<p>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.</p>

<p>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).</p>

<p>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:</p>

<p><a href="https://github.com/nanopore-wgs-consortium/NA12878/blob/master/RNA.md" rel="nofollow noreferrer">https://github.com/nanopore-wgs-consortium/NA12878/blob/master/RNA.md</a></p>

<p>Here's the consortium's graph from that summary document showing polyA length:</p>

<p><a href="https://i.sstatic.net/Sr1Fl.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/Sr1Fl.png" alt="direct RNA polyA lengths"></a></p>

CC BY-SA 3.0
2017-11-23T22:33:46.270Z
"> 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?
ShanZhengYang
CC BY-SA 3.0
2017-11-24T01:32:31.410Z
You've done direct-RNA sequencing on the MinION?
gringer
CC BY-SA 3.0
2017-11-24T02:27:46.040Z
No, I think I'm misunderstanding the reference to "public datasets"
ShanZhengYang
CC BY-SA 3.0
2017-11-24T06:26:54.467Z
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.
gringer
CC BY-SA 3.0
2017-11-23T22:12:06.313Z
2017-11-30T23:05:37.057Z
2017-11-30T23:05:37.057Z
gringer
<p>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).</p>

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

<p>Amongst others, Narry Kim’s lab developed a dedicated method — <a href="https://www.ncbi.nlm.nih.gov/pubmed/24582499" rel="nofollow noreferrer">TAIL-seq</a> — 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.</p>

<p>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 <em>before basecalling</em>. 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.</p>

CC BY-SA 3.0
2017-12-01T17:30:06.297Z
(This is a copy of [my answer from Reddit](https://www.reddit.com/r/bioinformatics/comments/7f2k8r/estimate_the_length_of_polya_tails_from/dq9zbrw/); I only now noticed this question here.)
Konrad Rudolph
CC BY-SA 3.0
2017-12-01T17:38:43.097Z
Thanks. I'll probably remove the Reddit question soon, so the link will be broken.
ShanZhengYang
CC BY-SA 3.0
2017-12-01T17:27:18.087Z
2017-12-01T17:27:18.087Z
Konrad Rudolph
<p>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.</p>

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

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

<p>Perhaps I would need to model this somehow? All suggestions appreciated</p>

CC BY-SA 3.0
2017-11-23T21:26:04.807Z
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?
llrs
CC BY-SA 3.0
2017-11-23T21:34:43.493Z
@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
ShanZhengYang
<rna-seq><software-recommendation><rna><algorithms>
Estimate the length of poly-A tails from randomly-primed RNAseq data
# Estimate the length of poly-A tails from randomly-primed RNAseq data

(Asked by: username_0 on 2017-11-23)

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

**Tags:** rna-seq, software-recommendation, rna, algorithms
> **Comments:**
> **username_1**: 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?  
> **username_0**: @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  

---

## 2 answers

### Answer by username_2 on 2017-11-23. Score: 3 | Accepted answer

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](https://i.sstatic.net/Sr1Fl.png)](https://i.sstatic.net/Sr1Fl.png)

> **Comments:**
> **username_0**: "> 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?  
> **username_2**: You've done direct-RNA sequencing on the MinION?  
> **username_0**: No, I think I'm misunderstanding the reference to "public datasets"  
> **username_2**: 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.  

---

### Answer by username_3 on 2017-12-01. Score: 4

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](https://www.ncbi.nlm.nih.gov/pubmed/24582499) — 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.

> **Comments:**
> **username_3**: (This is a copy of [my answer from Reddit](https://www.reddit.com/r/bioinformatics/comments/7f2k8r/estimate_the_length_of_polya_tails_from/dq9zbrw/); I only now noticed this question here.)  
> **username_0**: Thanks. I'll probably remove the Reddit question soon, so the link will be broken.  

---

