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Fastqc report for small RNA sequencing

Hai,

I am running fastQC on small RNAseq datasets, the per-base sequence content is peculiar (they are not parallel but all squiggly). I thought it might be because of my dataset and ran fastqc on another small RNAseq data but it looks the same. Any idea why this is happening??

rna fastqc smallrnaseq small sequencing
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written by Aiswarya • Aiswarya

1 answer

Should it be uniform? Probably not.

It is not randomly sheared DNA after all.

Even for DNA the start of the sequence is usually nonuniform because of non-random priming.

(PS. post an image with what you see instead of describing it as squiggly)

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written by Istvan Albert Istvan Albert

This is what it looks like:

enter image description here

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written by Aiswarya • Aiswarya

take known small RNA sequences, turn them into a fastq file

seqtk seq -F I smallrna.fa > smallrna.fq

now run fastqc on that file. I bet the plot will be squiggly as well

The small rna has a function, and the fragments start in the same position. the bases are not supposed to be random by position.

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written by Istvan Albert Istvan Albert

That makes perfect sense. And as you said the fastq for known small RNA seq (attached is fastqc for a list of piRNAs from piRNA enter image description herebank) looks all squiggly.

(Offtopic - I wonder why there are no 'G's)

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written by Aiswarya • Aiswarya

probably T is missing because you have RNA with uracil (U) instead

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written by Istvan Albert Istvan Albert

fear not you can fix it with another tool called seqkit, not to be confused with seqtk :-)

seqtk seq -F I rna.fa | seqkit seq --rna2dna > dna.fq
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written by Istvan Albert Istvan Albert

uh!! A reminder to remember the basics.

Thank you :)

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written by Aiswarya • Aiswarya

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