We used the new kit but our bioinformatician has been unsuccessful with the demux using Nugen's protocol. We are trying to figure out the source of the problem.
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Are the indices by chance not properly methylated or do they seem contaminated with unrelated sequences?
Originally posted by nucacidhunter View PostDemultiplexing rate with new kit is low with or without PhiX. Undetermined folder reads have index but it seems to have various sequences. NuGEN should have seen this issue if they sequenced libraries according to their recommendation.
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Sequences of part of NuGEN P5 adapter is not in public domain (they use custom read 1 sequencing primer). I think they ligate partial adapters (methylated) and then add index and restore full adapter sequence after conversion with PCR. Undetermined reads’ index seems to be random indicating either sequencing or synthesis error. Sequencing error and sequencing primer synthesis issues can be excluded because other lanes in flow cell are demultiplexing with higher rate (95-99%). That narrows down the possible cause to adapter or PCR primer synthesis error.
They are not from contamination because reads have very low C and high T content indicating conversion and map to reference.
Edit: Further analysis indicates that they use Y adapters in which P5 sequences are non-methylated, but P7 sequences are.
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We are now thinking that our demultiplexing issue is due to low diversity of the index pool. We had a pool of four libraries with the following indexes: GAGTCA, AGCATG, AAGAGG, and GGAGAA. According to their instructions, it shouldn't have been a problem but apparently, it is.
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I had cluster densities intentially at 700s well below max. My explanation at post #20 seems most probable. They either have a faulty design or oligo synthesis issues. These issues should be resolved before a product launch or kit shipment. Their techsupport has been silent too.Last edited by nucacidhunter; 09-30-2015, 10:52 AM.
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