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We have been running mRNAseq on an Illumina GAll. At cycle 36 we are seeing a spike in our error rate. Has anyone else observed this? We do not see it when we run genomic dna or in our standard.
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Bruce,
I have what may be a solution/explanation of your problem, but I'd strongly recommend that you contact Illumina's tech support team as they can help you work through the issue.
Our R&D group used to see higher error rates when they first starting running mRNA-Seq samples longer than 36 reads. It turns out they were sequencing libraries with relatively short inserts (<50 bases). They were reading across the entire insert and in to the adapter sequence on the other side (and since the adapter doesn't align to the genome, you get higher error rates). Libraries with consistently larger inserts are needed when sequencing out to 75 or 100 bases (or beyond). Hopefully this will help you with what you're seeing.
Sincerely,
Shawn C. Baker
Market Manager, Expression and Regulation
Illumina
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