Hello,
We are recently experiencing demultiplexing issues on HiSeq 4000, NextSeq 500 and MiSeq, where we have good cluster formations, high read PFs, and greater than 90% >Q30 scores but only a very small fraction <1% of the sequenced indexes passed PF and were mapped. Majority of our read output (>60%) were in the undetermined fastq file but each sample in our pooled library do get a fastq file containing very little reads. We spiked in about 5% PhiX as it is a low diversity library (predetermined viral peptide sequences).
We are using custom 7-nucleotide IS7 indexes which can be mapped (98%) if only one sample (one index) was ran. Library was gel and pcr purified and bioanalyzer showed a single sharp peak at the expected fragment size.
Any thoughts?
We are recently experiencing demultiplexing issues on HiSeq 4000, NextSeq 500 and MiSeq, where we have good cluster formations, high read PFs, and greater than 90% >Q30 scores but only a very small fraction <1% of the sequenced indexes passed PF and were mapped. Majority of our read output (>60%) were in the undetermined fastq file but each sample in our pooled library do get a fastq file containing very little reads. We spiked in about 5% PhiX as it is a low diversity library (predetermined viral peptide sequences).
We are using custom 7-nucleotide IS7 indexes which can be mapped (98%) if only one sample (one index) was ran. Library was gel and pcr purified and bioanalyzer showed a single sharp peak at the expected fragment size.
Any thoughts?
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