I have a bunch of sequence files fetched from the EBI ENA and I'm trying to find the end of the 5' UTR for specific genes.

The UTR itself doesn't seem to be annotated in most sequence files I have. My original idea was to simply use the CDS annotations, as the start of those should clearly identify the end of the 5' UTR. But I also noticed gene annotations in many of the sequences, most of them starting and ending at exactly the same locations as the corresponding CDS annotation. But there are also cases where there are small differences between the gene and the CDS annotation.

So I'm wondering how exactly the gene annotation is defined (if there is actually an agreed definition) and how the gene and CDS annotations are used in practice.

Which annotation would be the most reliable to identify the end of the 5' UTR?

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    $\begingroup$ Realistically speaking, there's no guarantee that UTRs are in a given gene definition unless you're looking at a well annotated species (e.g., mouse or human). There are many species for which there is simply very little information on UTR sequences. $\endgroup$ – Devon Ryan Feb 25 '15 at 9:42

CDS is based on ORF prediction and unless the protein product is known it is classified as an mRNA for a hypothetical protein (Refseq id: XM_*).

As Devon Ryan said sometimes UTR ends are not accurately annotated. If you are interested in finding the 3' end of 5'UTR then the problem is basically to find the translation start site. You can have a look at ribosome profiling experiments or try doing eIF2 CLIP-RNAseq (I don't think it has been done); peptide sequences may not be of help because N-terminal region in many cases, gets clipped.

If you are interested in finding the 5' end of 5'UTR then the problem is relatively simple. You just need to do a 5'-RACE followed by Sanger sequencing (this would work in most cases or you can do a deep-RNAseq).

There are official GTF files that provide the most reliable annotations. For human I would recommend GENCODE.

In the annotations the term Gene denotes the entire DNA region that is responsible for all of its RNA products (splice variants, alternate start/termination etc). Other features include Transcript, Exon, CDS, UTR and in some cases translation start site is separately annotated. CDS comprises of just the protein coding region.

You should read more about the GTF/GFF annotation format.


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