SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
Questions about Nextera kit/tagmentation kobeho24 Sample Prep / Library Generation 4 10-27-2016 07:01 PM
Failed tagmentation reaction for Nextera library? sweetph3 Sample Prep / Library Generation 14 07-21-2016 07:18 AM
Nextera XT tagmentation problem nao Sample Prep / Library Generation 3 03-21-2016 02:11 PM
Possibility of tagmentation without fragmentation with Nextera XT kit Ahmed_BH Sample Prep / Library Generation 3 07-11-2014 06:11 AM
step- limited pcr after tagmentation with Nextera XT... how many cycles? Synthetica RNA Sequencing 0 06-10-2014 08:37 AM

Reply
 
Thread Tools
Old 09-20-2018, 10:31 PM   #1
kblake
Junior Member
 
Location: California

Join Date: Mar 2016
Posts: 2
Default Nextera DNA Exome Tagmentation Peaks

I'm new to the library prep side of sequencing and I've been having trouble getting my post-tagmentation, post-amplification libraries to match the profile in the Nextera DNA Exome protocol.

I've repeated the process a few times with increasing care (using low-bind tubes, checking input concentrations with Qubit, testing beads on a DNA ladder, etc.) and I'm finding that I have a flat, right skewed bioanalyzer peak (the peak should be between 200-500bps and mine hangs over 1000bps - see attached trace). Illumina's FAQ claims that a secondary, larger peak is common and should be of no concern since it's often an amplification product but I'm not convinced that's what I'm seeing here.

Has anyone else had a pre-enrichment library prep with a similar profile? Is this cause for concern (over- or under-tagmentation)? If so, do you have any tips on how the protocol can be adjusted to account for it?
Attached Images
File Type: png Screen Shot 2018-09-20 at 11.12.02 PM.png (62.3 KB, 10 views)
kblake is offline   Reply With Quote
Old 09-21-2018, 01:13 AM   #2
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,179
Default

The library profile indicates input DNA overload and it can affect on target capture (enrichment efficiency).

Exon length on average are around 200bp and a 1Kb fragment might have hybridized to target in the middle so sequencing 100 cycles from ends will result in off target reads.
nucacidhunter is offline   Reply With Quote
Old 09-21-2018, 10:17 AM   #3
kblake
Junior Member
 
Location: California

Join Date: Mar 2016
Posts: 2
Default

Is it common to have to use less input DNA than the kit recommends? I'm worried about reducing my library complexity and I don't know how much the input concentration influences that.
kblake is offline   Reply With Quote
Old 09-21-2018, 10:21 PM   #4
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,179
Default

You should not use less DNA. Your thermocycler or kit also could be faulty (expired, exposed to high temperatures). You can test lower DNA input and see if you get shorter fragments.
nucacidhunter is offline   Reply With Quote
Reply

Tags
exome sequencing, sample preparation, tagmentation

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 05:51 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO