[maker-devel] maker annotation with cufflinks output
Carson Holt
carsonhh at gmail.com
Wed Feb 5 13:38:44 MST 2014
Protein data doesn’t have to be from that closely a related species. This
is because genes maintain homology at the amino acid level across even very
large evolutionary distances. Having a closer related species just ensures
that genome contents are similar (fewer losses/gains relative to each
other). And use the entire proteome of at least one related species (just
using a database like swiss-prot is not sufficient).
Using translated mRNA-seq data will not give you any new information that
was not already available from the untranslated sequence. Plus it will
introduce the complicating artifacts that mRNA-seq generates into the
protein part of the pipeline (gene merging, incorrect assembly, and false
calls caused by background transcription). A big gotcha with mRNA-seq is
that all of your genome gets transcribed at a low level, not just the genes,
so you will always have contamination that does not represent real gene
models. Also in the end you really only expect to capture about 50% of the
genes with mRNA-seq (maybe 70% if you are fortunate - and most of those will
be partial). So using the proteins from another species, is important to
improve sensitivity, and fix many of the issues that arise from the noisy
nature of mRNA-seq. In fact if you were forced to use only one (either
protein evidence or mRNA-seq) you will actually get better annotations from
the protein evidence in most cases. You get better annotations when you use
both, but if using only one of them, the proteins from another species are
better, and noisy mRNA-seq will be the primary source of annotation error.
Thanks,
Carson
From: dhivya arasappan <darasappan at gmail.com>
Date: Wednesday, February 5, 2014 at 1:13 PM
To: Daniel Ence <dence at genetics.utah.edu>
Cc: Carson Holt <carsonhh at gmail.com>, "maker-devel at yandell-lab.org"
<maker-devel at yandell-lab.org>
Subject: Re: [maker-devel] maker annotation with cufflinks output
Hello Daniel and Carson,
Thanks for your replies.
Yes I used the the protein sequences resulting from annotation of trinity
assembly (using trinotate). I'll try using protein sequences from related
species (though there arent sequences from closely related orgs). Could you
tell me a little about why protein data from annotating my rnaseq data would
not work best here?
Thanks
Dhivya
On Feb 5, 2014, at 1:28 PM, Daniel Ence wrote:
> Hi Dhivya, Are the protein matches in your results coming from your
> annotations of the transcriptome? You should really use amino-acid sequences
> from related organisms and some kind of omnibus source like SwissProt.
>
> Thanks,
> Daniel
>
> Daniel Ence
> Graduate Student
> Eccles Institute of Human Genetics
> University of Utah
> 15 North 2030 East, Room 2100
> Salt Lake City, UT 84112-5330
>
> From: Carson Holt [carsonhh at gmail.com]
> Sent: Wednesday, February 05, 2014 11:38 AM
> To: dhivya arasappan; Daniel Ence
> Cc: maker-devel at yandell-lab.org
> Subject: Re: [maker-devel] maker annotation with cufflinks output
>
> Do you have any features of type snap in your results from step 3? We’ve had
> a couple of recent posts where after training snap was giving no results, and
> as a result maker couldn’t give any genes. One cause of something like that
> may be your step 2. Make sure the ZFF wasn’t empty you used to train with.
> The maker2zff script uses filters to only put the best genes in the off file,
> and if all your genes fail the filtering then you are training with an empty
> ZFF.
>
> Also you should use proteins from a related species as your protein file. I
> see that you protein marches are varying wildly from run to run? So is your
> contig count? Were the subset of contigs you have results for long enough to
> contain genes?
>
> —Carson
>
> From: dhivya arasappan <darasappan at gmail.com>
> Date: Monday, February 3, 2014 at 9:31 AM
> To: Daniel Ence <dence at genetics.utah.edu>
> Cc: "maker-devel at yandell-lab.org" <maker-devel at yandell-lab.org>
> Subject: Re: [maker-devel] maker annotation with cufflinks output
>
> Hi Daniel,
>
> I was able to check on some of those questions.
>
> 1. From trinity assembly: I started with 102000 contigs. I used trinotate to
> annotate proteins in this.
>
> I ran maker on this data with est2genome set to 1. The output looks like this
> (most important parts on top):
>
> 6653 gene
> 46675 exon
> 280534 protein_match
> 59934 CDS
> 969 contig
> 105388 expressed_sequence_match
> 12584 five_prime_UTR
> 78565 match
> 1401369 match_part
> 10180 mRNA
> 11545 three_prime_UTR
>
> 2. From cufflinks assembly: I started with 133380 entries (out of which there
> are 29,000 transcripts). I used the protein sequences from trinity assembly.
>
> I ran maker on this data with est2genome set to 1. The output looks like this:
> 29 gene
> 75 exon
> 573659 protein_match
> 67 CDS
> 1099 contig
> 269298 expressed_sequence_match
> 23 five_prime_UTR
> 173844 match
> 2221846 match_part
> 29 mRNA
> 23 three_prime_UTR
>
> The genes annotated using the trinity assembly is lower than expected, so I
> went the cufflinks route. I dont understand why when using the cufflinks
> transcripts, even less genes are being found.
>
> 3. Training SNAP: I used the results of maker from 1 to train SNAP. I then
> used that training set to rerun maker:
> snaphmm=/scratch/01184/daras/jansen/RHA/allpaths/maker_mpi_withAlltrinity/snap
> /RHA.hmm
> est2genome=0
>
> And again I got results with no entries for gene, exon, CDS etc.
> 957 contig
> 46555 expressed_sequence_match
> 43651 match
> 553633 match_part
> 113738 protein_match
>
> As I mentioned in another email, cegma results indicated that the genome was
> more than 90% complete. Any suggestions would be helpful.
>
> Thank you
> Dhivya
>
>
>
>
> On Jan 30, 2014, at 2:51 PM, Daniel Ence wrote:
>
>> Hi Dhivya,
>>
>> I think there a few numbers that could be helpful to understand what's
>> happening here.
>>
>> How many transcripts did Trinity assembly the RNA-seq data into? Also, you
>> had 29,000 transcripts from cufflinks, but fewer from MAKER when you gave it
>> the cufflinks data. How many transcripts did MAKER identify with the
>> cufflinks data? Did you still get more than the 10,000 transcripts that you
>> found with just the Trinity data?
>>
>> A key part of MAKER's approach to genome annotation that might be affecting
>> it's performance is that it only annotates a gene where there is both
>> evidence (like your RNA-seq data) and an ab-initio prediction. If a
>> prediction is unsupported by the evidence, then MAKER won't annotate a gene
>> and if evidence aligns where there's no prediction, MAKER won't annotate a
>> gene either. What ab-initio predictors are you using and have they been
>> trained specific genome?
>>
>> You can force MAKER to automatically promote evidence alignments to a gene
>> model by setting the est2genome option to 1, but that will usually give you
>> many false positives.
>>
>> Try rerunning it with either the Trinity data or the Cufflinks data and with
>> est2genome set to 1, and let us know how that affects the MAKER results.
>>
>> Thanks,
>> Daniel
>>
>> Daniel Ence
>> Graduate Student
>> Eccles Institute of Human Genetics
>> University of Utah
>> 15 North 2030 East, Room 2100
>> Salt Lake City, UT 84112-5330
>> ________________________________________
>> From: maker-devel [maker-devel-bounces at yandell-lab.org] on behalf of dhivya
>> arasappan [darasappan at gmail.com]
>> Sent: Thursday, January 30, 2014 11:18 AM
>> To: maker-devel at yandell-lab.org
>> Subject: [maker-devel] maker annotation with cufflinks output
>>
>> Hello,
>>
>> I am trying to annotate a 200 mb plant genome for which I have a very
>> good assembly.
>>
>> I tried to denovo assemble RNA-seq data using trinity and ran maker
>> using my genome assembly and the trinity results. I did not get as
>> many transcripts as expected, around 10,000 transcripts.
>>
>> So, I decided to try a different approach. I did a genome assisted
>> assembly of the RNA-seq data using tophat/cufflinks. This pipeline
>> generated 21,000 genes, 29,000 transcripts. I then ran maker using my
>> genome assembly and the cufflinks result. I get much less number of
>> transcripts as a result.
>>
>> If cufflinks found 29000 transcripts by mapping to the genome, I'm
>> confused as to why maker is not finding the same.
>>
>> Any suggestions would be appreciated.
>>
>> Thanks
>> Dhivya
>>
>>
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