Fastqc report for small RNA sequencing

Question

[Aiswarya]

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??

Answer 1

[Istvan Albert]

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)

[Aiswarya]

This is what it looks like:

[Istvan Albert]

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.

[Aiswarya]

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 bank) looks all squiggly.

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

[Istvan Albert]

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

[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

[Aiswarya]

uh!! A reminder to remember the basics.

Thank you :)