SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
adaptors and primers Nextera kit: modified or not? Simone78 Sample Prep / Library Generation 7 07-29-2016 12:03 AM
TruSeq or Nextera kit for library prep? rooty Sample Prep / Library Generation 12 10-29-2013 07:02 AM
Nextera Exome Enrichment kit dummyseq Illumina/Solexa 8 10-03-2013 06:46 AM
Can Truseq RNA kit compatiblity with Truseq exome-enrichment kit? gemzhanglinlin Sample Prep / Library Generation 0 07-22-2013 02:16 PM
cDNA prep for Illumina using the Nextera kit fgoetz Sample Prep / Library Generation 3 07-09-2012 07:34 PM

Reply
 
Thread Tools
Old 02-19-2014, 09:57 AM   #1
cnicolet
Member
 
Location: Los Angeles

Join Date: Dec 2008
Posts: 35
Default Nextera exome kit

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
cnicolet is offline   Reply With Quote
Old 02-19-2014, 04:04 PM   #2
Bukowski
Senior Member
 
Location: UK

Join Date: Jan 2010
Posts: 324
Default

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?
Bukowski is offline   Reply With Quote
Old 02-20-2014, 11:57 AM   #3
cnicolet
Member
 
Location: Los Angeles

Join Date: Dec 2008
Posts: 35
Default

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.
cnicolet is offline   Reply With Quote
Old 07-21-2014, 08:03 PM   #4
Genohub
Registered Vendor
 
Location: genohub.com

Join Date: Mar 2013
Posts: 208
Default

We've outlined each currently available capture technology in a comparison table here:
https://genohub.com/exome-sequencing...y-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
Genohub is offline   Reply With Quote
Old 01-10-2017, 03:12 AM   #5
evakoe
Member
 
Location: Italia

Join Date: Jul 2012
Posts: 22
Default

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.
evakoe is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 05:39 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO