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Trinotate Annotation Report: No PFAM #58

Closed brittonstrickland closed 2 years ago

brittonstrickland commented 3 years ago

Hi all,

I have successfully annotated my transcriptome of interest, and all of the individual data files look great and populated. However, when generating the sqlite database, the PFAM column is left empty. Is there any particular reason this is left out?

I am using Trinotate-v3.2.2 and hmmer-3.3.2, with a freshly generated Pfam-A.hmm as described in the manual.

brianjohnhaas commented 3 years ago

Hi,

It's important to use the pfam database that trinotate pulls for you during the boilerplate database construction process (as well as the corresponding swissprot fasta file). This is the only way that the identifiers are all sync'd up in the database. It's the number one reason for not seeing results in the final data table. If you're using the files generated by Trinotate, then there must be something else going on.

~b

On Wed, Sep 29, 2021 at 1:22 PM brittonstrickland @.***> wrote:

Hi all,

I have successfully annotated my transcriptome of interest, and all of the individual data files look great and populated. However, when generating the sqlite database, the PFAM column is left empty. Is there any particular reason this is left out?

I am using Trinotate-v3.2.2 and hmmer-3.3.2, with a freshly generated Pfam-A.hmm as described in the manual.

— You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub https://github.com/Trinotate/Trinotate.github.io/issues/58, or unsubscribe https://github.com/notifications/unsubscribe-auth/ABZRKX3ECJ7AX4WXX5U2FS3UENDMTANCNFSM5FAMQUBQ . Triage notifications on the go with GitHub Mobile for iOS https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675 or Android https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign%3Dnotification-email%26utm_medium%3Demail%26utm_source%3Dgithub.

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kishor2019 commented 3 years ago

Hi @brianjohnhaas I am also having similar type of issues. I also didnt receive the any Pfam or GO or any other functional description or ids. I have loaded all the blastx, blastp, hmmscan and signalp hits to Trinotate.sqlite and also didnt found any error message. Here i am giving the cmd line for your convenience:

loading transcripts and proteins

/home/softwares/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite init --gene_trans_map ../../trinity_out_dir/Trinity.fasta.gene_trans_map --transcript_fasta ../../trinity_out_dir/Trinity.fasta --transdecoder_pep ../Trinity.RSEM.retained.clustered.fasta.transdecoder.pep

loading homologue hits

/home/softwares/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite LOAD_swissprot_blastx swissprot.blastx.outfmt6.log1 /home/softwares/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite LOAD_swissprot_blastp swissprot.blastp.outfmt6.log1 /home/softwares/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite LOAD_pfam PFAM.out /home/softwares/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite LOAD_signalp singnalp.out /home/softwares/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite report > Trinotate.xls

Here i am also sharing a part of my xls (in txt format) output for your convenience. It will be a great help for me if i get some suggestions to solve the problem. Thanks. trinotate.txt

I

brianjohnhaas commented 3 years ago

hi - just checking - did you search the pfam and swissprot databases that Trinotate provided during the boilerplate database construction step?

On Thu, Sep 30, 2021 at 4:43 PM kishor2019 @.***> wrote:

Hi @brianjohnhaas https://github.com/brianjohnhaas I am also having similar type of issues. I also didnt receive the any Pfam or GO or any other functional description or ids. I have loaded all the blastx, blastp, hmmscan and signalp hits to Trinotate.sqlite and also didnt found any error message. Here i am giving the cmd line for your convenience:

loading transcripts and proteins

/home/softwares/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite init --gene_trans_map ../../trinity_out_dir/Trinity.fasta.gene_trans_map --transcript_fasta ../../trinity_out_dir/Trinity.fasta --transdecoder_pep ../Trinity.RSEM.retained.clustered.fasta.transdecoder.pep

loading homologue hits

/home/softwares/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite LOAD_swissprot_blastx swissprot.blastx.outfmt6.log1 /home/softwares/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite LOAD_swissprot_blastp swissprot.blastp.outfmt6.log1 /home/softwares/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite LOAD_pfam PFAM.out /home/softwares/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite LOAD_signalp singnalp.out /home/softwares/Trinotate-Trinotate-v3.2.2/Trinotate Trinotate.sqlite report > Trinotate.xls

Here i am also sharing a part of my xls (in txt format) output for your convenience. It will be a great help for me if i get some suggestions to solve the problem. Thanks. trinotate.txt https://github.com/Trinotate/Trinotate.github.io/files/7262370/trinotate.txt

I

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brittonstrickland commented 3 years ago

My Trinotate annotation report looks exactly the same as @kishor2019. I am using the Pfam and swissprot databases from the boilerplate construction command. I have also redownloaded it all and got the same results. Everything else looks great, except the Pfam column.

kishor2019 commented 3 years ago

Hi @brianjohnhaas After running ./runMe.sh I copied the Trinotate.boilerplate.sqlite as Trinotate.sqlite to working dir and run like as above. I have manually downloaded Pfam and swissprot before so that i did not run Build_Trinotate_Boilerplate_SQLite_db.pl. I thought that as i have these databases i dont need to download again. Anyway, is it a problem? But i have loaded all the hit of my transcript from blastx, blastp, signalp and hmmscan to Trinotate.sqlite. But i didnt get any GO, COG or any functional information.

Btw, when i run, runMe.sh i have also found some note like as below: ........ Done processing, writing output.

Finished.

CMD: /mnt/genome3/Lab_Users/Kishor/DISK_2/softwares/Trinotate-Trinotate-v3.2.2/util/report_summary/../gene_ontology/Trinotate_GO_to_SLIM.pl Trinotate_report_stats.GO > Trinotate_report_stats.GO.slim CMD: /mnt/genome3/Lab_Users/Kishor/DISK_2/softwares/Trinotate-Trinotate-v3.2.2/util/report_summary/util/make_cXp_html.pl Trinotate_report_stats > Trinotate_report_stats.cXp_summary.html Copy failed: No such file or directory at ../util/report_summary/trinotate_report_summary.pl line 104, <$fh> line 688.

Can you please help me to fix the problem?Thanks.

brianjohnhaas commented 3 years ago

brittonstrickland - if you want to share your sqlite database with me, I'll take a look. It could be that something has changed in the data downloads that's now causing an issue.

You can share with @.***

best,

~brian

On Fri, Oct 1, 2021 at 12:02 PM brittonstrickland @.***> wrote:

My Trinotate annotation report looks exactly the same as @kishor2019 https://github.com/kishor2019. I am using the Pfam and swissprot databases from the boilerplate construction command. I have also redownloaded it all and got the same results. Everything else looks great, except the Pfam column.

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brianjohnhaas commented 3 years ago

kishor2019 - you'd need to use the boilerplate database that's constructed at the time of the boilerplate construction is run. You can't use the one that's part of the sample data execution - that one is overly contrived for demo purposes.

On Fri, Oct 1, 2021 at 1:17 PM Brian Haas @.***> wrote:

brittonstrickland - if you want to share your sqlite database with me, I'll take a look. It could be that something has changed in the data downloads that's now causing an issue.

You can share with @.***

best,

~brian

On Fri, Oct 1, 2021 at 12:02 PM brittonstrickland < @.***> wrote:

My Trinotate annotation report looks exactly the same as @kishor2019. I am using the Pfam and swissprot databases from the boilerplate construction command. I have also redownloaded it all and got the same results. Everything else looks great, except the Pfam column.

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub, or unsubscribe. Triage notifications on the go with GitHub Mobile for iOS or Android.

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brittonstrickland commented 3 years ago

@brianjohnhaas The email address is blanked out, but I set to your @gmail account listed on your profile. Thanks for your help.

kishor2019 commented 3 years ago

@brianjohnhaas I have used Build_Trinotate_Boilerplate_SQLite_db.pl the script to generate the SQlite. And this time i have got Pfam and GO ids. But yet to receive eggnog and kegg ids. For your convenience i am adding a part of result with you. Trinotate.xlsx

brianjohnhaas commented 3 years ago

kishor2019 - I see in your sprot top hit column accessions like 'sp|Q8BTY2|S4A7_MOUSE'

this leads me to think that your blast results are against a protein fasta file that wasn't generated by the Trinotate build process.

When you run the 'Build_Trinotate_Boilerplate_SQLite_db.pl' script to build the boilerplate database, it will generate file 'uniprot_sprot.pep', and this is the file that must be searched using blast.

The kegg and eggnog annotations come from these blast hits.

The GO info you're seeing is likely just based on the pfam results and not considering the blast hits.

hope this helps,

~b

On Sat, Oct 2, 2021 at 1:43 AM kishor2019 @.***> wrote:

@brianjohnhaas I have used Build_Trinotate_Boilerplate_SQLite_db.pl the script to generate the SQlite. And this time i have got Pfam and GO ids. But yet to receive eggnog and kegg ids. For your convenience i am adding a part of result with you. Trinotate.xlsx

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kishor2019 commented 3 years ago

@brianjohnhaas Your suggestion works well. A small clarification will be great. All the ID for KEGG i received, are entry number. Is there any way within the trinotate package to convert the entries to KOG number? Thank you very much.

brianjohnhaas commented 2 years ago

Glad it's working for you now!

The KEGG support is pretty limited. I think there are other tools that will do a more rigorous job for kegg annotations if you want to explore that.

On Sun, Oct 3, 2021 at 1:51 PM kishor2019 @.***> wrote:

@brianjohnhaas https://github.com/brianjohnhaas Your suggestion works well. A small clarification will be great. All the ID for KEGG i received, are entry number. Is there any way within the trinotate package to convert the entries to KOG number? Thank you very much.

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kishor2019 commented 2 years ago

@brianjohnhaas Thanks a lot to you.

brianjohnhaas commented 2 years ago

Sure thing.

Also, wrt brittonstrickland's issue, his sqlite db looked fine and I was able to generate the report including the pfam data. It's not clear why this didn't happen during his report-writing... remains a mystery at the moment.

~b

On Mon, Oct 4, 2021 at 7:29 AM kishor2019 @.***> wrote:

@brianjohnhaas https://github.com/brianjohnhaas Thanks a lot to you.

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kishor2019 commented 2 years ago

@brianjohnhaas It also halted for me during the Build_Trinotate_Boilerplate_SQLite_db.pl running. May be around 10/12 times even my space, speed and other requirement was absolutely fine. But finally it was generated. And i run it suceesfully.

And another thing to report you that may be installation with conda has some issue as i initially tried with conda and faced trouble to work. But later with manual installation, it works great!!

brianjohnhaas commented 2 years ago

conda is super useful for a lot of things, but I don't support it directly for my software - others tend to integrate it into conda. I just provide the manual installations and support docker/singularity where possible.

On Mon, Oct 4, 2021 at 7:55 AM kishor2019 @.***> wrote:

@brianjohnhaas https://github.com/brianjohnhaas It also halted for me during the Build_Trinotate_Boilerplate_SQLite_db.pl running. May be around 10/12 times even my space, speed and other requirement was absolutely fine. But finally it was generated. And i run it suceesfully.

And another thing to report you that may be installation with conda has some issue as i initially tried with conda and faced trouble to work. But later with manual installation, it works great!!

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/Trinotate/Trinotate.github.io/issues/58#issuecomment-933409772, or unsubscribe https://github.com/notifications/unsubscribe-auth/ABZRKX3PKP3C7QVUT2IBB6LUFGI3HANCNFSM5FAMQUBQ . Triage notifications on the go with GitHub Mobile for iOS https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675 or Android https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign%3Dnotification-email%26utm_medium%3Demail%26utm_source%3Dgithub.

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margaretc-ho commented 1 year ago

I also made the same initial mistake as these guys, thanks for raising the issue and providing a solution.

Summary is don't use the trinotate-sample-data boilerplate sqlite file to generate the SQL file for the trinotate annotation report, use the one generated by ./Build_Trinotate_Boilerplate_SQLite_db.pl

I now have PFAM hits, yay!

phoebecunningham93 commented 1 year ago

I am having the same problem. I have assembled the transciptome de novo and run all the transdecoder commands which seems to have workers. Now I have run all the commands laid out in the Trinotate github and created the myTrinotate.sqlite file but when I try to initialise it i get this error:

Use of uninitialized value $blast_type in concatenation (.) or string at Trinotate-Trinotate-v4.0.0/Trinotate line 200.

I think I may have misunderstood how to load in the databases - SQlite, NCBI, infernal and HMMER etc - I loaded these into my current working directory and then created an empty directory for the --trinotate_data_dir and then ran the create command. If I run through all the commands to the end I get no data in the myTrinotate.tsv report except column names and gene id's and identifiers. Can you help? I am quite a novice at this kind of bioinformatics.

brianjohnhaas commented 1 year ago

Hi,

The warning "Use of uninitialized value $blast_type in concatenation (.) or string at Trinotate-Trinotate-v4.0.0/Trinotate line 200." is just an annoyance that I overlooked earlier - shouldn't impact anything.

I'll be putting a patch release out shortly.

With v4.0.0, use the --run parameter with the list of analyses to compute or 'ALL' to run what it can (including tmhmm and signalpv6 if you have them installed). It should load results into the database on its own.

If you're experiencing other errors as part of the process, let me know - they should provide more insights.

best,

~b

On Thu, Jun 1, 2023 at 7:53 AM phoebecunningham93 @.***> wrote:

I am having the same problem. I have assembled the transciptome de novo and run all the transdecoder commands which seems to have workers. Now I have run all the commands laid out in the Trinotate github and created the myTrinotate.sqlite file but when I try to initialise it i get this error:

Use of uninitialized value $blast_type in concatenation (.) or string at Trinotate-Trinotate-v4.0.0/Trinotate line 200.

I think I may have misunderstood how to load in the databases - SQlite, NCBI, infernal and HMMER etc - I loaded these into my current working directory and then created an empty directory for the --trinotate_data_dir and then ran the create command. If I run through all the commands to the end I get no data in the myTrinotate.tsv report except column names and gene id's and identifiers. Can you help? I am quite a novice at this kind of bioinformatics.

— Reply to this email directly, view it on GitHub https://github.com/Trinotate/Trinotate.github.io/issues/58#issuecomment-1571908797, or unsubscribe https://github.com/notifications/unsubscribe-auth/ABZRKX5NPMY6OU432OSZRPTXJB7B5ANCNFSM5FAMQUBQ . You are receiving this because you were mentioned.Message ID: @.***>

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phoebecunningham93 commented 1 year ago

Hi Brian,

Thanks for getting back to me. The problem is that although the directory seems to be populated with databases and a boilerplate after the step Trinotate --create --db myTrinotate.sqlite --trinotate_data_dir TrinData, the myTrinotate.sqlite file is apparently not created? When I run the initialisation command I don’t get anything and it runs really quickly, and then when I run the next command to do the sequence analysis it just comes up with the trinotate commands page and again very quickly. When I then try to create a report, its runs but I just get data missing in every column.

I’m not sure how best to share the problem but could you clarify the steps to creating the myTrinotate.sqlite file and what I need to have downloaded prior to this and to where? i.e. should SQlite and ncbi-blast be downloaded into the current working directory?

Thanks so much, Phoebe

From: Brian Haas @.> Date: Thursday, 1 June 2023 at 13:31 To: Trinotate/Trinotate.github.io @.> Cc: Phoebe Hazel Mary Cunningham @.>, Comment @.> Subject: Re: [Trinotate/Trinotate.github.io] Trinotate Annotation Report: No PFAM (#58) Hi,

The warning "Use of uninitialized value $blast_type in concatenation (.) or string at Trinotate-Trinotate-v4.0.0/Trinotate line 200." is just an annoyance that I overlooked earlier - shouldn't impact anything.

I'll be putting a patch release out shortly.

With v4.0.0, use the --run parameter with the list of analyses to compute or 'ALL' to run what it can (including tmhmm and signalpv6 if you have them installed). It should load results into the database on its own.

If you're experiencing other errors as part of the process, let me know - they should provide more insights.

best,

~b

On Thu, Jun 1, 2023 at 7:53 AM phoebecunningham93 @.***> wrote:

I am having the same problem. I have assembled the transciptome de novo and run all the transdecoder commands which seems to have workers. Now I have run all the commands laid out in the Trinotate github and created the myTrinotate.sqlite file but when I try to initialise it i get this error:

Use of uninitialized value $blast_type in concatenation (.) or string at Trinotate-Trinotate-v4.0.0/Trinotate line 200.

I think I may have misunderstood how to load in the databases - SQlite, NCBI, infernal and HMMER etc - I loaded these into my current working directory and then created an empty directory for the --trinotate_data_dir and then ran the create command. If I run through all the commands to the end I get no data in the myTrinotate.tsv report except column names and gene id's and identifiers. Can you help? I am quite a novice at this kind of bioinformatics.

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brianjohnhaas commented 1 year ago

Hi Phoebe,

I just put out a new software release moments ago. Could you try running that in a new working directory so no intermediates will be reused? Let's see if that works for you. If it doesn't, it should give more meaningful error messages to help guide things forward.

best,

~b

On Thu, Jun 1, 2023 at 11:24 AM phoebecunningham93 @.***> wrote:

Hi Brian,

Thanks for getting back to me. The problem is that although the directory seems to be populated with databases and a boilerplate after the step Trinotate --create --db myTrinotate.sqlite --trinotate_data_dir TrinData, the myTrinotate.sqlite file is apparently not created? When I run the initialisation command I don’t get anything and it runs really quickly, and then when I run the next command to do the sequence analysis it just comes up with the trinotate commands page and again very quickly. When I then try to create a report, its runs but I just get data missing in every column.

I’m not sure how best to share the problem but could you clarify the steps to creating the myTrinotate.sqlite file and what I need to have downloaded prior to this and to where? i.e. should SQlite and ncbi-blast be downloaded into the current working directory?

Thanks so much, Phoebe

From: Brian Haas @.> Date: Thursday, 1 June 2023 at 13:31 To: Trinotate/Trinotate.github.io @.> Cc: Phoebe Hazel Mary Cunningham @.>, Comment @.> Subject: Re: [Trinotate/Trinotate.github.io] Trinotate Annotation Report: No PFAM (#58) Hi,

The warning "Use of uninitialized value $blast_type in concatenation (.) or string at Trinotate-Trinotate-v4.0.0/Trinotate line 200." is just an annoyance that I overlooked earlier - shouldn't impact anything.

I'll be putting a patch release out shortly.

With v4.0.0, use the --run parameter with the list of analyses to compute or 'ALL' to run what it can (including tmhmm and signalpv6 if you have them installed). It should load results into the database on its own.

If you're experiencing other errors as part of the process, let me know - they should provide more insights.

best,

~b

On Thu, Jun 1, 2023 at 7:53 AM phoebecunningham93 @.***> wrote:

I am having the same problem. I have assembled the transciptome de novo and run all the transdecoder commands which seems to have workers. Now I have run all the commands laid out in the Trinotate github and created the myTrinotate.sqlite file but when I try to initialise it i get this error:

Use of uninitialized value $blast_type in concatenation (.) or string at Trinotate-Trinotate-v4.0.0/Trinotate line 200.

I think I may have misunderstood how to load in the databases - SQlite, NCBI, infernal and HMMER etc - I loaded these into my current working directory and then created an empty directory for the --trinotate_data_dir and then ran the create command. If I run through all the commands to the end I get no data in the myTrinotate.tsv report except column names and gene id's and identifiers. Can you help? I am quite a novice at this kind of bioinformatics.

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phoebecunningham93 commented 1 year ago

Hi Brian,

So that now seems to run smoother and I make the myTrinotate.sqlite file, however when I run the sequence analysis command like so:

Trinotate-Trinotate-v4.0.1/Trinotate --db myTrinotate.sqlite --CPU __init.ok \ --transcript_fasta Trinity.fasta \ --transdecoder_pep longest_orfs.pep \ --trinotate_data_dir TrinData \ --run "ALL"

It doesn’t give me an error but immediately gives me what looks like the help page with the logo and then usage: Trinotate-Trinotate-v4.0.1/Trinotate --db <--command ...> etc. so I am not confident this has run. When I the create the report, it is completely empty again.

All help is really appreciated! Phoebe

From: Brian Haas @.> Date: Thursday, 1 June 2023 at 16:29 To: Trinotate/Trinotate.github.io @.> Cc: Phoebe Hazel Mary Cunningham @.>, Comment @.> Subject: Re: [Trinotate/Trinotate.github.io] Trinotate Annotation Report: No PFAM (#58) Hi Phoebe,

I just put out a new software release moments ago. Could you try running that in a new working directory so no intermediates will be reused? Let's see if that works for you. If it doesn't, it should give more meaningful error messages to help guide things forward.

best,

~b

On Thu, Jun 1, 2023 at 11:24 AM phoebecunningham93 @.***> wrote:

Hi Brian,

Thanks for getting back to me. The problem is that although the directory seems to be populated with databases and a boilerplate after the step Trinotate --create --db myTrinotate.sqlite --trinotate_data_dir TrinData, the myTrinotate.sqlite file is apparently not created? When I run the initialisation command I don’t get anything and it runs really quickly, and then when I run the next command to do the sequence analysis it just comes up with the trinotate commands page and again very quickly. When I then try to create a report, its runs but I just get data missing in every column.

I’m not sure how best to share the problem but could you clarify the steps to creating the myTrinotate.sqlite file and what I need to have downloaded prior to this and to where? i.e. should SQlite and ncbi-blast be downloaded into the current working directory?

Thanks so much, Phoebe

From: Brian Haas @.> Date: Thursday, 1 June 2023 at 13:31 To: Trinotate/Trinotate.github.io @.> Cc: Phoebe Hazel Mary Cunningham @.>, Comment @.> Subject: Re: [Trinotate/Trinotate.github.io] Trinotate Annotation Report: No PFAM (#58) Hi,

The warning "Use of uninitialized value $blast_type in concatenation (.) or string at Trinotate-Trinotate-v4.0.0/Trinotate line 200." is just an annoyance that I overlooked earlier - shouldn't impact anything.

I'll be putting a patch release out shortly.

With v4.0.0, use the --run parameter with the list of analyses to compute or 'ALL' to run what it can (including tmhmm and signalpv6 if you have them installed). It should load results into the database on its own.

If you're experiencing other errors as part of the process, let me know - they should provide more insights.

best,

~b

On Thu, Jun 1, 2023 at 7:53 AM phoebecunningham93 @.***> wrote:

I am having the same problem. I have assembled the transciptome de novo and run all the transdecoder commands which seems to have workers. Now I have run all the commands laid out in the Trinotate github and created the myTrinotate.sqlite file but when I try to initialise it i get this error:

Use of uninitialized value $blast_type in concatenation (.) or string at Trinotate-Trinotate-v4.0.0/Trinotate line 200.

I think I may have misunderstood how to load in the databases - SQlite, NCBI, infernal and HMMER etc - I loaded these into my current working directory and then created an empty directory for the --trinotate_data_dir and then ran the create command. If I run through all the commands to the end I get no data in the myTrinotate.tsv report except column names and gene id's and identifiers. Can you help? I am quite a novice at this kind of bioinformatics.

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brianjohnhaas commented 1 year ago

Hi Phoebe,

It might just be this setting: --CPU __init.ok

where you'll want to indicate the number of cores to use (eg. 4)

best,

~brian

On Thu, Jun 1, 2023 at 12:51 PM phoebecunningham93 @.***> wrote:

Hi Brian,

So that now seems to run smoother and I make the myTrinotate.sqlite file, however when I run the sequence analysis command like so:

Trinotate-Trinotate-v4.0.1/Trinotate --db myTrinotate.sqlite --CPU __init.ok \ --transcript_fasta Trinity.fasta \ --transdecoder_pep longest_orfs.pep \ --trinotate_data_dir TrinData \ --run "ALL"

It doesn’t give me an error but immediately gives me what looks like the help page with the logo and then usage: Trinotate-Trinotate-v4.0.1/Trinotate --db <--command ...> etc. so I am not confident this has run. When I the create the report, it is completely empty again.

All help is really appreciated! Phoebe

From: Brian Haas @.> Date: Thursday, 1 June 2023 at 16:29 To: Trinotate/Trinotate.github.io @.> Cc: Phoebe Hazel Mary Cunningham @.>, Comment @.> Subject: Re: [Trinotate/Trinotate.github.io] Trinotate Annotation Report: No PFAM (#58) Hi Phoebe,

I just put out a new software release moments ago. Could you try running that in a new working directory so no intermediates will be reused? Let's see if that works for you. If it doesn't, it should give more meaningful error messages to help guide things forward.

best,

~b

On Thu, Jun 1, 2023 at 11:24 AM phoebecunningham93 @.***> wrote:

Hi Brian,

Thanks for getting back to me. The problem is that although the directory seems to be populated with databases and a boilerplate after the step Trinotate --create --db myTrinotate.sqlite --trinotate_data_dir TrinData, the myTrinotate.sqlite file is apparently not created? When I run the initialisation command I don’t get anything and it runs really quickly, and then when I run the next command to do the sequence analysis it just comes up with the trinotate commands page and again very quickly. When I then try to create a report, its runs but I just get data missing in every column.

I’m not sure how best to share the problem but could you clarify the steps to creating the myTrinotate.sqlite file and what I need to have downloaded prior to this and to where? i.e. should SQlite and ncbi-blast be downloaded into the current working directory?

Thanks so much, Phoebe

From: Brian Haas @.> Date: Thursday, 1 June 2023 at 13:31 To: Trinotate/Trinotate.github.io @.> Cc: Phoebe Hazel Mary Cunningham @.>, Comment @.> Subject: Re: [Trinotate/Trinotate.github.io] Trinotate Annotation Report: No PFAM (#58) Hi,

The warning "Use of uninitialized value $blast_type in concatenation (.) or string at Trinotate-Trinotate-v4.0.0/Trinotate line 200." is just an annoyance that I overlooked earlier - shouldn't impact anything.

I'll be putting a patch release out shortly.

With v4.0.0, use the --run parameter with the list of analyses to compute or 'ALL' to run what it can (including tmhmm and signalpv6 if you have them installed). It should load results into the database on its own.

If you're experiencing other errors as part of the process, let me know

they should provide more insights.

best,

~b

On Thu, Jun 1, 2023 at 7:53 AM phoebecunningham93 @.***> wrote:

I am having the same problem. I have assembled the transciptome de novo and run all the transdecoder commands which seems to have workers. Now I have run all the commands laid out in the Trinotate github and created the myTrinotate.sqlite file but when I try to initialise it i get this error:

Use of uninitialized value $blast_type in concatenation (.) or string at Trinotate-Trinotate-v4.0.0/Trinotate line 200.

I think I may have misunderstood how to load in the databases - SQlite, NCBI, infernal and HMMER etc - I loaded these into my current working directory and then created an empty directory for the --trinotate_data_dir and then ran the create command. If I run through all the commands to the end I get no data in the myTrinotate.tsv report except column names and gene id's and identifiers. Can you help? I am quite a novice at this kind of bioinformatics.

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