Problems with NEXTERA DNA FLEX ENRICHMENT

[HelenaSC]

Hi there!
Anyone working with Illumina Nextera DNA Flex for Enrichment?

We are experiencing some problems related to the hybridization or capture step. We don't know why, but after pooling the samples (12 samples in one pool, total 8 pools) and doing the capture, there is always some pools that don't perform correctly and gave very low yield and concentration.

We have performed 6 96-plates and in each of the processing of these plates, there is always this issue, and it seems to be at random.

We pool by mass, and we always quantify the samples with Qubit before pooling them, so, it has no relation with the hibridized amount. I mean, we hybridize the same total nanograms on each pool, but some of them work properly and others just don't...all the samples are fresh gDNA.

Any idea??

Thanks a lot in advance!!!

[luc]

What QC information do you have for the libraries before pooling and capture?
After sequencing, how is the read number within the pools?

[HelenaSC]

Dear Luc,

THanks for your comments. Before polling the libraries, they are quantified by Qubit and analyzed with TapeStation , so we can be sure that the amount and the profile is ok before pooling them. We pool the same amount of library on each pool...That's why we don't understand why some pools are not giving good results after capture process.

Those pools that give low yield are not sequenced (for now), but the others, that are processed exactly the same way, are good while sequencing....

Thanks!