From 59af7906ab3f707f591f717a7405017e91727347 Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Wed, 22 May 2019 16:04:34 +0200 Subject: [PATCH 01/22] improve tests --- bin/run_tests.sh | 37 +++++++++++++++++++++++++------------ 1 file changed, 25 insertions(+), 12 deletions(-) diff --git a/bin/run_tests.sh b/bin/run_tests.sh index 1a19312a68..8b9c49c4f0 100755 --- a/bin/run_tests.sh +++ b/bin/run_tests.sh @@ -2,6 +2,7 @@ set -xeuo pipefail CPUS=2 +LOGS='' PROFILE=docker TEST=ALL TRAVIS_BUILD_DIR=${TRAVIS_BUILD_DIR:-.} @@ -12,9 +13,12 @@ while [[ $# -gt 0 ]] do key=$1 case $key in - -t|--test) - TEST=$2 - shift # past argument + -c|--cpus) + CPUS=$2 + shift # past value + ;; + -n|--no-logs) + LOGS=true shift # past value ;; -p|--profile) @@ -22,12 +26,13 @@ do shift # past argument shift # past value ;; - -v|--verbose) - VERBOSE="-ansi-log false -dump-channels" + -t|--test) + TEST=$2 + shift # past argument shift # past value ;; - -c|--cpus) - CPUS=$2 + -v|--verbose) + VERBOSE="-ansi-log false -dump-channels" shift # past value ;; *) # unknown option @@ -36,6 +41,13 @@ do esac done +function manage_logs() { + if [[ $LOGS ]] + then + rm -rf .nextflow* results/ work/ + fi +} + function run_sarek() { nextflow run ${TRAVIS_BUILD_DIR}/main.nf -profile test,${PROFILE} ${VERBOSE} --monochrome_logs $@ } @@ -47,19 +59,20 @@ then run_sarek --tools=false --sample data/testdata/tiny/normal --noReports run_sarek --tools=false --sample results/Preprocessing/TSV/duplicateMarked.tsv --step recalibrate --noReports run_sarek --tools HaplotypeCaller,Strelka --sample results/Preprocessing/TSV/recalibrated.tsv --step variantCalling --noReports - rm -rf .nextflow* results/ work/ + rm -rf data/ + manage_logs fi if [[ ALL,SOMATIC =~ $TEST ]] then run_sarek --tools FreeBayes,HaplotypeCaller,Manta,Strelka,Mutect2 --noReports - rm -rf .nextflow* results/ work/ + manage_logs fi if [[ ALL,TARGETED =~ $TEST ]] then run_sarek --tools FreeBayes,HaplotypeCaller,Manta,Strelka,Mutect2 --noReports --targetBED https://github.com/nf-core/test-datasets/raw/sarek/testdata/target.bed - rm -rf .nextflow* results/ work/ + manage_logs fi if [[ ALL,ANNOTATEALL,ANNOTATESNPEFF,ANNOTATEVEP =~ $TEST ]] @@ -75,11 +88,11 @@ then ANNOTATOR=merge,snpEFF,VEP fi run_sarek --step annotate --tools ${ANNOTATOR} --sample https://github.com/nf-core/test-datasets/raw/sarek/testdata/vcf/Strelka_1234N_variants.vcf.gz --noReports - rm -rf .nextflow* results/ work/ + manage_logs fi if [[ MULTIPLE =~ $TEST ]] then run_sarek --sample https://github.com/nf-core/test-datasets/raw/sarek/testdata/tsv/tiny-multiple-https.tsv --tools FreeBayes,HaplotypeCaller,Manta,Strelka,Mutect2 --noReports - rm -rf .nextflow* results/ work/ + manage_logs fi From 678abc259bcfbffe969726f0c6e6b988d34af452 Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Thu, 23 May 2019 14:55:03 +0200 Subject: [PATCH 02/22] put VEP summary in Reports --- main.nf | 6 ++---- 1 file changed, 2 insertions(+), 4 deletions(-) diff --git a/main.nf b/main.nf index 44cbb28c91..e0b0c73828 100644 --- a/main.nf +++ b/main.nf @@ -1707,8 +1707,7 @@ process Snpeff { val snpeffDb from Channel.value(params.genomes[params.genome].snpeffDb) output: - set file("${reducedVCF}_snpEff.txt"), file("${reducedVCF}_snpEff.html") into snpeffOut - file("${reducedVCF}_snpEff.csv") into snpeffReport + set file("${reducedVCF}_snpEff.txt"), file("${reducedVCF}_snpEff.html"), file("${reducedVCF}_snpEff.csv") into snpeffReport set val("snpEff"), variantCaller, idSample, file("${reducedVCF}_snpEff.ann.vcf") into snpeffVCF when: 'snpeff' in tools || 'merge' in tools @@ -1732,7 +1731,6 @@ process Snpeff { """ } -snpeffOut = snpeffOut.dump(tag:'snpEff output') snpeffReport = snpeffReport.dump(tag:'snpEff report') if ('merge' in tools) { @@ -1754,7 +1752,7 @@ process VEP { tag {"${idSample} - ${variantCaller} - ${vcf}"} publishDir params.outdir, mode: params.publishDirMode, saveAs: { - if (it == "${reducedVCF}_VEP.summary.html") "Annotation/${idSample}/VEP/${it}" + if (it == "${reducedVCF}_VEP.summary.html") "Reports/${idSample}/VEP/${it}" else null } From 14c62b6951a5cfa0865cec1280c0e31c64316a30 Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Thu, 23 May 2019 15:37:34 +0200 Subject: [PATCH 03/22] better docs --- docs/input.md | 79 +++++++++++++++++++++++++++++++-------------------- 1 file changed, 48 insertions(+), 31 deletions(-) diff --git a/docs/input.md b/docs/input.md index 49ec48d344..8856e0cdc7 100644 --- a/docs/input.md +++ b/docs/input.md @@ -31,37 +31,12 @@ Multiple TSV files can be specified if the path is enclosed in quotes. Somatic variant calling output will be in a specific directory for each normal/tumor pair. -# Example TSV file for a normal/tumor pair with FASTQ files +# Path to a FASTQ directory for a single normal sample (step mapping) -In this sample for the normal case there are 3 read groups, and 2 for the tumor. - -``` -G15511 XX 0 C09DFN C09DF_1 pathToFiles/C09DFACXX111207.1_1.fastq.gz pathToFiles/C09DFACXX111207.1_2.fastq.gz -G15511 XX 0 C09DFN C09DF_2 pathToFiles/C09DFACXX111207.2_1.fastq.gz pathToFiles/C09DFACXX111207.2_2.fastq.gz -G15511 XX 0 C09DFN C09DF_3 pathToFiles/C09DFACXX111207.3_1.fastq.gz pathToFiles/C09DFACXX111207.3_2.fastq.gz -G15511 XX 1 D0ENMT D0ENM_1 pathToFiles/D0ENMACXX111207.1_1.fastq.gz pathToFiles/D0ENMACXX111207.1_2.fastq.gz -G15511 XX 1 D0ENMT D0ENM_2 pathToFiles/D0ENMACXX111207.2_1.fastq.gz pathToFiles/D0ENMACXX111207.2_2.fastq.gz -``` - -# Example TSV file for a normal/tumor pair with BAM files - -In this sample for the normal case there are 3 read groups, and 2 for the tumor. - -``` -G15511 XX 0 C09DFN C09DF_1 pathToFiles/C09DFAC_1.bam -G15511 XX 0 C09DFN C09DF_2 pathToFiles/C09DFAC_2.bam -G15511 XX 0 C09DFN C09DF_3 pathToFiles/C09DFAC_3.bam -G15511 XX 1 D0ENMT D0ENM_1 pathToFiles/D0ENMAC_1.bam -G15511 XX 1 D0ENMT D0ENM_2 pathToFiles/D0ENMAC_2.bam -``` - -# Example TSV file for a normal/tumor pair with recalibrated BAM files - -The same way, if you have recalibrated BAMs and their indexes, you should use a structure like: +Input files for Sarek can be specified using the path to a FASTQ directory given to the `--sample` command only with the `mapping` step. -``` -G15511 XX 0 C09DFN pathToFiles/G15511.C09DFN.md.real.bam pathToFiles/G15511.C09DFN.md.real.bai -G15511 XX 1 D0ENMT pathToFiles/G15511.D0ENMT.md.real.bam pathToFiles/G15511.D0ENMT.md.real.bai +```bash +nextflow run nf-core/sarek --sample pathToDirectory ... ``` ## Input FASTQ file name best practices @@ -105,11 +80,53 @@ Read group information will be parsed from fastq file names according to this: - `PU` = sample - `RGLB` = lib -# Path to a FASTQ directory for a single normal sample +# Example TSV file for a normal/tumor pair with FASTQ files (step mapping) -Input files for Sarek can be specified using the path to a FASTQ directory given to the `--sample` command only with the `mapping` step. +In this sample for the normal case there are 3 read groups, and 2 for the tumor. + +``` +G15511 XX 0 C09DFN C09DF_1 pathToFiles/C09DFACXX111207.1_1.fastq.gz pathToFiles/C09DFACXX111207.1_2.fastq.gz +G15511 XX 0 C09DFN C09DF_2 pathToFiles/C09DFACXX111207.2_1.fastq.gz pathToFiles/C09DFACXX111207.2_2.fastq.gz +G15511 XX 0 C09DFN C09DF_3 pathToFiles/C09DFACXX111207.3_1.fastq.gz pathToFiles/C09DFACXX111207.3_2.fastq.gz +G15511 XX 1 D0ENMT D0ENM_1 pathToFiles/D0ENMACXX111207.1_1.fastq.gz pathToFiles/D0ENMACXX111207.1_2.fastq.gz +G15511 XX 1 D0ENMT D0ENM_2 pathToFiles/D0ENMACXX111207.2_1.fastq.gz pathToFiles/D0ENMACXX111207.2_2.fastq.gz +``` + +# Example TSV file for a normal/tumor pair with uBAM files (step mapping) + +In this sample for the normal case there are 3 read groups, and 2 for the tumor. + +``` +G15511 XX 0 C09DFN C09DF_1 pathToFiles/C09DFAC_1.bam +G15511 XX 0 C09DFN C09DF_2 pathToFiles/C09DFAC_2.bam +G15511 XX 0 C09DFN C09DF_3 pathToFiles/C09DFAC_3.bam +G15511 XX 1 D0ENMT D0ENM_1 pathToFiles/D0ENMAC_1.bam +G15511 XX 1 D0ENMT D0ENM_2 pathToFiles/D0ENMAC_2.bam +``` + +# Example TSV file for a normal/tumor pair with non recalibrated BAM files (step recalibrate) + +The same way, if you have non recalibrated BAMs, their indexes and their recalibration tables, you should use a structure like: + +``` +G15511 XX 0 C09DFN pathToFiles/G15511.C09DFN.md.bam pathToFiles/G15511.C09DFN.md.bai pathToFiles/G15511.C09DFN.md.recal.table +G15511 XX 1 D0ENMT pathToFiles/G15511.D0ENMT.md.bam pathToFiles/G15511.D0ENMT.md.bai pathToFiles/G15511.D0ENMT.md.recal.table +``` + +# Example TSV file for a normal/tumor pair with recalibrated BAM files (step variantcalling) + +The same way, if you have recalibrated BAMs and their indexes, you should use a structure like: + +``` +G15511 XX 0 C09DFN pathToFiles/G15511.C09DFN.md.recal.bam pathToFiles/G15511.C09DFN.md.recal.bai +G15511 XX 1 D0ENMT pathToFiles/G15511.D0ENMT.md.recal.bam pathToFiles/G15511.D0ENMT.md.recal.bai +``` # VCF files for annotation Input files for Sarek can be specified using the path to a VCF directory given to the `--sample` command only with the `annotate` step. Multiple VCF files can be specified if the path is enclosed in quotes. + +```bash +nextflow run nf-core/sarek --step annotate --sample "results/VariantCalling/*/.vcf.gz" ... +``` From cb3fe49876dd8a6a9f3c23981051a3c54cd3087d Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Thu, 23 May 2019 15:38:57 +0200 Subject: [PATCH 04/22] add docs about annotation --- docs/output.md | 47 ++++++++++++++++++++++++++++++++++++++++++++--- 1 file changed, 44 insertions(+), 3 deletions(-) diff --git a/docs/output.md b/docs/output.md index ec70888d40..c8a83974f2 100644 --- a/docs/output.md +++ b/docs/output.md @@ -98,11 +98,42 @@ BAM files with Recalibration tables can also be used as an input to start with t ## Annotation +This directory contains results from the final annotation steps: two software are used for annotation, [VEP][vep-link] and [snpEff][snpeff-link]. +Only a subset of the VCF files are annotated, and only variants that have a PASS filter. +FreeBayes results are not annotated in the moment yet as we are lacking a decent somatic filter. +For HaplotypeCaller the germline variations are annotated for both the tumour and the normal sample. + ### snpEff -[snpeff](http://snpeff.sourceforge.net/) +[snpeff](http://snpeff.sourceforge.net/) is a genetic variant annotation and effect prediction toolbox. +It annotates and predicts the effects of variants on genes (such as amino acid changes) using multiple databases for annotations. +The generated VCF header contains the software version and the used command line. + +**Output directory: `results/Annotation/[SAMPLE]/snpEff`** + +* `VariantCaller_Sample_snpEff.ann.vcf.gz` and `VariantCaller_Sample_snpEff.ann.vcf.gz.tbi` + * VCF with Tabix index ### VEP -[VEP (Variant Effect Predictor)](https://www.ensembl.org/info/docs/tools/vep/index.html) +[VEP (Variant Effect Predictor)](https://www.ensembl.org/info/docs/tools/vep/index.html), based on Ensembl, is a tools to determine the effects of all sorts of variants, including SNPs, indels, structural variants, CNVs. +The generated VCF header contains the software version, also the version numbers for additional databases like Clinvar or dbSNP used in the "VEP" line. +The format of the [consequence annotations](https://www.ensembl.org/info/genome/variation/prediction/predicted_data.html) is also in the VCF header describing the INFO field. +In the moment it contains: +* Consequence: impact of the variation, if there is any +* Codons: the codon change, i.e. cGt/cAt +* Amino_acids: change in amino acids, i.e. R/H if there is any +* Gene: ENSEMBL gene name +* SYMBOL: gene symbol +* Feature: actual transcript name +* EXON: affected exon +* PolyPhen: prediction based on [PolyPhen](http://genetics.bwh.harvard.edu/pph2/) +* SIFT: prediction by [SIFT](http://sift.bii.a-star.edu.sg/) +* Protein_position: Relative position of amino acid in protein +* BIOTYPE: Biotype of transcript or regulatory feature + +**Output directory: `results/Annotation/[SAMPLE]/VEP`** + +* `VariantCaller_Sample_VEP.ann.vcf.gz` and `VariantCaller_Sample_VEP.ann.vcf.gz.tbi` + * VCF with Tabix index ## QC and reports @@ -189,7 +220,8 @@ For more information about how to use VCFtools reports, see [VCFtools manual](ht ### snpEff reports [snpeff](http://snpeff.sourceforge.net/) is a genetic variant annotation and effect prediction toolbox. -It annotates and predicts the effects of variants on genes (such as amino acid changes). +It annotates and predicts the effects of variants on genes (such as amino acid changes) using multiple databases for annotations. + Plots will shows : * locations of detected variants in the genome and the number of variants for each location. * the putative impact of detected variants and the number of variants for each impact. @@ -206,6 +238,15 @@ Plots will shows : For more information about how to use snpEff reports, see [snpEff manual](http://snpeff.sourceforge.net/SnpEff_manual.html#outputSummary) +### VEP reports +[VEP (Variant Effect Predictor)](https://www.ensembl.org/info/docs/tools/vep/index.html), based on Ensembl, is a tools to determine the effects of all sorts of variants, including SNPs, indels, structural variants, CNVs. + +**Output directory: `results/Reports/[SAMPLE]/VEP`** +* `VariantCaller_Sample_VEP.summary.html` + * Summary of the VEP run to be visualised with a web browser + +For more information about how to use VEP reports, see [VEP manual](https://www.ensembl.org/info/docs/tools/vep/index.html) + ### MultiQC [MultiQC](http://multiqc.info) is a visualisation tool that generates a single HTML report summarising all samples in your project. Most of the pipeline QC results are visualised in the report and further statistics are available in within the report data directory. From db9495427a3cea559a43d6ea9dcb4350196735bb Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Thu, 23 May 2019 16:51:12 +0200 Subject: [PATCH 05/22] move helper scripts to scripts --- {bin => scripts}/build_reference.sh | 0 {bin => scripts}/download_docker.sh | 0 {bin => scripts}/run_tests.sh | 0 3 files changed, 0 insertions(+), 0 deletions(-) rename {bin => scripts}/build_reference.sh (100%) rename {bin => scripts}/download_docker.sh (100%) rename {bin => scripts}/run_tests.sh (100%) diff --git a/bin/build_reference.sh b/scripts/build_reference.sh similarity index 100% rename from bin/build_reference.sh rename to scripts/build_reference.sh diff --git a/bin/download_docker.sh b/scripts/download_docker.sh similarity index 100% rename from bin/download_docker.sh rename to scripts/download_docker.sh diff --git a/bin/run_tests.sh b/scripts/run_tests.sh similarity index 100% rename from bin/run_tests.sh rename to scripts/run_tests.sh From ff94a0230297a9732f2b098a9f4d1348a175eec3 Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Thu, 23 May 2019 16:51:35 +0200 Subject: [PATCH 06/22] add helper script to filter freebayes output --- scripts/speedseq.filter.awk | 50 +++++++++++++++++++++++++++++++++++++ 1 file changed, 50 insertions(+) create mode 100644 scripts/speedseq.filter.awk diff --git a/scripts/speedseq.filter.awk b/scripts/speedseq.filter.awk new file mode 100644 index 0000000000..d5d9ab0020 --- /dev/null +++ b/scripts/speedseq.filter.awk @@ -0,0 +1,50 @@ +# somatic filter for FreeBayes VCF files, based on SpeedSeq: https://github.com/hall-lab/speedseq +# recommended to filter the large VCF files like: +# vcfsamplediff -s VT normal tumour freebayesresult.vcf | \ +# awk '/^#/{print}!/^#/&&!/germline/' |\ +# vcffilter -f "QUAL > 20" | vcfflatten |\ +# awk -f speedseq.filter.awk -v NORMAL_IDX=10 -v TUMOUR_IDX=11 > filtered.vcf +# where "normal" and "tumour" are the sample names in the VCF respectively, and the IDX variables are their 1-based column numbers in the #CHROM line of the VCF +# also check the index for these sample names also +BEGIN{ + MINQUAL=1; + SSC_THRES=1; # somatic score threshold ssc = LOD_T + LOD_N, log of odds (LOD) is the genotype quality ratio (http://www.nature.com/nmeth/journal/v12/n10/pdf/nmeth.3505.pdf) + ONLY_SOMATIC=1; # prints out only somatic lines if not zero + GL_IDX=0; # GL in the original: PL is the Normalized, Phred-scaled likelihoods for genotypes +} +{ + OFS="\t"; + + if ($0~"^#" && $0!~"^#CHROM") { print ; next; } + # add extra header line + if ($0~"^#CHROM") { + print "##INFO=" + print ; + } + if (! GL_IDX) { + split($9,fmt,":") + for (i=1;i<=length(fmt);++i) { if (fmt[i]=="GL") GL_IDX=i } + } + split($NORMAL_IDX,N,":"); # split field 10 and put values into + split(N[GL_IDX],NGL,","); + split($TUMOR_IDX,T,":"); + split(T[GL_IDX],TGL,","); + LOD_NORM=NGL[1]-NGL[2]; + LOD_TUMOR_HET=TGL[2]-TGL[1]; + LOD_TUMOR_HOM=TGL[3]-TGL[1]; + + if (LOD_TUMOR_HET > LOD_TUMOR_HOM) { LOD_TUMOR=LOD_TUMOR_HET } + else { LOD_TUMOR=LOD_TUMOR_HOM } + + DQUAL=LOD_TUMOR+LOD_NORM; + + if (DQUAL>=SSC_THRES && $NORMAL_IDX~"^0/0") { + $7="PASS" + $8="DQUAL="DQUAL";"$8 + print + } + else if (!ONLY_SOMATIC && $6>=MINQUAL && $NORMAL_IDX~"^0/0" && ! match($TUMOUR_IDX,"^0/0")) { + $8="DQUAL="DQUAL";"$8 + print + } +} From 7ad53ec5c1a0ceaa559718d353b0a6f29e4d505b Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Thu, 23 May 2019 16:51:53 +0200 Subject: [PATCH 07/22] update path to helper scripts --- .travis.yml | 6 +++--- Jenkinsfile | 14 +++++++------- 2 files changed, 10 insertions(+), 10 deletions(-) diff --git a/.travis.yml b/.travis.yml index f0dc2b8adb..08f48f99a3 100644 --- a/.travis.yml +++ b/.travis.yml @@ -27,7 +27,7 @@ before_install: # PRs to master are only ok if coming from dev branch - '[ $TRAVIS_PULL_REQUEST = "false" ] || [ $TRAVIS_BRANCH != "master" ] || ([ $TRAVIS_PULL_REQUEST_SLUG = $TRAVIS_REPO_SLUG ] && [ $TRAVIS_PULL_REQUEST_BRANCH = "dev" ])' # Pull the docker image first so the test doesn't wait for this - - "travis_retry ./bin/download_docker.sh --test $TEST" + - "travis_retry ./scripts/download_docker.sh --test $TEST" install: # Install Nextflow @@ -39,8 +39,8 @@ install: # Build references if needed before_script: - - "${TRAVIS_BUILD_DIR}/bin/build_reference.sh --test $TEST --verbose" + - "${TRAVIS_BUILD_DIR}/scripts/build_reference.sh --test $TEST --verbose" # Actual tests script: - - "${TRAVIS_BUILD_DIR}/bin/run_tests.sh --test $TEST --verbose" + - "${TRAVIS_BUILD_DIR}/scripts/run_tests.sh --test $TEST --verbose" diff --git a/Jenkinsfile b/Jenkinsfile index e210d56d0b..42d6074d46 100644 --- a/Jenkinsfile +++ b/Jenkinsfile @@ -8,41 +8,41 @@ pipeline { stages { stage('Docker setup') { steps { - sh "./bin/download_docker.sh" + sh "./scripts/download_docker.sh" } } stage('Build references') { steps { sh "rm -rf references/" - sh "./bin/build_reference.sh" + sh "./scripts/build_reference.sh" } } stage('Germline') { steps { sh "rm -rf data/" sh "git clone --single-branch --branch sarek https://github.com/nf-core/test-datasets.git data" - sh "./bin/run_tests.sh --test GERMLINE" + sh "./scripts/run_tests.sh --test GERMLINE" sh "rm -rf data/" } } stage('Somatic') { steps { - sh "./bin/run_tests.sh --test SOMATIC" + sh "./scripts/run_tests.sh --test SOMATIC" } } stage('Targeted') { steps { - sh "./bin/run_tests.sh --test TARGETED" + sh "./scripts/run_tests.sh --test TARGETED" } } stage('Annotation') { steps { - sh "./bin/run_tests.sh --test ANNOTATEALL" + sh "./scripts/run_tests.sh --test ANNOTATEALL" } } stage('Multiple') { steps { - sh "./bin/run_tests.sh --test MULTIPLE" + sh "./scripts/run_tests.sh --test MULTIPLE" } } } From e3558276b06d76ec2a290fecdc1710da78d2df4b Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Thu, 23 May 2019 16:52:22 +0200 Subject: [PATCH 08/22] add docs about Variant Calling and Preprocessing --- docs/output.md | 127 ++++++++++++++++++++++++++++++++++++++++--------- 1 file changed, 105 insertions(+), 22 deletions(-) diff --git a/docs/output.md b/docs/output.md index c8a83974f2..b595221905 100644 --- a/docs/output.md +++ b/docs/output.md @@ -9,13 +9,13 @@ and processes data using the following steps: 1. **Preprocessing** _(based on [GATK best practices](https://software.broadinstitute.org/gatk/best-practices/))_ * Map reads to Reference - * [BWA](#BWA) + * BWA mem * Mark Duplicates - * [GATK MarkDuplicates](#MarkDuplicates) + * GATK MarkDuplicates * Base (Quality Score) Recalibration - * [GATK BaseRecalibrator](#BaseRecalibrator) - * [GATK GatherBQSRReports](#GatherBQSRReports) - * [GATK ApplyBQSR](#ApplyBQSR) + * GATK BaseRecalibrator + * GATK GatherBQSRReports + * GATK ApplyBQSR 2. **Variant calling** * SNVs and small indels * [Freebayes](#Freebayes) @@ -48,49 +48,132 @@ and processes data using the following steps: * [MultiQC](#MultiQC) ## Preprocessing -Sarek preprocessing raw FastQ files or unmapped BAM files is based on [GATK best practices](https://software.broadinstitute.org/gatk/best-practices/)). +Sarek preprocessing raw FastQ files or unmapped BAM files is based on [GATK best practices](https://software.broadinstitute.org/gatk/best-practices/). BAM files with Recalibration tables can also be used as an input to start with the recalibration of said BAM files. -### BWA -[BWA](http://bio-bwa.sourceforge.net/) +### Duplicate Marked BAM file(s) with Recalibration Table(s) -### MarkDuplicates -[GATK MarkDuplicates](https://github.com/broadinstitute/gatk) +**Output directory: `results/Preprocessing/[SAMPLE]/DuplicateMarked`** -### BaseRecalibrator -[GATK BaseRecalibrator](https://github.com/broadinstitute/gatk) +* `Sample.md.bam`, `Sample.md.bai` and `Sample.recal.table` + * BAM file and index with Recalibration Table -### GatherBQSRReports -[GATK GatherBQSRReports](https://github.com/broadinstitute/gatk) +### Recalibrated BAM file(s) -### ApplyBQSR -[GATK ApplyBQSR](https://github.com/broadinstitute/gatk) +This directory is usually empty, it is the location for the final recalibrated files in the preprocessing pipeline: recalibrated BAM files are usually 2-3 times larger than the duplicate marked BAM files. +To re-generate recalibrated BAM file you have to apply the recalibration table delivered to the `DuplicateMarked` directory either within sarek, or doing this [recalibration step] yourself. + +**Output directory: `results/Preprocessing/[SAMPLE]/Recalibrated`** + +* `Sample.recal.bam` and `Sample.recal.bai` + * BAM file and index + +### TSV files +The TSV files are autogenerated and can be used by Sarek for further processing and/or variant calling. + +**Output directory: `results/Preprocessing/TSV`** + +* `duplicateMarked.tsv` and `recalibrated.tsv` + * TSV files to start Sarek from `recalibration` or `variantcalling` steps. +* `duplicateMarked_Sample.tsv` and `recalibrated_Sample.tsv` + * TSV files to start Sarek from `recalibration` or `variantcalling` steps for a specific sample. ## Variant Calling +All the results regarding variant-calling are collected in this directory. ### Freebayes -[Freebayes](https://github.com/ekg/freebayes) +[Freebayes](https://github.com/ekg/freebayes) is a Bayesian genetic variant detector designed to find small polymorphisms, specifically SNPs, indels, MNPs, and complex events smaller than the length of a short-read sequencing alignment.. +The single VCF file generated by `FreeBayes` being huge, it is recommended to flatten and filter this VCF, i.e. using the provided [SpeedSeq](https://github.com/nf-core/sarek/blob/master/scripts/speedseq.filter.awk) filter. + +**Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/FreeBayes`** + +* `FreeBayes_TumorSample_vs_NormalSample.vcf.gz` and `FreeBayes_TumorSample_vs_NormalSample.vcf.gz.tbi` + * VCF with Tabix index ### HaplotypeCaller -[GATK HaplotypeCaller](https://github.com/broadinstitute/gatk) +[GATK HaplotypeCaller](https://software.broadinstitute.org/gatk/documentation/tooldocs/4.1.2.0/org_broadinstitute_hellbender_tools_walkers_haplotypecaller_HaplotypeCaller.php) calls germline SNPs and indels via local re-assembly of haplotypes. + +**Output directory: `results/VariantCalling/[SAMPLE]/HaploTypeCaller`** + +* `HaplotypeCaller_Sample.vcf.gz` and `HaplotypeCaller_Sample.vcf.gz.tbi` + * VCF with Tabix index ### GenotypeGVCFs -[GATK GenotypeGVCFs](https://github.com/broadinstitute/gatk) +[GATK GenotypeGVCFs](https://software.broadinstitute.org/gatk/documentation/tooldocs/4.1.2.0/org_broadinstitute_hellbender_tools_walkers_GenotypeGVCFs.php) performs joint genotyping on one or more samples pre-called with HaplotypeCaller. + +**Output directory: `results/VariantCalling/[SAMPLE]/HaplotypeCallerGVCF`** + +* `HaplotypeCaller_Sample.g.vcf.gz` and `HaplotypeCaller_Sample.g.vcf.gz.tbi` + * VCF with Tabix index ### MuTect2 -[MuTect2](https://github.com/broadinstitute/gatk) +[MuTect2](https://software.broadinstitute.org/gatk/documentation/tooldocs/4.1.2.0/org_broadinstitute_hellbender_tools_walkers_mutect_Mutect2.php) calls somatic SNVs and indels via local assembly of haplotypes. + +**Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/MuTect2`** + +* `MuTect2_TumorSample_vs_NormalSample.vcf.gz` and `MuTect2_TumorSample_vs_NormalSample.vcf.gz.tbi` + * VCF with Tabix index ### Strelka2 -[Strelka2](https://github.com/Illumina/strelka) +[Strelka2](https://github.com/Illumina/strelka) is a fast and accurate small variant caller optimized for analysis of germline variation in small cohorts and somatic variation in tumor/normal sample pairs. + +**Output directory: `results/VariantCalling/[SAMPLE]/Strelka`** + +* `Strelka_Sample_genome.vcf.gz` and `Strelka_Sample_genome.vcf.gz.tbi` + * VCF with Tabix index +* `Strelka_Sample_variants.vcf.gz` and `Strelka_Sample_variants.vcf.gz.tbi` + * VCF with Tabix index + +For a Tumor/Normal pair: +**Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/Strelka`** + +* `Strelka_TumorSample_vs_NormalSample_somatic_indels.vcf.gz` and `Strelka_TumorSample_vs_NormalSample_somatic_indels.vcf.gz.tbi` + * VCF with Tabix index +* `Strelka_TumorSample_vs_NormalSample_somatic_snvs.vcf.gz` and `Strelka_TumorSample_vs_NormalSample_somatic_snvs.vcf.gz.tbi` + * VCF with Tabix index +* `StrelkaBP_TumorSample_vs_NormalSample_somatic_indels.vcf.gz` and `StrelkaBP_TumorSample_vs_NormalSample_somatic_indels.vcf.gz.tbi` + * VCF with Tabix index +* `StrelkaBP_TumorSample_vs_NormalSample_somatic_snvs.vcf.gz` and `StrelkaBP_TumorSample_vs_NormalSample_somatic_snvs.vcf.gz.tbi` + * VCF with Tabix index ### Manta -[Manta](https://github.com/Illumina/manta) +[Manta](https://github.com/Illumina/manta) calls structural variants (SVs) and indels from mapped paired-end sequencing reads. +It is optimized for analysis of germline variation in small sets of individuals and somatic variation in tumor/normal sample pairs. + +**Output directory: `results/VariantCalling/[SAMPLE]/Manta`** + +* `Manta_Sample.candidateSmallIndells.vcf.gz` and `Manta_Sample.candidateSmallIndells.vcf.gz.tbi` + * VCF with Tabix index +* `Manta_Sample.candidateSV.vcf.gz` and `Manta_Sample.candidateSV.vcf.gz.tbi` + * VCF with Tabix index + +For Normal sample: +* `Manta_NormalSample.diploidSV.vcf.gz` and `Manta_NormalSample.diploidSV.vcf.gz.tbi` + * VCF with Tabix index + +For a Tumor sample: +* `Manta_TumorSample.tumorSV.vcf.gz` and `Manta_TumorSample.tumorSV.vcf.gz.tbi` + * VCF with Tabix index + +For a Tumor/Normal pair: +**Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/Manta`** + +* `Manta_TumorSample_vs_NormalSample.candidateSmallIndels.vcf.gz` and `Manta_TumorSample_vs_NormalSample.candidateSmallIndels.vcf.gz.tbi` + * VCF with Tabix index +* `Manta_TumorSample_vs_NormalSample.candidateSV.vcf.gz` and `Manta_TumorSample_vs_NormalSample.candidateSV.vcf.gz.tbi` + * VCF with Tabix index +* `Manta_TumorSample_vs_NormalSample.diploidSV.vcf.gz` and `Manta_TumorSample_vs_NormalSample.diploidSV.vcf.gz.tbi` + * VCF with Tabix index +* `Manta_TumorSample_vs_NormalSample.somaticSV.vcf.gz` and `Manta_TumorSample_vs_NormalSample.somaticSV.vcf.gz.tbi` + * VCF with Tabix index ### alleleCounter ### ConvertAlleleCounts ### ASCAT +[ASCAT](https://www.crick.ac.uk/research/a-z-researchers/researchers-v-y/peter-van-loo/software/) is a method to derive copy number profiles of tumour cells, accounting for normal cell admixture and tumour aneuploidy. +ASCAT infers tumour purity and ploidy and calculates whole-genome allele-specific copy number profiles. ### samtools mpileup From 9247f883b107bbfd34d3b3980af8ea91f4028568 Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Fri, 24 May 2019 15:17:49 +0200 Subject: [PATCH 09/22] add scripts for ASCAT --- bin/convertAlleleCounts.r | 93 +++++++++++++++++++++++++++++++++++++++ bin/run_ascat.r | 58 ++++++++++++++++++++++++ {bin => scripts}/ascat.R | 0 3 files changed, 151 insertions(+) create mode 100644 bin/convertAlleleCounts.r create mode 100644 bin/run_ascat.r rename {bin => scripts}/ascat.R (100%) diff --git a/bin/convertAlleleCounts.r b/bin/convertAlleleCounts.r new file mode 100644 index 0000000000..302e45384c --- /dev/null +++ b/bin/convertAlleleCounts.r @@ -0,0 +1,93 @@ +#!/bin/env Rscript +# Description: +# R-script for converting output from AlleleCount to BAF and LogR values. +# +# Input: +# AlleleCounter output file for tumor and normal samples +# The first line should contain a header describing the data +# The following columns and headers should be present: +# CHR POS Count_A Count_C Count_G Count_T Good_depth +# +# Output: +# BAF and LogR tables (tab delimited text files) +################################################################################ + +##First read in the arguments listed at the command line +args = commandArgs(trailingOnly=TRUE) + +## args is now a list of character vectors +## First check to see if arguments are passed. +if(length(args)<5){ + stop("No input files supplied\n\nUsage:\nRscript convertAlleleCounts.r tumorid tumorac normalid normalac gender\nWhere:\ntumorid - id of tumor sample\ntumorac - output from AlleleCount for the tumor\nnormalid - id of normal sample\nnormalac - output from AlleleCount for the normal\ngender - XX or XY\n\n") +} else{ + tumorid = args[1] + tumorac = args[2] + normalid = args[3] + normalac = args[4] + gender = args[5] +} + +tumorcounts = read.table(tumorac, header=F, sep="\t") +normalcounts = read.table(normalac, header=F, sep="\t") + +SNPpos = matrix(nrow = dim(normalcounts)[1],ncol = 2) + +rownames(SNPpos) = paste("snp",1:dim(SNPpos)[1],sep="") + +#Change rownames to "chr_pos" instead, such as 1_44552 +#This does not exactly work: +#rownames(SNPpos) = apply(cbind(tumorcounts[,1], tumorcounts[,2]), 1, paste, collapse="_") +#This is for compatibility with gc correction file + +colnames(SNPpos) = c("Chr","Position") +SNPpos[,1] = as.vector(normalcounts[,1]) +SNPpos[,2] = normalcounts[,2] + +#Caclulate BAF +Tumor_BAF = matrix(nrow = dim(normalcounts)[1],ncol = 1) +rownames(Tumor_BAF) = rownames(SNPpos) +colnames(Tumor_BAF) = c(tumorid) +acgt = tumorcounts[,c(3:6)] +acgts = t(apply(acgt,1,sort)) +Tumor_BAF[,1] = acgts[,4]/(acgts[,3]+acgts[,4]) +Tumor_BAF[,1] = ifelse(runif(length(Tumor_BAF[,1]))<0.5,Tumor_BAF[,1],1-Tumor_BAF[,1]) +Tumor_BAF[is.nan(Tumor_BAF)]=NA + +Germline_BAF = matrix(nrow = dim(normalcounts)[1],ncol = 1) +rownames(Germline_BAF) = rownames(SNPpos) +colnames(Germline_BAF) = c(normalid) +acgt = normalcounts[,c(3:6)] +acgts = t(apply(acgt,1,sort)) +Germline_BAF[,1] = acgts[,4]/(acgts[,3]+acgts[,4]) +Germline_BAF[,1] = ifelse(runif(length(Germline_BAF[,1]))<0.5,Germline_BAF[,1],1-Germline_BAF[,1]) +Germline_BAF[is.nan(Germline_BAF)]=NA + + +Tumor_LogR = matrix(nrow = dim(normalcounts)[1],ncol = 1) +Germline_LogR = matrix(nrow = dim(normalcounts)[1],ncol = 1) +rownames(Tumor_LogR) = rownames(SNPpos) +colnames(Tumor_LogR) = c(tumorid) +rownames(Germline_LogR) = rownames(SNPpos) +colnames(Germline_LogR) = c(normalid) +Tumor_LogR[,1] = log(tumorcounts[,7]/normalcounts[,7],2) +Germline_LogR[,1] = 0 +Tumor_LogR[is.infinite(Tumor_LogR)]=NA +if(gender=="XY") { + Tumor_LogR[SNPpos[,1]=="X",1] = Tumor_LogR[SNPpos[,1]=="X",1]-1 + Germline_LogR[SNPpos[,1]=="X",1] = Germline_LogR[SNPpos[,1]=="X",1]-1 +} +Tumor_LogR[,1] = Tumor_LogR[,1] - median(Tumor_LogR[,1],na.rm=T) +# set regions with 0 reads in tumor and normal to a LogR of 0. +Tumor_LogR[is.na(Tumor_LogR[,1]),1] = 0 + +# limit the number of digits: +Tumor_LogR = round(Tumor_LogR,4) +Tumor_BAF = round(Tumor_BAF,4) +Germline_LogR = round(Germline_LogR,4) +Germline_BAF = round(Germline_BAF,4) + +# write output to files +write.table(cbind(SNPpos,Tumor_LogR),paste(tumorid,".LogR",sep=""),sep="\t",row.names=T,col.names=NA,quote=F) +write.table(cbind(SNPpos,Tumor_BAF),paste(tumorid,".BAF",sep=""),sep="\t",row.names=T,col.names=NA,quote=F) +write.table(cbind(SNPpos,Germline_LogR),paste(normalid,".LogR",sep=""),sep="\t",row.names=T,col.names=NA,quote=F) +write.table(cbind(SNPpos,Germline_BAF),paste(normalid,".BAF",sep=""),sep="\t",row.names=T,col.names=NA,quote=F) diff --git a/bin/run_ascat.r b/bin/run_ascat.r new file mode 100644 index 0000000000..6921f98c82 --- /dev/null +++ b/bin/run_ascat.r @@ -0,0 +1,58 @@ +#!/bin/env Rscript +args = commandArgs(trailingOnly=TRUE) +if(length(args)<6){ + stop("No input files supplied\n\nUsage:\nRscript run_ascat.r tumor_baf tumor_logr normal_baf normal_logr tumor_sample_name baseDir gcfile\n\n") +} else{ + tumorbaf = args[1] + tumorlogr = args[2] + normalbaf = args[3] + normallogr = args[4] + tumorname = args[5] + baseDir = args[6] + gcfile = args[7] +} + +source(paste(baseDir,"/scripts/ascat.R", sep="")) + +if(!require(RColorBrewer)){ + source("http://bioconductor.org/biocLite.R") + biocLite("RColorBrewer", suppressUpdates=TRUE, lib="$baseDir/scripts") + library(RColorBrewer) +} +options(bitmapType='cairo') + +#Load the data +ascat.bc <- ascat.loadData(Tumor_LogR_file=tumorlogr, Tumor_BAF_file=tumorbaf, Germline_LogR_file=normallogr, Germline_BAF_file=normalbaf) + +#GC wave correction +ascat.bc = ascat.GCcorrect(ascat.bc, gcfile) + +#Plot the raw data +ascat.plotRawData(ascat.bc) + +#Segment the data +ascat.bc <- ascat.aspcf(ascat.bc) + +#Plot the segmented data +ascat.plotSegmentedData(ascat.bc) + +#Run ASCAT to fit every tumor to a model, inferring ploidy, normal cell contamination, and discrete copy numbers +ascat.output <- ascat.runAscat(ascat.bc, gamma=1) + +#Write out segmented regions (including regions with one copy of each allele) +#write.table(ascat.output$segments, file=paste(tumorname, ".segments.txt", sep=""), sep="\t", quote=F, row.names=F) + +#Write out CNVs in bed format +cnvs=ascat.output$segments[2:6] +write.table(cnvs, file=paste(tumorname,".cnvs.txt",sep=""), sep="\t", quote=F, row.names=F, col.names=T) + +#Write out purity and ploidy info +summary <- tryCatch({ + matrix(c(ascat.output$aberrantcellfraction, ascat.output$ploidy), ncol=2, byrow=TRUE)}, error = function(err) { + # error handler picks up where error was generated + print(paste("Could not find optimal solution: ",err)) + return(matrix(c(0,0),nrow=1,ncol=2,byrow = TRUE)) + } +) +colnames(summary) <- c("AberrantCellFraction","Ploidy") +write.table(summary, file=paste(tumorname,".purityploidy.txt",sep=""), sep="\t", quote=F, row.names=F, col.names=T) diff --git a/bin/ascat.R b/scripts/ascat.R similarity index 100% rename from bin/ascat.R rename to scripts/ascat.R From abc584251e8c3a0fef36c1e7475b31ca87ea568a Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Fri, 24 May 2019 15:18:29 +0200 Subject: [PATCH 10/22] fix publishDir for MergeMpileup --- main.nf | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/main.nf b/main.nf index e0b0c73828..b51f47b62c 100644 --- a/main.nf +++ b/main.nf @@ -1409,7 +1409,7 @@ mpileupMerge = mpileupMerge.groupTuple(by:[0,1]) process MergeMpileup { tag {idSample} - publishDir params.outdir, mode: params.publishDirMode, saveAs: { it == "${idSample}.pileup.gz" ? "VariantCalling/${idSampleTumor}_vs_${idSampleNormal}/mpileup/${it}" : '' } + publishDir params.outdir, mode: params.publishDirMode, saveAs: { it == "${idSample}.pileup.gz" ? "VariantCalling/${idSample}/mpileup/${it}" : '' } input: set idPatient, idSample, file(mpileup) from mpileupMerge From 42695b7f57ed99a6854d71bcd1c4f0f5a689e1fe Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Fri, 24 May 2019 16:00:08 +0200 Subject: [PATCH 11/22] update docs --- docs/input.md | 31 +++++------ docs/output.md | 137 ++++++++++++++++++++++++++++++++++++------------- 2 files changed, 116 insertions(+), 52 deletions(-) diff --git a/docs/input.md b/docs/input.md index 8856e0cdc7..3b4502f039 100644 --- a/docs/input.md +++ b/docs/input.md @@ -1,5 +1,6 @@ -# TSV file for sample(s) +# Input Documentation +## Information about the TSV files Input files for Sarek can be specified using a TSV file given to the `--sample` command. The TSV file is a Tab Separated Value file with columns: * `subject gender status sample lane fastq1 fastq2` for step `mapping` with paired-end FASTQs @@ -11,8 +12,8 @@ The content of these columns is quite straight-forward: * `subject` designate the subject, it should be the ID of the Patient, and it must design only one patient * `gender` is the gender of the Patient, (XX or XY) * `status` is the status of the Patient, (0 for Normal or 1 for Tumor) -* `sample` designate the Sample, it should be the ID of the Sample (it is possible to have more than one tumor sample for each patient), it should design only one sample -* `lane` is used when the sample is multiplexed on several lanes +* `sample` designate the Sample, it should be the ID of the sample (it is possible to have more than one tumor sample for each patient, i.e. a tumor and a relapse), it must design only one sample +* `lane` is used when the sample is multiplexed on several lanes, it must be unique for each lane in the same sample * `fastq1` is the path to the first pair of the fastq file * `fastq2` is the path to the second pair of the fastq file * `bam` is the bam file @@ -31,6 +32,18 @@ Multiple TSV files can be specified if the path is enclosed in quotes. Somatic variant calling output will be in a specific directory for each normal/tumor pair. +# Example TSV file for a normal/tumor pair with FASTQ files (step mapping) + +In this sample for the normal case there are 3 read groups, and 2 for the tumor. + +``` +G15511 XX 0 C09DFN C09DF_1 pathToFiles/C09DFACXX111207.1_1.fastq.gz pathToFiles/C09DFACXX111207.1_2.fastq.gz +G15511 XX 0 C09DFN C09DF_2 pathToFiles/C09DFACXX111207.2_1.fastq.gz pathToFiles/C09DFACXX111207.2_2.fastq.gz +G15511 XX 0 C09DFN C09DF_3 pathToFiles/C09DFACXX111207.3_1.fastq.gz pathToFiles/C09DFACXX111207.3_2.fastq.gz +G15511 XX 1 D0ENMT D0ENM_1 pathToFiles/D0ENMACXX111207.1_1.fastq.gz pathToFiles/D0ENMACXX111207.1_2.fastq.gz +G15511 XX 1 D0ENMT D0ENM_2 pathToFiles/D0ENMACXX111207.2_1.fastq.gz pathToFiles/D0ENMACXX111207.2_2.fastq.gz +``` + # Path to a FASTQ directory for a single normal sample (step mapping) Input files for Sarek can be specified using the path to a FASTQ directory given to the `--sample` command only with the `mapping` step. @@ -80,18 +93,6 @@ Read group information will be parsed from fastq file names according to this: - `PU` = sample - `RGLB` = lib -# Example TSV file for a normal/tumor pair with FASTQ files (step mapping) - -In this sample for the normal case there are 3 read groups, and 2 for the tumor. - -``` -G15511 XX 0 C09DFN C09DF_1 pathToFiles/C09DFACXX111207.1_1.fastq.gz pathToFiles/C09DFACXX111207.1_2.fastq.gz -G15511 XX 0 C09DFN C09DF_2 pathToFiles/C09DFACXX111207.2_1.fastq.gz pathToFiles/C09DFACXX111207.2_2.fastq.gz -G15511 XX 0 C09DFN C09DF_3 pathToFiles/C09DFACXX111207.3_1.fastq.gz pathToFiles/C09DFACXX111207.3_2.fastq.gz -G15511 XX 1 D0ENMT D0ENM_1 pathToFiles/D0ENMACXX111207.1_1.fastq.gz pathToFiles/D0ENMACXX111207.1_2.fastq.gz -G15511 XX 1 D0ENMT D0ENM_2 pathToFiles/D0ENMACXX111207.2_1.fastq.gz pathToFiles/D0ENMACXX111207.2_2.fastq.gz -``` - # Example TSV file for a normal/tumor pair with uBAM files (step mapping) In this sample for the normal case there are 3 read groups, and 2 for the tumor. diff --git a/docs/output.md b/docs/output.md index b595221905..db0b52ffa1 100644 --- a/docs/output.md +++ b/docs/output.md @@ -1,13 +1,11 @@ # nf-core/sarek: Output This document describes the output produced by the pipeline. -Most of the plots are taken from the MultiQC report, which summarises results at the end of the pipeline. ## Pipeline overview -The pipeline is built using [Nextflow](https://www.nextflow.io/) -and processes data using the following steps: +The pipeline processes data using the following steps: -1. **Preprocessing** _(based on [GATK best practices](https://software.broadinstitute.org/gatk/best-practices/))_ +1. [**Preprocessing**](#Preprocessing) _(based on [GATK best practices](https://software.broadinstitute.org/gatk/best-practices/))_ * Map reads to Reference * BWA mem * Mark Duplicates @@ -16,9 +14,9 @@ and processes data using the following steps: * GATK BaseRecalibrator * GATK GatherBQSRReports * GATK ApplyBQSR -2. **Variant calling** +2. [**Variant calling**](#Variant-Calling) * SNVs and small indels - * [Freebayes](#Freebayes) + * [FreeBayes](#FreeBayes) * [GATK HaplotypeCaller](#HaplotypeCaller) * [GATK GenotypeGVCFs](#GenotypeGVCFs) * [GATK MuTect2](#MuTect2) @@ -26,16 +24,16 @@ and processes data using the following steps: * Structural variants * [Manta](#Manta) * Sample heterogeneity, ploidy and CNVs - * [alleleCounter](#alleleCounter) + * alleleCounter * [ConvertAlleleCounts](#ConvertAlleleCounts) * [ASCAT](#ASCAT) * [samtools mpileup](#mpileup) * [Control-FREEC](#Control-FREEC) -3. **Annotation** +3. [**Annotation**](#Annotation) * Variant annotation * [snpEff](#snpEff) * [VEP (Variant Effect Predictor)](#VEP) -4. **QC and Reporting** +4. [**QC and Reporting**](#QC-and-reporting) * QC * [FastQC](#FastQC) * [Qualimap bamqc](#bamQC) @@ -48,11 +46,16 @@ and processes data using the following steps: * [MultiQC](#MultiQC) ## Preprocessing -Sarek preprocessing raw FastQ files or unmapped BAM files is based on [GATK best practices](https://software.broadinstitute.org/gatk/best-practices/). -BAM files with Recalibration tables can also be used as an input to start with the recalibration of said BAM files. +Sarek preprocesses raw FastQ files or unmapped BAM files, based on [GATK best practices](https://software.broadinstitute.org/gatk/best-practices/). + +BAM files with Recalibration tables can also be used as an input to start with the recalibration of said BAM files, for more information see [TSV files output information](#TSV-files) ### Duplicate Marked BAM file(s) with Recalibration Table(s) +This directory is the location for the BAM files delivered to users. Besides the duplicate marked BAM files, the recalibration tables (`*.recal.table`) are also stored, and can be used to create base recalibrated files. + +For further reading and documentation see the [data pre-processing workflow from the GATK best practices](https://software.broadinstitute.org/gatk/best-practices/workflow?id=11165). + **Output directory: `results/Preprocessing/[SAMPLE]/DuplicateMarked`** * `Sample.md.bam`, `Sample.md.bai` and `Sample.recal.table` @@ -60,8 +63,11 @@ BAM files with Recalibration tables can also be used as an input to start with t ### Recalibrated BAM file(s) -This directory is usually empty, it is the location for the final recalibrated files in the preprocessing pipeline: recalibrated BAM files are usually 2-3 times larger than the duplicate marked BAM files. -To re-generate recalibrated BAM file you have to apply the recalibration table delivered to the `DuplicateMarked` directory either within sarek, or doing this [recalibration step] yourself. +This directory is usually empty, it is the location for the final recalibrated BAM files. +Recalibrated BAM files are usually 2-3 times larger than the duplicate marked BAM files. +To re-generate recalibrated BAM file you have to apply the recalibration table delivered to the `DuplicateMarked` directory either within Sarek, or doing this recalibration step yourself. + +For further reading and documentation see the [data pre-processing workflow from the GATK best practices](https://software.broadinstitute.org/gatk/best-practices/workflow?id=11165). **Output directory: `results/Preprocessing/[SAMPLE]/Recalibrated`** @@ -71,6 +77,8 @@ To re-generate recalibrated BAM file you have to apply the recalibration table d ### TSV files The TSV files are autogenerated and can be used by Sarek for further processing and/or variant calling. +For further reading and documentation see the [input documentation](https://github.com/nf-core/sarek/blob/master/docs/input.md). + **Output directory: `results/Preprocessing/TSV`** * `duplicateMarked.tsv` and `recalibrated.tsv` @@ -81,17 +89,25 @@ The TSV files are autogenerated and can be used by Sarek for further processing ## Variant Calling All the results regarding variant-calling are collected in this directory. -### Freebayes -[Freebayes](https://github.com/ekg/freebayes) is a Bayesian genetic variant detector designed to find small polymorphisms, specifically SNPs, indels, MNPs, and complex events smaller than the length of a short-read sequencing alignment.. +Recalibrated BAM files can also be used as an input to start the Variant Calling, for more information see [TSV files output information](#TSV-files) + +### FreeBayes +[FreeBayes](https://github.com/ekg/freebayes) is a Bayesian genetic variant detector designed to find small polymorphisms, specifically SNPs, indels, MNPs, and complex events smaller than the length of a short-read sequencing alignment.. The single VCF file generated by `FreeBayes` being huge, it is recommended to flatten and filter this VCF, i.e. using the provided [SpeedSeq](https://github.com/nf-core/sarek/blob/master/scripts/speedseq.filter.awk) filter. +For further reading and documentation see the [FreeBayes manual](https://github.com/ekg/freebayes/blob/master/README.md#user-manual-and-guide). + **Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/FreeBayes`** * `FreeBayes_TumorSample_vs_NormalSample.vcf.gz` and `FreeBayes_TumorSample_vs_NormalSample.vcf.gz.tbi` * VCF with Tabix index ### HaplotypeCaller -[GATK HaplotypeCaller](https://software.broadinstitute.org/gatk/documentation/tooldocs/4.1.2.0/org_broadinstitute_hellbender_tools_walkers_haplotypecaller_HaplotypeCaller.php) calls germline SNPs and indels via local re-assembly of haplotypes. +[GATK HaplotypeCaller](https://github.com/broadinstitute/gatk) calls germline SNPs and indels via local re-assembly of haplotypes. + +Germline calls are provided for all samples, to able comparison of both tumor and normal for possible mixup. + +For further reading and documentation see the [HaplotypeCaller manual](https://software.broadinstitute.org/gatk/documentation/tooldocs/4.1.2.0/org_broadinstitute_hellbender_tools_walkers_haplotypecaller_HaplotypeCaller.php). **Output directory: `results/VariantCalling/[SAMPLE]/HaploTypeCaller`** @@ -99,7 +115,11 @@ The single VCF file generated by `FreeBayes` being huge, it is recommended to fl * VCF with Tabix index ### GenotypeGVCFs -[GATK GenotypeGVCFs](https://software.broadinstitute.org/gatk/documentation/tooldocs/4.1.2.0/org_broadinstitute_hellbender_tools_walkers_GenotypeGVCFs.php) performs joint genotyping on one or more samples pre-called with HaplotypeCaller. +[GATK GenotypeGVCFs](https://github.com/broadinstitute/gatk) performs joint genotyping on one or more samples pre-called with HaplotypeCaller. + +Germline calls are provided for all samples, to able comparison of both tumor and normal for possible mixup. + +For further reading and documentation see the [GenotypeGVCFs manual](https://software.broadinstitute.org/gatk/documentation/tooldocs/4.1.2.0/org_broadinstitute_hellbender_tools_walkers_GenotypeGVCFs.php). **Output directory: `results/VariantCalling/[SAMPLE]/HaplotypeCallerGVCF`** @@ -107,7 +127,9 @@ The single VCF file generated by `FreeBayes` being huge, it is recommended to fl * VCF with Tabix index ### MuTect2 -[MuTect2](https://software.broadinstitute.org/gatk/documentation/tooldocs/4.1.2.0/org_broadinstitute_hellbender_tools_walkers_mutect_Mutect2.php) calls somatic SNVs and indels via local assembly of haplotypes. +[GATK MuTect2](https://github.com/broadinstitute/gatk) calls somatic SNVs and indels via local assembly of haplotypes. + +For further reading and documentation see the [MuTect2 manual](https://software.broadinstitute.org/gatk/documentation/tooldocs/4.1.2.0/org_broadinstitute_hellbender_tools_walkers_mutect_Mutect2.php). **Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/MuTect2`** @@ -117,6 +139,8 @@ The single VCF file generated by `FreeBayes` being huge, it is recommended to fl ### Strelka2 [Strelka2](https://github.com/Illumina/strelka) is a fast and accurate small variant caller optimized for analysis of germline variation in small cohorts and somatic variation in tumor/normal sample pairs. +For further reading and documentation see the [Strelka2 user guide](https://github.com/Illumina/strelka/blob/v2.9.x/docs/userGuide/README.md). + **Output directory: `results/VariantCalling/[SAMPLE]/Strelka`** * `Strelka_Sample_genome.vcf.gz` and `Strelka_Sample_genome.vcf.gz.tbi` @@ -131,6 +155,9 @@ For a Tumor/Normal pair: * VCF with Tabix index * `Strelka_TumorSample_vs_NormalSample_somatic_snvs.vcf.gz` and `Strelka_TumorSample_vs_NormalSample_somatic_snvs.vcf.gz.tbi` * VCF with Tabix index + +Using Strelka Best Practices with the `candidateSmallIndels` from Manta: +**Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/Strelka`** * `StrelkaBP_TumorSample_vs_NormalSample_somatic_indels.vcf.gz` and `StrelkaBP_TumorSample_vs_NormalSample_somatic_indels.vcf.gz.tbi` * VCF with Tabix index * `StrelkaBP_TumorSample_vs_NormalSample_somatic_snvs.vcf.gz` and `StrelkaBP_TumorSample_vs_NormalSample_somatic_snvs.vcf.gz.tbi` @@ -139,10 +166,13 @@ For a Tumor/Normal pair: ### Manta [Manta](https://github.com/Illumina/manta) calls structural variants (SVs) and indels from mapped paired-end sequencing reads. It is optimized for analysis of germline variation in small sets of individuals and somatic variation in tumor/normal sample pairs. +Manta provides a candidate list for small indels also that can be fed to Strelka. + +For further reading and documentation see the [Manta user guide](https://github.com/Illumina/manta/blob/master/docs/userGuide/README.md). **Output directory: `results/VariantCalling/[SAMPLE]/Manta`** -* `Manta_Sample.candidateSmallIndells.vcf.gz` and `Manta_Sample.candidateSmallIndells.vcf.gz.tbi` +* `Manta_Sample.candidateSmallIndels.vcf.gz` and `Manta_Sample.candidateSmallIndels.vcf.gz.tbi` * VCF with Tabix index * `Manta_Sample.candidateSV.vcf.gz` and `Manta_Sample.candidateSV.vcf.gz.tbi` * VCF with Tabix index @@ -167,21 +197,53 @@ For a Tumor/Normal pair: * `Manta_TumorSample_vs_NormalSample.somaticSV.vcf.gz` and `Manta_TumorSample_vs_NormalSample.somaticSV.vcf.gz.tbi` * VCF with Tabix index -### alleleCounter - ### ConvertAlleleCounts +[ConvertAlleleCounts](https://github.com/nf-core/sarek/blob/master/bin/convertAlleleCounts.r) is a R-script for converting output from AlleleCount to BAF and LogR values. + +**Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/ASCAT`** + +* `TumorSample.BAF` and `NormalSample.BAF` + * file with beta allele frequencies +* `TumorSample.LogR` and `NormalSample.LogR` + * file with total copy number on a logarithmic scale ### ASCAT -[ASCAT](https://www.crick.ac.uk/research/a-z-researchers/researchers-v-y/peter-van-loo/software/) is a method to derive copy number profiles of tumour cells, accounting for normal cell admixture and tumour aneuploidy. +[ASCAT](https://github.com/Crick-CancerGenomics/ascat) is a method to derive copy number profiles of tumour cells, accounting for normal cell admixture and tumour aneuploidy. ASCAT infers tumour purity and ploidy and calculates whole-genome allele-specific copy number profiles. -### samtools mpileup +For further reading and documentation see the [ASCAT manual](https://www.crick.ac.uk/research/labs/peter-van-loo/software). + +**Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/ASCAT`** + +### mpileup +[samtools mpileup](https://www.htslib.org/doc/samtools.html) generate pileup for a BAM file. + +For further reading and documentation see the [samtools manual](https://www.htslib.org/doc/samtools.html#COMMANDS_AND_OPTIONS). + +**Output directory: `results/VariantCalling/[SAMPLE]/mpileup`** +* `Sample.pileup.gz` + * The pileup format is a text-based format for summarizing the base calls of aligned reads to a reference sequence. Alignment records are grouped by sample (SM) identifiers in @RG header lines. ### Control-FREEC +[Control-FREEC](https://github.com/BoevaLab/FREEC) is a tool for detection of copy-number changes and allelic imbalances (including LOH) using deep-sequencing data. +Control-FREEC automatically computes, normalizes, segments copy number and beta allele frequency profiles, then calls copy number alterations and LOH. +And also detects subclonal gains and losses and evaluate the likeliest average ploidy of the sample. + +For further reading and documentation see the [Control-FREEC manual](http://boevalab.com/FREEC/tutorial.html). + +**Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/ControlFREEC`** +* `TumorSample_vs_NormalSample.config.txt` + * Configuration file used to run Control-FREEC +* `TumorSample.pileup.gz_CNVs` and `TumorSample.pileup.gz_normal_CNVs` + * file with coordinates of predicted copy number alterations +* `TumorSample.pileup.gz_ratio.txt` and `TumorSample.pileup.gz_normal_ratio.txt` + * file with ratios and predicted copy number alterations for each window +* `TumorSample.pileup.gz_BAF.txt` and `NormalSample.pileup.gz_BAF.txt` + * file with beta allele frequencies for each possibly heterozygous SNP position ## Annotation -This directory contains results from the final annotation steps: two software are used for annotation, [VEP][vep-link] and [snpEff][snpeff-link]. +This directory contains results from the final annotation steps: two software are used for annotation, [snpEff](http://snpeff.sourceforge.net/) and [VEP](https://www.ensembl.org/info/docs/tools/vep/index.html). Only a subset of the VCF files are annotated, and only variants that have a PASS filter. FreeBayes results are not annotated in the moment yet as we are lacking a decent somatic filter. For HaplotypeCaller the germline variations are annotated for both the tumour and the normal sample. @@ -191,6 +253,8 @@ For HaplotypeCaller the germline variations are annotated for both the tumour an It annotates and predicts the effects of variants on genes (such as amino acid changes) using multiple databases for annotations. The generated VCF header contains the software version and the used command line. +For further reading and documentation see the [snpEff manual](http://snpeff.sourceforge.net/SnpEff_manual.html#outputSummary) + **Output directory: `results/Annotation/[SAMPLE]/snpEff`** * `VariantCaller_Sample_snpEff.ann.vcf.gz` and `VariantCaller_Sample_snpEff.ann.vcf.gz.tbi` @@ -213,12 +277,14 @@ In the moment it contains: * Protein_position: Relative position of amino acid in protein * BIOTYPE: Biotype of transcript or regulatory feature +For further reading and documentation see the [VEP manual](https://www.ensembl.org/info/docs/tools/vep/index.html) + **Output directory: `results/Annotation/[SAMPLE]/VEP`** * `VariantCaller_Sample_VEP.ann.vcf.gz` and `VariantCaller_Sample_VEP.ann.vcf.gz.tbi` * VCF with Tabix index -## QC and reports +## QC and reporting ### FastQC [FastQC](http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) gives general quality metrics about your reads. @@ -246,21 +312,18 @@ Plot will show: For more information about how to use Qualimap bamqc reports, see [Qualimap bamqc manual](http://qualimap.bioinfo.cipf.es/doc_html/analysis.html#id7) -### MarkDuplicates -reports +### MarkDuplicates reports [GATK MarkDuplicates](https://github.com/broadinstitute/gatk) locates and tags duplicate reads in a BAM or SAM file, where duplicate reads are defined as originating from a single fragment of DNA. Duplicates can arise during sample preparation e.g. library construction using PCR. Duplicate reads can also result from a single amplification cluster, incorrectly detected as multiple clusters by the optical sensor of the sequencing instrument. These duplication artifacts are referred to as optical duplicates. -Plot will show: -* Duplication metrics - **Output directory: `results/Reports/[SAMPLE]/MarkDuplicates`** * `Sample.bam.metrics` * RAW statistics used by MultiQC -For more information about how to use MarkDuplicates reports, see [MarkDuplicates manual](https://software.broadinstitute.org/gatk/documentation/tooldocs/4.1.2.0/picard_sam_markduplicates_MarkDuplicates.php) +For further reading and documentation see the [MarkDuplicates manual](https://software.broadinstitute.org/gatk/documentation/tooldocs/4.1.2.0/picard_sam_markduplicates_MarkDuplicates.php). ### samtools stats [samtools stats](https://www.htslib.org/doc/samtools.html) collects statistics from BAM files and outputs in a text format. @@ -271,10 +334,10 @@ Plots will show: * `Sample.bam.samtools.stats.out` * RAW statistics used by MultiQC -For more information about how to use samtools stats reports, see [samtools stats manual](http://www.htslib.org/doc/samtools.html#COMMANDS_AND_OPTIONS) +For further reading and documentation see the [samtools manual](https://www.htslib.org/doc/samtools.html#COMMANDS_AND_OPTIONS) ### bcftools stats -[bcftools stats](https://samtools.github.io/bcftools/) is a program for variant calling and manipulating files in the Variant Call Format. +[bcftools](https://samtools.github.io/bcftools/) is a program for variant calling and manipulating files in the Variant Call Format. Plot will show: * Stats by non-reference allele frequency, depth distribution, stats by quality and per-sample counts, singleton stats, etc. @@ -282,7 +345,7 @@ Plot will show: * `VariantCaller_Sample.bcf.tools.stats.out` * RAW statistics used by MultiQC -For more information about how to use bcftools stats reports, see [bcftools stats manual](https://samtools.github.io/bcftools/bcftools.html#stats) +For further reading and documentation see the [bcftools stats manual](https://samtools.github.io/bcftools/bcftools.html#stats) ### VCFtools [VCFtools](https://vcftools.github.io/) is a program package designed for working with VCF files. @@ -299,7 +362,7 @@ Plots will show: * `VariantCaller_Sample.TsTv.qual` * RAW statistics used by MultiQC -For more information about how to use VCFtools reports, see [VCFtools manual](https://vcftools.github.io/man_latest.html#OUTPUT%20OPTIONS) +For further reading and documentation see the [VCFtools manual](https://vcftools.github.io/man_latest.html#OUTPUT%20OPTIONS) ### snpEff reports [snpeff](http://snpeff.sourceforge.net/) is a genetic variant annotation and effect prediction toolbox. @@ -319,7 +382,7 @@ Plots will shows : * `VariantCaller_Sample_snpEff.txt` * TXT (tab separated) summary counts for variants affecting each transcript and gene -For more information about how to use snpEff reports, see [snpEff manual](http://snpeff.sourceforge.net/SnpEff_manual.html#outputSummary) +For further reading and documentation see the [snpEff manual](http://snpeff.sourceforge.net/SnpEff_manual.html#outputSummary) ### VEP reports [VEP (Variant Effect Predictor)](https://www.ensembl.org/info/docs/tools/vep/index.html), based on Ensembl, is a tools to determine the effects of all sorts of variants, including SNPs, indels, structural variants, CNVs. @@ -328,7 +391,7 @@ For more information about how to use snpEff reports, see [snpEff manual](http:/ * `VariantCaller_Sample_VEP.summary.html` * Summary of the VEP run to be visualised with a web browser -For more information about how to use VEP reports, see [VEP manual](https://www.ensembl.org/info/docs/tools/vep/index.html) +For further reading and documentation see the [VEP manual](https://www.ensembl.org/info/docs/tools/vep/index.html) ### MultiQC [MultiQC](http://multiqc.info) is a visualisation tool that generates a single HTML report summarising all samples in your project. @@ -343,4 +406,4 @@ The pipeline has special steps which allow the software versions used to be repo * `multiqc_data/` * Directory containing parsed statistics from the different tools used in the pipeline -For more information about how to use MultiQC reports, see [http://multiqc.info](http://multiqc.info) +For further reading and documentation see the [MultiQC website](http://multiqc.info) From 95c26a6a0b06b1ef1167581b5271b6e71304181c Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Fri, 24 May 2019 16:04:47 +0200 Subject: [PATCH 12/22] fix intra link --- docs/output.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/docs/output.md b/docs/output.md index db0b52ffa1..caaf882e48 100644 --- a/docs/output.md +++ b/docs/output.md @@ -39,7 +39,7 @@ The pipeline processes data using the following steps: * [Qualimap bamqc](#bamQC) * [GATK MarkDuplicates](#MarkDuplicates-reports) * [samtools stats](#Samtools-stats) - * [bcftools stats](#BCFtools) + * [bcftools stats](#bcftools-stats) * [VCFtools](#VCFtools) * [snpeff](#snpEff-reports) * Reporting From 433deebfb9229ff732c8957fbd04b7452a1aaefa Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Fri, 24 May 2019 16:06:02 +0200 Subject: [PATCH 13/22] add link to VEP --- docs/output.md | 1 + 1 file changed, 1 insertion(+) diff --git a/docs/output.md b/docs/output.md index caaf882e48..4e1f0c2f91 100644 --- a/docs/output.md +++ b/docs/output.md @@ -42,6 +42,7 @@ The pipeline processes data using the following steps: * [bcftools stats](#bcftools-stats) * [VCFtools](#VCFtools) * [snpeff](#snpEff-reports) + * [VEP](#snpEff-reports) * Reporting * [MultiQC](#MultiQC) From 4e91262fa323489f2ac7da0de9dac1951d9fdf76 Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Fri, 24 May 2019 16:21:32 +0200 Subject: [PATCH 14/22] add extra info --- docs/output.md | 154 ++++++++++++++++++++++++++++--------------------- 1 file changed, 89 insertions(+), 65 deletions(-) diff --git a/docs/output.md b/docs/output.md index 4e1f0c2f91..1f861cefe6 100644 --- a/docs/output.md +++ b/docs/output.md @@ -7,44 +7,44 @@ The pipeline processes data using the following steps: 1. [**Preprocessing**](#Preprocessing) _(based on [GATK best practices](https://software.broadinstitute.org/gatk/best-practices/))_ * Map reads to Reference - * BWA mem + * `BWA mem` * Mark Duplicates - * GATK MarkDuplicates + * `GATK MarkDuplicates` * Base (Quality Score) Recalibration - * GATK BaseRecalibrator - * GATK GatherBQSRReports - * GATK ApplyBQSR + * `GATK BaseRecalibrator` + * `GATK GatherBQSRReports` + * `GATK ApplyBQSR` 2. [**Variant calling**](#Variant-Calling) * SNVs and small indels - * [FreeBayes](#FreeBayes) - * [GATK HaplotypeCaller](#HaplotypeCaller) - * [GATK GenotypeGVCFs](#GenotypeGVCFs) - * [GATK MuTect2](#MuTect2) - * [Strelka2](#Strelka2) + * [`FreeBayes`](#FreeBayes) + * [`GATK HaplotypeCaller`](#HaplotypeCaller) + * [`GATK GenotypeGVCFs`](#GenotypeGVCFs) + * [`GATK MuTect2`](#MuTect2) + * [`Strelka2`](#Strelka2) * Structural variants - * [Manta](#Manta) + * [`Manta`](#Manta) * Sample heterogeneity, ploidy and CNVs - * alleleCounter - * [ConvertAlleleCounts](#ConvertAlleleCounts) - * [ASCAT](#ASCAT) - * [samtools mpileup](#mpileup) - * [Control-FREEC](#Control-FREEC) + * `alleleCounter` + * [`ConvertAlleleCounts`](#ConvertAlleleCounts) + * [`ASCAT`](#ASCAT) + * [`samtools mpileup`](#mpileup) + * [`Control-FREEC`](#Control-FREEC) 3. [**Annotation**](#Annotation) * Variant annotation - * [snpEff](#snpEff) - * [VEP (Variant Effect Predictor)](#VEP) + * [`snpEff`](#snpEff) + * [`VEP` (Variant Effect Predictor)](#VEP) 4. [**QC and Reporting**](#QC-and-reporting) * QC - * [FastQC](#FastQC) - * [Qualimap bamqc](#bamQC) - * [GATK MarkDuplicates](#MarkDuplicates-reports) - * [samtools stats](#Samtools-stats) - * [bcftools stats](#bcftools-stats) - * [VCFtools](#VCFtools) - * [snpeff](#snpEff-reports) - * [VEP](#snpEff-reports) + * [`FastQC`](#FastQC) + * [`Qualimap bamqc`](#bamQC) + * [`GATK MarkDuplicates`](#MarkDuplicates-reports) + * [`samtools stats`](#Samtools-stats) + * [`bcftools stats`](#bcftools-stats) + * [`VCFtools`](#VCFtools) + * [`snpeff`](#snpEff-reports) + * [`VEP`](#snpEff-reports) * Reporting - * [MultiQC](#MultiQC) + * [`MultiQC`](#MultiQC) ## Preprocessing Sarek preprocesses raw FastQ files or unmapped BAM files, based on [GATK best practices](https://software.broadinstitute.org/gatk/best-practices/). @@ -57,9 +57,10 @@ This directory is the location for the BAM files delivered to users. Besides the For further reading and documentation see the [data pre-processing workflow from the GATK best practices](https://software.broadinstitute.org/gatk/best-practices/workflow?id=11165). +For all samples: **Output directory: `results/Preprocessing/[SAMPLE]/DuplicateMarked`** -* `Sample.md.bam`, `Sample.md.bai` and `Sample.recal.table` +* `[SAMPLE].md.bam`, `[SAMPLE].md.bai` and `[SAMPLE].recal.table` * BAM file and index with Recalibration Table ### Recalibrated BAM file(s) @@ -70,9 +71,10 @@ To re-generate recalibrated BAM file you have to apply the recalibration table d For further reading and documentation see the [data pre-processing workflow from the GATK best practices](https://software.broadinstitute.org/gatk/best-practices/workflow?id=11165). +For all samples: **Output directory: `results/Preprocessing/[SAMPLE]/Recalibrated`** -* `Sample.recal.bam` and `Sample.recal.bai` +* `[SAMPLE].recal.bam` and `[SAMPLE].recal.bai` * BAM file and index ### TSV files @@ -80,11 +82,12 @@ The TSV files are autogenerated and can be used by Sarek for further processing For further reading and documentation see the [input documentation](https://github.com/nf-core/sarek/blob/master/docs/input.md). +For all samples: **Output directory: `results/Preprocessing/TSV`** * `duplicateMarked.tsv` and `recalibrated.tsv` * TSV files to start Sarek from `recalibration` or `variantcalling` steps. -* `duplicateMarked_Sample.tsv` and `recalibrated_Sample.tsv` +* `duplicateMarked_[SAMPLE].tsv` and `recalibrated_[SAMPLE].tsv` * TSV files to start Sarek from `recalibration` or `variantcalling` steps for a specific sample. ## Variant Calling @@ -98,9 +101,10 @@ The single VCF file generated by `FreeBayes` being huge, it is recommended to fl For further reading and documentation see the [FreeBayes manual](https://github.com/ekg/freebayes/blob/master/README.md#user-manual-and-guide). +For a Tumor/Normal pair only: **Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/FreeBayes`** -* `FreeBayes_TumorSample_vs_NormalSample.vcf.gz` and `FreeBayes_TumorSample_vs_NormalSample.vcf.gz.tbi` +* `FreeBayes_[TUMORSAMPLE]_vs_[NORMALSAMPLE].vcf.gz` and `FreeBayes_[TUMORSAMPLE]_vs_[NORMALSAMPLE].vcf.gz.tbi` * VCF with Tabix index ### HaplotypeCaller @@ -110,9 +114,10 @@ Germline calls are provided for all samples, to able comparison of both tumor an For further reading and documentation see the [HaplotypeCaller manual](https://software.broadinstitute.org/gatk/documentation/tooldocs/4.1.2.0/org_broadinstitute_hellbender_tools_walkers_haplotypecaller_HaplotypeCaller.php). +For all samples: **Output directory: `results/VariantCalling/[SAMPLE]/HaploTypeCaller`** -* `HaplotypeCaller_Sample.vcf.gz` and `HaplotypeCaller_Sample.vcf.gz.tbi` +* `HaplotypeCaller_[SAMPLE].vcf.gz` and `HaplotypeCaller_[SAMPLE].vcf.gz.tbi` * VCF with Tabix index ### GenotypeGVCFs @@ -122,9 +127,10 @@ Germline calls are provided for all samples, to able comparison of both tumor an For further reading and documentation see the [GenotypeGVCFs manual](https://software.broadinstitute.org/gatk/documentation/tooldocs/4.1.2.0/org_broadinstitute_hellbender_tools_walkers_GenotypeGVCFs.php). +For all samples: **Output directory: `results/VariantCalling/[SAMPLE]/HaplotypeCallerGVCF`** -* `HaplotypeCaller_Sample.g.vcf.gz` and `HaplotypeCaller_Sample.g.vcf.gz.tbi` +* `HaplotypeCaller_[SAMPLE].g.vcf.gz` and `HaplotypeCaller_[SAMPLE].g.vcf.gz.tbi` * VCF with Tabix index ### MuTect2 @@ -132,9 +138,10 @@ For further reading and documentation see the [GenotypeGVCFs manual](https://sof For further reading and documentation see the [MuTect2 manual](https://software.broadinstitute.org/gatk/documentation/tooldocs/4.1.2.0/org_broadinstitute_hellbender_tools_walkers_mutect_Mutect2.php). +For a Tumor/Normal pair only: **Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/MuTect2`** -* `MuTect2_TumorSample_vs_NormalSample.vcf.gz` and `MuTect2_TumorSample_vs_NormalSample.vcf.gz.tbi` +* `MuTect2_[TUMORSAMPLE]_vs_[NORMALSAMPLE].vcf.gz` and `MuTect2_[TUMORSAMPLE]_vs_[NORMALSAMPLE].vcf.gz.tbi` * VCF with Tabix index ### Strelka2 @@ -142,6 +149,7 @@ For further reading and documentation see the [MuTect2 manual](https://software. For further reading and documentation see the [Strelka2 user guide](https://github.com/Illumina/strelka/blob/v2.9.x/docs/userGuide/README.md). +For all samples: **Output directory: `results/VariantCalling/[SAMPLE]/Strelka`** * `Strelka_Sample_genome.vcf.gz` and `Strelka_Sample_genome.vcf.gz.tbi` @@ -152,60 +160,62 @@ For further reading and documentation see the [Strelka2 user guide](https://gith For a Tumor/Normal pair: **Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/Strelka`** -* `Strelka_TumorSample_vs_NormalSample_somatic_indels.vcf.gz` and `Strelka_TumorSample_vs_NormalSample_somatic_indels.vcf.gz.tbi` +* `Strelka_[TUMORSAMPLE]_vs_[NORMALSAMPLE]_somatic_indels.vcf.gz` and `Strelka_[TUMORSAMPLE]_vs_[NORMALSAMPLE]_somatic_indels.vcf.gz.tbi` * VCF with Tabix index -* `Strelka_TumorSample_vs_NormalSample_somatic_snvs.vcf.gz` and `Strelka_TumorSample_vs_NormalSample_somatic_snvs.vcf.gz.tbi` +* `Strelka_[TUMORSAMPLE]_vs_[NORMALSAMPLE]_somatic_snvs.vcf.gz` and `Strelka_[TUMORSAMPLE]_vs_[NORMALSAMPLE]_somatic_snvs.vcf.gz.tbi` * VCF with Tabix index -Using Strelka Best Practices with the `candidateSmallIndels` from Manta: +Using [Strelka Best Practices](https://github.com/Illumina/strelka/blob/v2.9.x/docs/userGuide/README.md#somatic-configuration-example) with the `candidateSmallIndels` from `Manta`: **Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/Strelka`** -* `StrelkaBP_TumorSample_vs_NormalSample_somatic_indels.vcf.gz` and `StrelkaBP_TumorSample_vs_NormalSample_somatic_indels.vcf.gz.tbi` +* `StrelkaBP_[TUMORSAMPLE]_vs_[NORMALSAMPLE]_somatic_indels.vcf.gz` and `StrelkaBP_[TUMORSAMPLE]_vs_[NORMALSAMPLE]_somatic_indels.vcf.gz.tbi` * VCF with Tabix index -* `StrelkaBP_TumorSample_vs_NormalSample_somatic_snvs.vcf.gz` and `StrelkaBP_TumorSample_vs_NormalSample_somatic_snvs.vcf.gz.tbi` +* `StrelkaBP_[TUMORSAMPLE]_vs_[NORMALSAMPLE]_somatic_snvs.vcf.gz` and `StrelkaBP_[TUMORSAMPLE]_vs_[NORMALSAMPLE]_somatic_snvs.vcf.gz.tbi` * VCF with Tabix index ### Manta [Manta](https://github.com/Illumina/manta) calls structural variants (SVs) and indels from mapped paired-end sequencing reads. It is optimized for analysis of germline variation in small sets of individuals and somatic variation in tumor/normal sample pairs. -Manta provides a candidate list for small indels also that can be fed to Strelka. +`Manta` provides a candidate list for small indels also that can be fed to `Strelka` following [Strelka Best Practices](https://github.com/Illumina/strelka/blob/v2.9.x/docs/userGuide/README.md#somatic-configuration-example. For further reading and documentation see the [Manta user guide](https://github.com/Illumina/manta/blob/master/docs/userGuide/README.md). +For all samples: **Output directory: `results/VariantCalling/[SAMPLE]/Manta`** -* `Manta_Sample.candidateSmallIndels.vcf.gz` and `Manta_Sample.candidateSmallIndels.vcf.gz.tbi` +* `Manta_[SAMPLE].candidateSmallIndels.vcf.gz` and `Manta_[SAMPLE].candidateSmallIndels.vcf.gz.tbi` * VCF with Tabix index -* `Manta_Sample.candidateSV.vcf.gz` and `Manta_Sample.candidateSV.vcf.gz.tbi` +* `Manta_[SAMPLE].candidateSV.vcf.gz` and `Manta_[SAMPLE].candidateSV.vcf.gz.tbi` * VCF with Tabix index -For Normal sample: -* `Manta_NormalSample.diploidSV.vcf.gz` and `Manta_NormalSample.diploidSV.vcf.gz.tbi` +For Normal sample only: +* `Manta_[NORMALSAMPLE].diploidSV.vcf.gz` and `Manta_[NORMALSAMPLE].diploidSV.vcf.gz.tbi` * VCF with Tabix index -For a Tumor sample: -* `Manta_TumorSample.tumorSV.vcf.gz` and `Manta_TumorSample.tumorSV.vcf.gz.tbi` +For a Tumor sample only: +* `Manta_Tumor[SAMPLE].tumorSV.vcf.gz` and `Manta_Tumor[SAMPLE].tumorSV.vcf.gz.tbi` * VCF with Tabix index -For a Tumor/Normal pair: +For a Tumor/Normal pair only: **Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/Manta`** -* `Manta_TumorSample_vs_NormalSample.candidateSmallIndels.vcf.gz` and `Manta_TumorSample_vs_NormalSample.candidateSmallIndels.vcf.gz.tbi` +* `Manta_[TUMORSAMPLE]_vs_[NORMALSAMPLE].candidateSmallIndels.vcf.gz` and `Manta_[TUMORSAMPLE]_vs_[NORMALSAMPLE].candidateSmallIndels.vcf.gz.tbi` * VCF with Tabix index -* `Manta_TumorSample_vs_NormalSample.candidateSV.vcf.gz` and `Manta_TumorSample_vs_NormalSample.candidateSV.vcf.gz.tbi` +* `Manta_[TUMORSAMPLE]_vs_[NORMALSAMPLE].candidateSV.vcf.gz` and `Manta_[TUMORSAMPLE]_vs_[NORMALSAMPLE].candidateSV.vcf.gz.tbi` * VCF with Tabix index -* `Manta_TumorSample_vs_NormalSample.diploidSV.vcf.gz` and `Manta_TumorSample_vs_NormalSample.diploidSV.vcf.gz.tbi` +* `Manta_[TUMORSAMPLE]_vs_[NORMALSAMPLE].diploidSV.vcf.gz` and `Manta_[TUMORSAMPLE]_vs_[NORMALSAMPLE].diploidSV.vcf.gz.tbi` * VCF with Tabix index -* `Manta_TumorSample_vs_NormalSample.somaticSV.vcf.gz` and `Manta_TumorSample_vs_NormalSample.somaticSV.vcf.gz.tbi` +* `Manta_[TUMORSAMPLE]_vs_[NORMALSAMPLE].somaticSV.vcf.gz` and `Manta_[TUMORSAMPLE]_vs_[NORMALSAMPLE].somaticSV.vcf.gz.tbi` * VCF with Tabix index ### ConvertAlleleCounts [ConvertAlleleCounts](https://github.com/nf-core/sarek/blob/master/bin/convertAlleleCounts.r) is a R-script for converting output from AlleleCount to BAF and LogR values. +For a Tumor/Normal pair only: **Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/ASCAT`** -* `TumorSample.BAF` and `NormalSample.BAF` +* `Tumor[SAMPLE].BAF` and `[NORMALSAMPLE].BAF` * file with beta allele frequencies -* `TumorSample.LogR` and `NormalSample.LogR` +* `Tumor[SAMPLE].LogR` and `[NORMALSAMPLE].LogR` * file with total copy number on a logarithmic scale ### ASCAT @@ -214,6 +224,7 @@ ASCAT infers tumour purity and ploidy and calculates whole-genome allele-specifi For further reading and documentation see the [ASCAT manual](https://www.crick.ac.uk/research/labs/peter-van-loo/software). +For a Tumor/Normal pair only: **Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/ASCAT`** ### mpileup @@ -221,8 +232,9 @@ For further reading and documentation see the [ASCAT manual](https://www.crick.a For further reading and documentation see the [samtools manual](https://www.htslib.org/doc/samtools.html#COMMANDS_AND_OPTIONS). +For all samples: **Output directory: `results/VariantCalling/[SAMPLE]/mpileup`** -* `Sample.pileup.gz` +* `[SAMPLE].pileup.gz` * The pileup format is a text-based format for summarizing the base calls of aligned reads to a reference sequence. Alignment records are grouped by sample (SM) identifiers in @RG header lines. ### Control-FREEC @@ -232,14 +244,15 @@ And also detects subclonal gains and losses and evaluate the likeliest average p For further reading and documentation see the [Control-FREEC manual](http://boevalab.com/FREEC/tutorial.html). +For a Tumor/Normal pair only: **Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/ControlFREEC`** -* `TumorSample_vs_NormalSample.config.txt` +* `[TUMORSAMPLE]_vs_[NORMALSAMPLE].config.txt` * Configuration file used to run Control-FREEC -* `TumorSample.pileup.gz_CNVs` and `TumorSample.pileup.gz_normal_CNVs` +* `Tumor[SAMPLE].pileup.gz_CNVs` and `Tumor[SAMPLE].pileup.gz_normal_CNVs` * file with coordinates of predicted copy number alterations -* `TumorSample.pileup.gz_ratio.txt` and `TumorSample.pileup.gz_normal_ratio.txt` +* `Tumor[SAMPLE].pileup.gz_ratio.txt` and `Tumor[SAMPLE].pileup.gz_normal_ratio.txt` * file with ratios and predicted copy number alterations for each window -* `TumorSample.pileup.gz_BAF.txt` and `NormalSample.pileup.gz_BAF.txt` +* `Tumor[SAMPLE].pileup.gz_BAF.txt` and `[NORMALSAMPLE].pileup.gz_BAF.txt` * file with beta allele frequencies for each possibly heterozygous SNP position ## Annotation @@ -256,6 +269,7 @@ The generated VCF header contains the software version and the used command line For further reading and documentation see the [snpEff manual](http://snpeff.sourceforge.net/SnpEff_manual.html#outputSummary) +For all samples: **Output directory: `results/Annotation/[SAMPLE]/snpEff`** * `VariantCaller_Sample_snpEff.ann.vcf.gz` and `VariantCaller_Sample_snpEff.ann.vcf.gz.tbi` @@ -280,6 +294,7 @@ In the moment it contains: For further reading and documentation see the [VEP manual](https://www.ensembl.org/info/docs/tools/vep/index.html) +For all samples: **Output directory: `results/Annotation/[SAMPLE]/VEP`** * `VariantCaller_Sample_VEP.ann.vcf.gz` and `VariantCaller_Sample_VEP.ann.vcf.gz.tbi` @@ -294,6 +309,7 @@ You get information about adapter contamination and other overrepresented sequen For further reading and documentation see the [FastQC help](http://www.bioinformatics.babraham.ac.uk/projects/fastqc/Help/). +For all samples: **Output directory: `results/Reports/[SAMPLE]/fastqc`** * `sample_R1_XXX_fastqc.html` and `sample_R2_XXX_fastqc.html` @@ -307,8 +323,9 @@ For further reading and documentation see the [FastQC help](http://www.bioinform Plot will show: * Stats by non-reference allele frequency, depth distribution, stats by quality and per-sample counts, singleton stats, etc. +For all samples: **Output directory: `results/Reports/[SAMPLE]/bamQC`** -* `VariantCaller_Sample.bcf.tools.stats.out` +* `VariantCaller_[SAMPLE].bcf.tools.stats.out` * RAW statistics used by MultiQC For more information about how to use Qualimap bamqc reports, see [Qualimap bamqc manual](http://qualimap.bioinfo.cipf.es/doc_html/analysis.html#id7) @@ -320,8 +337,9 @@ library construction using PCR. Duplicate reads can also result from a single amplification cluster, incorrectly detected as multiple clusters by the optical sensor of the sequencing instrument. These duplication artifacts are referred to as optical duplicates. +For all samples: **Output directory: `results/Reports/[SAMPLE]/MarkDuplicates`** -* `Sample.bam.metrics` +* `[SAMPLE].bam.metrics` * RAW statistics used by MultiQC For further reading and documentation see the [MarkDuplicates manual](https://software.broadinstitute.org/gatk/documentation/tooldocs/4.1.2.0/picard_sam_markduplicates_MarkDuplicates.php). @@ -331,8 +349,9 @@ For further reading and documentation see the [MarkDuplicates manual](https://so Plots will show: * Alignment metrics. +For all samples: **Output directory: `results/Reports/[SAMPLE]/SamToolsStats`** -* `Sample.bam.samtools.stats.out` +* `[SAMPLE].bam.samtools.stats.out` * RAW statistics used by MultiQC For further reading and documentation see the [samtools manual](https://www.htslib.org/doc/samtools.html#COMMANDS_AND_OPTIONS) @@ -342,8 +361,9 @@ For further reading and documentation see the [samtools manual](https://www.htsl Plot will show: * Stats by non-reference allele frequency, depth distribution, stats by quality and per-sample counts, singleton stats, etc. +For all samples: **Output directory: `results/Reports/[SAMPLE]/BCFToolsStats`** -* `VariantCaller_Sample.bcf.tools.stats.out` +* `VariantCaller_[SAMPLE].bcf.tools.stats.out` * RAW statistics used by MultiQC For further reading and documentation see the [bcftools stats manual](https://samtools.github.io/bcftools/bcftools.html#stats) @@ -355,12 +375,13 @@ Plots will show: * the transition to transversion ratio as a function of alternative allele count (using only bi-allelic SNPs). * the transition to transversion ratio as a function of SNP quality threshold (using only bi-allelic SNPs). +For all samples: **Output directory: `results/Reports/[SAMPLE]/VCFTools`** -* `VariantCaller_Sample.FILTER.summary` +* `VariantCaller_[SAMPLE].FILTER.summary` * RAW statistics used by MultiQC -* `VariantCaller_Sample.TsTv.count` +* `VariantCaller_[SAMPLE].TsTv.count` * RAW statistics used by MultiQC -* `VariantCaller_Sample.TsTv.qual` +* `VariantCaller_[SAMPLE].TsTv.qual` * RAW statistics used by MultiQC For further reading and documentation see the [VCFtools manual](https://vcftools.github.io/man_latest.html#OUTPUT%20OPTIONS) @@ -375,6 +396,7 @@ Plots will shows : * the effect of variants at protein level and the number of variants for each effect type. * the quantity as function of the variant quality score. +For all samples: **Output directory: `results/Reports/[SAMPLE]/snpEff`** * `VariantCaller_Sample_snpEff.csv` * RAW statistics used by MultiQC @@ -388,6 +410,7 @@ For further reading and documentation see the [snpEff manual](http://snpeff.sour ### VEP reports [VEP (Variant Effect Predictor)](https://www.ensembl.org/info/docs/tools/vep/index.html), based on Ensembl, is a tools to determine the effects of all sorts of variants, including SNPs, indels, structural variants, CNVs. +For all samples: **Output directory: `results/Reports/[SAMPLE]/VEP`** * `VariantCaller_Sample_VEP.summary.html` * Summary of the VEP run to be visualised with a web browser @@ -400,6 +423,7 @@ Most of the pipeline QC results are visualised in the report and further statist The pipeline has special steps which allow the software versions used to be reported in the MultiQC output for future traceability. +For the whole Sarek run: **Output directory: `results/Reports/MultiQC`** * `multiqc_report.html` From c76cc949f2ccb31bfc569aac695904ea6f616a5e Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Fri, 24 May 2019 16:33:24 +0200 Subject: [PATCH 15/22] complete information about ASCAT --- docs/output.md | 37 ++++++++++++++++++++++++++++--------- 1 file changed, 28 insertions(+), 9 deletions(-) diff --git a/docs/output.md b/docs/output.md index 1f861cefe6..c5d7009415 100644 --- a/docs/output.md +++ b/docs/output.md @@ -192,7 +192,7 @@ For Normal sample only: * VCF with Tabix index For a Tumor sample only: -* `Manta_Tumor[SAMPLE].tumorSV.vcf.gz` and `Manta_Tumor[SAMPLE].tumorSV.vcf.gz.tbi` +* `Manta_[TUMORSAMPLE].tumorSV.vcf.gz` and `Manta_[TUMORSAMPLE].tumorSV.vcf.gz.tbi` * VCF with Tabix index For a Tumor/Normal pair only: @@ -213,20 +213,39 @@ For a Tumor/Normal pair only: For a Tumor/Normal pair only: **Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/ASCAT`** -* `Tumor[SAMPLE].BAF` and `[NORMALSAMPLE].BAF` +* `[TUMORSAMPLE].BAF` and `[NORMALSAMPLE].BAF` * file with beta allele frequencies -* `Tumor[SAMPLE].LogR` and `[NORMALSAMPLE].LogR` +* `[TUMORSAMPLE].LogR` and `[NORMALSAMPLE].LogR` * file with total copy number on a logarithmic scale ### ASCAT -[ASCAT](https://github.com/Crick-CancerGenomics/ascat) is a method to derive copy number profiles of tumour cells, accounting for normal cell admixture and tumour aneuploidy. -ASCAT infers tumour purity and ploidy and calculates whole-genome