Filter out unstranded annotations and log warning [CW-6125]
This commit is contained in:
Sarah Griffiths
parent
9f18c3f0d5
commit
85b767358c
@ -54,7 +54,7 @@ docker-run:
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"only_differential_expression", "differential_expression_gff3",
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"ncbi_gzip", "ncbi_no_gene_id", "ensembl_with_versions",
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"differential_expression_mouse", "no_ref_annotation",
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"unstranded_annotation_error", "igv", "igv_fai_gz",
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"unstranded_annotation_warn", "igv", "igv_fai_gz",
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"mismatch_seqid_error","mismatch_seqid_warn"
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]
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rules:
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@ -68,21 +68,21 @@ docker-run:
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NF_BEFORE_SCRIPT: "mkdir -p ${CI_PROJECT_NAME}/data/ && wget -O ${CI_PROJECT_NAME}/data/wf-isoforms_test_data.tar.gz https://ont-exd-int-s3-euwst1-epi2me-labs.s3.amazonaws.com/wf-isoforms/wf-isoforms_test_data.tar.gz && tar -xzvf ${CI_PROJECT_NAME}/data/wf-isoforms_test_data.tar.gz -C ${CI_PROJECT_NAME}/data/ && wget -O ${CI_PROJECT_NAME}/data/demo.nextflow.config https://ont-exd-int-s3-euwst1-epi2me-labs.s3.amazonaws.com/wf-isoforms/demo.nextflow.config;"
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NF_WORKFLOW_OPTS: "--fastq ${CI_PROJECT_NAME}/data/ERR6053095_chr20.fastq --transcriptome_source reference-guided \
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--ref_genome ${CI_PROJECT_NAME}/data/chr20/hg38_chr20.fa --ref_annotation ${CI_PROJECT_NAME}/data/chr20/gencode.v22.annotation.chr20.gtf --pychopper_backend phmm"
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NF_IGNORE_PROCESSES: preprocess_reads,validate_ref_annotation,faidx,gz_faidx,check_annotation_strand,merge_transcriptomes,decompress_annotation,decompress_ref,decompress_transcriptome,preprocess_ref_transcriptome
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NF_IGNORE_PROCESSES: preprocess_reads,filter_unstranded_annotation,validate_ref_annotation,faidx,gz_faidx,merge_transcriptomes,decompress_annotation,decompress_ref,decompress_transcriptome,preprocess_ref_transcriptome
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- if: $MATRIX_NAME == "isoforms_bam"
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variables:
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NF_BEFORE_SCRIPT: "mkdir -p ${CI_PROJECT_NAME}/data/ && wget -O ${CI_PROJECT_NAME}/data/wf-isoforms_test_data.tar.gz https://ont-exd-int-s3-euwst1-epi2me-labs.s3.amazonaws.com/wf-isoforms/wf-isoforms_test_data.tar.gz && tar -xzvf ${CI_PROJECT_NAME}/data/wf-isoforms_test_data.tar.gz -C ${CI_PROJECT_NAME}/data/ && wget -O ${CI_PROJECT_NAME}/data/demo.nextflow.config https://ont-exd-int-s3-euwst1-epi2me-labs.s3.amazonaws.com/wf-isoforms/demo.nextflow.config;"
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NF_WORKFLOW_OPTS: "--bam ${CI_PROJECT_NAME}/data/ERR6053095_chr20.bam --transcriptome_source reference-guided \
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--ref_genome ${CI_PROJECT_NAME}/data/chr20/hg38_chr20.fa --ref_annotation ${CI_PROJECT_NAME}/data/chr20/gencode.v22.annotation.chr20.gtf --pychopper_backend phmm \
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-c ${CI_PROJECT_NAME}/data/demo.nextflow.config "
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NF_IGNORE_PROCESSES: preprocess_reads,validate_ref_annotation,faidx,gz_faidx,check_annotation_strand,merge_transcriptomes,decompress_annotation,decompress_ref,decompress_transcriptome,preprocess_ref_transcriptome
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NF_IGNORE_PROCESSES: preprocess_reads,filter_unstranded_annotation,validate_ref_annotation,faidx,gz_faidx,merge_transcriptomes,decompress_annotation,decompress_ref,decompress_transcriptome,preprocess_ref_transcriptome
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- if: $MATRIX_NAME == "no_ref_annotation"
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variables:
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NF_BEFORE_SCRIPT: "mkdir -p ${CI_PROJECT_NAME}/data/ && wget -O ${CI_PROJECT_NAME}/data/wf-isoforms_test_data.tar.gz https://ont-exd-int-s3-euwst1-epi2me-labs.s3.amazonaws.com/wf-isoforms/wf-isoforms_test_data.tar.gz && tar -xzvf ${CI_PROJECT_NAME}/data/wf-isoforms_test_data.tar.gz -C ${CI_PROJECT_NAME}/data/ && wget -O ${CI_PROJECT_NAME}/data/demo.nextflow.config https://ont-exd-int-s3-euwst1-epi2me-labs.s3.amazonaws.com/wf-isoforms/demo.nextflow.config;"
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NF_WORKFLOW_OPTS: "--fastq ${CI_PROJECT_NAME}/data/ERR6053095_chr20.fastq --transcriptome_source reference-guided \
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--ref_genome ${CI_PROJECT_NAME}/data/chr20/hg38_chr20.fa \
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-c ${CI_PROJECT_NAME}/data/demo.nextflow.config "
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NF_IGNORE_PROCESSES: run_gffcompare,validate_ref_annotation,check_annotation_strand,preprocess_reads,faidx,gz_faidx,merge_transcriptomes,decompress_annotation,decompress_ref,decompress_transcriptome,preprocess_ref_transcriptome
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NF_IGNORE_PROCESSES: run_gffcompare,filter_unstranded_annotation,validate_ref_annotation,preprocess_reads,faidx,gz_faidx,merge_transcriptomes,decompress_annotation,decompress_ref,decompress_transcriptome,preprocess_ref_transcriptome
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- if: $MATRIX_NAME == "differential_expression"
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variables:
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NF_BEFORE_SCRIPT: "mkdir -p ${CI_PROJECT_NAME}/data/ && wget -O ${CI_PROJECT_NAME}/data/differential_expression.tar.gz https://ont-exd-int-s3-euwst1-epi2me-labs.s3.amazonaws.com/wf-isoforms/differential_expression.tar.gz && tar -xzvf ${CI_PROJECT_NAME}/data/differential_expression.tar.gz -C ${CI_PROJECT_NAME}/data/ && wget -O ${CI_PROJECT_NAME}/data/demo.nextflow.config https://ont-exd-int-s3-euwst1-epi2me-labs.s3.amazonaws.com/wf-isoforms/demo.nextflow.config;"
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@ -179,7 +179,7 @@ docker-run:
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NF_IGNORE_PROCESSES: >
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preprocess_reads,validate_ref_annotation,faidx,gz_faidx,merge_transcriptomes,assemble_transcripts,decompress_annotation,
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build_minimap_index,get_transcriptome,merge_gff_bundles,run_gffcompare,build_minimap_index,split_bam
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- if: $MATRIX_NAME == "unstranded_annotation_error"
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- if: $MATRIX_NAME == "unstranded_annotation_warn"
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variables:
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NF_BEFORE_SCRIPT: "mkdir -p ${CI_PROJECT_NAME}/data/ && wget -O ${CI_PROJECT_NAME}/data/differential_expression.tar.gz https://ont-exd-int-s3-euwst1-epi2me-labs.s3.amazonaws.com/wf-transcriptomes/differential_expression.tar.gz && tar -xzvf ${CI_PROJECT_NAME}/data/differential_expression.tar.gz -C ${CI_PROJECT_NAME}/data/ && wget -O ${CI_PROJECT_NAME}/data/demo.nextflow.config https://ont-exd-int-s3-euwst1-epi2me-labs.s3.amazonaws.com/wf-isoforms/demo.nextflow.config;"
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NF_WORKFLOW_OPTS: "--fastq ${CI_PROJECT_NAME}/data/differential_expression/differential_expression_fastq \
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@ -189,8 +189,7 @@ docker-run:
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--direct_rna --minimap2_index_opts '-k 15' --sample_sheet ${CI_PROJECT_NAME}/data/differential_expression/sample_sheet.csv \
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-c ${CI_PROJECT_NAME}/data/demo.nextflow.config "
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NF_IGNORE_PROCESSES: preprocess_reads,faidx,gz_faidx,merge_transcriptomes,decompress_annotation,decompress_ref,decompress_transcriptome,preprocess_ref_transcriptome
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ASSERT_NEXTFLOW_FAILURE: "test_fail" # set to any non-zero length str to allow the nextflow CMD to fail
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ASSERT_NEXTFLOW_FAILURE_REXP: "In ref_annotation, transcript features must have a strand of either '+' or '-'"
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AFTER_NEXTFLOW_CMD: "grep 'Unstranded entries found and excluded from differential expression analysis.' '.nextflow.log'"
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- if: $MATRIX_NAME == "igv"
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variables:
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NF_BEFORE_SCRIPT: "mkdir -p ${CI_PROJECT_NAME}/data/ && wget -O ${CI_PROJECT_NAME}/data/differential_expression.tar.gz https://ont-exd-int-s3-euwst1-epi2me-labs.s3.amazonaws.com/wf-isoforms/differential_expression.tar.gz && tar -xzvf ${CI_PROJECT_NAME}/data/differential_expression.tar.gz -C ${CI_PROJECT_NAME}/data/ && wget -O ${CI_PROJECT_NAME}/data/demo.nextflow.config https://ont-exd-int-s3-euwst1-epi2me-labs.s3.amazonaws.com/wf-isoforms/demo.nextflow.config;"
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@ -18,6 +18,8 @@ and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0
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- Sequence summary read length N50 incorrectly displayed minimum read length, it now correctly shows the N50.
