Error running demo data in wf-transcriptomes after removing --direct_rna

[bioshendi]

Ask away!

nextflow run epi2me-labs/wf-transcriptomes --de_analysis --fastq 'wf-transcriptomes-demo/differential_expression_fastq' --minimap2_index_opts '-k 15' --ref_annotation 'wf-transcriptomes-demo/gencode.v22.annotation.chr20.gtf' --ref_genome 'wf-transcriptomes-demo/hg38_chr20.fa' --sample_sheet 'wf-transcriptomes-demo/sample_sheet.csv' -profile standard

N E X T F L O W ~ version 25.10.0

Launching https://github.com/epi2me-labs/wf-transcriptomes [elated_agnesi] DSL2 - revision: 27d12e3 [master]

|||||||||| _____ ____ ___ ____ __ __ _____
|||||||||| | | _ _ | | / | |
||||| | | | |) | | ) | |/| | |
||||| | || /| | / /| | | | |
|||||||||| |___|| ||_____|| ||_____|
|||||||||| wf-transcriptomes v1.7.2-g27d12e3

Core Nextflow options
revision : master
runName : elated_agnesi
containerEngine : docker
container : [withLabel:isoforms:ontresearch/wf-transcriptomes:shaaaf20a5a0e76f9e18bad21af639a6b69e4a31a2f, withLabel:wf_common:ontresearch/wf-common:sha72f3517dd994984e0e2da0b97cb3f23f8540be4b]
launchDir : /home/dell/jia/epi2me
workDir : /home/dell/jia/epi2me/work
projectDir : /home/dell/.nextflow/assets/epi2me-labs/wf-transcriptomes
userName : dell
profile : standard
configFiles : /home/dell/.nextflow/assets/epi2me-labs/wf-transcriptomes/nextflow.config

Input Options
fastq : wf-transcriptomes-demo/differential_expression_fastq
ref_genome : wf-transcriptomes-demo/hg38_chr20.fa
ref_annotation : wf-transcriptomes-demo/gencode.v22.annotation.chr20.gtf

Sample Options
sample_sheet : wf-transcriptomes-demo/sample_sheet.csv

Options for reference-based workflow
minimap2_index_opts: -k 15

Differential Expression Options
de_analysis : true

!! Only displaying parameters that differ from the pipeline defaults !!

If you use epi2me-labs/wf-transcriptomes for your analysis please cite:

• The nf-core framework
  https://doi.org/10.1038/s41587-020-0439-x

---

This is epi2me-labs/wf-transcriptomes v1.7.2-g27d12e3.

Searching input for [.fastq, .fastq.gz, .fq, .fq.gz] files.
executor > local (109)
[ff/68689a] process > validate_sample_sheet [100%] 1 of 1 ✔
[2d/fdad3e] process > fastcat (4) [100%] 6 of 6 ✔
[ed/ba281d] process > pipeline:preprocess_ref_annotation [100%] 1 of 1 ✔
[4b/b2f719] process > pipeline:collectFastqIngressResultsInDir (6) [100%] 6 of 6 ✔
[44/c142f8] process > pipeline:getVersions [100%] 1 of 1 ✔
[25/d18b98] process > pipeline:getParams [100%] 1 of 1 ✔
[72/9a127d] process > pipeline:preprocess_reads (2) [100%] 6 of 6 ✔
[85/4cbbae] process > pipeline:build_minimap_index (1) [100%] 1 of 1 ✔
[a7/a13cdf] process > pipeline:reference_assembly:map_reads (5) [100%] 6 of 6 ✔
[69/55bbe0] process > pipeline:split_bam (6) [100%] 6 of 6 ✔
[fc/af0afe] process > pipeline:assemble_transcripts (6) [100%] 6 of 6 ✔
[cf/e07378] process > pipeline:merge_gff_bundles (1) [100%] 6 of 6 ✔
[d6/24c363] process > pipeline:run_gffcompare (5) [100%] 6 of 6 ✔
[9c/57df6a] process > pipeline:get_transcriptome (6) [100%] 6 of 6 ✔
[25/821b31] process > pipeline:validate_ref_annotation (1) [100%] 1 of 1 ✔
[c1/f69f19] process > pipeline:merge_transcriptomes (1) [100%] 1 of 1 ✔
[01/2161c6] process > pipeline:filter_unstranded_annotation (1) [100%] 1 of 1 ✔
[97/95da76] process > pipeline:differential_expression:checkSampleSheetCondition (1) [100%] 1 of 1 ✔
[a3/101738] process > pipeline:differential_expression:build_minimap_index_transcriptome (1) [100%] 1 of 1 ✔
[4a/48f86e] process > pipeline:differential_expression:map_transcriptome (5) [100%] 6 of 6 ✔
[9d/a0b588] process > pipeline:differential_expression:count_transcripts (6) [100%] 6 of 6 ✔
[a8/d582d1] process > pipeline:differential_expression:mergeCounts [100%] 1 of 1 ✔
[4e/946a28] process > pipeline:differential_expression:mergeTPM [100%] 1 of 1 ✔
[45/04ff42] process > pipeline:differential_expression:deAnalysis (1) [ 0%] 0 of 1
[- ] process > pipeline:differential_expression:plotResults -
[- ] process > pipeline:makeReport -
[c8/122d57] process > publish_results (30) [100%] 30 of 30
ERROR ~ Error executing process > 'pipeline:differential_expression:deAnalysis (1)'

Caused by:
Process pipeline:differential_expression:deAnalysis (1) terminated with an error exit status (1)

executor > local (109)
[ff/68689a] process > validate_sample_sheet [100%] 1 of 1 ✔
[2d/fdad3e] process > fastcat (4) [100%] 6 of 6 ✔
[ed/ba281d] process > pipeline:preprocess_ref_annotation [100%] 1 of 1 ✔
[4b/b2f719] process > pipeline:collectFastqIngressResultsInDir (6) [100%] 6 of 6 ✔
[44/c142f8] process > pipeline:getVersions [100%] 1 of 1 ✔
[25/d18b98] process > pipeline:getParams [100%] 1 of 1 ✔
[72/9a127d] process > pipeline:preprocess_reads (2) [100%] 6 of 6 ✔
[85/4cbbae] process > pipeline:build_minimap_index (1) [100%] 1 of 1 ✔
[a7/a13cdf] process > pipeline:reference_assembly:map_reads (5) [100%] 6 of 6 ✔
[69/55bbe0] process > pipeline:split_bam (6) [100%] 6 of 6 ✔
[fc/af0afe] process > pipeline:assemble_transcripts (6) [100%] 6 of 6 ✔
[cf/e07378] process > pipeline:merge_gff_bundles (1) [100%] 6 of 6 ✔
[d6/24c363] process > pipeline:run_gffcompare (5) [100%] 6 of 6 ✔
[9c/57df6a] process > pipeline:get_transcriptome (6) [100%] 6 of 6 ✔
[25/821b31] process > pipeline:validate_ref_annotation (1) [100%] 1 of 1 ✔
[c1/f69f19] process > pipeline:merge_transcriptomes (1) [100%] 1 of 1 ✔
[01/2161c6] process > pipeline:filter_unstranded_annotation (1) [100%] 1 of 1 ✔
[97/95da76] process > pipeline:differential_expression:checkSampleSheetCondition (1) [100%] 1 of 1 ✔
[a3/101738] process > pipeline:differential_expression:build_minimap_index_transcriptome (1) [100%] 1 of 1 ✔
[4a/48f86e] process > pipeline:differential_expression:map_transcriptome (5) [100%] 6 of 6 ✔
[9d/a0b588] process > pipeline:differential_expression:count_transcripts (6) [100%] 6 of 6 ✔
[a8/d582d1] process > pipeline:differential_expression:mergeCounts [100%] 1 of 1 ✔
[4e/946a28] process > pipeline:differential_expression:mergeTPM [100%] 1 of 1 ✔
[45/04ff42] process > pipeline:differential_expression:deAnalysis (1) [ 0%] 0 of 1 ✘
[- ] process > pipeline:differential_expression:plotResults -
[- ] process > pipeline:makeReport [ 0%] 0 of 1
[c8/122d57] process > publish_results (30) [100%] 30 of 30
ERROR ~ Error executing process > 'pipeline:differential_expression:deAnalysis (1)'

Caused by:
Process pipeline:differential_expression:deAnalysis (1) terminated with an error exit status (1)