allele-specific copy number profiles. +[ASCAT](https://github.com/Crick-CancerGenomics/ascat) is a method to derive copy number profiles of tumor cells, accounting for normal cell admixture and tumor aneuploidy. +ASCAT infers tumor purity and ploidy and calculates whole-genome allele-specific copy number profiles. For further reading and documentation see the [ASCAT manual](https://www.crick.ac.uk/research/labs/peter-van-loo/software). For a Tumor/Normal pair only: **Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/ASCAT`** +* `[TUMORSAMPLE].aberrationreliability.png` + * Image with information about aberration reliability +* `[TUMORSAMPLE].ASCATprofile.png` + * Image with information about ASCAT profile +* `[TUMORSAMPLE].ASPCF.png` + * Image with information about ASPCF +* `[TUMORSAMPLE].rawprofile.png` + * Image with information about raw profile +* `[TUMORSAMPLE].sunrise.png` + * Image with information about sunrise +* `[TUMORSAMPLE].tumour.png` + * Image with information about tumor +* `[TUMORSAMPLE].cnvs.txt` + * file with information about CNVS +* `[TUMORSAMPLE].LogR.PCFed.txt` + * file with information about LogR +* `[TUMORSAMPLE].purityploidy.txt` + * file with information about purity ploidy + ### mpileup [samtools mpileup](https://www.htslib.org/doc/samtools.html) generate pileup for a BAM file. @@ -248,11 +267,11 @@ For a Tumor/Normal pair only: **Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/ControlFREEC`** * `[TUMORSAMPLE]_vs_[NORMALSAMPLE].config.txt` * Configuration file used to run Control-FREEC -* `Tumor[SAMPLE].pileup.gz_CNVs` and `Tumor[SAMPLE].pileup.gz_normal_CNVs` +* `[TUMORSAMPLE].pileup.gz_CNVs` and `[TUMORSAMPLE].pileup.gz_normal_CNVs` * file with coordinates of predicted copy number alterations -* `Tumor[SAMPLE].pileup.gz_ratio.txt` and `Tumor[SAMPLE].pileup.gz_normal_ratio.txt` +* `[TUMORSAMPLE].pileup.gz_ratio.txt` and `[TUMORSAMPLE].pileup.gz_normal_ratio.txt` * file with ratios and predicted copy number alterations for each window -* `Tumor[SAMPLE].pileup.gz_BAF.txt` and `[NORMALSAMPLE].pileup.gz_BAF.txt` +* `[TUMORSAMPLE].pileup.gz_BAF.txt` and `[NORMALSAMPLE].pileup.gz_BAF.txt` * file with beta allele frequencies for each possibly heterozygous SNP position ## Annotation @@ -260,7 +279,7 @@ For a Tumor/Normal pair only: This directory contains results from the final annotation steps: two software are used for annotation, [snpEff](http://snpeff.sourceforge.net/) and [VEP](https://www.ensembl.org/info/docs/tools/vep/index.html). Only a subset of the VCF files are annotated, and only variants that have a PASS filter. FreeBayes results are not annotated in the moment yet as we are lacking a decent somatic filter. -For HaplotypeCaller the germline variations are annotated for both the tumour and the normal sample. +For HaplotypeCaller the germline variations are annotated for both the tumor and the normal sample. ### snpEff [snpeff](http://snpeff.sourceforge.net/) is a genetic variant annotation and effect prediction toolbox. From f711a47a6d8873bd6773bee0ad327e1d7b25d11d Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Fri, 24 May 2019 16:43:42 +0200 Subject: [PATCH 16/22] completed docs about ASCAT --- docs/ascat.md | 138 +++++++++++++++++++++++++++++++++++++++++++++++++ docs/output.md | 2 +- 2 files changed, 139 insertions(+), 1 deletion(-) create mode 100644 docs/ascat.md diff --git a/docs/ascat.md b/docs/ascat.md new file mode 100644 index 0000000000..695795f60b --- /dev/null +++ b/docs/ascat.md @@ -0,0 +1,138 @@ +# ASCAT + +## Introduction + +ASCAT is a software for performing allele-specific copy number analysis of tumor samples and for estimating tumor ploidy and purity (normal contamination). +ASCAT is written in R and available here: [github.com/Crick-CancerGenomics/ascat](https://github.com/Crick-CancerGenomics/ascat). + +To run ASCAT on NGS data we need BAM files for the tumor and normal samples, as well as a loci file with SNP positions. +If ASCAT is run on SNP array data, the loci file contains the SNPs on the chip. +When runnig ASCAT on NGS data we can use the same loci file, for exampe the one corresponding to the AffymetrixGenome-Wide Human SNP Array 6.0, but we can also choose a loci file of our choice with i.e. SNPs detected in the 1000 Genomes project. + +### BAF and LogR values + +Running ASCAT on NGS data requires that the BAM files are converted into BAF and LogR values. +This can be done using the software [AlleleCount](https://github.com/cancerit/alleleCount) followed by a simple R script. +AlleleCount extracts the number of reads in a BAM file supporting each allele at specified SNP positions. +Based on this, BAF and logR can be calculated for every SNP position i as: + +``` +BAFi(tumor)=countsBi(tumor)/(countsAi(tumor)+countsBi(tumor)) +BAFi(normal)=countsBi(normal)/(countsAi(normal)+countsBi(normal)) +LogRi(tumor)=log2((countsAi(tumor)+countsBi(tumor))/(countsAi(normal)+countsBi(normal)) - median(log2((countsA(tumor)+countsB(tumor))/(countsA(normal)+countsB(normal))) +LogRi(normal)=0 +``` + +For male samples, the X chromosome markers have special treatment: + +``` +LogRi(tumor)=log2((countsAi(tumor)+countsBi(tumor))/(countsAi(normal)+countsBi(normal))-1 - median(log2((countsA(tumor)+countsB(tumor))/(countsA(normal)+countsB(normal))-1) +``` + +where: +*i* corresponds to the postions of all SNPs in the loci file. +*CountsA* and *CountsB* are vectors containing number of reads supporting the *A* and *B* alleles of all SNPs +*A* = the major allele +*B* = the minor allele +*Minor* and *major* alleles are defined in the loci file (it actually doesn't matter which one is defied as A and B in this application) + +Calculation of LogR and BAF based on AlleleCount output is done as in [runASCAT.R](https://github.com/cancerit/ascatNgs/tree/dev/perl/share/ascat/runASCAT.R) in the ascatNgs repository on Github. + +### Loci file + +The loci file was created based on the 1000Genomes latest release (phase 3, releasedate 20130502), available [here](ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp//release/20130502/ALL.wgs.phase3_shapeit2_mvncall_integrated_v5b.20130502.sites.vcf.gz). +The following filter was applied: Only bi-allelc SNPs with minor allele frequencies > 0.3. + +The loci file was originally generated for GRCh37. +It was translated into GRCh38 using the tool liftOver available at the UCSC Genome Browser. +To run liftOver the loci file was first written in bed format: + +```bash +awk '{print "chr"$1":"$2"-"$2}' 1000G_phase3_20130502_SNP_maf0.3.loci > 1000G_phase3_20130502_SNP_maf0.3.bed +``` + +Using the web interface to liftOver at [genome.ucsc.edu](https://genome.ucsc.edu/cgi-bin/hgLiftOver) the file was translated into GRCh38 coordinates. +LiftOver was possible for 3261270 out of 3268043 SNPs. +The converted SNP positions were printed in the format required by AlleleCounter by: + +```bash +more hglft_genome_5834_13aba0.bed | awk 'BEGIN{FS="chr"} {print $2}' | awk 'BEGIN{FS="-"} {print $1}' | awk 'BEGIN{FS=":";OFS="\t"} {print $1,$2}' > 1000G_phase3_GRCh38_maf0.3.loci +``` + +### GC correction file +Input files for ASCAT's GC correction were created for the above loci files, using the scripts and instructions for this on [ASCAT's github repository](https://github.com/Crick-CancerGenomics/ascat/tree/master/gcProcessing) + +#### scripts and data for generating the GC correction file +The following scripts were downloaded from https://github.com/Crick-CancerGenomics/ascat/tree/master/gcProcessing: +- *createGCcontentFile.R* +- *createWindowBed.pl* +- *GCfileCreation.sh*. +To generate the GC correction file additional files are needed: +- *locifile* described above. +- *reference.fasta* is the genome reference file in fasta format. +The files are descibed in [Genomes and reference files documentation](REFERENCES.md). +- *chromosomesizes.txt* is a tab delimited text file containing the size of all chromosomes included in the loci file. +An example file is available in https://github.com/Crick-CancerGenomics/ascat/tree/master/gcProcessing/hg19.chrom.sizes + +#### Modification of createWindowBed.pl for our GRCh37 loci file +The genomc reference file we use in Sarek for GRCh37 is coded without "chr" in the chromosome names, while the genomcd referece file we use in Sarek for GRCh38 includes "chr" in the chromosome names. +The script https://github.com/Crick-CancerGenomics/ascat/tree/master/gcProcessing/createWindowBed.pl assumes that "chr" is included in the chromosome names of the reference file, so a small modification of this script was done for the process to work on our GRCh37 loci file. + +These two lines in createWindowBed.pl generate output (lines 61 and 64): +```perl +(61) print OUT "chr".$tab[1]."\t".$start."\t".$stop."\t".$tab[0]."\t".$tab[2]."\t".($w*2+1)."\n"; +(64) print OUT "chr".$tab[1]."\t".$start."\t".$stop."\t".$tab[0]."\t".$tab[2]."\t".($w*2)."\n"; +``` +and were changed to: +```perl +(61) print OUT $tab[1]."\t".$start."\t".$stop."\t".$tab[0]."\t".$tab[2]."\t".($w*2+1)."\n"; +(64) print OUT $tab[1]."\t".$start."\t".$stop."\t".$tab[0]."\t".$tab[2]."\t".($w*2)."\n"; +``` +After this modification the script works for our GRCh37 loci file. + +#### Process +The following sbatch script was run on the Uppmax cluster Rackham, to generate the CG correction files: +```bash +#!/bin/bash -l +#SBATCH -A projid +#SBATCH -p node +#SBATCH -t 24:00:00 +#SBATCH -J createGCfile +module load bioinfo-tools +module load BEDTools +module load R/3.5.0 +module load R_packages/3.5.0 +./GCfileCreation.sh 1000G_phase3_20130502_SNP_maf0.3.loci chrom.sizes 19 human_g1k_v37_decoy.fasta +``` +where: +*1000G_phase3_20130502_SNP_maf0.3.loci* is the loci file for GRCh37 described above +*human_g1k_v37_decoy.fasta* is the genome reference file used for GRCh37 +*chrom.sizes* is the list of the chromosome lengths in GRCh37. +Names of the chromosomes in chrom.sizes file must be the same as in the genome reference, so in case of GRCh37 we used "1", "2" etc and in GRCh38 we used "chr1", "chr2" etc. +*19* means that 19 cores are available for the script. + +This created GC correction files with the following column headers: +Chr Position 25 50 100 200 500 1000 2000 5000 10000 20000 50000 100000 200000 500000 1M 2M 5M 10M +This file gave an error when running ASCAT, and the error message suggested that it had to do with the column headers. +The Readme.txt in https://github.com/Crick-CancerGenomics/ascat/tree/master/gcProcessing suggested that the column headers should be: +Chr Position 25bp 50bp 100bp 200bp 500bp 1000bp 2000bp 5000bp 10000bp 20000bp 50000bp 100000bp 200000bp 500000bp 1M 2M 5M 10M +The column headers headers of the generated GC correction files were therefore manually edited. + +#### Format of GC correction file +The final files are tab-delimited with the following columns (and some example data): +Chr Position 25bp 50bp 100bp 200bp 500bp 1000bp 2000bp 5000bp 10000bp 20000bp 50000bp 100000bp 200000bp 500000bp 1M 2M 5M 10M +snp1 1 14930 0.541667 0.58 0.61 0.585 0.614 0.62 0.6 0.5888 0.588 0.4277 0.395041 0.380702 0.383259 0.341592 0.339747 0.386343 0.500537 0.511514 +snp2 1 15211 0.625 0.64 0.67 0.63 0.61 0.612 0.6135 0.591 0.5922 0.4358 0.39616 0.380411 0.383167 0.34163 0.339771 0.386417 0.500558 0.511511 +snp3 1 15820 0.541667 0.56 0.62 0.655 0.65 0.612 0.5885 0.5936 0.5797 0.4511 0.397771 0.379945 0.382999 0.341791 0.339832 0.386554 0.500579 0.511504 + +### Output +The ASCAT process gives several images as output, described in detail in this [book chapter](http://www.ncbi.nlm.nih.gov/pubmed/22130873). +The script also gives out a text file (*tumor.cnvs.txt*) with information about copy number state for all the segments predicted by ASCAT. +The output is a tab delimited text file with the following columns: chr startpos endpos nMajor nMinor +Where: +*chr* is the chromosome number +*startpos* is the start position of the segment +*endpos* is the end position of the segment +*nMajor* is number of copies of one of the allels (for example the chromosome inherited from the father) +*nMinor* is the number of copies of the other allele (for example the chromosome inherited of the mother) +The file *tumor.cnvs.txt* contains all segments predicted by ASCAT, both those with normal copy number (nMinor = 1 and nMajor =1) and those corresponding to copy number aberrations. diff --git a/docs/output.md b/docs/output.md index c5d7009415..08bd9b3b66 100644 --- a/docs/output.md +++ b/docs/output.md @@ -222,7 +222,7 @@ For a Tumor/Normal pair only: [ASCAT](https://github.com/Crick-CancerGenomics/ascat) is a method to derive copy number profiles of tumor cells, accounting for normal cell admixture and tumor aneuploidy. ASCAT infers tumor purity and ploidy and calculates whole-genome allele-specific copy number profiles. -For further reading and documentation see the [ASCAT manual](https://www.crick.ac.uk/research/labs/peter-van-loo/software). +For further reading and documentation see [the Sarek documentation about ASCAT](https://github.com/nf-core/sarek/blob/master/docs/ascat.md) or the [ASCAT manual](https://www.crick.ac.uk/research/labs/peter-van-loo/software). For a Tumor/Normal pair only: **Output directory: `results/VariantCalling/[TUMOR_vs_NORMAL]/ASCAT`** From 813aede36589b33473683d65eda406b13a8c263d Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Mon, 27 May 2019 11:36:26 +0200 Subject: [PATCH 17/22] add building container for dogs --- .circleci/config.yml | 34 ++++++++++++++++++++++++++++++++++ 1 file changed, 34 insertions(+) diff --git a/.circleci/config.yml b/.circleci/config.yml index c7fa3812d4..2a805a0ea4 100644 --- a/.circleci/config.yml +++ b/.circleci/config.yml @@ -48,6 +48,22 @@ jobs: echo "$DOCKERHUB_PASS" | docker login -u "$DOCKERHUB_USERNAME" --password-stdin docker push nfcore/sareksnpeff:dev.${GENOME} + snpeffcanfam3_1: + docker: + - image: circleci/buildpack-deps:stretch + environment: + GENOME: CanFam3.1 + SNPEFF_CACHE_VERSION: 86 + steps: + - checkout + - setup_remote_docker + - run: + command: docker build -t nfcore/sareksnpeff:dev.${GENOME} containers/snpeff/. --build-arg GENOME=${GENOME} --build-arg SNPEFF_CACHE_VERSION=${SNPEFF_CACHE_VERSION} + - run: + command: | + echo "$DOCKERHUB_PASS" | docker login -u "$DOCKERHUB_USERNAME" --password-stdin + docker push nfcore/sareksnpeff:dev.${GENOME} + vepgrch37: docker: - image: circleci/buildpack-deps:stretch @@ -96,13 +112,31 @@ jobs: - run: command: echo "$DOCKERHUB_PASS" | docker login -u "$DOCKERHUB_USERNAME" --password-stdin ; docker push nfcore/sarekvep:dev.${GENOME} + vepcanfam3_1: + docker: + - image: circleci/buildpack-deps:stretch + environment: + GENOME: CanFam3.1 + SPECIES: canis_familiaris + VEP_VERSION: 95 + steps: + - checkout + - setup_remote_docker + - run: + command: docker build -t nfcore/sarekvep:dev.${GENOME} containers/vep/. --build-arg GENOME=${GENOME} --build-arg SPECIES=${SPECIES} --build-arg VEP_VERSION=${VEP_VERSION} + no_output_timeout: 1h + - run: + command: echo "$DOCKERHUB_PASS" | docker login -u "$DOCKERHUB_USERNAME" --password-stdin ; docker push nfcore/sarekvep:dev.${GENOME} + workflows: version: 2 build: jobs: + - snpeffcanfam3_1 - snpeffgrch37 - snpeffgrch38 - snpeffgrcm38 + - vepcanfam3_1 - vepgrch37 - vepgrch38 - vepgrcm38 From 784f710b529a3a52cb7f1c7366169f40b59b3011 Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Mon, 27 May 2019 11:36:44 +0200 Subject: [PATCH 18/22] change example to GRCh38 --- docs/usage.