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- Sequence summary component alignment and coverage plots failed to plot under some conditions.
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- Error in `deAnalysis` process - `mode(counts) %in% "numeric" is not TRUE` - caused by hyphens in sample sheet aliases.
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- Error in `deAnalysis` process - `values in 'transcripts$tx_strand' must be "+" or "-"`.
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- The workflow will now filter out any unstranded annotations from downstream analysis and log a warning.
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## [v1.7.0]
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### Changed
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47
main.nf
47
main.nf
@ -540,16 +540,37 @@ process publish_results {
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// Check ref_annotation transcript strand column for "." if in de_analysis mode
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process check_annotation_strand {
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process filter_unstranded_annotation {
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label "isoforms"
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cpus 1
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memory "2 GB"
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input:
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path "ref_annotation.gtf"
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output:
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tuple stdout, path("ref_annotation.gtf")
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tuple stdout, path("ref_annotation_stranded.gtf")
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script:
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"""
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awk '{if (\$3=="transcript" && \$7 != "+" && \$7 != "-") print \$3, \$7}' "ref_annotation.gtf"
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awk '
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BEGIN { OFS = "\\t" }
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/^#/ { print; next }
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# Valid strands
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(\$7 == "+" || \$7 == "-") { print; next }
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# Invalid strand
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{
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print \$0 >> "unstranded.gtf"
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}
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' ref_annotation.gtf > ref_annotation_stranded.gtf
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# Check and log warning if unstranded entries exist
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if [ -s unstranded.gtf ]; then
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echo "Warning: Unstranded entries found and excluded from differential expression analysis.
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If running with reference-guided transcriptome source, consider increasing read depth to reduce unstranded annotations.
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If running with precomputed transcriptome source, ensure your ref_annotation gtf file contains only '+' or '-' strand entries."
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echo "A sample of unstranded entries:"
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head -n 20 unstranded.gtf
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fi
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"""
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}
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@ -727,12 +748,6 @@ workflow pipeline {
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}
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if (params.de_analysis){
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sample_sheet = file(params.sample_sheet, type:"file")
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// check ref annotation contains only + or - strand as DE analysis will error on .
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check_annotation_strand(ref_annotation).map { stdoutput, annotation ->
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// check if there was an error message
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if (stdoutput) error "In ref_annotation, transcript features must have a strand of either '+' or '-'."
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stdoutput
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}
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if (!params.ref_transcriptome){
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validate_ref_annotation(ref_annotation, ref_genome).map { stdoutput ->
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if (stdoutput) {
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@ -751,7 +766,19 @@ workflow pipeline {
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transcriptome = preprocess_ref_transcriptome(transcriptome)
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gtf = ref_annotation
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}
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de = differential_expression(transcriptome, full_len_reads.map{ sample_id, reads -> [[alias:sample_id], reads]}, sample_sheet, gtf)
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// Filter out any .(unstranded) records
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// only + or - strand allowed for DE analysis.
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stranded_annotation = filter_unstranded_annotation(gtf)
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.map{ stdoutput, annotation -> annotation
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if (stdoutput) {
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log.warn(stdoutput)
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}
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annotation
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}
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de = differential_expression(
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transcriptome,
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full_len_reads.map{ sample_id, fq_reads -> [[alias:sample_id], fq_reads]},
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sample_sheet, stranded_annotation)
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de_report = de.all_de
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de_outputs = de.de_outputs
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de_alignment_stats = de.de_alignment_stats
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