Hello, I'm puzzled over an issue we are having with an Illumina HiSeq x run we completed recently for a number of reasons.
We were following the protocol for bisulfite padlock probe sequencing which we have completed successfully recently on a novaSeq 6000 without issue.
This time, unbenknownst to us, the company sequenced our libraries on a HiSeq X, which has different chemistry, and we had serious issues with the index reads.
the i7 read seems to have read correctly, but the i5 reads are all very strange, and very few reads actually demultiplexed to the i5 indexes that we used. When troubleshooting we found that many of the i5 indexes with the highest reads assigned were actually the exact reverse complement of the i7 index for those reads. Many other i5 index reads appear to be random sequences not used in indexes.
Has anyone ever heard of an issue like this? I'm at my wits end here.
We were following the protocol for bisulfite padlock probe sequencing which we have completed successfully recently on a novaSeq 6000 without issue.
This time, unbenknownst to us, the company sequenced our libraries on a HiSeq X, which has different chemistry, and we had serious issues with the index reads.
the i7 read seems to have read correctly, but the i5 reads are all very strange, and very few reads actually demultiplexed to the i5 indexes that we used. When troubleshooting we found that many of the i5 indexes with the highest reads assigned were actually the exact reverse complement of the i7 index for those reads. Many other i5 index reads appear to be random sequences not used in indexes.
Has anyone ever heard of an issue like this? I'm at my wits end here.
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