Hello all. We've recently done some HiSeq sequencing of pooled haloplex libraries. We have sequenced 6 lanes so far from 4 different captures. 4 of those lanes have behaved themselves perfectly, but 2 have very low read numbers, around 5% of the good lanes. We looked at the run QC, and for the poor lanes we only have around 14% of clusters passing filters. We initially thought the lanes were overloaded, so we reduced the amount of input library from 10pM to 8 but it didn't have much affect. Also, although the cluster density was a bit on the high side 8-900K/mm2, one of the good lanes was over 1000K/mm2.
We don't think it is an adapter issue, as some of the good lanes had more adapter than the failed lanes. We are investigating with the manufacturers whether we have been sent a faulty kit. I was wondering if anyone else had seen these issues. Are haloplex kits particularly sensitive to overloading (have we just been lucky with our successful lanes). Have people seen some designs being more sensitive to this than others.
Any thoughts from the collective experience?
We don't think it is an adapter issue, as some of the good lanes had more adapter than the failed lanes. We are investigating with the manufacturers whether we have been sent a faulty kit. I was wondering if anyone else had seen these issues. Are haloplex kits particularly sensitive to overloading (have we just been lucky with our successful lanes). Have people seen some designs being more sensitive to this than others.
Any thoughts from the collective experience?
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