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Hi,
I fund some surprizing result with pooled libraries. The standarad 454 RLMID kit has 12 barcodes. I used RLMIDs 1-6 to prepare 6 libraries, then did Nimblegen sequence enrichment and then sequenced the enriched sample in two separate runs on GS Jr. The resulting two .sff files were combined in one .sff file and split by barcodes (sfffile -o -s). To much of my surprize I got 12 separate .sff files (*.RL1.*.sff to *.RL12.*.sff)!!! While numbers of reads with unexpected RL7 to RL12 were on average about 10% of reads with expected RL1 to RL6, where these RL7-RL12 reads have come from? Anyone experienced this?
Also I am paranoid about crosscontamination and try to minimize its possibility, I cannot exclude this completely. For this reason I ask those of you who has used pooled barcoded libraries take a few previous .sff files and reprocess them with sfffile -s [SomeDataFile.sff] to see if you get RMID reads that are not supposed to be there. Having statistics from different labs would indicate if this is something common or prather peculiar.
I fund some surprizing result with pooled libraries. The standarad 454 RLMID kit has 12 barcodes. I used RLMIDs 1-6 to prepare 6 libraries, then did Nimblegen sequence enrichment and then sequenced the enriched sample in two separate runs on GS Jr. The resulting two .sff files were combined in one .sff file and split by barcodes (sfffile -o -s). To much of my surprize I got 12 separate .sff files (*.RL1.*.sff to *.RL12.*.sff)!!! While numbers of reads with unexpected RL7 to RL12 were on average about 10% of reads with expected RL1 to RL6, where these RL7-RL12 reads have come from? Anyone experienced this?
Also I am paranoid about crosscontamination and try to minimize its possibility, I cannot exclude this completely. For this reason I ask those of you who has used pooled barcoded libraries take a few previous .sff files and reprocess them with sfffile -s [SomeDataFile.sff] to see if you get RMID reads that are not supposed to be there. Having statistics from different labs would indicate if this is something common or prather peculiar.