Question for 3 prime bias in rna short read and long read (illumina, ont, pacbio)
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14 months ago
shinyjj ▴ 50

Hello, I know rna seq in general has 3 prime bias. When I say rna-seq, I am talking about illumina, oxford nanopore, and pacBio. Here are my questions.

  1. As I know, illumina begins sequencing from both 5 prime and 3 prime but why are there 3 prime bias?
  2. I heard ONT has 3 prime bias the most, then illumina, and then pacbio. Is this because pacbio is longer (2k bp) than ONT (660bp) and begins sequencing from 3 prime end?

Can someone explain these two questions?

rna-seq short-read long-read • 991 views
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illumina begins sequencing from both 5 prime and 3 prime but why are there 3 prime bias?

This is largely due to the way libraries are made and the biology of RNA. As you recall libraries are made from fragmented cDNA's. Additionally since RNA's start degrading from 5'-end as soon as you break a cell open there is a better chance of 3'-ends surviving longer. If poly-A enrichment is used for library prep then you will preferentially get 3'-ends being captured (the case even with 10x scRNA). So while any sequencing happens from 5'-end it is only going to start with the part of the original cDNA that is present in library fragment.

You have specific techniques (IsoSeq for Pacbio) that sequence full length cDNA's and then you have the ability of directly sequencing RNA with ONT. So these are specialized applications.

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