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/docs/usage.md b/docs/usage.md index bf63ec88fb..48d5864794 100644 --- a/docs/usage.md +++ b/docs/usage.md @@ -194,7 +194,7 @@ The syntax for this reference configuration is as follows: ```nextflow params { genomes { - 'GRCh37' { + 'GRCh38' { acLoci = '' acLociGC = '' bwaIndex = '' From 5c3b26345a9bdc8ad4bc038180d382d4aacbe15a Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Tue, 28 May 2019 15:19:41 +0200 Subject: [PATCH 19/22] add Abstracts --- docs/abstracts/2016-09-KICR.md | 44 ++++++++++++++++++++++++ docs/abstracts/2017-05-ESHG.md | 31 +++++++++++++++++ docs/abstracts/2018-05-PMC.md | 48 ++++++++++++++++++++++++++ docs/abstracts/2018-06-EACR25.md | 59 ++++++++++++++++++++++++++++++++ docs/abstracts/2018-06-NPMI.md | 50 +++++++++++++++++++++++++++ docs/abstracts/2018-07-JOBIM.md | 43 +++++++++++++++++++++++ 6 files changed, 275 insertions(+) create mode 100644 docs/abstracts/2016-09-KICR.md create mode 100644 docs/abstracts/2017-05-ESHG.md create mode 100644 docs/abstracts/2018-05-PMC.md create mode 100644 docs/abstracts/2018-06-EACR25.md create mode 100644 docs/abstracts/2018-06-NPMI.md create mode 100644 docs/abstracts/2018-07-JOBIM.md diff --git a/docs/abstracts/2016-09-KICR.md b/docs/abstracts/2016-09-KICR.md new file mode 100644 index 0000000000..3b3df35eec --- /dev/null +++ b/docs/abstracts/2016-09-KICR.md @@ -0,0 +1,44 @@ +# The XVth KICancer Retreat 2016 + +## Cancer Analysis Workflow Of Tumor/Normal Pairs At The National Genomics Infrastructure Of SciLifeLab + +Maxime Garcia +Pelin Akan, +Teresita Díaz de Ståhl, +Jesper Eisfeldt, +Szilveszter Juhos, +Malin Larsson, +Björn Nystedt, +Pall Olason, +Monica Nistér, +Max Käller + +BarnTumörBanken, Department of Oncology Pathology, Karolinska Institutet, Science for Life Laboratory + +One of the most prominent usage of NGS is whole genome sequencing (WGS). The +National Genomics Infrastructure (NGI) at Science for Life Laboratory is today +providing WGS and germ line variant analysis. However, building a robust and +reliable bioinformatics workflow to find somatic mutations is challenging: +tumor samples are heterogeneous, likely contain structural variants and +multiple sub-clones besides the normal tissue. + +We are presenting our workflow that is designed to analyze WGS tumor/normal +data in a high-throughput environment. The framework is based on the Nextflow +domain-specific language on top of Java/Groovy. Using Nextflow we are able to +utilize both the Slurm load balancing environment and local execution, +implementing data flow forks and joins, call external software etc. Individual +sub-steps of a complex flow can be connected and restarted after failure from +the last execution point. + +The actual preprocessing workflow is based on BWA as an aligner and GATK best +practice steps. To achieve a consensus variant call different variant callers +can be added, currently MuTect2, Strelka and VarDict are supported, more to be +added. Structural variants are going to be estimated by Manta, ploidy and +sample heterogeneity is measured by ASCAT. The expected output of the workflow +is a VCF file presenting filtered, prioritized and annotated polymorphisms. + +As the Nextflow environment is flexible, we can add other tools or remove +obsolete ones as development progresses. The goal is to build a workflow for +cancer genome analysis that can be deployed to both research and clinical +environments and are going to be included as a standard workflow at NGI during +the fall of 2016. diff --git a/docs/abstracts/2017-05-ESHG.md b/docs/abstracts/2017-05-ESHG.md new file mode 100644 index 0000000000..d87f473a96 --- /dev/null +++ b/docs/abstracts/2017-05-ESHG.md @@ -0,0 +1,31 @@ +# European Human Genetics Conference 2017 + +## CAW - Cancer Analysis Workflow to process normal/tumor WGS data + +Maxime Garcia 1, +Szilveszter Juhos 2, +Malin Larsson 3, +Teresita Díaz de Ståhl 4, +Jesper Eisfeldt 5, +Sebastian DiLorenzo 6, +Pall Olason 7, +Björn Nystedt 7, +Monica Nistér 4, +Max Käller 8 + +1. BarnTumörBanken, Department of Oncology Pathology, Science for Life Laboratory, Karolinska Institutet +2. Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University +3. Science for Life laboratory, Department of Physics, Chemistry and Biology, Linköping University +4. BarnTumörBanken, Department of Oncology Pathology, Karolinska Institutet +5. Clinical Genetics, Department of Molecular Medicine and Surgery, Karolinska Institutet +6. Department of Medical Sciences, National Bioinformatics Infrastructure Sweden, Science for Life Laboratory, Uppsala University +7. Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University +8. Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, Royal Institute of Technology + +As whole genome sequencing is getting cheaper, it is viable to compare NGS data from normal and tumor samples of numerous patients. There are still many challenges, mostly regarding bioinformatics: datasets are huge, workflows are complex, and there are multiple tools to choose from for somatic and structural variants and quality control. + +We are presenting CAW (Cancer Analysis Workflow) a complete open source pipeline to resolve somatic variants from WGS data: it is written in Nextflow, a domain specific language for workflow building. We are utilizing GATK best practices to align, realign and recalibrate short-read data in parallel for both tumor and normal sample. After these preprocessing steps several somatic variant callers scan the resulting BAM files; MuTect1, MuTect2 and Strelka are used to find somatic SNVs and small indels.For structural variants we use Manta. Furthermore, we are applying ASCAT to estimate sample heterogeneity, ploidy and CNVs. + +The software can start the analysis from raw FASTQ files, from the realignment step, or directly with any subset of variant callers. At the end of the analysis the resulting VCF files are merged to facilitate further downstream processing, though the individual results are also retained. The flow is capable of accommodating further variant calling software or CNV callers. It is also prepared to process normal - tumor - and several relapse samples. + +Besides variant calls, the workflow provides quality controls presented by MultiQC. A docker image is also available, the open source software can be downloaded from https://github.com/SciLifeLab/CAW . diff --git a/docs/abstracts/2018-05-PMC.md b/docs/abstracts/2018-05-PMC.md new file mode 100644 index 0000000000..7e25e269b4 --- /dev/null +++ b/docs/abstracts/2018-05-PMC.md @@ -0,0 +1,48 @@ +# Keystone Symposia - Precision Medicine in Cancer + +## Sarek, a workflow for WGS analysis of germline and somatic mutations + +Maxime Garcia 123*, +Szilveszter Juhos 123*, +Malin Larsson 456, +Teresita Díaz de Ståhl 13, +Johanna Sandgren 13, +Jesper Eisfeldt 73, +Sebastian DiLorenzo 85A, +Marcel Martin B3C, +Pall Olason 95A, +Phil Ewels B2C, +Björn Nystedt 95A*, +Monica Nistér 13, +Max Käller 2D, +*Corresponding Author + +1. Barntumörbanken, Dept. of Oncology Pathology; +2. Science for Life Laboratory; +3. Karolinska Institutet; +4. Dept. of Physics, Chemistry and Biology; +5. National Bioinformatics Infrastructure Sweden, Science for Life Laboratory; +6. Linköping University; +7. Clinical Genetics, Dept. of Molecular Medicine and Surgery; +8. Dept. of Medical Sciences; +9. Dept. of Cell and Molecular Biology; +A. Uppsala University; +B. Dept. of Biochemistry and Biophysics; +C. Stockholm University; +D. School of Biotechnology, Division of Gene Technology, Royal Institute of Technology + +We present Sarek, a complete Open Source pipeline to resolve germline and somatic variants from WGS data: it is written in Nextflow, a domain-specific language for workflow building. +Sarek is based on GATK best practices to prepare short-read data, in parallel for a tumor/normal pair sample. +After these preprocessing steps several variant callers scan the resulting BAM files; For structural variants we use Manta. +Strelka and GATK HaplotypeCaller are used to find germline variants and for somatic calls we use MuTect2 and Strelka. +Finally, we apply ASCAT to estimate sample heterogeneity, ploidy and CNVs. +Checkpoints allow to start the software from different states. +At the end of the analysis the resulting VCF files are annotated to facilitate further downstream processing. +The flow is capable of accommodating further variant callers. +It can also process only the normal sample, tumor/normal pairs or even normal, tumor and several relapse samples. +Besides variant calls, the workflow provides quality controls presented by MultiQC. +For easy sharing, installation, and to ensure reproducibility, containers (Docker and Singularity) are available. +The MIT licensed open source code can be downloaded from GitHub. + +The authors thank the Swedish Childhood Cancer Foundation for the funding of Barntumörbanken. +We would like to acknowledge support from Science for Life Laboratory, the National Genomics Infrastructure, NGI, and UPPMAX for providing assistance in massive parallel sequencing and computational infrastructure. diff --git a/docs/abstracts/2018-06-EACR25.md b/docs/abstracts/2018-06-EACR25.md new file mode 100644 index 0000000000..ca86f2e658 --- /dev/null +++ b/docs/abstracts/2018-06-EACR25.md @@ -0,0 +1,59 @@ +# 25th Biennial Congress Of The European Association For Cancer Research 2018 + +## Somatic and germline calls from tumour/normal whole genome data: bioinformatics workflow for reproducible research + + +Szilveszter Juhos 1, +Maxime Garcia 2, +Teresita Díaz de Ståhl 3, +Johanna Sandgren 3, +Markus Mayrhofer 4, +Max Käller 5, +Björn Nystedt 6, +Monica Nistér 3 + +1. Karolinska Institutet, Department of Oncology Pathology, Stockholm, Sweden. +2. Karolinska Institutet- Science for Life Laboratory, Department of Oncology-Pathology, Stockholm, Sweden. +3. Karolinska Institutet, Department of Oncology-Pathology, Stockholm, Sweden. +4. Science for Life Laboratory, Uppsala University, Uppsala, Sweden. +5. Science for Life Laboratory, Royal Institute of Technology- School of Biotechnology- Division of Gene Technology, Stockholm, Sweden. +6. Science for Life Laboratory, Department of Cell and Molecular Biology- National Bioinformatics Infrastructure Sweden- Uppsala University, Uppsala, Sweden. + +### Introduction + +Whole-genome sequencing of cancer tumours is more a research tool nowadays, but going to be used in clinical settings in +the near future to facilitate precision medicine. While large institutions have built up in-house bioinformatics +solutions for their own data analysis, robust and portable workflows combining multiple software have been lacking, +making it difficult for individual research groups to utilise the potential of this research field. Here we present +Sarek, a robust, easy-to-install workflow for identification of both somatic and germline mutations from paired +tumour/normal/relapse samples. + +### Material and Methods + +Sarek is open source and implemented in Nextflow; a domain specific programming language to enable portability and +reproducibility. With the help of docker containers the versions of the underlying software can be maintained. +Furthermore, with Singularity it is possible to run the workflow on protected clusters with no internet connection. + +The workflow starts from raw FASTQ files, and follows the GATK best practices to prepare the recalibrated files with +joint realignment around indels for both the tumour and the normal data. Reads are alignment to the GRCh38 human +reference in an ALT-aware settings using BWA, however, it is possible to assign other references. HaplotypeCaller and +Strelka2 germline calls are collected for both the tumour and the normal sample, and Manta provides germline structural +variants. The somatic variations are calculated by running MuTect2, Strelka and FreeBayes (and MuTect1 optionally). +Somatic structural variants are delivered by Manta, and ASCAT estimates ploidy, tumour heterogeneity and CNVs. The +resulting variant call files are annotated by SnpEff and Ensembl-VEP. The annotated calls are further filtered and +prioritised by our custom methods. During running the workflow quality control metrics are also calculated and +aggregated by MultiQC. + +### Results and Discussions + +Sarek was validated on a real dataset with known golden set of somatic mutations. In a real settings, whole-genome +sequencing (WGS, 45-60x coverage) of patient-matched tumor and blood derived-DNA is being performed on a set of 80 +pediatric brain tumor samples of the Swedish Childhood Tumor Biobank. The workflow helps to produce, filter, prioritise +and characterise both germline and somatic variations. + +### Conclusion + +Sarek is a portable bioinformatics pipeline for WGS normal/tumour matched samples, aiding precision medicine by improved +subtyping and to gain novel functional insights in a reproducible framework. + + diff --git a/docs/abstracts/2018-06-NPMI.md b/docs/abstracts/2018-06-NPMI.md new file mode 100644 index 0000000000..ef15cc035f --- /dev/null +++ b/docs/abstracts/2018-06-NPMI.md @@ -0,0 +1,50 @@ +# The Nordic Precision Medicine Initiative - Meeting No 5 + +## Sarek, a portable workflow for WGS analysis of germline and somatic mutations + +Maxime Garcia 123*, +Szilveszter Juhos 123*, +Malin Larsson 456, +Teresita Díaz de Ståhl 13, +Johanna Sandgren 13, +Jesper Eisfeldt 73, +Sebastian DiLorenzo 85A, +Marcel Martin B5C, +Pall Olason 95A, +Phil Ewels B2C, +Björn Nystedt 95A*, +Monica Nistér 13, +Max Käller 2D, +*Corresponding Author + +1. Barntumörbanken, Dept. of Oncology Pathology; +2. Science for Life Laboratory; +3. Karolinska Institutet; +4. Dept. of Physics, Chemistry and Biology; +5. National Bioinformatics Infrastructure Sweden, Science for Life Laboratory; +6. Linköping University; +7. Clinical Genetics, Dept. of Molecular Medicine and Surgery; +8. Dept. of Medical Sciences; +9. Dept. of Cell and Molecular Biology; +A. Uppsala University; +B. Dept. of Biochemistry and Biophysics; +C. Stockholm University; +D. School of Biotechnology, Division of Gene Technology, Royal Institute of Technology + +We present Sarek, a portable Open Source pipeline to resolve germline and somatic variants from WGS data: it is written in Nextflow, a domain-specific language for workflow building. +It processes normal samples or normal/tumor pairs (with the option to include matched relapses). + +Sarek is based on GATK best practices to prepare short-read data, which is done in parallel for a tumor/normal pair sample. +After these preprocessing steps several variant callers scan the resulting BAM files: Manta for structural variants; Strelka and GATK HaplotypeCaller for germline variants; Freebayes, MuTect1, MuTect2 and Strelka for somatic variants; ASCAT to estimate sample heterogeneity, ploidy and CNVs. +At the end of the analysis the resulting VCF files can be annotated by SNPEff and/or VEP to facilitate further downstream processing. +Our ongoing effort focuses in filtering and prioritizing the annotated variants. + +Sarek is based on Docker and Singularity containers, enabling version tracking, reproducibility and handling sensitive data. +It is designed with flexible environments in mind, like running on a local fat node, a HTC cluster or in a cloud environment like AWS. +The workflow is capable of accommodating further variant callers. +Besides variant calls, the workflow provides quality controls presented by MultiQC. +Checkpoints allow the software to be started from FastQ, BAM or VCF. +Besides WGS data, it is capable to process inputs from WES or gene panels. +The pipeline currently use GRCh37 or GRCh38 as a reference genome, it is also possible to add custom genomes. +It has been successfully used to analyze more than two hundred WGS samples sent to National Genomics Infrastructure (Science for Life Laboratory) from different users. +The MIT licensed Open Source code can be downloaded from GitHub. diff --git a/docs/abstracts/2018-07-JOBIM.md b/docs/abstracts/2018-07-JOBIM.md new file mode 100644 index 0000000000..19e42d372c --- /dev/null +++ b/docs/abstracts/2018-07-JOBIM.md @@ -0,0 +1,43 @@ +# Journées Ouvertes en Biologie, Informatique et Mathématiques 2018 + +## Sarek, a portable workflow for WGS analysis of germline and somatic mutations + +Maxime Garcia 123, +Szilveszter Juhos 123, +Malin Larsson 456, +Teresita Díaz de Ståhl 13, +Johanna Sandgren 13, +Jesper Eisfeldt 73, +Sebastian DiLorenzo 85A, +Marcel Martin B5C, +Pall Olason 95A, +Phil Ewels B2C, +Björn Nystedt 95A, +Monica Nistér 13, +Max Käller 2D + + Max Käller + +1. Barntumörbanken, Dept. of Oncology Pathology; +2. Science for Life Laboratory; +3. Karolinska Institutet; +4. Dept. of Physics, Chemistry and Biology; +5. National Bioinformatics Infrastructure Sweden, Science for Life Laboratory; +6. Linköping University; +7. Clinical Genetics, Dept. of Molecular Medicine and Surgery; +8. Dept. of Medical Sciences; +9. Dept. of Cell and Molecular Biology; +A. Uppsala University; +B. Dept. of Biochemistry and Biophysics; +C. Stockholm University; +D. School of Biotechnology, Division of Gene Technology, Royal Institute of Technology + +We present Sarek, a portable Open Source pipeline to resolve germline and somatic variants from WGS data: it is written in Nextflow, a domain-specific language for workflow building. It processes normal samples or normal/tumor pairs (with the option to include matched relapses). + +Sarek is based on GATK best practices to prepare short-read data, which is done in parallel for a tumor/normal pair sample. After these preprocessing steps several variant callers scan the resulting BAM files: Manta for structural variants; Strelka and GATK HaplotypeCaller for germline variants; Freebayes, MuTect2 and Strelka for somatic variants; ASCAT and Control-FREEC to estimate sample heterogeneity, ploidy and CNVs. At the end of the analysis the resulting VCF files can be annotated by SNPEff and/or VEP to facilitate further downstream processing. Our ongoing effort focuses in filtering and prioritizing the annotated variants. + +Sarek is based on Docker and Singularity containers, enabling version tracking, reproducibility and handling sensitive data. It is designed with flexible environments in mind, like running on a local fat node, a HTC cluster or in a cloud environment like AWS. The workflow is modular and capable of accommodating further variant callers. Besides variant calls, the workflow provides quality controls presented by MultiQC. Checkpoints allow the software to be started from FastQ, BAM or VCF. Besides WGS data, it is capable to process inputs from WES or gene panels. + +The pipeline currently uses GRCh37 or GRCh38 as a reference genome, it is also possible to add custom genomes. It has been successfully used to analyze more than two hundred WGS samples sent to National Genomics Infrastructure (Science for Life Laboratory) from different users. The MIT licensed Open Source code can be downloaded from GitHub. + +The authors thank the Swedish Childhood Cancer Foundation for the funding of Barntumörbanken. We would like to acknowledge support from Science for Life Laboratory, the National Genomics Infrastructure, NGI, and UPPMAX for providing assistance in massive parallel sequencing and computational infrastructure. 