<ol>
<li>Genes, particularly transcribed genes in Eukaryotes, can have alternate start and stop sites (and splicing).  Alternate transcriptional start sites may be found in different tissues or at different developmental periods due to alternate transcription factor binding sites being used.  The only reliable way to determine the transcriptional start and stop sites is by read coverage.</li>
<li>Genome annotations are generally the product of bioinformatic pipelines and are mostly predictions based upon computational models.  Different gene-finders may call different boundaries; for example, in eukaryotic genomes, an exon may be assigned to the wrong up/down-stream gene.  Arabadopsis, as model organism, has had it's gene boundaries considerably refined/corrected/validated, but even so, there could still exist errors in the annotation or, more likely, the boundaries indicated are the most common start/stop sites (see point 1 above).</li>
</ol>

<p>In short, your UTRs are whatever your experimental data (reads) show it to be.  Don't worry too much about it not matching up to the standard annotation boundaries.</p>

CC BY-SA 3.0
2018-03-01T05:26:09.037Z
2018-03-01T05:26:09.037Z
Edward Kirton
<p>I am studying the transcriptome of Arabidopsis. Interestingly, the 5' UTR of the latest annotation is usually too long. 
Here is an example (this gene is right to left).
<a href="https://i.sstatic.net/Ig3t6.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/Ig3t6.png" alt="enter image description here"></a>
You can see both EST (orange bars) and the RNA-seq reads cover a much smaller region of the annotated 5' UTR. Unfortunately, many 5'UTR of the genes have the same problem. Is there a way to fix that? I was wondering if any assembly tools can help me to get the correct 5'UTR ranges. Thanks!!!</p>

CC BY-SA 3.0
2018-02-26T03:18:49.903Z
What evidence do you have that the gene stops earlier? This observation could just be due to selective amplification of the 3' end in the RNASeq reads.
gringer
CC BY-SA 3.0
2018-02-26T03:23:30.793Z
I think it starts late (not stop early) as I mentioned 5'UTR, not 3'UTR.
l0110
CC BY-SA 3.0
2018-02-26T07:38:10.597Z
Do you really think standard RNAseq is perfect enough to judge the start of a transcript with single-base resolution? There's a reason that people have developed methods explicitly for that.
Devon Ryan
CC BY-SA 3.0
2018-02-26T14:02:28.810Z
It is not only my data. It is also from many people's data. There is no read at 5'UTR in the old EST data, and in >100 set of RNA seq data. There is just nothing there. I do not understand why those upstream sequences are annotated as 5'UTR, and it could mislead many sequence analysis.
l0110
CC BY-SA 3.0
2018-02-26T14:18:53.373Z
@Devon Ryan, Could you please enlighten me the reasoning (why did they give a long 5'UTR with no evidence?). I am asking this because there are lots of genes behaving like this. I am afraid it will cause a problem in motif finding. Thanks! 
I updated the image to include the EST data.
l0110
CC BY-SA 3.0
2018-02-26T14:28:50.117Z
To be frank, you need to ask the people who made the annotation.
Devon Ryan
CC BY-SA 3.0
2018-02-26T14:32:21.977Z
So is there any tool that can help me to reannotate the 5'UTR based on RNAseq data? I am asking this because, as I checked, most cases RNAseq data are quite consistent with the EST data. Thanks!
l0110
CC BY-SA 3.0
2018-02-26T15:18:41.503Z
@l0110 Could you [edit] your comments in the question. I think I would get a better idea of what do you want.
llrs
CC BY-SA 3.0
2018-02-26T17:26:53.477Z
@Llopis, I cannot edit my comments after 5 minutes. Is there any part unclear? Please let me know. Thanks!
l0110
CC BY-SA 3.0
2018-02-26T23:47:33.737Z
In the case of light-grown seedling it looks like there actually are reads (very few) that are actually the entire length of the 5' UTR. I think what you're after is "what is the absolute 5' transcription start site of my genes?" Right? If that is the case then you're better off doing a few SMRT IsoSEQ or ONT cDNA sequencing flowcells to get the starts and stops. Keep in mind that when you select for polyA you are going to get RNAs that have undergone more processing and may have lost some 5' UTR.
conchoecia
<rna-seq><sequence-annotation>
How to fix 5'UTR annotation with RNA-seq data?
# How to fix 5'UTR annotation with RNA-seq data?

