Nextera exome kit

[cnicolet]

Hi:
Recently we tested the nextera exome enrichment libe kit. In our analysis pipeline we have a metric that calculates exonic reads, intronic reads, and intergenic reads. Our nextera libes all had about 50% match to the exons. That seemed low, I went back and looked at the same metric from the original illumina kits, those also had only about 50% exonic reads. Does this seem low? It throws a real wrench into coverage calculations

[Bukowski]

What is the on-target specification of Nextera? I don't use Nextera, I use SureSelect, which will generally give ~65% 'on target' reads on variable quality samples, but the Agilent literature claims 70%. For custom capture regions on SureSelect, 50% on target is all you will get, so it depends. Also are you feeding a list of all exons to your coverage metric calculator? Or just the exons in the capture kit?

[cnicolet]

Thanks for your comments. That is an excellent point, I'm sure we are feeding all known exons so that could be a big part of it. I'll know a lot more when we send the sureselect material info through the same pipeline, but it does sound as though those numbers are to be expected. Just looking around on the browser it is clear that are abundant areas of off target seqeuncing.

[Genohub]

We've outlined each currently available capture technology in a comparison table here:

https://genohub.com/exome-sequencing-library-preparation/

In our guide for determining the number of sequencing reads one needs for a targeted capture expt, we estimate on-target rates to be between 65-75%. I'd be interested in hearing other people's experiences and their rates.

- Genohub

[evakoe]

In my lab we are using the Nextera Rapid Capture Exome kit and I also get only slightly above 50% of all reads mapping on the target region. Unfortunately, this number has been low and stable over multiple runs. In my experience, the on-target or coverage estimations given by the vendors are always to high and cannot be reproduced.