library prep and sequencing run using the standard GS FLX kit and protocol. The sequence output looked fantastic but when I went to assemble it (gsAssembler v 2.0) a large fraction of the reads were rejected by the assembler. After a lot of head scratching and further analysis I discovered that 90% of the reads were plasmid DNA; that plasmid being some pBR derivative. Being on the bioinformtics side I didn't know the full details of the library prep but discovered the source of the problem after discussing it with the lab folks. For those who haven't done this protocol, at one step you are supposed to add 3 ug of pUC19 as a "carrier". You are supposed to separate your DNA of interest a few steps later via a biotin moiety on the paired end adaptor. Obviously our cleanup was not very efficient.
My question is, has anyone else experienced this plasmid carry over with the long tag protocol? If so what have you done to address the problem?
Thanks,
Kevin
My question is, has anyone else experienced this plasmid carry over with the long tag protocol? If so what have you done to address the problem?
Thanks,
Kevin
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