using randomized adaptors as UMI in small RNA seq

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4.0 years ago

Paulina • 0

Hi there,

I apologize if this subject was already discussed.

I prepared my small RNA library using NEXTflex Small RNA-seq kit, which has 4 randomized nucleotides at the ends of both adaptors (to reduce ligation bias). After looking at my alignments, I am pretty sure that some of my IP samples contain plenty of PCR duplicates. I am trying to figure out a way to use the randomized adaptors ends as UMIs. Is it OK to do it this way? Could I get a recommendation for a tool to do it?

Thanks! Paulina

RNA-Seq • 808 views

ADD COMMENT • link 4.0 years ago by Paulina • 0

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4 randomized nucleotides at the ends of both adaptors

That is not a lot of UMI's correct? Their utility may thus be limited.

Check out clumpify.sh. It should give you an idea of the duplicates (PCR and optical). While you can't be 100% sure they are PCR dups at least you would get an idea of the prevalence. See: A: Introducing Clumpify: Create 30% Smaller, Faster Gzipped Fastq Files

ADD REPLY • link 4.0 years ago by GenoMax 142k

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Thank you, I'll check it out! While 4 nts is not long, both adaptors are randomized, so I was hoping to combine the information so together it would be 8 randomized nts. Do you think it's still not enough? Thanks! Paulina

ADD REPLY • link 4.0 years ago by Paulina • 0

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It may be but you will need to check with your data to see how many unique combinations you are able to come up with and how many reads get assigned to each combination. This may need custom code since you are not following a standard protocol here.