Recently, we made 4 Illumina paired-end libraries using the standard protocol with a brand new Illumina paired-end sample prep kit. The libraries looked great on a bioanalyzer but when we sequenced, we noticed few odd things.
1. Read 1 was "unusually" high in "A" bases. "Unusually" since this is not typical for this organism. Read 2 was unusually high in "T"s. We blasted the reads for possible contaminants but the reads did not match to other organisms. After further digging, we noticed that the same "AT" rich regions are over amplified compared to other regions.
2. Read 2 had a 0.3% higher error rate than read 1. Usually we see just a 0.1% difference.
3. Read 2 had a 10% less % align than read 1. Usually we see maybe a 1% difference.
What will cause the above? What step in the Illumina protocol went wrong? Has anyone else seen this?
We know it is not the cluster station, GAII or any of the cluster or sequencing reagents. Other samples on the flowcell including the control looked fine. I can not rule out Illumina sample prep kit yet.
I have not been able to get any answers for the above questions from Illumina. So any feedback would be appreciated.
1. Read 1 was "unusually" high in "A" bases. "Unusually" since this is not typical for this organism. Read 2 was unusually high in "T"s. We blasted the reads for possible contaminants but the reads did not match to other organisms. After further digging, we noticed that the same "AT" rich regions are over amplified compared to other regions.
2. Read 2 had a 0.3% higher error rate than read 1. Usually we see just a 0.1% difference.
3. Read 2 had a 10% less % align than read 1. Usually we see maybe a 1% difference.
What will cause the above? What step in the Illumina protocol went wrong? Has anyone else seen this?
We know it is not the cluster station, GAII or any of the cluster or sequencing reagents. Other samples on the flowcell including the control looked fine. I can not rule out Illumina sample prep kit yet.
I have not been able to get any answers for the above questions from Illumina. So any feedback would be appreciated.