Hi, I've been trying to get a protocol automated using pooled samples and indexing. We've had some initial success when performing library prep on individual samples and pooling postcapture and limited success pooling before hybridisation at the precapture stage. However, in our last expt we set up 32 samples using 3 primer system with barcodes, performed precapture PCR and then pooled the samples in sizes x8, x24 and x32. All subsequent steps (hyb using Nimblegen probes, clean up, post capPCR) were performed on the pooled samples.
These were run on an illumina GA II but our results were very strange in that for all the pools there were 2 barcodes (the same in each pool) that comprised ~90% of the reads (total reads about 90 million). Each of the pools were prepared separately so the two samples corresponding to the over represented indexes were always added in the same proportion as the other samples.
Has anyone had a similar experience to this and if so do they know what went wrong or does anyone have an idea of what could cause such a result?
Thanks in advance - H
These were run on an illumina GA II but our results were very strange in that for all the pools there were 2 barcodes (the same in each pool) that comprised ~90% of the reads (total reads about 90 million). Each of the pools were prepared separately so the two samples corresponding to the over represented indexes were always added in the same proportion as the other samples.
Has anyone had a similar experience to this and if so do they know what went wrong or does anyone have an idea of what could cause such a result?
Thanks in advance - H