SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
FastQC per base sequence content analyst Bioinformatics 14 02-15-2017 06:25 AM
Strange fastqc per base sequence content 3'end kirstyn Bioinformatics 16 01-05-2017 09:58 AM
FastQC,kmer content, per base sequence content: is this good enough mgg Bioinformatics 10 11-06-2013 10:45 PM
What might cause the "Sequence Duplication Levels" failures in FastQC report? elrohir610 Bioinformatics 6 05-07-2012 09:38 PM
FastQC - strange 'per base sequence content' graph gconcepcion Bioinformatics 11 10-31-2011 12:39 AM

Reply
 
Thread Tools
Old 10-22-2013, 07:59 PM   #1
leekb
Junior Member
 
Location: Hong Kong

Join Date: Jul 2013
Posts: 7
Default FastQC Report: A horn in Per sequence GC content ?!

Hi Everyone,

Nice to meet you guys here!
I'm doing ChIP-Seq using HiSeq 1500 on SR 50 cycles, multiplexed condition.
And I got a funny curve in my FastQC report on Per sequence GC content and Sequence Duplication Levels as well as overrepresented sequence.
Anyone please give a help
1) Why my Per sequence GC content curve got a horn?
2) How come such high sequence duplication level like 71.09% ? Due to overrepresented sequence?
3) As attached BioAnalyzer, there's totally absence of adapter, why still overrepresented sequence like Index 7?

Library prep condition: 12cycles PCR amplification

Cheers!
kb
Attached Images
File Type: gif Lane1_per sequence gc content.gif (131.7 KB, 43 views)
File Type: gif Lane1_sequence duplication levels.gif (74.4 KB, 14 views)
File Type: gif Lane1_overrepresented sequence.gif (36.7 KB, 16 views)
File Type: gif Lane1_BioA_Index007.gif (11.1 KB, 12 views)
leekb is offline   Reply With Quote
Old 10-22-2013, 09:36 PM   #2
leekb
Junior Member
 
Location: Hong Kong

Join Date: Jul 2013
Posts: 7
Default One more strange FastQC report

Quote:
Originally Posted by leekb View Post
Hi Everyone,

Nice to meet you guys here!
I'm doing ChIP-Seq using HiSeq 1500 on SR 50 cycles, multiplexed condition.
And I got a funny curve in my FastQC report on Per sequence GC content and Sequence Duplication Levels as well as overrepresented sequence.
Anyone please give a help
1) Why my Per sequence GC content curve got a horn?
2) How come such high sequence duplication level like 71.09% ? Due to overrepresented sequence?
3) As attached BioAnalyzer, there's totally absence of adapter, why still overrepresented sequence like Index 7?

Library prep condition: 12cycles PCR amplification

Cheers!
kb
Hi all,

More strange FastQC report.
This's time I got problems on
1)Per base sequence quality: why quality drop dramatically in base 23-25? Is there a bubble inside the lane?
2)per base N content : a small peak shows up in base 23, any relationship with quality drop in per base sequence result?
3)sequence duplication levels : a little bit difference from above one, but more serious in duplication.

In general, is't a fair good result or in very pool quality? Should I keep continue the analysis?

kb
Attached Images
File Type: gif Lane4_per base sequence quality.gif (104.6 KB, 25 views)
File Type: gif Lane4_per sequence gc content.gif (86.3 KB, 13 views)
File Type: gif Lane4_per base N content.gif (63.8 KB, 10 views)
File Type: gif Lane4_sequence duplication levels.gif (40.8 KB, 10 views)
leekb is offline   Reply With Quote
Old 10-23-2013, 03:26 AM   #3
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 6,976
Default

Quote:
Originally Posted by leekb View Post
Hi all,

More strange FastQC report.
This's time I got problems on
1)Per base sequence quality: why quality drop dramatically in base 23-25? Is there a bubble inside the lane?
2)per base N content : a small peak shows up in base 23, any relationship with quality drop in per base sequence result?
3)sequence duplication levels : a little bit difference from above one, but more serious in duplication.

In general, is't a fair good result or in very pool quality? Should I keep continue the analysis?

kb
It is possible that there was a bubble in the lane (or some other issue) around cycle 23. It does not appear to be a severe problem though since most of bases are still appear to be of good quality. Hopefully during alignment these reads would not align and will be discarded.

Since you are doing ChIP-seq you may be enriching a binding sequence that may have a GC predominance. You should continue with the analysis.

Just to be safe run your sequences through "trimmomatic" (http://www.usadellab.org/cms/?page=trimmomatic) to make sure that there are no primer dimer and other odd contaminants.

Last edited by GenoMax; 10-23-2013 at 03:28 AM.
GenoMax is offline   Reply With Quote
Old 10-23-2013, 09:07 PM   #4
leekb
Junior Member
 
Location: Hong Kong

Join Date: Jul 2013
Posts: 7
Default

Quote:
Originally Posted by GenoMax View Post
It is possible that there was a bubble in the lane (or some other issue) around cycle 23. It does not appear to be a severe problem though since most of bases are still appear to be of good quality. Hopefully during alignment these reads would not align and will be discarded.

Since you are doing ChIP-seq you may be enriching a binding sequence that may have a GC predominance. You should continue with the analysis.

Just to be safe run your sequences through "trimmomatic" (http://www.usadellab.org/cms/?page=trimmomatic) to make sure that there are no primer dimer and other odd contaminants.
Thanks GenoMax!

Yes you're right, though cycle 23-25 drop quality but still stays in green zone.

How about my duplication levels issue? It's crazy high and looks like not much unique reads left for me to analysis. Any way to improve next time?
Many thanks!

kb
leekb is offline   Reply With Quote
Reply

Tags
chip-seq, duplication, fastqc, illumina

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 11:28 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO