SEQanswers

Go Back   SEQanswers > Bioinformatics > Bioinformatics



Similar Threads
Thread Thread Starter Forum Replies Last Post
Scripting Help - Common Elements Giorgio C Bioinformatics 10 10-05-2013 10:53 AM
Common elements within two set of miRNAs Giorgio C Bioinformatics 1 01-05-2012 09:35 AM
How to extract Common Genes from 2 spreadsheets byou678 Bioinformatics 4 10-04-2011 07:42 PM
Fastq sorting and common part? stoker Bioinformatics 0 07-07-2011 12:53 AM
very common mutation not in SNP databases fpepin Bioinformatics 17 02-11-2011 05:14 PM

Reply
 
Thread Tools
Old 11-25-2012, 10:23 AM   #1
knostrov
Junior Member
 
Location: Boston

Join Date: Oct 2012
Posts: 7
Default How much adapter contamination is common?

Hi everybody,

I am looking at my Fastq QC stats with much surprise: 25% of my reads contain Trueseq adapter sequences. That seems like a lot. I am completely new to NGS data analysis, so I have no point of comparison... is this very unusual? What type of analysis should I do to find out more about the roots of this problem?

This is a 50bp PE run on a HiSeq 2000, target enrichment using Haloplex.

Any comments would be appreciated.

Thanks!
knostrov is offline   Reply With Quote
Old 11-26-2012, 07:47 AM   #2
kopi-o
Senior Member
 
Location: Stockholm, Sweden

Join Date: Feb 2008
Posts: 319
Default

It sounds like a lot. Is it a special protocol, like small RNA sequencing? With very short insert sizes, you expect to see a lot of adapters in the reads. Maybe you could start by obtaining fragment size statistics from the lab.
kopi-o is offline   Reply With Quote
Old 11-26-2012, 08:27 AM   #3
Wallysb01
Senior Member
 
Location: San Francisco, CA

Join Date: Feb 2011
Posts: 286
Default

That is a lot. In my experience it is <1%.
Wallysb01 is offline   Reply With Quote
Old 11-26-2012, 03:17 PM   #4
jimmybee
Senior Member
 
Location: Adelaide, Australia

Join Date: Sep 2010
Posts: 119
Default

Its very protocol specific. Some can have massive amounts of primer left after sequencing, so maybe fill us in on the sequencing experiment and what you did before sequencing?
jimmybee is offline   Reply With Quote
Old 11-27-2012, 04:14 AM   #5
knostrov
Junior Member
 
Location: Boston

Join Date: Oct 2012
Posts: 7
Default

Thank you for the quick replies. Actually I have by now realized that almost all of these contaminating sequences are reads that consist of nothing but adapter sequence, e.g. 50bp of read2-primersite--tag--adapter. I imagine those must be two linked adapters with no insert in between. One would think such constructs would be eliminated by size selection? Also their abundance varies per sample. They make up between 2-25% of all reads, depending on the sample.

So the specifics of this sequencing experiment are (I did not do the experimental part myself...)
- FFPE tissue samples, good DNA quality according to size distribution on gel
- target enrichment using the Haloplex protocol (custom probes that hybridize to the two ends of a desired region to make a circle --> ligation --> circle amplification)
- 50 bp PE run on HiSeq 2000
knostrov is offline   Reply With Quote
Old 03-06-2013, 06:57 PM   #6
MWN
Junior Member
 
Location: CA

Join Date: Aug 2011
Posts: 8
Default

Quote:
Originally Posted by knostrov View Post
Thank you for the quick replies. Actually I have by now realized that almost all of these contaminating sequences are reads that consist of nothing but adapter sequence, e.g. 50bp of read2-primersite--tag--adapter. I imagine those must be two linked adapters with no insert in between. One would think such constructs would be eliminated by size selection? Also their abundance varies per sample. They make up between 2-25% of all reads, depending on the sample.

So the specifics of this sequencing experiment are (I did not do the experimental part myself...)
- FFPE tissue samples, good DNA quality according to size distribution on gel
- target enrichment using the Haloplex protocol (custom probes that hybridize to the two ends of a desired region to make a circle --> ligation --> circle amplification)
- 50 bp PE run on HiSeq 2000
Any update?
How much FFPE DNA do you use? Appreciate.
MWN is offline   Reply With Quote
Old 03-07-2013, 10:49 AM   #7
swNGS
Member
 
Location: SW UK

Join Date: Nov 2011
Posts: 83
Default

For Haloplex, you really need to do adapter trimming before alignment, then clip 5 bases of the ends of each read, otherwise it suffers from biasing of the wild type allele if you get a variant at a restriction siteor probe binding site.
Yes, you should be able to reduce them by size selection, but as a previous person said, have a look at the range of insert sizes (after establishing that trimming was done correctly, otherwise you'll lose a lot of potentially useful sequence). We are using Halo with 150 bp reads and I suspect we've got a bigger adapter read-through problem than you!
swNGS 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 12:55 PM.


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