executor > local (109)
[ff/68689a] process > validate_sample_sheet [100%] 1 of 1 ✔
[2d/fdad3e] process > fastcat (4) [100%] 6 of 6 ✔
[ed/ba281d] process > pipeline:preprocess_ref_annotation [100%] 1 of 1 ✔
[4b/b2f719] process > pipeline:collectFastqIngressResultsInDir (6) [100%] 6 of 6 ✔
[44/c142f8] process > pipeline:getVersions [100%] 1 of 1 ✔
[25/d18b98] process > pipeline:getParams [100%] 1 of 1 ✔
[72/9a127d] process > pipeline:preprocess_reads (2) [100%] 6 of 6 ✔
[85/4cbbae] process > pipeline:build_minimap_index (1) [100%] 1 of 1 ✔
[a7/a13cdf] process > pipeline:reference_assembly:map_reads (5) [100%] 6 of 6 ✔
[69/55bbe0] process > pipeline:split_bam (6) [100%] 6 of 6 ✔
[fc/af0afe] process > pipeline:assemble_transcripts (6) [100%] 6 of 6 ✔
[cf/e07378] process > pipeline:merge_gff_bundles (1) [100%] 6 of 6 ✔
[d6/24c363] process > pipeline:run_gffcompare (5) [100%] 6 of 6 ✔
[9c/57df6a] process > pipeline:get_transcriptome (6) [100%] 6 of 6 ✔
[25/821b31] process > pipeline:validate_ref_annotation (1) [100%] 1 of 1 ✔
[c1/f69f19] process > pipeline:merge_transcriptomes (1) [100%] 1 of 1 ✔
[01/2161c6] process > pipeline:filter_unstranded_annotation (1) [100%] 1 of 1 ✔
[97/95da76] process > pipeline:differential_expression:checkSampleSheetCondition (1) [100%] 1 of 1 ✔
[a3/101738] process > pipeline:differential_expression:build_minimap_index_transcriptome (1) [100%] 1 of 1 ✔
[4a/48f86e] process > pipeline:differential_expression:map_transcriptome (5) [100%] 6 of 6 ✔
[9d/a0b588] process > pipeline:differential_expression:count_transcripts (6) [100%] 6 of 6 ✔
[a8/d582d1] process > pipeline:differential_expression:mergeCounts [100%] 1 of 1 ✔
[4e/946a28] process > pipeline:differential_expression:mergeTPM [100%] 1 of 1 ✔
[45/04ff42] process > pipeline:differential_expression:deAnalysis (1) [ 0%] 0 of 1 ✘
[- ] process > pipeline:differential_expression:plotResults -
[- ] process > pipeline:makeReport [ 0%] 0 of 1
[c8/122d57] process > publish_results (30) [100%] 30 of 30
ERROR ~ Error executing process > 'pipeline:differential_expression:deAnalysis (1)'

Caused by:
Process pipeline:differential_expression:deAnalysis (1) terminated with an error exit status (1)

Command executed:

de_analysis.R --annotation annotation.gtf --min_samps_gene_expr 3 --min_samps_feature_expr 1 --min_gene_expr 10 --min_feature_expr 3 --sample_sheet sample_sheet.csv --all_counts all_counts.tsv --de_out_dir de_analysis --merged_out_dir merged

Check that the original aliases in the input TSV have not been mangled by R's read.csv or other functions

Sample column order should be in the same order as the input sample sheet

alias_col=$(awk -v RS=',' '/alias/{print NR; exit}' "sample_sheet.csv")
cut -d',' -f3 sample_sheet.csv | tail -n +2 | paste -sd ' ... ' - | sed 's/ *$//' > expected_colnames

head -1 de_analysis/cpm_gene_counts.tsv | cut -f2- > cpm_gene_counts_colnames
head -1 merged/all_gene_counts.tsv > merged_counts_colnames
head -1 merged/filtered_transcript_counts_with_genes.tsv | cut -f3- > merged_filtered_colnames

Check for mismatches in sample column names

for file in cpm_gene_counts_colnames merged_counts_colnames merged_filtered_colnames; do
if ! diff -q $file expected_colnames > /dev/null; then
echo "Column names in $file do not match expected aliases."
exit 70
fi
done

Command exit status:
1

Command output:
Loading counts, conditions and parameters.
Checking annotation file type.
Annotation file type is gtf.
Checking annotation file for presence of transcript_id versions.
Annotation file transcript_ids include versions.
Loading annotation database.
Filtering counts using DRIMSeq.

Command error:
Loading counts, conditions and parameters.
Checking annotation file type.
Annotation file type is gtf.
Checking annotation file for presence of transcript_id versions.
Annotation file transcript_ids include versions.
Loading annotation database.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
'select()' returned 1:many mapping between keys and columns
Filtering counts using DRIMSeq.
Error in dmDS_filter(counts = x@counts, min_samps_gene_expr = min_samps_gene_expr, :
!No genes left after filtering!
Calls: dmFilter -> dmFilter -> .local -> dmDS_filter
Execution halted

Work dir:
/home/dell/jia/epi2me/work/45/04ff42dbb44735356b6d18230da852

Container:
ontresearch/wf-transcriptomes:shaaaf20a5a0e76f9e18bad21af639a6b69e4a31a2f

Tip: when you have fixed the problem you can continue the execution adding the option -resume to the run command line

-- Check '.nextflow.log' file for details

Think you