[maker-devel] maker annotation with cufflinks output
Carson Holt
carsonhh at gmail.com
Tue Feb 11 11:55:38 MST 2014
I wouldn’t use mpi_evaluator. It is buggy and has virtually no
documentation. The AED values are the best way to identify which genes are
higher and lower quality. You can also run interproscan to identify protein
domain content as an independent evaluation. Look at this paper here —>
http://www.biomedcentral.com/1471-2105/12/491
Figure 4 has a nice example of how AED, domain content, and gene orthology
correlate to show the quality of different subsets of genes in seven ant
genomes.
If you choose to try mpi_evaluator it uses the -CTL option to generate empty
files that you then fill in.
Thanks,
Carson
From: dhivya arasappan <darasappan at gmail.com>
Date: Tuesday, February 11, 2014 at 11:48 AM
To: Carson Holt <carsonhh at gmail.com>
Cc: Daniel Ence <dence at genetics.utah.edu>, <maker-devel at yandell-lab.org>
Subject: Re: [maker-devel] maker annotation with cufflinks output
With your suggested changes (using a protein file not derived from the
RNA-seq data and fixing the gff file for training SNAP), I was able to
increase the number of genes from 6000+ to 18116.
I'm now trying to evaluate the quality of the annotation. I have a question
about the usage for mpi_evaluator.
In the maker tutorial, the usage is given as:
mpi_evaluator [options] <eval_opts> <eval_bopts> <eval_exe>
What files are being referred to in the input parameters: eval_opts,
eval_bopts and eval_exe?
Thanks
Dhivya
On Feb 6, 2014, at 11:47 AM, Carson Holt wrote:
> Ok. Content looks good. Just make sure to use gff3_merge to join the GFF3’s
> without stripping out the fasta sequence at the end when training SNAP.
>
> Thanks,
> Carson
>
>
> From: dhivya arasappan <darasappan at gmail.com>
> Date: Thursday, February 6, 2014 at 10:29 AM
> To: Carson Holt <carsonhh at gmail.com>
> Cc: Daniel Ence <dence at genetics.utah.edu>
> Subject: Re: [maker-devel] maker annotation with cufflinks output
>
> Sorry I was just trying to make it small enough to be approved by the mailing
> list.
>
> Here is the whole file:
>
>
> cat.formatted.gff.tgz
> <https://docs.google.com/file/d/0B3fACsJDXQi6VEE1VG5tWEh5M1U/edit?usp=drive_we
> b>
>
>
>
> On Thu, Feb 6, 2014 at 11:04 AM, Carson Holt <carsonhh at gmail.com> wrote:
>> Could you give me the file without using 'head’ to trim it, its cutting it
>> before it reaches the part I’m interested in.
>>
>> —Carson
>>
>>
>> From: dhivya arasappan <darasappan at gmail.com>
>> Date: Thursday, February 6, 2014 at 10:01 AM
>>
>> To: Carson Holt <carsonhh at gmail.com>
>> Cc: Daniel Ence <dence at genetics.utah.edu>, "maker-devel at yandell-lab.org"
>> <maker-devel at yandell-lab.org>
>> Subject: Re: [maker-devel] maker annotation with cufflinks output
>>
>> Oh yes I did- I took just the non sequence entries in the gff file and used
>> that as my input. I will rerun snap with the gff file containing the
>> sequences as well.
>>
>> I'm attaching a snippet of the gff file that I used as input to maker2zff.
>>
>> Thanks for your help
>> Dhivya
>>
>>
>>
>>
>> On Feb 6, 2014, at 10:05 AM, Carson Holt wrote:
>>
>>> Your genome.dna file has no sequence? Did you by any chance strip the fasta
>>> sequence from the GFF3 you are using as input to maker2zff? There should be
>>> fasta sequence at the end of that file. Also can I see the GFF3 file you
>>> are using as input to maker2zff.
>>>
>>> Thanks,
>>> Carson
>>>
>>> From: dhivya arasappan <darasappan at gmail.com>
>>> Date: Thursday, February 6, 2014 at 7:47 AM
>>> To: Carson Holt <carsonhh at gmail.com>
>>> Cc: Daniel Ence <dence at genetics.utah.edu>, "maker-devel at yandell-lab.org"
>>> <maker-devel at yandell-lab.org>
>>> Subject: Re: [maker-devel] maker annotation with cufflinks output
>>>
>>> Hello,
>>>
>>> I does appear than my genome.ann file from maker2zff script has data in it.
>>> However, the SNAP steps after that have created empty files. The following
>>> are all empty:
>>>
>>> alt.dna err.dna export.dna genome.dna olp.dna uni.dna wrn.dna
>>> alt.ann err.ann export.ann genome.ann olp.ann uni.ann wrn.ann
>>>
>>> When I tried to get gene stats or validate genome.ann, I get errors like
>>> this for all of them:
>>>
>>> fathom genome.ann genome.dna -gene-stats |more
>>> MODEL5547 1 1 6 + errors(6): exon-1:out_of_bounds exon-2:out_of_bounds
>>> exon-3:out_of_bounds exon-4:out_of_bounds exon-5:out_of_bounds
>>> exon-6:out_of_bounds
>>> MODEL5568 1 1 6 - errors(6): exon-6:out_of_bounds exon-5:out_of_bounds
>>> exon-4:out_of_bounds exon-3:out_of_bounds exon-2:out_of_bounds
>>> exon-1:out_of_bounds
>>> MODEL5589 1 1 5 + errors(5): exon-1:out_of_bounds exon-2:out_of_bounds
>>> exon-3:out_of_bounds exon-4:out_of_bounds exon-5:out_of_bounds
>>> MODEL5195 1 1 21 + errors(21): exon-1:out_of_bounds exon-2:out_of_bounds
>>> exon-3:out_of_bounds exon-4:out_of_bounds exon-5:out_of_bounds
>>> exon-6:out_of_bounds exon-7:out_of_bounds exon-8:out_of_bounds
>>> exon-9:out_of_bounds exon-10:out_of_bounds exon-11:out_of_bounds
>>> exon-12:out_of_bounds exon-13:out_of_bounds exon-14:out_of_bounds
>>> exon-15:out_of_bounds exon-16:out_of_bounds exon-17:out_of_bounds
>>> exon-18:out_of_bounds exon-19:out_of_bounds exon-20:out_of_bounds
>>> exon-21:out_of_bounds
>>>
>>> I'm not sure why the annotation I'm seeing in genome.ann are all showing up
>>> as errors. I realize this may be an issue with snap, but are you familiar
>>> with anything like this? My genome.ann file is attached for reference.
>>>
>>> Thanks
>>> Dhivya
>>>
>>> On Feb 5, 2014, at 12:38 PM, Carson Holt wrote:
>>>
>>>> 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/s
>>>> nap/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
>>>>>
>>>>>
>>>>> _______________________________________________
>>>>> maker-devel mailing list
>>>>> maker-devel at box290.bluehost.com
>>>>> http://box290.bluehost.com/mailman/listinfo/maker-devel_yandell-lab.org
>>>>
>>>> _______________________________________________ maker-devel mailing list
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>>>> http://box290.bluehost.com/mailman/listinfo/maker-devel_yandell-lab.org
>>>
>>
>
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