(Asked by: username_0 on 2018-02-26)

I am studying the transcriptome of Arabidopsis. Interestingly, the 5' UTR of the latest annotation is usually too long.
Here is an example (this gene is right to left).
[![enter image description here](https://i.sstatic.net/Ig3t6.png)](https://i.sstatic.net/Ig3t6.png)
You can see both EST (orange bars) and the RNA-seq reads cover a much smaller region of the annotated 5' UTR. Unfortunately, many 5'UTR of the genes have the same problem. Is there a way to fix that? I was wondering if any assembly tools can help me to get the correct 5'UTR ranges. Thanks!!!

**Tags:** rna-seq, sequence-annotation
> **Comments:**
> **username_1**: What evidence do you have that the gene stops earlier? This observation could just be due to selective amplification of the 3' end in the RNASeq reads.  
> **username_0**: I think it starts late (not stop early) as I mentioned 5'UTR, not 3'UTR.  
> **username_2**: Do you really think standard RNAseq is perfect enough to judge the start of a transcript with single-base resolution? There's a reason that people have developed methods explicitly for that.  
> **username_0**: It is not only my data. It is also from many people's data. There is no read at 5'UTR in the old EST data, and in >100 set of RNA seq data. There is just nothing there. I do not understand why those upstream sequences are annotated as 5'UTR, and it could mislead many sequence analysis.  
> **username_0**: @Devon Ryan, Could you please enlighten me the reasoning (why did they give a long 5'UTR with no evidence?). I am asking this because there are lots of genes behaving like this. I am afraid it will cause a problem in motif finding. Thanks!  I updated the image to include the EST data.  
> **username_2**: To be frank, you need to ask the people who made the annotation.  
> **username_0**: So is there any tool that can help me to reannotate the 5'UTR based on RNAseq data? I am asking this because, as I checked, most cases RNAseq data are quite consistent with the EST data. Thanks!  
> **username_3**: @l0110 Could you [edit] your comments in the question. I think I would get a better idea of what do you want.  
> **username_0**: @Llopis, I cannot edit my comments after 5 minutes. Is there any part unclear? Please let me know. Thanks!  
> **username_4**: In the case of light-grown seedling it looks like there actually are reads (very few) that are actually the entire length of the 5' UTR. I think what you're after is "what is the absolute 5' transcription start site of my genes?" Right? If that is the case then you're better off doing a few SMRT IsoSEQ or ONT cDNA sequencing flowcells to get the starts and stops. Keep in mind that when you select for polyA you are going to get RNAs that have undergone more processing and may have lost some 5' UTR.  

---

## 1 answers

### Answer by username_5 on 2018-03-01. Score: 1

1. Genes, particularly transcribed genes in Eukaryotes, can have alternate start and stop sites (and splicing). Alternate transcriptional start sites may be found in different tissues or at different developmental periods due to alternate transcription factor binding sites being used. The only reliable way to determine the transcriptional start and stop sites is by read coverage.
2. Genome annotations are generally the product of bioinformatic pipelines and are mostly predictions based upon computational models. Different gene-finders may call different boundaries; for example, in eukaryotic genomes, an exon may be assigned to the wrong up/down-stream gene. Arabadopsis, as model organism, has had it's gene boundaries considerably refined/corrected/validated, but even so, there could still exist errors in the annotation or, more likely, the boundaries indicated are the most common start/stop sites (see point 1 above).

In short, your UTRs are whatever your experimental data (reads) show it to be. Don't worry too much about it not matching up to the standard annotation boundaries.


---

