Error when running run_GOseq.pl script

[scandyLuna]

Dear Trinity&Trinotate users,

I have gone through the whole pipeline of Trinity and Trinotate and everything is working nicely for me now!
Yesterday I also wanted to perform gene ontology enrichment and I was following this pipeline : http://trinityrnaseq.sourceforge.net/analysis/run_GOseq.html
But for some reason I get this error and I cannot figure out what is wrong :

Error in gene_lengths[features_with_GO, ] : subscript out of bounds
Execution halted
Error, cmd: R --vanilla -q < __runGOseq.R died with ret 256 at /home/ilona/bin/trinityrnaseq-2.0.6/Analysis/DifferentialExpression/run_GOseq.pl line 161.

I tried to change the headings of the files and also the order of columns - as it said in one feed : http://sourceforge.net/p/trinityrnaseq/mailman/message/32838167/ , but it did not help.
Some of you have an idea what can be wrong?? I would be very thankful for any input!!

With best regards,
Ilona

[Brian Haas]

Hi,

If you can privately send me your inputs to this step, I'll debug it right away.

Best,

-Brian

(by iPhone)

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[Brian Haas]

Hi Ilona,

It appears the problem has to do with the feature lengths file provided.  The one you're using corresponds to the transcripts, but you need to use one based on the gene identifiers - which you can get from the RSEM gene-estimates file. (RSEM.genes.results file)

best of luck,

~brian

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[Nico Posnien]

Hi Brian,

I seem to have the same error as described by Ilona. My files are not a result of the Trinity pipeline. I rather used a set of transcripts (extracted from a gff file of a genome) to run the complete Trinotate pipeline successfully. I extracted the GO assignments per gene as described here: http://trinityrnaseq.sourceforge.net/analysis/run_GOseq.html

That ran smoothly. Since I did not run RSEM on my dataset I extracted the gene length using samtools samtools faidx aug3.1_mrna.fasta and subsequently cut -f1-2 aug3.1_mrna.fasta.fai). Below I paste the heads of the three files used:

factor labeling:

small_peak aug3.g12.t1

small_peak aug3.g55.t1

small_peak aug3.g57.t1

small_peak aug3.g69.t1

length file:

aug3.g1.t1 453

aug3.g2.t1 530

aug3.g3.t1 1362

aug3.g4.t1 2066

GO assignment:

aug3.g1.t1 GO:0003674,

aug3.g10.t1 GO:0005575,...

aug3.g1000.t1 GO:0000166,...

aug3.g10002.t1 GO:0003674,...

aug3.g10003.t1 GO:0003674,...

aug3.g10004.t1 GO:0003674,...

Any idea, why this does not work? Any hint on what exactly the R script requires at that step wold be probably helpful to solve the problem.

Thanks in advance!

Cheers,

nico

Am Samstag, 7. März 2015 22:16:46 UTC+1 schrieb Brian Haas:

Hi Ilona,

It appears the problem has to do with the feature lengths file provided.  The one you're using corresponds to the transcripts, but you need to use one based on the gene identifiers - which you can get from the RSEM gene-estimates file. (RSEM.genes.results file)

best of luck,

~brian

On Fri, Mar 6, 2015 at 4:34 AM, scandyLuna <urbarov...@gmail.com> wrote:

Dear Trinity&Trinotate users,

I have gone through the whole pipeline of Trinity and Trinotate and everything is working nicely for me now!
Yesterday I also wanted to perform gene ontology enrichment and I was following this pipeline : http://trinityrnaseq.sourceforge.net/analysis/run_GOseq.html
But for some reason I get this error and I cannot figure out what is wrong :

Error in gene_lengths[features_with_GO, ] : subscript out of bounds
Execution halted
Error, cmd: R --vanilla -q < __runGOseq.R died with ret 256 at /home/ilona/bin/trinityrnaseq-2.0.6/Analysis/DifferentialExpression/run_GOseq.pl line 161.

I tried to change the headings of the files and also the order of columns - as it said in one feed : http://sourceforge.net/p/trinityrnaseq/mailman/message/32838167/ , but it did not help.
Some of you have an idea what can be wrong?? I would be very thankful for any input!!

With best regards,
Ilona

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[Brian Haas]

Hi Nico,

If you can send me your inputs (privately), I'll give it a whirl and see if I can figure out what the issue is.

best,

~b

[Brian Haas]

Hi Nico,

Thanks for sending the files!

The only thing you need to do is to add a header line to your lengths.txt file.

In this case, I just put:

gene_name(tab)length

and then it ran without a hitch.

best,

~brian

[Carlos Prada]

Hi All trinity crowd,

I am a VERY happy trinity user. Everything has run smoothly until I run  "analyze_diff_expr.pl" with the "--examine_GO_enrichment   --GO_annots  --gene_lengths". It runs fine but at the end the *depleted *enriched files are empty. Only contain the header: "category over_represented_pvalue under_represented_pvalue numDEInCat numInCat term ontology over_represented_FDR". I generated the GO file with Trinotate and looks like:

TR100008|c1_g1 GO:0000785,GO:0003674,GO:0003824,GO:0005575,GO:0005634,GO:0005737,GO:0006139,GO:0006259,GO:0006260,GO:0006725,GO:0006807,GO:0008150,GO:0008152,GO:0009058,GO:0009059,GO:0009987,GO:0032991,GO:0034641,GO:0034645,GO:0043170,GO:0043226,GO:0043227,GO:0043229,GO:0043231,GO:0043900,GO:0043901,GO:0043903,GO:0044237,GO:0044238,GO:0044249,GO:0044260,GO:0044422,GO:0044424,GO:0044427,GO:0044446,GO:0044464,GO:0045069,GO:0045071,GO:0046483,GO:0048519,GO:0048523,GO:0048525,GO:0050789,GO:0050792,GO:0050794,GO:0065007,GO:0071704,GO:0090304,GO:1901360,GO:1901576

I also extracted the lengths by using "cat genes.results | cut -f 1,3 > genes.lengths.txt" on one of my replicates. The file looks like:

gene_id length

TR100008|c0_g1 1387.00

TR100008|c1_g1 804.00

TR10000|c1_g2 231.00

TR10000|c3_g1 230.00

TR100011|c0_g1 225.00

TR100015|c0_g2 247.00

 I wonder what I am doing incorrectly. Any advise is VERY welcome!

Carlos

[Tiago Hori]

That probably means it found no significant results, what FDR cutoff are you using?

T.

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[Carlos Prada]

I used 0.001

It is weird because in some comparisons I have differentially expressed genes judging by the *results files. One of them looks like:

logFC logCPM PValue FDR

TR61407|c1_g2_i1 10.7794117122046 5.54549404738264 1.93029079183305e-10 2.7246587716805e-06

TR61407|c2_g1_i1 9.6897068955846 7.01554257108975 2.03910999227698e-10 2.7246587716805e-06

TR14515|c0_g1_i1 5.72812630054466 4.91432398185298 7.11552590248611e-10 6.33851047393463e-06

TR23706|c0_g1_i1 10.3775129841346 7.46483778172027 1.45045178514368e-09 9.6904683765449e-06

TR61407|c1_g1_i1 9.04818981884657 5.40926805964082 3.02051591525955e-09 1.61440534638793e-05

TR22297|c0_g1_i1 2.32159289479599 6.22478289636004 6.55696590990134e-09 2.92047261627005e-05

TR42483|c0_g1_i1 9.16817099715287 3.99742127439487 8.57595108618224e-09 3.27405309753049e-05

TR49871|c2_g1_i4 -4.91830184292399 1.95459937825247 1.38109523734571e-08 4.61354864035333e-05

TR61407|c5_g1_i1 8.27315129618878 4.86635696785049 1.95143038007636e-08 5.79444727524008e-05

TR132039|c1_g1_i1 -7.51431853307473 2.60009351990992 2.97722317365807e-08 7.95633120928383e-05

TR47141|c0_g1_i4 -3.31336864138287 1.75675773966102 6.30254509328648e-08 0.000153117468248171

TR19949|c1_g2_i1 7.62456026880291 4.35254627640026 7.19207221942148e-08 0.000160167448326516

TR30828|c6_g1_i1 -2.12558452674748 6.28453628479908 8.85926954415436e-08 0.000180020794634837

TR51025|c2_g1_i1 6.06542186160964 1.86341526871085 9.43081546507901e-08 0.000180020794634837

TR128003|c0_g1_i1 7.85304822795101 2.79691598119741 1.04922410442534e-07 0.000186929766444419

TR14833|c0_g1_i1 7.51596470183021 2.47416131451395 1.24863314116512e-07 0.000202617829684844

TR6140

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[Tiago Hori]

The fact you have DE genes does not mean you will be able to detect enrichment, especially if you are working with a poorly annotated genome. 

The enrichment calculates the deviation of the proportions of GO terms in each list from a expected distribution, which can be estimated.

Depending in how many genes actually have annotations at 0,001, not how many are DE, it could be very hard for the Fisher test or whatever is going on there to find significance. 

You can try BLAST2Go or DAVID. Reactome can be useful too.

T.

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[Carlos Prada]

Thanks Tiago. I will try Blast2go and DAVID.

[Ken Field]

Dear Carlos-

You didn't mention what species you are studying. If it is not a model organism or human, then Blast2go and DAVID (or GOrilla, which is my favorite) will require some extra work to first identify the best ortholog for each of your differentially expressed transcripts. For my study on bats, I used BLAST to align each transcript to the human database, which is the best annotated, of course. This has obvious limitations, but it allowed me to use the gene ontology tools available.

Good luck,

Ken

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[Tiago Hori]

You used to be able to extract the GO from the hits on BLAST2Go without extra work, is that not the case anymore? In David you have a few options depending on what you are working it, for example, when I work in fish, I use the zebrafish refeseq to get my putative orthologs, but in the case of Ken using human makes perfect sense. You will face the same challenge with Reactome.

 

T.

[Carlos Prada]

Thanks Tiago and Ken. I see what I can do with Blast2go and David. I work on corals so I am sure it is going to be even more challenging but I will give it a try and report back. Thanks you again for the advice!

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[Tiago Hori]

Yeah… you can try the c elegans or the drosophila. You can download the refeseqs from NCBI and the use blast locally.

 

T.

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[Carlos Prada]

Hi all,

After a full-redo of all my analysis Trinity identified enriched and depleted GO for each treatment. I however have a very naive question. In one of the treatments, I have about 50 genes differentially expressed but  have over 80 different GO-ennriched numbers. I am guessing that for each gene there is more than one GO term associated and thus the reason I have this difference. Is this the reason? Thanks for any comment/advice.

Best,

Carlos

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[Brian Haas]

Hi Carlos,

Each GO assignment exists in a graph structure and all parent terms are tested in addition to the final (highest resolution) identifier assigned.

best,

~brian

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[Carlos Prada]

Thanks Brian!

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[Kyle Martin]

Hi Carlos,

Can you recall what you did to solve this problem?  I am facing the same challenge - all of my .enriched and .depleted files contain only the column headers.

Thanks for any advice!
Kyle

[Susan fogelson]

Hello,

I am having a similar problem as Ilona.  I am trying to run the analyze_diff_expr.pl with GO enrichment and I get a similar error....see below.  I made sure that I ran the extraction of feature lengths on the RSEM.genes.results file and it is still giving me this error.  Any help would be much appreciated. 

Error in gene_lengths[features_with_GO, ] : subscript out of bounds

Execution halted

Error, cmd: R --vanilla -q < __runGOseq.R died with ret 256 at /panfs/pstor.storage/rcclocal/zcluster/trinity/2.0.6/Analysis/DifferentialExpression/run_GOseq.pl line 161.

The original code that I executed was:

 /usr/local/trinity/latest/Analysis/DifferentialExpression/analyze_diff_expr.pl --matrix Final.TMM.counts.wAnnot.matrix -P 1E-3 -C 2 --samples Erectus_samples_described.txt --examine_GO_enrichment --GO_annots All_go_annotation.txt --gene_lengths All2_genes.lengths.txt 

-Susan

On Friday, March 6, 2015 at 8:07:30 AM UTC-5, Brian Haas wrote:

Hi,

If you can privately send me your inputs to this step, I'll debug it right away.

Best,

-Brian

(by iPhone)

On Mar 6, 2015, at 4:34 AM, scandyLuna <urbarov...@gmail.com> wrote:

Dear Trinity&Trinotate users,

I have gone through the whole pipeline of Trinity and Trinotate and everything is working nicely for me now!
Yesterday I also wanted to perform gene ontology enrichment and I was following this pipeline : http://trinityrnaseq.sourceforge.net/analysis/run_GOseq.html
But for some reason I get this error and I cannot figure out what is wrong :

Error in gene_lengths[features_with_GO, ] : subscript out of bounds
Execution halted
Error, cmd: R --vanilla -q < __runGOseq.R died with ret 256 at /home/ilona/bin/trinityrnaseq-2.0.6/Analysis/DifferentialExpression/run_GOseq.pl line 161.

I tried to change the headings of the files and also the order of columns - as it said in one feed : http://sourceforge.net/p/trinityrnaseq/mailman/message/32838167/ , but it did not help.
Some of you have an idea what can be wrong?? I would be very thankful for any input!!

With best regards,
Ilona

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[Tiago Hori]

That usually means no enriched terms were detected....

T.

Tiago Hori, Ph.D

Associate Director of Genomics 

The Center for Aquaculture Technologies 

Sent from my iPhone

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[Susan fogelson]

I can run DE on an annotated file and it gives me differentially expressed GO annotated transcripts that have a P value as low as 2E-16, which leads me to believe that there is something wrong with the file format.  Am I totally off base?

-S

[Brian Haas]

I've seen this happen when the gene lengths file was missing the column headers.  If you run

 head  All2_genes.lengths.txt 

Do you see a column heading?

From: 

https://github.com/trinityrnaseq/trinityrnaseq/wiki/Running-GOSeq

The gene.lengths.txt file has the format

 ${gene_name} (tab) ${gene_length}

Note, be sure to include column header "gene(tab)length" at the top of your gene.lengths.txt file.

~b

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[Brian Haas]

Hi Susan,

The file looks formatted correctly, but I wonder if there's a disconnect between transcripts vs. genes.  Those are transcript identifiers, whereas if you're going to do gene-based analysis, then you'll need a similar file with the gene identifiers incorporated.

best,

~brian

On Tue, Aug 23, 2016 at 12:18 PM, Susan fogelson <susanfog...@gmail.com> wrote:

Hi Brian,

Thank you for responding to my post.  When I perform the 'head' command, I receive the column headers gene_id (tab) length.  Is it formatted properly?

gene_id length

TR100000|c0_g1_i1       332.00

TR100001|c0_g1_i1       244.00

TR100002|c0_g1_i1       337.00

TR100003|c0_g1_i1       263.00

TR100004|c0_g1_i1       243.00

TR100005|c0_g1_i1       224.00

TR100006|c0_g1_i1       283.00

TR100007|c0_g1_i1       309.00

-S

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[hoppers]

Hi Brian-

Trying to solve the same error, and I can tell that there is a transcript vs gene identifier file problem. But I can't seem to generate the right file.

The RSEM.genes.results file was made multiple times for each treatment (using abundance_estimates_to_matrix.pl) in my RNAseq experiment, and there doesn't seem to be one for the dataset as a whole. The previous step, align_and_estimate_abundance.pl, does not seem to provide one, unless I am doing something wrong.

Can you suggest a way forward? I saw the rsem-prepare-reference command, but that also didn't seem to make the right file.

Thanks very much-

On Saturday, March 7, 2015 at 3:16:46 PM UTC-6, Brian Haas wrote:

Hi Ilona,

It appears the problem has to do with the feature lengths file provided.  The one you're using corresponds to the transcripts, but you need to use one based on the gene identifiers - which you can get from the RSEM gene-estimates file. (RSEM.genes.results file)

best of luck,

~brian

On Fri, Mar 6, 2015 at 4:34 AM, scandyLuna <urbarov...@gmail.com> wrote:

Dear Trinity&Trinotate users,

I have gone through the whole pipeline of Trinity and Trinotate and everything is working nicely for me now!
Yesterday I also wanted to perform gene ontology enrichment and I was following this pipeline : http://trinityrnaseq.sourceforge.net/analysis/run_GOseq.html
But for some reason I get this error and I cannot figure out what is wrong :

Error in gene_lengths[features_with_GO, ] : subscript out of bounds
Execution halted
Error, cmd: R --vanilla -q < __runGOseq.R died with ret 256 at /home/ilona/bin/trinityrnaseq-2.0.6/Analysis/DifferentialExpression/run_GOseq.pl line 161.

I tried to change the headings of the files and also the order of columns - as it said in one feed : http://sourceforge.net/p/trinityrnaseq/mailman/message/32838167/ , but it did not help.
Some of you have an idea what can be wrong?? I would be very thankful for any input!!

With best regards,
Ilona

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[Brian Haas]

Hi,

Can you give me a little more info on what the issue is?   If you could share your command and the top few lines of each of the inputs, it'll give me some useful hints too.

many thanks!

~b

[hoppers]

Thank you, appreciate your time.

As I was looking at my commands, I saw that I used the RSEM.isoforms.results as follows:

./trinityrnaseq-master/util/abundance_estimates_to_matrix.pl --est_method RSEM --out_prefix trans_counts  --name_sample_by_basedir control_1/RSEM.isoforms.results control_2/RSEM.isoforms.results cold_1/RSEM.isoforms.results cold_2/RSEM.isoforms.results heat_1/RSEM.isoforms.results heat_2/RSEM.isoforms.results

Perhaps should have used the RSEM.gene.results, and/or used the isoforms to get my gene_lengths file.

I'll run that down, and get back to you if this doesn't solve my problem.