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
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:
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 So you’ll need to find a machine that runs an outdated firmware.
