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Per base sequence content: "A" underrepresented

Dear all,

my RNA-seq reads seem to be slighly biased to show more 'T' than 'A' (see image). This is unexpected for me. Does anyone have a suggestion why that might be (and how to proceed)?

enter image description here

The data are paired-end, sequenced with Illumina HiSeq 2500. (The read direction is reverse.)

thanks for your help!

rna-seq fastqc
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updated by GenoMax GenoMax • written by Wöps • Wöps

Your image doesn't work but also something to think about is that the most RNA-seq technologies sequence the cDNA (reverse transcribed copy) of your mRNA molecules. Thus assuming the method is strand-specific the poly-A tails will often look like stretches of T's; potentially explaining your results.

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written by benformatics benformatics

Fixed the link to the image. Please use the image button and paste in the full link incl. the suffix (e.g. .png) into the field that pops up:

enter image description here

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written by ATpoint ATpoint

Thank you! Will do next time.

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written by Wöps • Wöps

Have you trimmed off poly-A sections?

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written by russhh russhh

I hadn't trimmed poly-A sections so far. I now quickly tried running afterQC (https://github.com/OpenGene/AfterQC) to remove any reads with polyX above length 20. It does not seem to change the situation though:

FastQC after removing polyX

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written by Wöps • Wöps

As genomax says, what you see is normal. Just proceed with your analysis.

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written by ATpoint ATpoint

1 answer

This is a fairly classic example of RNAseq libraries. See this blog post from authors of FastQC for more details.

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written by GenoMax GenoMax

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