Hi everyone,
Today I put on a MiSeq run using Nextera XT library preparation to be run on a V3 600 cartridge. After ~20 cycles it has given me a low cluster density of 473K/mm2. The quality however is fantastic (98.9% over 30).
The sample consisted of 6 uniquely barcoded libraries that were equalised in 24ul before adding to PhiX 6ul and HT1. I checked all samples using QF and my individual concentrations of DNA were between 0.6 and 3.5 ng/ul. is this a little low and part of the issue?
Does anyone know why this low cluster density may have come about so I can trouble shoot this in the future? So far I have looked into the NaOH concentration and confirmed this is not the case.
Thanks for any help you can provide,
Jamie
Hi Jamie,
Did you resolve this issue? I have done the same thing today and my cluster density is ~400K/mm2 but 99% over 30.
It'd be good to know what to do to improve the cluster density, if possible.
Thanks,
George
Hi George,
Yes, I managed to solve the problem in the end. What it turned out to be was the initial DNA concentration being inaccurate and slightly above the correct concentration required. I found that the use of a quantas flourometer helped to better assess the original concentration of DNA and made the dilution more accurate for the tagmentation.
Secondly the use of a bioanalyser allowed a more accurate use of the library prep to work out the correct concentration and volume to include in the final mixture for analysis.
I hope that helps,
Jamie