SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
High Mol. Weight Peak from Library on Agilent Bioanalyzer MWN Illumina/Solexa 1 11-09-2013 07:46 AM
Ideas on Bioanalyzer trace for ChIP-seq library? orlatron Sample Prep / Library Generation 0 05-14-2012 10:02 PM
Ladder-like Bioanalyzer trace on TruSeq RNA libraries egunth Sample Prep / Library Generation 2 12-01-2011 11:53 AM
Truseq RNA library has 2 peak on bioanalyzer houda Sample Prep / Library Generation 7 07-06-2011 10:39 AM
Truseq library has dimer/trimer peak on bioanalyzer after amplfication sehrrot Sample Prep / Library Generation 4 06-15-2011 03:38 PM

Reply
 
Thread Tools
Old 06-09-2014, 05:06 PM   #1
Acacianpunk
Junior Member
 
Location: Providence, RI

Join Date: May 2014
Posts: 3
Default Library peak-let contamination at high end of TruSeq BioAnalyzer trace

Hi everyone,
We are constructing ddRad-seq libraries using a homebrew variant of the TruSeq system. The library prep was pooled and run on a BioAnalyzer before Kapa quantification and running. The resulting BioAnalyzer profile is below.

The majority of our library is centered at the selected size of 300bp with minor adapter dimer contamination in the top sample. Both samples show some variant sized products at very low concentrations ranging from 800-6000bp. Does anyone know what these could be?

Based on reading threads my assumptions are either beads or bubbled molecules but any suggestions would be appreciated before we sequence.

Thank you
Matt
Attached Images
File Type: png Rad-seq_lib_060914_peaks.png (30.2 KB, 61 views)
Acacianpunk is offline   Reply With Quote
Old 06-09-2014, 06:19 PM   #2
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,232
Default

They do not look like bubbles that you are reffering. They could be remaining non-target digested DNA themselves or their amplification products or both. If size-selection was done on Pippin instrument, my previous statement would not be true. Maybe the bioanalyser reagents and run (specially ladder) should be inspected more closely for further explanation in first step. The answer might be in the modified protocol that have been used to prep libraries.

Last edited by nucacidhunter; 06-09-2014 at 06:24 PM.
nucacidhunter is offline   Reply With Quote
Old 06-10-2014, 06:04 AM   #3
Acacianpunk
Junior Member
 
Location: Providence, RI

Join Date: May 2014
Posts: 3
Default

Great point. Size selection was gel based so any DNA coming through without adapters possibly could be amplified from at the last step.

This is becoming problematic in quantifying the amount of material to load. We might try a weighted average and see how the cluster generation goes.
Acacianpunk is offline   Reply With Quote
Old 06-10-2014, 06:13 PM   #4
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,232
Default

I would first rerun the sample on Bioanlyser to see if the trace is consistent or is due to anomaly in that particular run. If the trace is repeated the next step would be to see if those off target fragments are amplifiable or not by running a PCR with diluted library. If they are not amplified, their presence can be ignored and a qPCR quantification method should result in consistent cluster numbers. If you are using Qubit for quantification, those fragments would interfere with quantification and likely source of them should be explored. In a gel run in optimum condition and extraction you should not get those large fragments after PCR (assuming size selection is before PCR on digested-ligated DNA), although smaller fragments can be expected.

Quote:
Size selection was gel based so any DNA coming through without adapters possibly could be amplified from at the last step.
This is less likely as the fragments without adapters will lack priming site for amplification.
nucacidhunter 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 03:38 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO