add lncRNAs annotation workflow #579

rlibouba · 2024-10-22T08:32:29Z

Hello,
I would like to propose this workflow for annotating long non-coding RNAs from the GTN "Long non-coding RNAs (lncRNAs) annotation with FEELnc".

Thank you! Have a nice day!

pavanvidem

It looks good but seems to follow the GTN tutorial only. The idea here is to have some generic workflows. Please make all the important parameters available to the user to set although they are not used in the tutorial.

make the strandness param available to set by the user
take both GFF3/GTF as genome annotation inputs and make the gffread step as optional if already a GTF is provided
Use a tiny test data

workflows/genome_annotation/lncRNAs_annotation/Galaxy-Workflow-lncRNAs_annotation_workflow.ga

workflows/genome_annotation/lncRNAs_annotation/.dockstore.yml

workflows/genome_annotation/lncRNAs_annotation/Galaxy-Workflow-lncRNAs_annotation_workflow.ga

...s/genome_annotation/lncRNAs_annotation/Galaxy-Workflow-lncRNAs_annotation_workflow-tests.yml

rlibouba · 2025-01-16T12:24:14Z

Hi @pavanvidem, thank you!
I've made some changes in response to your comments.

...s/genome_annotation/lncRNAs_annotation/Galaxy-Workflow-lncRNAs_annotation_workflow-tests.yml

pavanvidem · 2025-01-16T16:54:22Z

thanks @rlibouba Can you please also address the following points:

Currently, the strandness param of the StringTie is set to "unstranded". But the input data can also be strand-specific. Please expose this parameter to the user
Wouldn't it possible to take both GFF3 and GTF as genome annotation inputs? StringTie can work with both types.
the current test data is too large (>5GB BAM file). Please subset (all the input files) to only one of the smaller scaffolds.

rlibouba · 2025-01-31T16:14:05Z

Hello @pavanvidem,
Thanks for your feedback.
I managed to reduce the bam file a bit. I can't reduce it any more, because FEELnc doesn't have enough elements to analyze. I was able to fix the problem with the strandness parameter.

Concerning the question with StringTie, you can give it both a GFF3 and a GTF file, but this generates two outputs, one for the GFF3 and another for the GTF.

github-actions · 2025-01-31T17:38:06Z

Test Results (powered by Planemo)

Test Summary

Test State	Count
Total	1
Passed	1
Error	0
Failure	0
Skipped	0

Passed Tests

✅ Galaxy-Workflow-lncRNAs_annotation_workflow.ga_0

Workflow invocation details

Invocation Messages

Steps

Step 1: strandedness:
- step_state: scheduled
Step 2: Genome assembly:
- step_state: scheduled
Step 3: Genome annotation:
- step_state: scheduled
Step 4: Alignments from RNA-seq:
- step_state: scheduled

Step 5: toolshed.g2.bx.psu.edu/repos/iuc/map_param_value/map_param_value/0.2.0:

step_state: scheduled

Jobs

Job 1:

Job state is ok

Command Line:

```
cd ../; python _evaluate_expression_.py
```

Exit Code:

```
0
```

Traceback:

Job Parameters:

Job parameter	Parameter value
__input_ext	`"input"`
__workflow_invocation_uuid__	`"84803768dff311efb9ab0022482354f6"`
chromInfo	`"/tmp/tmpdfutzrj0/galaxy-dev/tool-data/shared/ucsc/chrom/?.len"`
dbkey	`"?"`
input_param_type	`{"__current_case__": 0, "input_param": "unstranded", "mappings": [{"__index__": 0, "from": "stranded - forward", "to": "--fr"}, {"__index__": 1, "from": "stranded - reverse", "to": "--rf"}, {"__index__": 2, "from": "unstranded", "to": ""}], "type": "text"}`
output_param_type	`"text"`
unmapped	`{"__current_case__": 1, "on_unmapped": "fail"}`

Step 6: gffread:

step_state: scheduled

Jobs

Job 1:

Job state is ok

Command Line:

gffread '/tmp/tmpdfutzrj0/files/7/a/7/dataset_7a7291f4-216d-4b2a-8f72-cc0245fea1bb.dat'       -T -o output.gtf

Exit Code:

```
0
```

Traceback:

Job Parameters:

Job parameter	Parameter value
__input_ext	`"input"`
__workflow_invocation_uuid__	`"84803768dff311efb9ab0022482354f6"`
chr_replace	`None`
chromInfo	`"/tmp/tmpdfutzrj0/galaxy-dev/tool-data/shared/ucsc/chrom/?.len"`
dbkey	`"?"`
decode_url	`false`
expose	`false`
filtering	`None`
full_gff_attribute_preservation	`false`
gffs	`{"__current_case__": 2, "gff_fmt": "gtf", "tname": ""}`
maxintron	`None`
merging	`{"__current_case__": 0, "merge_sel": "none"}`
reference_genome	`{"__current_case__": 0, "source": "none"}`
region	`{"__current_case__": 0, "region_filter": "none"}`

Step 7: Assembly step with StringTie:

step_state: scheduled

Jobs

Job 1:

Job state is ok

Command Line:

mkdir -p ./special_de_output/sample1/ &&   ln -s '/tmp/tmpdfutzrj0/files/7/a/7/dataset_7a7291f4-216d-4b2a-8f72-cc0245fea1bb.dat' guide.gff &&  stringtie '/tmp/tmpdfutzrj0/files/a/3/e/dataset_a3ee76b1-007e-4967-a7eb-91c86b0a5580.dat'  -o '/tmp/tmpdfutzrj0/job_working_directory/000/6/outputs/dataset_f91a24f4-a7a7-4948-b344-b5a82b8757f4.dat' -p "${GALAXY_SLOTS:-1}"    -G guide.gff  -b ./special_de_output/sample1/  -f '0.01' -m '200' -a '10' -j '1' -c '1' -g '50' -M '1.0'

Exit Code:

```
0
```

Traceback:

Job Parameters:

Job parameter	Parameter value
__input_ext	`"input"`
__workflow_invocation_uuid__	`"84803768dff311efb9ab0022482354f6"`
adv	`{"abundance_estimation": false, "bdist": "50", "bundle_fraction": "1.0", "disable_trimming": false, "fraction": "0.01", "min_anchor_cov": "1", "min_anchor_len": "10", "min_bundle_cov": "1", "min_tlen": "200", "multi_mapping": false, "name_prefix": null, "omit_sequences": "", "point_features": null}`
chromInfo	`"/tmp/tmpdfutzrj0/galaxy-dev/tool-data/shared/ucsc/chrom/?.len"`
dbkey	`"?"`
guide	`{"__current_case__": 1, "coverage_file": false, "guide_source": {"__current_case__": 1, "guide_gff_select": "history", "ref_hist": {"values": [{"id": 2, "src": "hda"}]}}, "input_estimation": false, "special_outputs": {"__current_case__": 2, "special_outputs_select": "no"}, "use_guide": "yes"}`
input_options	`{"__current_case__": 0, "input_bam": {"values": [{"id": 3, "src": "hda"}]}, "input_mode": "short_reads"}`
rna_strandness	`""`

Step 8: lncRNA annotation step with FEELnc:

step_state: scheduled

Jobs

Job 1:

Job state is ok

Command Line:

export FEELNCPATH=$(dirname $(command -v FEELnc_pipeline.sh))/../ && ln -s '/tmp/tmpdfutzrj0/files/8/1/3/dataset_813eb582-14c0-44d3-9ec5-8dc0d7b50433.dat' genome_fa && FEELnc_pipeline.sh --candidate='/tmp/tmpdfutzrj0/files/f/9/1/dataset_f91a24f4-a7a7-4948-b344-b5a82b8757f4.dat' --reference='/tmp/tmpdfutzrj0/files/1/b/5/dataset_1b554f89-8220-4974-9e44-8f574ac9d678.dat' --genome=genome_fa --outname='candidate_lncRNA' --outdir='out_feelnc'  &&  cat out_feelnc/codpot/candidate_lncRNA.codpot_RF_summary.txt out_feelnc/classifier/candidate_lncRNA.classifier.log

Exit Code:

```
0
```

Standard Error:

Possible precedence issue with control flow operator at /usr/local/lib/site_perl/5.26.2/Bio/DB/IndexedBase.pm line 805.
Filtered transcripts will be available in file: 'out_feelnc/filter/candidate_lncRNA.filter.log'
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	Your input GTF file 'candidate_lncRNA.filter.gtf' contains *113* transcripts
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	Extracted '106' ORF/cDNAs sequences on '113'.
> Run random Forest on '/tmp//413_candidate_lncRNA.codpot.test_rna.fa'
	1. Compute the size of each sequence and ORF
	2. Compute the kmer ratio for each kmer and put the output file name in a list
	3. Compute the kmer score for each kmer size on learning and test ORF
	4. Merge the score and size files into one file for each type
	5. Make the model on learning sequences and apply it on test sequences
		Running 10-fold cross-validation on learning

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		10-fold cross-validation step is finish. Best threshold found: '0.6356'.
		Write the coding label for '413_candidate_lncRNA.codpot.modelTest.out' in 'out_feelnc/codpot//candidate_lncRNA.codpot_RF.txt'.
> Writing the GTF output files

Transcripts without ORF found: check file 'out_feelnc/codpot//candidate_lncRNA.codpot.noORF.gtf'.
	These transcripts correspond probably to lncRNAs since no ORF was found. You might also want to change the --testorftype option.
window : 10000 - max window : 100000 - lncrna : out_feelnc/codpot//candidate_lncRNA.codpot.lncRNA.gtf - mrna : /tmp/tmpdfutzrj0/files/1/b/5/dataset_1b554f89-8220-4974-9e44-8f574ac9d678.dat - biotype: 0
Possible precedence issue with control flow operator at /usr/local/lib/site_perl/5.26.2/Bio/DB/IndexedBase.pm line 805.
 Importation  merge GTF file : 106 lncRNA done 
 Importation  merge GTF file : 13795 mRNA done 
 ... Looking for interactions, currently eq to 300

Standard Output:

####################
Create output directory out_feelnc

####################
Step 1/3: Running the FEELnc filter module:

commande line:
	FEELnc_filter.pl --infile=/tmp/tmpdfutzrj0/files/f/9/1/dataset_f91a24f4-a7a7-4948-b344-b5a82b8757f4.dat --mRNAfile=/tmp/tmpdfutzrj0/files/1/b/5/dataset_1b554f89-8220-4974-9e44-8f574ac9d678.dat --biotype=transcript_biotype=protein_coding --monoex=-1 --outlog=out_feelnc/filter/candidate_lncRNA.filter.log > out_feelnc/filter/candidate_lncRNA.filter.gtf


####################
Step 2/3: Running the FEELnc codpot module:

commande line:
	FEELnc_codpot.pl --infile=out_feelnc/filter/candidate_lncRNA.filter.gtf --mRNAfile=/tmp/tmpdfutzrj0/files/1/b/5/dataset_1b554f89-8220-4974-9e44-8f574ac9d678.dat --genome=genome_fa -b transcript_biotype=protein_coding --mode=shuffle --outname=candidate_lncRNA.codpot --outdir=out_feelnc/codpot/


####################
Step 3/3: Running the FEELnc classifier module:

commande line:
	FEELnc_classifier.pl --lncrna=out_feelnc/codpot//candidate_lncRNA.codpot.lncRNA.gtf --mrna=/tmp/tmpdfutzrj0/files/1/b/5/dataset_1b554f89-8220-4974-9e44-8f574ac9d678.dat --log=out_feelnc/classifier/candidate_lncRNA.classifier.log > out_feelnc/classifier/candidate_lncRNA.classifier.txt



####################
Done: results in out_feelnc/{filter;codpot;classifier}

# Summary file:
-With_cutoff:	0.6356
-Nb_lncRNAs:	106
-Nb_mRNAs:	NA
#FEELnc Classification
#lncRNA file :  lncrna : out_feelnc/codpot//candidate_lncRNA.codpot.lncRNA.gtf 
#mRNA file : /tmp/tmpdfutzrj0/files/1/b/5/dataset_1b554f89-8220-4974-9e44-8f574ac9d678.dat
#Minimal window size : 10000
#Maximal window size : 100000
#Number of lncRNA : 106 
#Number of mRNA : 13795
#Number of interaction : 695 
#Number of lncRNA without interaction : 0
#List of lncRNA without interaction :

Traceback:

Job Parameters:

Job parameter	Parameter value
__input_ext	`"input"`
__workflow_invocation_uuid__	`"84803768dff311efb9ab0022482354f6"`
chromInfo	`"/tmp/tmpdfutzrj0/galaxy-dev/tool-data/shared/ucsc/chrom/?.len"`
dbkey	`"?"`

Step 9: Concatenation step:

step_state: scheduled

Jobs

Job 1:

Job state is ok

Command Line:

python /tmp/tmpdfutzrj0/galaxy-dev/tools/filters/catWrapper.py '/tmp/tmpdfutzrj0/job_working_directory/000/8/outputs/dataset_263acba7-c500-46ef-82e6-ddd2e8c74464.dat' '/tmp/tmpdfutzrj0/files/e/d/a/dataset_eda56677-43d0-43a3-bcbd-51e9507a3f2d.dat' '/tmp/tmpdfutzrj0/files/1/b/5/dataset_1b554f89-8220-4974-9e44-8f574ac9d678.dat'

Exit Code:

```
0
```

Traceback:

Job Parameters:

Job parameter	Parameter value
__input_ext	`"gtf"`
__workflow_invocation_uuid__	`"84803768dff311efb9ab0022482354f6"`
chromInfo	`"/tmp/tmpdfutzrj0/galaxy-dev/tool-data/shared/ucsc/chrom/?.len"`
dbkey	`"?"`
queries	`[{"__index__": 0, "input2": {"values": [{"id": 5, "src": "hda"}]}}]`

Other invocation details
- history_id
  - 5982786cc98d0e7f
- history_state
  - ok
- invocation_id
  - 5982786cc98d0e7f
- invocation_state
  - scheduled
- workflow_id
  - 5982786cc98d0e7f

pavanvidem · 2025-02-03T06:31:46Z

hi @rlibouba thanks for adapting the workflow.

please add a release
make sure that all the inputs have the annotation
please check that no underscrores are allowed in the inputs and output names

workflows/genome_annotation/lncRNAs-annotation/CHANGELOG.md

mvdbeek

Thanks so much @rlibouba!

add lncRNAs annotation workflow

c2cf176

rlibouba marked this pull request as draft October 22, 2024 08:32

rlibouba added 5 commits October 22, 2024 10:54

add release and change license

b48793a

error test

3ac89f0

url error

2174cfa

url error

5ccad2a

addition of coloured borders and titles

cc83d92

rlibouba marked this pull request as ready for review November 29, 2024 10:44

update README

0704b9b

pavanvidem reviewed Jan 15, 2025

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adding label and correcting test

943ec1a

pavanvidem reviewed Jan 16, 2025

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...s/genome_annotation/lncRNAs_annotation/Galaxy-Workflow-lncRNAs_annotation_workflow-tests.yml Outdated Show resolved Hide resolved

rename RNA-Seq by Alignments from RNA-seq

5547f3e

dataet reduced and script modification

8778893

add release

0a0bef1

mvdbeek reviewed Mar 5, 2025

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workflows/genome_annotation/lncRNAs-annotation/CHANGELOG.md Outdated Show resolved Hide resolved

update CHANGELOG.md

d0a4bbf

mvdbeek approved these changes Mar 6, 2025

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mvdbeek merged commit 3a1a9d0 into galaxyproject:main Mar 6, 2025
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add lncRNAs annotation workflow #579

add lncRNAs annotation workflow #579

rlibouba commented Oct 22, 2024

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rlibouba commented Jan 16, 2025

pavanvidem commented Jan 16, 2025 •

edited

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rlibouba commented Jan 31, 2025

github-actions bot commented Jan 31, 2025

Workflow invocation details

pavanvidem commented Feb 3, 2025

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add lncRNAs annotation workflow #579

add lncRNAs annotation workflow #579

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rlibouba commented Jan 16, 2025

pavanvidem commented Jan 16, 2025 • edited Loading

rlibouba commented Jan 31, 2025

github-actions bot commented Jan 31, 2025

Test Results (powered by Planemo)

Test Summary

Workflow invocation details

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pavanvidem commented Jan 16, 2025 •

edited

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