how to remove warning and failures observed inQuality report by Fastqc for RNA-Seq data

Question

[aiswaryabioinfo]

Hi All,

I ran FastQC software on RNA-Seq data of Plant sample generated from illumina HiSeq platform. It all appears good (green) except :

In case of Forward Reads

warning (Orange) : Per tile sequence quality, Per base GC content and Sequence duplicaiton level.

Fail (Red) : Per base sequence content.

In case of Reverse Reads

warning (Orange) : Per base GC content and Sequence duplicaiton level.

Fail (Red) : Per tile sequence quality, Per base sequence content.

1. How can I rectify this warnings and failures?
2. The Adapter content is showing green tick mark, So is it necessary to use trimmomatic again to remove the above said warnings and failures in the forward and reverse strands?
3. Will the above said warnings and failures have any effect on further downstream analysis?

Thank you.

[Ram]

I'd recommend you read up on FASTQC and understand each metric it measures. These metrics are useful to anticipate oddities you might see downstream and stop downstream processing in case something important to you scores low, there's no use trying to get a "perfect" FASTQC report IMO.

[lieven.sterck]

I completely agree with RamRS here, moreover I fear that you might harm you data much more by trying to rectify these things rather then improving it.

I've seen a substantial amount of FastQC reports and I don't seem to remember ever seen a complete green one ;)

[GenoMax]

Please read following two blog posts from authors of FastQC.

Positional bias
Sequence duplication