Entering edit mode
7.5 years ago
sommer
•
0
Hi, does anyone experienced after a SureSelect QXT libraryprep workflow and Miseq run an output of really good quality reads in forward direction and absolutely bad quality scores for reads in reverse direction? Thanks in advance
For sure with a V3 600 cycles kit.
Never with a V2 300 cycles kit.
Unfortunately, a V2 300 cycles kit which I used
Do you know the cluster density?
Yes, the Cluster density is really low, 604 +/- 16 K/mm2
That may be "low" based on what you are used to seeing but not bad at all.
What makes them "bad quality"? Can you post a screenshot (must be from FastQC)?
That does not look like a typical drop in quality one sees from 300 cycle kits.
Have you talked with your sequence provider to see if there was some issue with the run (that was not related to your samples)? If phiX was spiked in, did those reads look ok/had normal quality?
PhiX was spiked in and those reads do look ok/ had normal quality. I haven't spoken to my sequence provider so far due to my assumption that the problem is based on QXT sequence primer or maybe library prep? I also had a combined sequence run the first time, using nextera libraries which did look really ok and didn't show these weird output of reads in reverse direction. I will start another run on thuesday using a different panel/ sample prep kit to futher exclude any problems with teh MiSeq.