Hi,
I've recently switched away from Illumina supplied to DIY library preps. I've been using the NEB Quick ligation kit (M2200) to ligate on my adapters. I've recently discovered this is 2,000 units/ul. The Illumina protocol uses 1 unit/ul (5 units in total). We have seen some unexpected chimeric reads in these PE libraries, where read 1 is a different chromosome to read 2. I suspect the excess of ligase maybe the reason. Although others I've spoken to are using this 2000 x ligase and seem to find it ok. What liagases are other people using? Has anyone had problems with inserts ligating to each other rather than the adaptors?
Cheers, Nick
I've recently switched away from Illumina supplied to DIY library preps. I've been using the NEB Quick ligation kit (M2200) to ligate on my adapters. I've recently discovered this is 2,000 units/ul. The Illumina protocol uses 1 unit/ul (5 units in total). We have seen some unexpected chimeric reads in these PE libraries, where read 1 is a different chromosome to read 2. I suspect the excess of ligase maybe the reason. Although others I've spoken to are using this 2000 x ligase and seem to find it ok. What liagases are other people using? Has anyone had problems with inserts ligating to each other rather than the adaptors?
Cheers, Nick
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