Re: [Transdecoder-users] ORFs without ATG as start

[Brian H. <bh...@br...>]

Hi Andre,

Transdecoder doesn't have a start-codon finding function.  It finds the
longest ORFs, and in the case where there isn't an intervening in-frame
stop-codon between your optimal start codon and the 5' end of the sequence,
it's going to simply assume that the transcript is not full-length and give
you the full translation from the beginning of the transcript.

Usually, there's an in-frame stop codon in the 5' UTR that prevents most
full-length transcripts from being reported as 5' partials.  I suppose, if
you have a GC-rich target (rarer random stop codons), this could definitely
be more of an issue.

The high prevalence of 5' partials tends to be a general indicator of not
having full-length transcripts, resulting from the 3' coverage bias typical
of poly-A captured cDNA sequencing.  I'm sure there are occassions where a
genuine full-length transcript has an in-frame translatable 5' UTR, but in
my experience, these have generally been the rare exceptions.

cheers,

~brian


On Wed, May 14, 2014 at 5:57 PM, Andre Minoche <And...@cr...> wrote:

> Dear Matt,
>
> Thank you for your quick reply.
>
> The quality of the reference sequence from which I derived the cds is not
> an issue.
>
> I have full length transcript cDNA sequences I aligned to a high quality
> genome assembly.  From the alignment coordinates and using the genome
> assembly as source, I derived high quality mRNA sequences I used as input
> for transdecoder.
>
> Deep RNA-seq data confirms the full length nature of my cDNA sequences. At
> the locations where my full length transcripts align I also have Augustus
> gene models supported by RNA-seq data. The ORFs of the predicted gene
> models generally correspond to the ORFs of transdecoder, with the
> exception, that in many cases transdecoder ORFs start upstream of the
> Augustus start codon and not with an ATG (5prime_partial, ORFs).
>
> Best regards
> André
>
> On 14 May 2014, at 21:44, Matthew MacManes <mat...@un...>
> wrote:
>
> Andre,
>
> Alternatively, this could be a metric of assembly quality (e.g.
> http://journal.frontiersin.org/Journal/10.3389/fgene.2014.00013/abstract)
> which may be a function of the input data. Can you tell us a little bit
> about the type and quantity of data used in this assembly?
>
> Matt
>
> __________________________________
> *Matthew MacManes*, Ph.D.
> University of New Hampshire  I  Assistant Professor
> Department of Molecular, Cellular, & Biomedical Sciences
> Durham, NH  03824
> Phone: 603-862-4052  I  Twitter: @PeroMHC <https://twitter.com/PeroMHC>
> Web: genomebio.org
> Office: 189 Rudman Hall I Lab: 145 Rudman Hall
>
>
> On Wed, May 14, 2014 at 2:44 PM, Andre Minoche <And...@cr...>wrote:
>
>>
>> Hi,
>>
>> First of all thanks for providing a tool to predict ORFs.
>>
>> I am surprised to see, that so many of my ORFs predicted with
>> transdecoder do not start with ATG (5prime_partial, 650 out of 1807).
>>
>> To me it seems like a malfunction of the software.
>>
>> Is there an easy way to trim the ORFs to the nearest start codon?
>>
>> Thanks
>> André
>>
>> --
>> André Minoche, Postdoc
>>
>> Centre for Genomic Regulation (CRG)
>> Doctor Aiguader, 88, 4th floor
>> 08003 Barcelona
>>
>> http://seq.crg.es
>>
>>
>>
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--
Brian J. Haas
The Broad Institute
http://broad.mit.edu/~bhaas