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Hi All
I'm new in sequencing field, may ask some stupid questions. Hopefully get some suggestions on my troubles.
And actually I only prepare the libraries and samples, the sequencing and kapa qPCR were done in the core facility. So I really am not familiar with sequencer running, but got the fail results from the core running but can not figure what is wrong.
I have a set of sequencing running on HIseq2500 with 2 flow cells(2 plex, each plex contained 12 indexed samples). One plex pass through the sequencing with good cluster density and reads. But one plex failed which gave low cluster density (probably are all Phix clusters) and the limited reads it came out all aligned to Phix. It confused me is that before go into sequencing, we have those indexed samples running on qPCR and kapa qPCR to adjust the balance for all the samples to pool together. And kapa qPCR supposed gave us the quantification only PCR-competent DNA products which are indexed sequences. And there are not much differences between these two plex on kapa qPCR results. So I’m SO CONFUSED why one plex form clusters perfectly but the other totally fail, no any clusters and reads. One more question, I want to ask that we have 12 indexing sequeces in one plex. If one of the index didn’t go well, will it make the whole plex fail? We anyway prepare each sample library separately, only pool together when go on sequencer, kind of not possible 12 samples are all bad in one plex but all good in another plex accidently.
thank you for any suggestions and comments.
I'm new in sequencing field, may ask some stupid questions. Hopefully get some suggestions on my troubles.
And actually I only prepare the libraries and samples, the sequencing and kapa qPCR were done in the core facility. So I really am not familiar with sequencer running, but got the fail results from the core running but can not figure what is wrong.
I have a set of sequencing running on HIseq2500 with 2 flow cells(2 plex, each plex contained 12 indexed samples). One plex pass through the sequencing with good cluster density and reads. But one plex failed which gave low cluster density (probably are all Phix clusters) and the limited reads it came out all aligned to Phix. It confused me is that before go into sequencing, we have those indexed samples running on qPCR and kapa qPCR to adjust the balance for all the samples to pool together. And kapa qPCR supposed gave us the quantification only PCR-competent DNA products which are indexed sequences. And there are not much differences between these two plex on kapa qPCR results. So I’m SO CONFUSED why one plex form clusters perfectly but the other totally fail, no any clusters and reads. One more question, I want to ask that we have 12 indexing sequeces in one plex. If one of the index didn’t go well, will it make the whole plex fail? We anyway prepare each sample library separately, only pool together when go on sequencer, kind of not possible 12 samples are all bad in one plex but all good in another plex accidently.
thank you for any suggestions and comments.