Contamination? What does this per sequence GC content mean?

[ZoeG]

Hello, I am quite new to RNAseq analysis.
When I did the quality control for my Illumina HiSeq data, I found an abnormal per sequence GC content, as the picture shows.

http://zgan.weebly.com/uploads/8/9/3/5/8935569/2863876_orig.png

It seems some contamination?
Need help, thanks

[dnusol]

This link might be useful

http://bioinfo-core.org/index.php/9th_Discussion-28_October_2010

[rhinoceros]

Looks to me like the reads come from two very different sources (the two peaks). Perhaps even three sources.. I don't know. How are we supposed to tell since you didn't specify what was sequenced. Since the read counts (of the peaks) are somewhat similar, perhaps your source has two chromosomes, which have different GC (not unheard of).

[ZoeG]

This link might be useful

http://bioinfo-core.org/index.php/9t...8_October_2010

Thanks, dnusol. I went through the webpage, what I can tell is that the library seems something wrong. The quality score actually seems okay. When I mapped the data to its reference, >90% reads were mapped successfully. Do this result conflict with the conclusion of wrong library? Confused..

[ZoeG]

Looks to me like the reads come from two very different sources (the two peaks). Perhaps even three sources.. I don't know. How are we supposed to tell since you didn't specify what was sequenced. Since the read counts (of the peaks) are somewhat similar, perhaps your source has two chromosomes, which have different GC (not unheard of).

Hi, rhinoceros, here are more details about the samples.
The total RNA was extracted from one piece of mouse muscle by Qiagen RNeasy fibrous tissue mini kit. The quality of RNA were estimated by Agilent bioanalyzer. A260/A280 for all samples were around 2.0. RIN for all RNAs were around 10. It seems the quality of RNAs is excellent. And I didn't seem a chance to have other chromosomes.
The Libraries were prepared by a RNAseq center with an illumina TruSeq kit, but I know little about that...
Here is the whole FastQC report, which may give you more information. http://zgan.weebly.com/uploads/8/9/3...tqc_report.zip

More clues for me? Thanks

[dnusol]

Maybe RNAseq is biased by nature?
Check these other threads

http://seqanswers.com/forums/showthread.php?t=27911

http://seqanswers.com/forums/showthread.php?t=17601

http://seqanswers.com/forums/showthread.php?t=9046

[ZoeG]

dnuso, thanks, very useful information.

Maybe RNAseq is biased by nature?
Check these other threads

http://seqanswers.com/forums/showthread.php?t=27911

http://seqanswers.com/forums/showthread.php?t=17601

http://seqanswers.com/forums/showthread.php?t=9046