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So a poly-A tail is a long chain of adenine nucleotides that is added to a messenger RNA (mRNA) molecule during RNA processing to increase the stability of the molecule.

For my project, I would like to estimate the length of poly-A tails from randomly-primed RNAseq data.

Any ideas how this could be done? Are there algorithms/tools which currently do this?

Perhaps I would need to model this somehow? All suggestions appreciated

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  • $\begingroup$ Why do you have randomly primed RNAseq data? Which organisms are you working with (or it is a mix or a simulated data) ? What have you looked up for your project ? Is there any database of poly-A tail lengths maybe? $\endgroup$ – llrs Nov 23 '17 at 21:26
  • $\begingroup$ @Llopis HeLa cells. But I would like to do this for a few other organisms as well. "Is there any database of poly-A tail lengths maybe?" It would have to be a data-driven measurement $\endgroup$ – ShanZhengYang Nov 23 '17 at 21:34
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Just look for polyA tracts at the end of sequences, and count them if they're larger than ~18 bases. I've done this with MinION cDNA reads by mapping the polyA adapter sequence (with an elongated polyA sequence) to the reads using LAST, and working out the length of aligned sequence.

Unfortunately, because the cDNA adapter finishes with a TVN sequence, rather than TTT, binding of the adapter is not necessarily a good representation of the true polyA length. I expect that Illumina adapters will have similar problems, if not worse (due to the read length limits).

It might be possible to use direct-RNA sequencing to better categorise the polyA lengths, but I've yet to look at the public direct-RNA datasets. Just in case you're interested in looking yourself, here's a direct RNA dataset for NA12878 from the wonderful nanopore-WGS consortium:

https://github.com/nanopore-wgs-consortium/NA12878/blob/master/RNA.md

Here's the consortium's graph from that summary document showing polyA length:

direct RNA polyA lengths

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  • $\begingroup$ "> It might be possible to use direct-RNA sequencing to better categorise the polyA lengths, but I'm not familiar with any available public datasets for that." There's no way to infer this from my own dataset? $\endgroup$ – ShanZhengYang Nov 23 '17 at 22:33
  • $\begingroup$ You've done direct-RNA sequencing on the MinION? $\endgroup$ – gringer Nov 24 '17 at 1:32
  • $\begingroup$ No, I think I'm misunderstanding the reference to "public datasets" $\endgroup$ – ShanZhengYang Nov 24 '17 at 2:27
  • $\begingroup$ Direct RNA sequencing is a very new technology that can only be done on a MinION. There's not much data available for it yet. $\endgroup$ – gringer Nov 24 '17 at 6:26
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Can’t be done. If you already sequenced then I’m afraid the money is wasted (unless of course the data is good for something else).

The standard Illumina basecaller doesn’t deal well with homopolymers and will consequently call incorrect poly(A) lengths.

Amongst others, Narry Kim’s lab developed a dedicated method — TAIL-seq — to solve this problem. It’s a protocol for library prep using pulldown of biotin tagged tails (rather than random priming), plus dedicated basecalling software that uses spike-ins for calibration.

Be warned though: the protocol is reportedly technically challenging, and in order to use it you need an Illumina machine that gives you access to the raw image files before basecalling. Up-to-date Illumina machines won’t give you these files, full stop. So you’ll need to find a machine that runs an outdated firmware.

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  • $\begingroup$ (This is a copy of my answer from Reddit; I only now noticed this question here.) $\endgroup$ – Konrad Rudolph Dec 1 '17 at 17:30
  • $\begingroup$ Thanks. I'll probably remove the Reddit question soon, so the link will be broken. $\endgroup$ – ShanZhengYang Dec 1 '17 at 17:38

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