From e6f1e1f1a28d983fa1477c1c885fe9b1e91e24a7 Mon Sep 17 00:00:00 2001 From: Brad Langhorst Date: Mon, 13 Oct 2025 16:00:43 -0400 Subject: [PATCH 1/6] adds updated feature_cov workflow --- README.md | 16 +- conf/references.config | 41 +- conf/test.config | 6 +- feature_cov.nf | 192 + main.nf | 14 +- modules/feature_counts.nf | 53 + modules/feature_methylation.nf | 48 + modules/prepare_features.nf | 239 + prepare_refseq_test_features.sh | 50 + tests/feature_cov.nf.test | 30 + tests/fixtures/aligned_bam/emseq-test1.md.bai | Bin 0 -> 664 bytes tests/fixtures/aligned_bam/emseq-test1.md.bam | Bin 0 -> 162821 bytes tests/fixtures/features/epd_promoters.bed | 10 + tests/fixtures/features/grch38-chm13v2.chain | 708310 +++++++++++++++ .../fixtures/features/prepare_refseq_test.sh | 4 + .../features/test_assembly_report.txt | 50 + tests/fixtures/features/test_cpgIslandExt.bb | Bin 0 -> 25902 bytes tests/fixtures/features/test_dfam.out | 50 + tests/fixtures/features/test_refseq.gff.gz | Bin 0 -> 603314 bytes 19 files changed, 709097 insertions(+), 16 deletions(-) create mode 100644 feature_cov.nf create mode 100644 modules/feature_counts.nf create mode 100644 modules/feature_methylation.nf create mode 100644 modules/prepare_features.nf create mode 100644 prepare_refseq_test_features.sh create mode 100644 tests/feature_cov.nf.test create mode 100644 tests/fixtures/aligned_bam/emseq-test1.md.bai create mode 100644 tests/fixtures/aligned_bam/emseq-test1.md.bam create mode 100644 tests/fixtures/features/epd_promoters.bed create mode 100644 tests/fixtures/features/grch38-chm13v2.chain create mode 100644 tests/fixtures/features/prepare_refseq_test.sh create mode 100644 tests/fixtures/features/test_assembly_report.txt create mode 100644 tests/fixtures/features/test_cpgIslandExt.bb create mode 100644 tests/fixtures/features/test_dfam.out create mode 100644 tests/fixtures/features/test_refseq.gff.gz diff --git a/README.md b/README.md index cc7e72d..c39d9b7 100644 --- a/README.md +++ b/README.md @@ -27,7 +27,7 @@ nextflow run fastq_to_ubam.nf \ ## Quick Start 1. Install [miniforge](https://conda-forge.org/download/) and [bioconda](https://bioconda.github.io/) (see [Requirements]()) 2. Install [Nextflow](https://www.nextflow.io/) (e.g. conda install nextflow, or see [Nextflow installation guide](https://www.nextflow.io/docs/latest/getstarted.html#installation)) -3. Clone this repository (`git clone https://github.com/nebiolabs/EM-seq.git`). Modify `nextflow.config` as needed for your environment, e.g. if running locally, change executor block to 'local' and set, e.g. `--max_cpus 10 --max_memory 30.GB`. +3. Clone this repository (`git clone https://github.com/nebiolabs/EM-seq.git`). Modify `nextflow.config` as needed for your environment, e.g. if running locally, change executor block to 'local' and set, e.g. `--max_cpus 10 --max_memory 30.GB`. 4. Download or prepare a genome reference FASTA file (see [Reference Genomes]()) 5. Create a bwameth index for the fasta and add it to your references in conf/references.config 6. Run the pipeline with appropriate parameters (see [Basic Usage]()) @@ -59,10 +59,10 @@ nextflow run main.nf \ ### References Config Modify the conf/references.config file to specify your genome files -- `genome_fa` path to your genome fasta file -- `genome_fai` path to your genome fasta fai file -- `bwameth_index` path to your genome fasta file where bwameth indices exist -- `target_bed` BED file for targeted analysis, Optional +- `genome_fa` path to your genome fasta file +- `genome_fai` path to your genome fasta fai file +- `bwameth_index` path to your genome fasta file where bwameth indices exist +- `target_bed` BED file for targeted analysis, Optional ### Advanced Options - `--tmp_dir` - Temporary directory (default: `/tmp`) @@ -77,7 +77,8 @@ Pre-built reference genomes with methylation spike-in controls: - Create your own reference by appending the [control sequences](methylation_controls.fa) to your preferred genome fasta (e.g. `cat genome.fa methylation_controls.fa > genome+methylation_controls.fa`) ## Requirements -- [Nextflow](https://www.nextflow.io/) +- [Nextflow](https://www.nextflow.io/) (version 24.04 or later) + https://get.nextflow.io | bash` - [Miniforge](https://conda-forge.org/download/), [Micromamba](https://mamba.readthedocs.io/en/latest/installation/micromamba-installation.html), or [Conda](https://docs.conda.io/projects/conda/en/stable/) for dependency management - [Bioconda](https://bioconda.github.io/) channel configured - Sufficient computational resources (memory scales with input size) @@ -105,7 +106,7 @@ You may also be interested in the [nf-core methylseq project](https://nf-co.re/m - development workflow will run from master branch ### Testing: - - Tests are run using nf-test and are integrated into github actions + - Tests are run using [nf-test](https://www.nf-test.com/) and are integrated into github actions - install nf-test from bioconda using conda/mamba - To run all tests: ```bash @@ -115,4 +116,3 @@ nf-test test ```bash nf-test test --updateSnapshot ``` - diff --git a/conf/references.config b/conf/references.config index 28b4f1e..4bd8869 100644 --- a/conf/references.config +++ b/conf/references.config @@ -4,23 +4,58 @@ params { bwa_index: "/mnt/galaxy/galaxyworks/tool-data/T2T_chm13v2.0+bs_controls/bwameth_index/T2T_chm13v2.0+bs_controls/T2T_chm13v2.0+bs_controls.fa", genome_fa: "/mnt/galaxy/galaxyworks/tool-data/T2T_chm13v2.0+bs_controls/bwameth_index/T2T_chm13v2.0+bs_controls/T2T_chm13v2.0+bs_controls.fa", genome_fai: "/mnt/galaxy/galaxyworks/tool-data/T2T_chm13v2.0+bs_controls/bwameth_index/T2T_chm13v2.0+bs_controls/T2T_chm13v2.0+bs_controls.fa.fai", - target_bed: "/bioinfo/ref/t2t_chm13_v2+meth_controls+m13+phix/folded_windows/T2T_chm13v2.0+meth_controls_hairpins.s18w200nogt.bed" + target_bed: "/bioinfo/ref/t2t_chm13_v2+meth_controls+m13+phix/folded_windows/T2T_chm13v2.0+meth_controls_hairpins.s18w200nogt.bed", + ucsc_cpg_islands_url: "https://hgdownload.soe.ucsc.edu/gbdb/hs1/bbi/cpgIslandExt.bb", // bigBed format + refseq_gtf_url: "https://ftp.ncbi.nlm.nih.gov/genomes/refseq/vertebrate_mammalian/Homo_sapiens/annotation_releases/GCF_009914755.1-RS_2023_10/GCF_009914755.1_T2T-CHM13v2.0_genomic.gff.gz", + ncbi_assembly_report_url: "https://ftp.ncbi.nlm.nih.gov/genomes/refseq/vertebrate_mammalian/Homo_sapiens/annotation_releases/GCF_009914755.1-RS_2023_10/GCF_009914755.1_T2T-CHM13v2.0_assembly_report.txt", + dfam_url: "https://www.dfam.org/releases/annotations_exports/hs1_df39/hs1_df39-byacc-full_region.tsv", + epd_promoters_url: "https://epd.expasy.org/ftp/epdnew/H_sapiens/current/Hs_EPDnew.bed", + epd_liftover_url: "https://s3-us-west-2.amazonaws.com/human-pangenomics/T2T/CHM13/assemblies/chain/v1_nflo/grch38-chm13v2.chain" //GRCh38 version, no hs1 available + ], + 'grch38_core+bs_controls': [ + bwa_index: "/mnt/galaxy/data/genome/grch38_core+bs_controls/bwameth_index/grch38_core+bs_controls/grch38_core+bs_controls.fa", + genome_fa: "/mnt/galaxy/data/genome/grch38_core+bs_controls/sam_indexes/grch38_core+bs_controls/grch38_core+bs_controls.fa", + genome_fai: "/mnt/galaxy/data/genome/grch38_core+bs_controls/sam_indexes/grch38_core+bs_controls/grch38_core+bs_controls.fa.fai", + ucsc_cpg_islands_url: "https://hgdownload.soe.ucsc.edu/goldenPath/hg38/database/cpgIslandExt.txt.gz", + refseq_gtf_url: "https://ftp.ncbi.nlm.nih.gov/genomes/refseq/vertebrate_mammalian/Homo_sapiens/latest_assembly_versions/GCF_000001405.40_GRCh38.p14/GCF_000001405.40_GRCh38.p14_genomic.gff.gz", + ncbi_assembly_report_url: "https://ftp.ncbi.nlm.nih.gov/genomes/refseq/vertebrate_mammalian/Homo_sapiens/latest_assembly_versions/GCF_000001405.40_GRCh38.p14/GCF_000001405.40_GRCh38.p14_assembly_report.txt", + dfam_url: "https://www.dfam.org/releases/annotations_exports/hg38_df39/hg38_df39-byacc-full_region.tsv", + epd_promoters_url: "https://epd.expasy.org/ftp/epdnew/H_sapiens/current/Hs_EPDnew.bed", + epd_liftover_url: null ], 'grcm39+meth_controls': [ bwa_index: "/mnt/galaxy/data/genome/grcm39+meth_controls/bwameth_index/grcm39+meth_controls/grcm39+meth_controls.fa", genome_fa: "/mnt/galaxy/data/genome/grcm39+meth_controls/bwameth_index/grcm39+meth_controls/grcm39+meth_controls.fa", genome_fai: "/mnt/galaxy/data/genome/grcm39+meth_controls/bwameth_index/grcm39+meth_controls/grcm39+meth_controls.fa.fai", + ucsc_cpg_islands_url: "https://hgdownload.soe.ucsc.edu/goldenPath/mm39/database/cpgIslandExt.txt.gz", + refseq_gtf_url: "https://ftp.ncbi.nlm.nih.gov/genomes/refseq/vertebrate_mammalian/Mus_musculus/latest_assembly_versions/GCF_000001635.27_GRCm39/GCF_000001635.27_GRCm39_genomic.gff.gz", + ncbi_assembly_report_url: "https://ftp.ncbi.nlm.nih.gov/genomes/refseq/vertebrate_mammalian/Mus_musculus/latest_assembly_versions/GCF_000001635.27_GRCm39/GCF_000001635.27_GRCm39_assembly_report.txt", + dfam_url: null, + epd_promoters_url: "ftp://ccg.epfl.ch/epdnew/M_musculus/006/Mm_EPDnew_006_mm39.bed", + epd_liftover_url: null ], 't2t_2.0_eschColi_K12_bs_controls': [ bwa_index: "/mnt/galaxy/galaxyworks/tool-data/t2t_2.0_eschColi_K12_bs_controls/bwameth_index/t2t_2.0_eschColi_K12_bs_controls/t2t_2.0_eschColi_K12_bs_controls.fa", genome_fa: "/mnt/galaxy/galaxyworks/tool-data/t2t_2.0_eschColi_K12_bs_controls/bwameth_index/t2t_2.0_eschColi_K12_bs_controls/t2t_2.0_eschColi_K12_bs_controls.fa", genome_fai: "/mnt/galaxy/galaxyworks/tool-data/t2t_2.0_eschColi_K12_bs_controls/bwameth_index/t2t_2.0_eschColi_K12_bs_controls/t2t_2.0_eschColi_K12_bs_controls.fa.fai", + ucsc_cpg_islands_url: "https://hgdownload.soe.ucsc.edu/gbdb/hs1/bbi/cpgIslandExt.bb", + refseq_gtf_url: "https://ftp.ncbi.nlm.nih.gov/genomes/refseq/vertebrate_mammalian/Homo_sapiens/annotation_releases/GCF_009914755.1-RS_2023_10/GCF_009914755.1_T2T-CHM13v2.0_genomic.gff.gz", + ncbi_assembly_report_url: "https://ftp.ncbi.nlm.nih.gov/genomes/refseq/vertebrate_mammalian/Homo_sapiens/annotation_releases/GCF_009914755.1-RS_2023_10/GCF_009914755.1_T2T-CHM13v2.0_assembly_report.txt", + dfam_url: "https://www.dfam.org/releases/annotations_exports/hs1_df39/hs1_df39-byacc-full_region.tsv", + epd_promoters_url: "https://epd.expasy.org/ftp/epdnew/H_sapiens/current/Hs_EPDnew.bed", + epd_liftover_url: "https://s3-us-west-2.amazonaws.com/human-pangenomics/T2T/CHM13/assemblies/chain/v1_nflo/grch38-chm13v2.chain", //GRCh38 version, no hs1 available ], 'test': [ bwa_index: "${projectDir}/tests/fixtures/reference_files/reference.fa", genome_fa: "${projectDir}/tests/fixtures/reference_files/reference.fa", genome_fai: "${projectDir}/tests/fixtures/reference_files/reference.fa.fai", - target_bed: "${projectDir}/tests/fixtures/target_bed/emseq_test_regions.bed" + target_bed: "${projectDir}/tests/fixtures/target_bed/emseq_test_regions.bed", + ucsc_cpg_islands_url: "file://${projectDir}/tests/fixtures/features/test_cpgIslandExt.bb", + refseq_gtf_url: "file://${projectDir}/tests/fixtures/features/test_refseq.gff.gz", + ncbi_assembly_report_url: "file://${projectDir}/tests/fixtures/features/test_assembly_report.txt", + dfam_url: "file://${projectDir}/tests/fixtures/features/test_dfam.out", + epd_promoters_url: "file://${projectDir}/tests/fixtures/features/epd_promoters.bed", + epd_liftover_url: "file://${projectDir}/tests/fixtures/features/grch38-chm13v2.chain" ] ] -} \ No newline at end of file +} diff --git a/conf/test.config b/conf/test.config index 734ff99..c35cf95 100644 --- a/conf/test.config +++ b/conf/test.config @@ -24,8 +24,6 @@ params { max_time = '6.h' outputDir = "test_output" +} - } - - trace.overwrite = true - +trace.overwrite = true diff --git a/feature_cov.nf b/feature_cov.nf new file mode 100644 index 0000000..3595f04 --- /dev/null +++ b/feature_cov.nf @@ -0,0 +1,192 @@ +#!/usr/bin/env nextflow + +nextflow.enable.dsl=2 + +// Require Nextflow version 24.04 or later for topic channels support +// Topic channels are used for version tracking in subprocesses +if( !nextflow.version.matches('>=24.04') ) { + error "This workflow requires Nextflow version 24.04 or later. Please upgrade your Nextflow installation:\n" + + " Current version: ${nextflow.version}\n" + + " Required version: 24.04+\n" + + " Upgrade with: nextflow self-update\n" + + " Or install latest: curl -s https://get.nextflow.io | bash" +} + +// Enable topic preview flag for versions < 25.04 (graduated from preview in 25.04) +if( nextflow.version.matches('>=24.04') && nextflow.version.matches('<25.04') ) { + nextflow.preview.topic = true +} + + +include { download_epd_promoters; + download_liftover_chain; + crossmap_epd_promoters; + download_cpg_islands; + download_refseq_gtf; + download_assembly_report; + normalize_cpg_islands; + normalize_refseq_features; + download_dfam_annotations } from './modules/prepare_features' +include { calculate_feature_methylation; + combine_methylation } from './modules/feature_methylation' +include { calculate_feature_counts; + combine_counts } from './modules/feature_counts' + +params.genome = null +params.methylation_bed = null +params.bam_files_glob = '*.bam' // Indexes auto-detected: .bai, .csi, .crai +params.tmp_dir = '/tmp' +params.outputDir = 'output' + +params.ucsc_cpg_islands_url = null +params.refseq_gtf_url = null +params.ncbi_assembly_report_url = null +params.dfam_url = null +params.epd_promoters_url = null + +if (!params.genome) { + exit 1, "Error: --genome parameter is required" +} +if (!params.bam_files_glob) { + exit 1, "Error: --bam_files_glob parameter is required" +} + +if (params.genomes && params.genome && !params.genomes.containsKey(params.genome)) { + exit 1, "The provided genome '${params.genome}' is not available in the genomes file. Currently the available genomes are ${params.genomes.keySet().join(", ")}" +} + +genome = params.genome +params.reference_list = params.genomes ? params.genomes[genome] : [:] +genome_fa = params.reference_list.genome_fa ?: params.genome_fa +genome_fai = params.reference_list.genome_fai ?: params.genome_fai + +ucsc_cpg_islands_url = params.ucsc_cpg_islands_url ?: params.reference_list.ucsc_cpg_islands_url +refseq_gtf_url = params.refseq_gtf_url ?: params.reference_list.refseq_gtf_url +ncbi_assembly_report_url = params.ncbi_assembly_report_url ?: params.reference_list.ncbi_assembly_report_url +dfam_url = params.dfam_url ?: params.reference_list.dfam_url +epd_promoters_url = params.epd_promoters_url ?: params.reference_list.epd_promoters_url +epd_liftover_url = params.reference_list.epd_liftover_url ?: null + +refseq_feature_types = Channel.of( + 'promoter', + 'transcriptional_cis_regulatory_region', + 'enhancer', + 'silencer', + 'mobile_genetic_element', + 'lnc_RNA', + 'snRNA', + 'snoRNA', + 'primary_transcript', + 'tRNA', + 'exon', + 'mRNAexon1', + 'mRNAexon' +) + +workflow { + main: + bam_with_index = Channel.fromPath(params.bam_files_glob, checkIfExists: true) + .map { alignment -> + def base = alignment.toString().replaceAll(/\.(bam|cram)$/, '') + def idx = ["${alignment}.bai", "${base}.bai", + "${alignment}.csi", "${base}.csi", + "${alignment}.crai", "${base}.crai"] + .collect { file(it) } + .find { it.exists() } + if (!idx) error "No index found for: ${alignment}" + tuple(alignment, idx) + } + .collect() + + if (epd_promoters_url) { + download_epd_promoters(epd_promoters_url) + + if (epd_liftover_url) { + download_liftover_chain(epd_liftover_url) + crossmap_epd_promoters( + download_epd_promoters.out.gtf, + download_liftover_chain.out.chain_file + ) + + epd_gtf = crossmap_epd_promoters.out.gtf + .map { gtf -> tuple('epd_promoter', gtf) } + } else { + epd_gtf = download_epd_promoters.out.gtf + .map { gtf -> tuple('epd_promoter', gtf) } + } + } else { + epd_gtf = Channel.empty() + } + + if (ncbi_assembly_report_url) { + download_assembly_report(ncbi_assembly_report_url) + assembly_report = download_assembly_report.out.report + } else { + assembly_report = Channel.empty() + } + + if (ucsc_cpg_islands_url && ncbi_assembly_report_url) { + download_cpg_islands(ucsc_cpg_islands_url) + normalize_cpg_islands(download_cpg_islands.out.ucsc_file, assembly_report) + cpg_gtf = normalize_cpg_islands.out.gtf + .map { gtf -> tuple('cpg_island', gtf) } + } else { + cpg_gtf = Channel.empty() + } + + if (refseq_gtf_url && ncbi_assembly_report_url) { + download_refseq_gtf(refseq_gtf_url) + refseq_combined = download_refseq_gtf.out.gtf + .combine(assembly_report) + .combine(Channel.value(file(genome_fai, checkIfExists: true))) + .combine(refseq_feature_types) + + normalize_refseq_features(refseq_combined) + refseq_gtfs = normalize_refseq_features.out.gtf + } else { + refseq_gtfs = Channel.empty() + } + + if (dfam_url) { + download_dfam_annotations(dfam_url) + dfam_gtf = download_dfam_annotations.out.gtf + .map { gtf -> tuple('dfam_repeat', gtf) } + } else { + dfam_gtf = Channel.empty() + } + + if (params.methylation_bed) { + methylation_bed = Channel.fromPath(params.methylation_bed, checkIfExists: true) + + all_gtfs = epd_gtf + .mix(cpg_gtf) + .mix(refseq_gtfs) + .mix(dfam_gtf) + + calculate_feature_methylation(all_gtfs, methylation_bed) + + combine_methylation( + calculate_feature_methylation.out.methylation + .map { feature_type, tsv -> tsv } + .collect() + ) + } + + all_features_for_counts = epd_gtf + .map { type, file -> tuple(type, file, 'gene_id') } + .mix(cpg_gtf.map { type, file -> tuple(type, file, 'transcript_id') }) + .mix(refseq_gtfs.map { type, file -> tuple(type, file, 'gene_id') }) + .mix(dfam_gtf.map { type, file -> tuple(type, file, 'gene_id') }) + + calculate_feature_counts( + all_features_for_counts.map { type, file, attr -> tuple(type, file) }, + bam_with_index, + all_features_for_counts.map { type, file, attr -> attr } + ) + + combine_counts( + calculate_feature_counts.out.counts + .map { feature_type, tsv -> tsv } + .collect() + ) +} diff --git a/main.nf b/main.nf index 79069ce..74970be 100644 --- a/main.nf +++ b/main.nf @@ -1,4 +1,16 @@ -nextflow.preview.topic = true +// Require Nextflow version 24.04 or later for topic channels support +if( !nextflow.version.matches('>=24.04') ) { + error "This workflow requires Nextflow version 24.04 or later. Please upgrade your Nextflow installation:\n" + + " Current version: ${nextflow.version}\n" + + " Required version: 24.04+\n" + + " Upgrade with: nextflow self-update\n" + + " Or install latest: curl -s https://get.nextflow.io | bash" +} + +// Enable topic preview flag for versions < 25.04 (graduated from preview in 25.04) +if( nextflow.version.matches('>=24.04') && nextflow.version.matches('<25.04') ) { + nextflow.preview.topic = true +} include { createVersionsFile } from './lib/versions.nf' include { format_ngs_agg_opts } from './modules/aggregate_results' diff --git a/modules/feature_counts.nf b/modules/feature_counts.nf new file mode 100644 index 0000000..670a4b9 --- /dev/null +++ b/modules/feature_counts.nf @@ -0,0 +1,53 @@ +process calculate_feature_counts { + label 'high_cpu' + tag { feature_type } + conda "subread=2.1.1" + publishDir "${params.outputDir}/feature_counts" + + input: + tuple val(feature_type), path(feature_file) + path(bam_with_index) + val(feature_id_attr) // e.g., 'transcript_id', 'gene_id' + + output: + tuple val(feature_type), path("${feature_type}_counts.tsv"), emit: counts + tuple val("${task.process}"), val('featureCounts'), eval('featureCounts -v 2>&1 | grep "featureCounts" | sed \'s/featureCounts v//\''), topic: versions + + script: + """ + featureCounts --primary --ignoreDup -Q 10 -M -f -O --fraction -p -P -B -C \ + -a ${feature_file} -F GTF \ + -t ${feature_type} \ + -g ${feature_id_attr} \ + --tmpDir ${params.tmp_dir ?: '/tmp'} \ + -T ${task.cpus} \ + -o ${feature_type}_counts.tsv *.bam + """ +} + +process combine_counts { + label 'low_cpu' + publishDir "${params.outputDir}" + + input: + path(count_files) + + output: + path('combined_feature_counts.tsv'), emit: combined + + script: + """ + # Construct the header + echo -n 'File\t' > combined_feature_counts.tsv + grep -hve '^\\s*\$' -e '^#' ${count_files[0]} | head -n 1 >> combined_feature_counts.tsv + + # Add a column containing the name of the file being processed + for f in ${count_files}; do + filebase=\$(basename "\${f}" _counts.tsv) + lines=\$(wc -l < <(grep -ve '^\\s*\$' -e '^#' "\$f") | cut -f 1 -d ' ') + if [ \$lines -gt 1 ]; then + paste <(yes \${filebase} | head -n \$lines) <(grep -ve '^\\s*\$' -e '^#' "\$f") | tail -n +2 >> combined_feature_counts.tsv + fi + done + """ +} diff --git a/modules/feature_methylation.nf b/modules/feature_methylation.nf new file mode 100644 index 0000000..ad81fdb --- /dev/null +++ b/modules/feature_methylation.nf @@ -0,0 +1,48 @@ +process calculate_feature_methylation { + label 'medium_cpu' + tag { "${feature_type}" } + conda "bedtools=2.29.2 htslib=1.9" + publishDir "${params.outputDir}/feature_methylation" + + input: + tuple val(feature_type), path(feature_gtf) + path(methylation_bed) + + output: + tuple val(feature_type), path("${feature_type}_methylation.tsv"), emit: methylation + tuple val("${task.process}"), val('bedtools'), eval('bedtools --version | sed \'s/^bedtools v//\''), topic: versions + + script: + """ + bedtools intersect -nonamecheck \ + -wa -wb -loj \ + -a ${feature_gtf} -b <(bgzip -d < ${methylation_bed}) \ + | awk -v FS='\\t' -v OFS='\\t' '\$14>0 {print \$10,\$11,\$12,\$1":"\$4-1"-"\$5,(\$15*1.0)/\$14 }' \ + | bedtools groupby -g 4 -o mean -c 5 \ + > ${feature_type}_methylation.tsv + """ +} + +process combine_methylation { + label 'low_cpu' + publishDir "${params.outputDir}" + + input: + path(methylation_files) + + output: + path('combined_methylation.tsv'), emit: combined + + script: + """ + echo 'File\tLocus\tFrac Methylated' > combined_methylation.tsv + + for f in ${methylation_files}; do + filebase=\$(basename "\${f}" _methylation.tsv) + lines=\$(wc -l < <(grep -ve '^\\s*\$' -e '^#' "\$f") | cut -f 1 -d ' ') + if [ \$lines -gt 0 ]; then + paste <(yes \${filebase} | head -n \$lines) <(grep -ve '^\\s*\$' -e '^#' "\$f") >> combined_methylation.tsv + fi + done + """ +} diff --git a/modules/prepare_features.nf b/modules/prepare_features.nf new file mode 100644 index 0000000..b6692ff --- /dev/null +++ b/modules/prepare_features.nf @@ -0,0 +1,239 @@ +process download_epd_promoters { + label 'low_cpu' + conda "conda-forge::curl conda-forge::gawk bioconda::ucsc-bedtogenepred bioconda::ucsc-genepredtogtf " + publishDir "${params.outputDir}/features" + + input: + val(epd_url) + + output: + path('epd_promoter.gtf'), emit: gtf + tuple val("${task.process}"), val('bedToGenePred'), eval('bedToGenePred 2>&1 | grep "bedToGenePred" | sed \'s/bedToGenePred - //\''), topic: versions + + script: + """ + curl -fsSL "${epd_url}" \ + | tr ' ' '\t' \ + | bedToGenePred /dev/stdin /dev/stdout \ + | genePredToGtf file /dev/stdin /dev/stdout \ + | gawk -v OFS='\\t' -v FS='\\t' '\$3=="transcript" {\$3="epd_promoter"} {print}' \ + > epd_promoter.gtf + """ +} + +process download_liftover_chain { + label 'low_cpu' + conda "conda-forge::curl bioconda::htslib=1.22.1" + publishDir "${params.outputDir}/features" + + input: + val(chain_file_url) + + output: + path('hg38ToHs1.over.chain.gz'), emit: chain_file + tuple val("${task.process}"), val('curl'), eval('curl --version | head -n 1 | cut -d " " -f 2'), topic: versions + + script: + """ + curl -fsSL "${chain_file_url}" | bgzip > hg38ToHs1.over.chain.gz + """ +} + +process crossmap_epd_promoters { + label 'low_cpu' + conda "bioconda::crossmap=0.7.0" + publishDir "${params.outputDir}/features" + + input: + path(epd_orig_gtf) + path(chain_file) + + output: + path('epd_promoter_lifted.gtf'), emit: gtf + tuple val("${task.process}"), val('liftOver'), eval('liftOver 2>&1 | grep "liftOver" | head -n 1 || echo "v1"'), topic: versions + + script: + """ + CrossMap gff ${chain_file} ${epd_orig_gtf} epd_promoter_lifted.gtf + """ +} + +process download_cpg_islands { + label 'low_cpu' + conda "conda-forge::curl" + publishDir "${params.outputDir}/features" + + input: + val(cpg_file_url) + + output: + path('*cpgIslandExt*'), emit: ucsc_file + tuple val("${task.process}"), val('curl'), eval('curl --version | head -n 1 | cut -d " " -f 2'), topic: versions + + script: + """ + curl -fsSL -O "${cpg_file_url}" + """ +} + +process download_refseq_gtf { + label 'low_cpu' + conda "conda-forge::curl" + + input: + val(refseq_url) + + output: + path('refseq.gff.gz'), emit: gtf + tuple val("${task.process}"), val('curl'), eval('curl --version | head -n 1 | cut -d " " -f 2'), topic: versions + + script: + """ + curl -fsSL "${refseq_url}" -o refseq.gff.gz + """ +} + +process download_assembly_report { + label 'low_cpu' + conda "conda-forge::curl" + + input: + val(report_url) + + output: + path('assembly_report.txt'), emit: report + tuple val("${task.process}"), val('curl'), eval('curl --version | head -n 1 | cut -d " " -f 2'), topic: versions + + script: + """ + curl -fsSL "${report_url}" -o assembly_report.txt + """ +} + +process normalize_cpg_islands { + label 'low_cpu' + conda "bioconda::htslib=1.22.1 bioconda::ucsc-bigbedtobed bioconda::ucsc-bedtogenepred bioconda::ucsc-genepredtogtf" + publishDir "${params.outputDir}/features" + + input: + path(cpg_file) + path(assembly_report) + + output: + path('cpg_island.gtf'), emit: gtf + tuple val("${task.process}"), val('bigBedToBed'), eval('bigBedToBed 2>&1 | grep "bigBedToBed" | head -n 1 || echo "v4"'), topic: versions + + script: + def is_bigbed = cpg_file.name.endsWith('.bb') + def is_txt = cpg_file.name.endsWith('.txt.gz') + if (is_bigbed) + """ + # Create chromosome mapping from assembly report (RefSeq accession -> chr name) + grep -v '^#' ${assembly_report} \ + | awk -F'\t' '\$5 != "na" {print \$7"\\t"\$1}' \ + > chr_mapping.txt + + # Convert bigBed to BED + bigBedToBed ${cpg_file} cpg_islands_raw.bed + + # Map RefSeq accessions to chr names and convert to GTF + awk -v FS='\t' -v OFS='\t' 'NR==FNR {map[\$1]=\$2; next} + { + chrom = (\$1 in map) ? map[\$1] : \$1 + start = \$2; end = \$3; name = \$4 + id = chrom":"start"-"end + print chrom, "cpgIslands", "cpg_island", start+1, end, ".", "+", ".", "transcript_id \\""id"\\"; gene_id \\""id"\\";" + }' chr_mapping.txt cpg_islands_raw.bed > cpg_island.gtf + """ + else if (is_txt) + """ + # Handle text format (original logic) + zcat -f ${cpg_file} \ + | awk -v FS='\t' -v OFS='\t' '{print \$1,"cpgIslands","cpg_island",\$4,\$5,\$6,\$7,\$8,\$9}' \ + > cpg_island.gtf + """ + else + error "Unsupported file format for CpG islands: ${cpg_file.name}" +} +process normalize_refseq_features { + label 'medium_cpu' + tag { feature } + conda "bioconda::subread=2.1.1 bioconda::bedtools=2.31.1 conda-forge::grep conda-forge::gawk conda-forge::gzip" + publishDir "${params.outputDir}/features" + + input: + tuple path(refseq_gtf), path(assembly_report), path(genome_fai), val(feature) + + output: + tuple val(feature), path("${feature}.gtf"), emit: gtf + tuple val("${task.process}"), val('bedtools'), eval('bedtools --version | sed \'s/^bedtools v//\''), topic: versions + + script: + """ + # Translate chromosome names using NCBI assembly report + gawk -v OFS='\t' -v FS='\t' 'NR==FNR {dict[\$1]=\$2; next} {\$1=dict[\$1]; print}' \ + <(grep -v '^#' ${assembly_report} | cut -f 7,10 | tr -d '\\r') \ + <(zcat -f < ${refseq_gtf} | grep -v '^#') \ + | grep "GeneID:" \ + | grep -P -v "_alt\\t" \ + | grep -P -v "^na\\t" \ + | sed -r 's/;Dbxref(=[^;]*)GeneID:([^,;]+)([;,])/;gene_id=\\2;Dbxref\\1GeneID:\\2\\3/' \ + | gawk -v OFS='\\t' -v FS='\\t' \ + '(\$3=="exon") && (index(\$9,"gbkey=mRNA") > 0) && (index(\$9,"-1;Parent") > 0) \ + { print(\$1,\$2,"mRNAexon1",\$4,\$5,\$6,\$7,\$8,\$9); next } + (\$3=="exon") && (index(\$9,"gbkey=mRNA") > 0) \ + { print(\$1,\$2,"mRNAexon",\$4,\$5,\$6,\$7,\$8,\$9); next } + { print } + ' \ + > name_converted.gff + + # Flatten overlapping features to avoid 0 coverage from featureCounts + flattenGTF -a name_converted.gff -o flat_name_converted.saf -t ${feature} + + # Convert to BED for intersection + tail -n +2 flat_name_converted.saf \ + | gawk -v OFS='\\t' -v FS='\\t' '{print \$2,\$3-1,\$4,\$1,"-",\$5}' \ + | bedtools sort -faidx ${genome_fai} -i /dev/stdin > ${feature}_flat.bed + + # Filter by feature type + gawk -v type=${feature} -v OFS='\\t' -v FS='\\t' '(\$3==type) { print }' name_converted.gff \ + > ${feature}.gtf + + # Only include entries that intersect with the desired feature type + echo "GeneID\tChr\tStart\tEnd\tStrand" > ${feature}_flat.saf + bedtools intersect -a ${feature}_flat.bed -b ${feature}.gtf -u \ + | gawk -v OFS='\\t' -v FS='\\t' '{print \$4,\$1,\$2+1,\$3,\$6}' >> ${feature}_flat.saf + """ +} + +process download_dfam_annotations { + label 'low_cpu' + conda "conda-forge::curl conda-forge::grep conda-forge::gawk bioconda::ucsc-bedtogenepred bioconda::ucsc-genepredtogtf" + publishDir "${params.outputDir}/features" + + input: + val(dfam_url) + + output: + path('dfam.gtf'), emit: gtf + tuple val("${task.process}"), val('genePredToGtf'), eval('genePredToGtf 2>&1 | grep "genePredToGtf" | sed \'s/genePredToGtf - //\''), topic: versions + + script: + """ + curl -fsSL "${dfam_url}" \ + | grep -v '^#' \ + | gawk '\$17==1 { + score = int(\$16 * 20) + if (score < 0) score = 0 + if (score > 1000) score = 1000 + start = \$10 < \$11 ? \$10 - 1 : \$11 - 1 + end = \$10 < \$11 ? \$11 : \$10 + print \$1, start, end, \$3, score, \$9 + }' OFS="\t" \ + | bedToGenePred /dev/stdin /dev/stdout \ + | genePredToGtf file /dev/stdin /dev/stdout \ + | gawk '{print \$1,"dfam_repeats", \$3, \$4, \$5, \$6, \$7, \$8, \$9}' FS="\t" OFS="\t" \ + | gawk -v OFS='\\t' -v FS='\\t' '\$3=="transcript" {\$3="dfam_repeat"} {print}' \ + > dfam.gtf + """ +} diff --git a/prepare_refseq_test_features.sh b/prepare_refseq_test_features.sh new file mode 100644 index 0000000..1732373 --- /dev/null +++ b/prepare_refseq_test_features.sh @@ -0,0 +1,50 @@ +#!/bin/bash +# Extract one example of each required feature type from RefSeq GFF file +# for testing the feature_cov workflow + +set -euo pipefail + +# Feature types we need +FEATURE_TYPES=( + "promoter" + "transcriptional_cis_regulatory_region" + "enhancer" + "mobile_genetic_element" + "primary_transcript" + "lnc_RNA" + "exon" +) + +# Input/output files +REFSEQ_URL="https://ftp.ncbi.nlm.nih.gov/genomes/refseq/vertebrate_mammalian/Homo_sapiens/annotation_releases/GCF_009914755.1-RS_2023_10/GCF_009914755.1_T2T-CHM13v2.0_genomic.gff.gz" +OUTPUT_FILE="tests/fixtures/features/test_refseq.gff" +TMP_FILE=$(mktemp) + +echo "Downloading and filtering RefSeq annotations..." +echo "Looking for feature types: ${FEATURE_TYPES[*]}" + +# Download and extract header +curl -fsSL "$REFSEQ_URL" | gunzip | grep '^#' > "$OUTPUT_FILE" + +# Extract first occurrence of each feature type +for feature in "${FEATURE_TYPES[@]}"; do + echo " Extracting first '$feature'..." + curl -fsSL "$REFSEQ_URL" \ + | gunzip \ + | grep -v '^#' \ + | awk -F'\t' -v feat="$feature" '$3 == feat' \ + | head -1 \ + >> "$TMP_FILE" +done + +# Sort by chromosome and position, append to output +sort -k1,1 -k4,4n "$TMP_FILE" >> "$OUTPUT_FILE" + +# Compress +gzip -f "$OUTPUT_FILE" + +rm "$TMP_FILE" + +echo "Created tests/fixtures/features/test_refseq.gff.gz" +echo "Feature counts:" +gunzip -c tests/fixtures/features/test_refseq.gff.gz | grep -v '^#' | awk -F'\t' '{print $3}' | sort | uniq -c diff --git a/tests/feature_cov.nf.test b/tests/feature_cov.nf.test new file mode 100644 index 0000000..5bca9f3 --- /dev/null +++ b/tests/feature_cov.nf.test @@ -0,0 +1,30 @@ +nextflow_pipeline { + + name "Test feature_cov workflow" + script "feature_cov.nf" + tag "pipeline" + options "-stub-run" + + test("feature_cov workflow basic run") { + when { + params.genome = 'test' + params.bam_files_glob = "$projectDir/tests/fixtures/aligned_bam/*.md.bam" + params.outputDir = "${launchDir}/test_output/feature_cov" + } + + then { + def combined_counts = path("${launchDir}/test_output/feature_cov/combined_feature_counts.tsv").exists() + def epd_gtf = path("${launchDir}/test_output/feature_cov/features/epd_promoter_lifted.gtf").exists() + def cpg_gtf = path("${launchDir}/test_output/feature_cov/features/cpg_island.gtf").exists() + def dfam_gtf = path("${launchDir}/test_output/feature_cov/features/dfam.gtf").exists() + + assertAll( + { assert workflow.success }, + { assert combined_counts }, + { assert epd_gtf }, + { assert cpg_gtf }, + { assert dfam_gtf } + ) + } + } +} diff --git a/tests/fixtures/aligned_bam/emseq-test1.md.bai b/tests/fixtures/aligned_bam/emseq-test1.md.bai new file mode 100644 index 0000000000000000000000000000000000000000..315d928e246c20b95f3f04e855ab8568d64eb145 GIT binary patch literal 664 zcmZ>A^kigYU|-a?=^8=vbFn-r8 zhGp~~gB zTn@{n*ikuEVIo$=<%(h|Q7UCel`t`_k}W$$xs-~ktTOZe{oi}tuV;F;2S+uqyECt6 z_V)ee|NZsOCDUVXooqCoo{UbucyZr36P}!1+Q0GETX$~We)IahTMs`yd;a9?;{J^< z-f>P|xVZ1e{?NA{e)`Jze^msg;&*}e@moMxeUbyhW^zdSG^70Gf_j*B4JeW*gyci_UycC^r z&aA)v;Nt$pTVJ_${>?AEe*Mjx*UsI3>-FoOzx(jhm-(;!{8z4@e&g1?FP^-7v3N|I zwa>U`qLT*~_V>=daQD`oyJzXmU-;r%ckbRhd-u+bv*)L$@80_9vuAF+_2$j%XK%gv z<-7QH+K&DOz;^B42iJj}$<6-CrXFq@Y&9n5+;9oiO_Rm_U?^%v@ 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7 +5 0 1 +5 0 1 +9 0 1402 +54 0 83 +58 1 0 +50 0 1 +48 0 1 +77 1 0 +54 0 8 +49 1 0 +27 1 0 +28 + +chain 255 chrY 57227415 + 24782064 24792358 chrY 62460029 - 36771537 36779474 0 +1332 0 1 +4084 2 0 +508 4 0 +53 1 0 +67 1 0 +47 0 16 +26 3 0 +3 2373 0 +540 0 1 +31 0 6 +245 1 0 +45 0 8 +124 3 0 +478 1 0 +322 + +chain 255 chrY 57227415 + 24822587 24840294 chrY 62460029 - 36807327 36825033 0 +2193 1 0 +15513 + +chain 255 chrY 57227415 + 24840294 24894103 chrY 62460029 - 36835880 36875390 0 +4892 2 0 +1572 1 0 +4026 3 0 +352 1 0 +4481 0 1 +452 0 1 +1999 0 2 +4955 1 0 +1193 0 3 +5787 1 0 +891 1 0 +346 1 0 +77 4756 0 +6361 9539 0 +2119 + diff --git a/tests/fixtures/features/prepare_refseq_test.sh b/tests/fixtures/features/prepare_refseq_test.sh new file mode 100644 index 0000000..5cfab3a --- /dev/null +++ b/tests/fixtures/features/prepare_refseq_test.sh @@ -0,0 +1,4 @@ +#this window of refseq contains all the features we're interested in +REFSEQ_URL="https://ftp.ncbi.nlm.nih.gov/genomes/refseq/vertebrate_mammalian/Homo_sapiens/annotation_releases/GCF_009914755.1-RS_2023_10/GCF_009914755.1_T2T-CHM13v2.0_genomic.gff.gz" +curl -fsSL -O "$REFSEQ_URL" +cat < GCF_009914755.1_T2T-CHM13v2.0_genomic.gff.gz | head -n 85000 | tail -n 40000 | bgzip > test_refseq.gff.gz diff --git a/tests/fixtures/features/test_assembly_report.txt b/tests/fixtures/features/test_assembly_report.txt new file mode 100644 index 0000000..1e635eb --- /dev/null +++ b/tests/fixtures/features/test_assembly_report.txt @@ -0,0 +1,50 @@ +# Assembly name: T2T-CHM13v2.0 +# Description: T2T CHM13v2.0 Telomere-to-Telomere assembly of the CHM13 cell line, with chrY from NA24385 +# Organism name: Homo sapiens (human) +# Taxid: 9606 +# BioSample: SAMN03255769 +# BioProject: PRJNA559484 +# Submitter: T2T Consortium +# Date: 2022-01-24 +# Assembly type: haploid +# Release type: major +# Assembly level: Complete Genome +# Genome representation: full +# Expected final version: no +# Genome coverage: 30x +# GenBank assembly accession: GCA_009914755.4 +# RefSeq assembly accession: GCF_009914755.1 +# RefSeq assembly and GenBank assemblies identical: no +# +## Assembly-Units: +## GenBank Unit Accession RefSeq Unit Accession Assembly-Unit name +## GCA_009914825.4 GCF_009914825.1 Primary Assembly +## GCA_009914855.2 non-nuclear +# +# Ordered by chromosome/plasmid; the chromosomes/plasmids are followed by +# unlocalized scaffolds. +# Unplaced scaffolds are listed at the end. +# RefSeq is equal or derived from GenBank object. +# +# Sequence-Name Sequence-Role Assigned-Molecule Assigned-Molecule-Location/Type GenBank-Accn Relationship RefSeq-Accn Assembly-Unit Sequence-Length UCSC-style-name +1 assembled-molecule 1 Chromosome CP068277.2 = NC_060925.1 Primary Assembly 248387328 chr1 +2 assembled-molecule 2 Chromosome CP068276.2 = NC_060926.1 Primary Assembly 242696752 chr2 +3 assembled-molecule 3 Chromosome CP068275.2 = NC_060927.1 Primary Assembly 201105948 chr3 +4 assembled-molecule 4 Chromosome CP068274.2 = NC_060928.1 Primary Assembly 193574945 chr4 +5 assembled-molecule 5 Chromosome CP068273.2 = NC_060929.1 Primary Assembly 182045439 chr5 +6 assembled-molecule 6 Chromosome CP068272.2 = NC_060930.1 Primary Assembly 172126628 chr6 +7 assembled-molecule 7 Chromosome CP068271.2 = NC_060931.1 Primary Assembly 160567428 chr7 +8 assembled-molecule 8 Chromosome CP068270.2 = NC_060932.1 Primary Assembly 146259331 chr8 +9 assembled-molecule 9 Chromosome CP068269.2 = NC_060933.1 Primary Assembly 150617247 chr9 +10 assembled-molecule 10 Chromosome CP068268.2 = NC_060934.1 Primary Assembly 134758134 chr10 +11 assembled-molecule 11 Chromosome CP068267.2 = NC_060935.1 Primary Assembly 135127769 chr11 +12 assembled-molecule 12 Chromosome CP068266.2 = NC_060936.1 Primary Assembly 133324548 chr12 +13 assembled-molecule 13 Chromosome CP068265.2 = NC_060937.1 Primary Assembly 113566686 chr13 +14 assembled-molecule 14 Chromosome CP068264.2 = NC_060938.1 Primary Assembly 101161492 chr14 +15 assembled-molecule 15 Chromosome CP068263.2 = NC_060939.1 Primary Assembly 99753195 chr15 +16 assembled-molecule 16 Chromosome CP068262.2 = NC_060940.1 Primary Assembly 96330374 chr16 +17 assembled-molecule 17 Chromosome CP068261.2 = NC_060941.1 Primary Assembly 84276897 chr17 +18 assembled-molecule 18 Chromosome CP068260.2 = NC_060942.1 Primary Assembly 80542538 chr18 +19 assembled-molecule 19 Chromosome CP068259.2 = NC_060943.1 Primary Assembly 61707364 chr19 +20 assembled-molecule 20 Chromosome CP068258.2 = NC_060944.1 Primary Assembly 66210255 chr20 +21 assembled-molecule 21 Chromosome CP068257.2 = NC_060945.1 Primary Assembly 45090682 chr21 diff --git a/tests/fixtures/features/test_cpgIslandExt.bb b/tests/fixtures/features/test_cpgIslandExt.bb new file mode 100644 index 0000000000000000000000000000000000000000..b0cf56c6e4be36b0d7ff25b4ab68dff77cc23b3b GIT binary patch literal 25902 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