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Workflow toprocess normal/tumor WGS data + As WGS (whole genome sequencing), the broadly used research tool is getting cheaper and being introduced to clinics, it is now possible to compare data from normal and tumor samples of numerous patients. There are still many challenges, mostly regarding bioinformatics: datasets are huge, workflows are complex, and there are multiple tools to choose from for somatic and structural variant detection and quality control. The pipeline can use GRCh37 or GRCh38 as a reference genome. Docker containers are also available for easier deployment and testing purposes. CAW can be downloaded from our GitHub repository. The authors thank the Swedish Childhood Cancer Foundation for the funding of Barntumörbanken. We would like to acknowledge support from Science for Life Laboratory, the National Genomics Infrastructure, NGI, and UPPMAX for providing assistance in massive parallel sequencing and computational infrastructure.AknowledgementsReferencesLinkshttp://opensource.scilifelab.se/projects/cawhttps://github.com/SciLifeLab/CAWhttps://gitter.im/SciLifeLab/CAWhttps://www.scilifelab.se/facilities/genomics-applications/ +* : http://dx.doi.org/10.1038/nbt.3820 +Core principlesbstractAWACWGS normal/tumor pairs analysis workflow written inFollows GATK best practices. Provides SNVs, small indels, structural variants, heterogeneity, ploidy CNVs, annotation and QC reportsTools used:- ASCAT- Freebayes- HaplotypeCaller- Manta- MultiQC- MuTect1- MuTect2- snpEff- Strelka- VEPL;DRTEasily deployable, supports containersThe MIT License (MIT) Copyright © 2016 SciLifeLabCan be used onCan also analyse normal only samples1 - BarnTumörBanken, Dept. of Oncology Pathology, Science for Life Laboratory, Karolinska Institutet2 - Dept. of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University3 - Dept. of Physics, Chemistry and Biology, National Bioinformatics Infrastructure Sweden, Science for Life Laboratory, Linköping University4 - BarnTumörBanken, Dept. of Oncology Pathology, Karolinska Institutet5 - Clinical Genetics, Dept. of Molecular Medicine and Surgery, Karolinska Institutet6 - Dept. of Medical Sciences, National Bioinformatics Infrastructure Sweden, Science for Life Laboratory, Uppsala University7 - Dept. of Biochemistry and Biophysics, National Bioinformatics Infrastructure Sweden, Science for Life Laboratory, Stockholm University 8 - Dept. of Cell and Molecular Biology, National Bioinformatics Infrastructure Sweden, Science for Life Laboratory, Uppsala University9 - Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, Royal Institute of TechnologyJoin the chat on gitterOpen source, contribute on githubAknowledgementsUPPMAX Preprocessing takes arround 4 days on one Huawei XH620 V3 compute node with dual CPUs (Intel Xeon E5-2630 v3, each with 8 cores) with 45 coverage normal/tumor pairs. Further Variant Calling can take up to 10 hours for each variant caller. CAW is prepared to process normal/tumor pairs and can handle additional relapse samples. It can also be used to preprocess and analyse normal only samples using GATK best practices and HaplotypeCaller. CAW can start the analysis from raw FASTQ files, from the realignment step, or directly with any subset of variant callers. Resulting VCF files can be annotated using snpEff or VEP. At the end of the analysis the final VCF files are merged to facilitate further downstream processing, though the individual results are also retained. The flow is capable of accommodating further variant calling software or CNV callers. We are utilizing GATK best practices to align, realign and recalibrate short-read data in parallel for both tumor and normal samples. After preprocessing, several somatic variant callers scan the resulting BAM files; MuTect1, MuTect2 and Strelka are used to find somatic SNVs and small indels. For structural variants we use Manta. Furthermore, we are applying ASCAT to estimate sample heterogeneity, ploidy and CNVs. The workflow also provides quality controls presented by MultiQC.orkflow specificitiesW We are presenting CAW (Cancer Analysis Workflow), a complete open source pipeline to resolve somatic variants from WGS data: it is written in Nextflow*, a domain specific language for workflow building.Variant Calling +Structural +Variants +SNVs +CNV +Indels +Preprocessing +GATK +Best Practices +normal +BAM, bai +GATK +Best Practices +tumor +BAM, bai +GATK +Best Practices +relapse +BAM, bai +Configuration +Metadata +Statistics & QC +Results +Annotation +Pre-filter +Merge + \ No newline at end of file diff --git a/docs/posters/PMC_2018_Mgarcia.pdf b/docs/posters/PMC_2018_Mgarcia.pdf new file mode 100644 index 0000000000000000000000000000000000000000..cb7686ffd0dc16836f5cbcfc8a2eb168d4dda84b GIT binary patch literal 307620 zcmV(-K-|A2P((&8F)lR4?5av(28Y+-a|L}g=dWMv9IJ_>Vma%Ev{3V57^y}Pm`Ij|(A=U0?Wv+b>XXX^Zc zl_=B7xI4lb(srrv^9dl_Jv_2b-z&`=?*s~uKp+s$|2e(?+{gR8w%7On{ICD<`mg`z 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DiLorenzo5, Marcel Martin6, Pall Olason7, Phil Ewels8, Björn Nystedt7, Monica Nistér3, Max Käller9 + +Sarek, a workflow for WGS analysisof germline and somatic mutations +Portable WGS germline and normal/tumor pairs analysis workflow written inSummaryEasily deployable with containersThe MIT License (MIT) Copyright © 2016 SciLifeLabCan be used onJoin the chat on GitterOpen source, contribute on GitHubAcknowledgementsUPPMAX1 - BarnTumörBanken, Dept. of Oncology Pathology, Science for Life Laboratory, Karolinska Institutet2 - Dept. of Physics, Chemistry and Biology, National Bioinformatics Infrastructure Sweden, Science for Life Laboratory, Linköping University3 - BarnTumörBanken, Dept. of Oncology Pathology, Karolinska Institutet4 - Clinical Genetics, Dept. of Molecular Medicine and Surgery, Karolinska Institutet5 - Dept. of Medical Sciences, National Bioinformatics Infrastructure Sweden, Science for Life Laboratory, Uppsala University6 - Dept. of Biochemistry and Biophysics, National Bioinformatics Infrastructure Sweden, Science for Life Laboratory, Stockholm University 7 - Dept. of Cell and Molecular Biology, National Bioinformatics Infrastructure Sweden, Science for Life Laboratory, Uppsala University8 - Dept. of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University9 - Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, Royal Institute of Technology + +rThe authors thank the Swedish Childhood Cancer Foundation for the funding of Barntumörbanken. We would like to acknowledge support from Science for Life Laboratory, the National Genomics Infrastructure, NGI, and UPPMAX for providing assistance in massive parallel sequencing and computational infrastructure.AcknowledgementsSarek is based on Docker and Singularity2 containers, enabling version tracking, reproducibility and handling sensitive data.The workflow is capable of accommodating further variant callers.Besides variant calls, the workflow provides quality controls presented by MultiQC3.Checkpoints allow the software to be started from FastQ, BAM or VCF.The pipeline currently use GRCh37 or GRCh38 as a reference genome, it is also possible to add custom genomes.The MIT licensed Open Source code can be downloaded from GitHub.Linksopensource.scilifelab.se/projects/sarekgithub.com/SciLifeLab/Sarekgitter.im/SciLifeLab/Sarekngisweden.scilifelab.se +References1: doi.org/10.1038/nbt.38202: doi.org/10.1371/journal.pone.01774593: doi.org/10.1093/bioinformatics/btw354 +We present Sarek, a portable Open Source pipeline to resolve germline and somatic variants from WGS data: it is written in Nextflow1, a domain-specific language for workflow building.It processes normal samples or normal/tumor pairs (with the option to include matched relapses).Sarek is based on GATK best practices to prepare short-read data, which is done in parallel for a tumor/normal pair sample.After these preprocessing steps several variant callers scan the resulting BAM files:- Manta for structural variants- Strelka and GATK HaplotypeCaller for germline variants- Freebayes, MuTect1, MuTect2 and Strelka for somatic variants- ASCAT to estimate sample heterogeneity, ploidy and CNVsAt the end of the analysis the resulting VCF files can be annotated to facilitate further downstream processing.Fig1: CPU usage for 90x tumor/normal pair sample (hours)Map, merge, dedup - 27%Realign, recalibrate - 20%HaplotypeCaller - 8%Strelka - 1%Manta - 10%FreeBayes - 5%MuTect2 - 6%MuTect1 - 3%ASCAT - 5%PrintReads - 16%84621815302548159Preprocessing based on GATK best practicesVariant Calling with:- HaplotypeCaller- Manta- StrelkaVariant Calling with:- ASCAT- Freebayes- HaplotypeCaller- Manta- MuTect1- MuTect2- StrelkaAnnotation with:- snpEff- VEPReports aggregated byFig2: Workflow organizationhtmlorfastqbambambairecalvcfvcfvcfVariant Calling +- HaplotypeCaller, Strelka- MantaPreprocessing +Based on GATK Best PracticesfastqbamorfastqbambambairecalbambairecalvcfvcfvcfvcfvcfvcfvcfvcfPreprocessing +Based on GATK Best PracticesVariant Calling +- HaplotypeCaller, Freebayes MuTect1, MuTect2, Strelka- ASCAT- MantaAnnotation +snpEff, VEPReports +SarekSomaticSarekGermlineSarekSarekGermlineSarekSomatic \ No newline at end of file From 884f831520cd5716a5061ed1ec3c77d9dda7e67a Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Tue, 28 May 2019 15:20:09 +0200 Subject: [PATCH 21/22] update docs --- README.md | 14 ++---- docs/reference.md | 29 +++++++++++ docs/use_cases.md | 122 ++++++++++++++++++++++++++++++++++++++++++++++ 3 files changed, 156 insertions(+), 9 deletions(-) create mode 100644 docs/use_cases.md diff --git a/README.md b/README.md index 4dcdff4454..a055578752 100644 --- a/README.md +++ b/README.md @@ -1,7 +1,7 @@ # [![Sarek](docs/images/Sarek_logo.png "Sarek")](https://sarek.scilifelab.se/) [![nf-core](docs/images/nf-core_logo.png "Sarek")](https://nf-co.re/) -**An open-source analysis pipeline to detect germline or somatic variants from whole genome or targeted sequencing**. +**An open-source analysis pipeline to detect germline or somatic variants from whole genome or targeted sequencing** > :warning: This pipeline is a work in progress being ported to nf-core from [SciLifeLab/Sarek](https://github/SciLifeLab/Sarek/) @@ -21,7 +21,7 @@ Previously known as the Cancer Analysis Workflow (CAW), -Sarek is a workflow designed to run analyses on whole genome or targeted sequencing data from regular samples or tumour / normal pairs and could including additional relapses. +Sarek is a workflow designed to run analyses on whole genome or targeted sequencing data from regular samples or tumour / normal pairs and could include additional relapses. It's built using [Nextflow](https://www.nextflow.io), a domain specific language for workflow building, @@ -29,7 +29,7 @@ across multiple compute infrastructures in a very portable manner. Software dependencies are handled using [Conda](https://conda.io/), [Docker](https://www.docker.com) or [Singularity](https://www.sylabs.io/singularity/) - environment/container technologies that provide excellent reproducibility and ease of use. Thus making installation trivial and results highly reproducible. -It is listed on the [Elixir - Tools and Data Services Registry](https://bio.tools/Sarek), [Dockstore](https://dockstore.org/workflows/github.com/SciLifeLab/Sarek/) and [omicX - Bioinformatics tools](https://omictools.com/sarek-tool). +It's listed on the [Elixir - Tools and Data Services Registry](https://bio.tools/Sarek), [Dockstore](https://dockstore.org/workflows/github.com/SciLifeLab/Sarek/) and [omicX - Bioinformatics tools](https://omictools.com/sarek-tool). ## Documentation The nf-core/sarek pipeline comes with documentation about the pipeline, found in the `docs/` directory: @@ -41,17 +41,13 @@ The nf-core/sarek pipeline comes with documentation about the pipeline, found in * [Reference genomes](https://nf-co.re/usage/reference_genomes) * [Extra documentation on reference](docs/reference.md) 3. [Running the pipeline](docs/usage.md) + * [Examples](docs/use_cases.md) * [Input files documentation](docs/input.md) * [Extra documentation on variant calling](docs/variantcalling.md) * [Documentation about containers](docs/containers.md) * [Extra documentation for targeted sequencing](docs/targetseq.md) - - * [Intervals documentation](docs/INTERVALS.md) - * [Command line parameters](docs/PARAMETERS.md) - * [Examples](docs/USE_CASES.md) - * [Processes documentation](docs/PROCESS.md) 4. [Output and how to interpret the results](docs/output.md) - * [Complementary information about ASCAT](docs/ASCAT.md) + * [Complementary information about ASCAT](docs/ascat.md) * [Extra documentation on annotation](docs/annotation.md) 5. [Troubleshooting](https://nf-co.re/usage/troubleshooting) diff --git a/docs/reference.md b/docs/reference.md index 55d267d023..eb77557ddc 100644 --- a/docs/reference.md +++ b/docs/reference.md @@ -7,6 +7,35 @@ Both `GRCh37` and `GRCh38` are available with `--genome GRCh37` or `--genome GRC Use `--genome smallGRCh37` to map against a small reference genome based on GRCh37. Settings in `igenomes.config` can be tailored to your needs. +### Intervals + +To speed up the variant calling processes, the reference is chopped into smaller pieces. +The intervals are chromosomes cut at their centromeres (so each chromosome arm processed separately) also additional unassigned contigs. +We are ignoring the hs37d5 contig that contains concatenated decoy sequences. +Parts of preprocessing and variant calling are done by this intervals, and the different resulting files are then merged. +This can parallelize processes, and push down wall clock time significantly. + +The calling intervals can be defined using a `.list` or a `.bed` file. +A `.list` file contains one interval per line in the format `chromosome:start-end` (1-based coordinates). + +When the intervals file is in BED format, the file must be a tab-separated text file with one interval per line. +There must be at least three columns: chromosome, start, and end. +In BED format, the coordinates are 0-based, so the interval `chrom:1-10` becomes `chrom010`. + +Additionally, the "score" column of the BED file can be used to provide an estimate of how many seconds it will take to call variants on that interval. +The fourth column remains unused. +Example (the fields would actually be tab-separated, this is not shown here): + +`chr1 10000 207666 NA 47.3` + +This indicates that variant calling on the interval chr1:10001-207666 takes approximately 47.3 seconds. + +The runtime estimate is used in two different ways. +First, when there are multiple consecutive intervals in the file that take little time to compute, they are processed as a single job, thus reducing the number of processes that needs to be spawned. +Second, the jobs with largest processing time are started first, which reduces wall-clock time. +If no runtime is given, a time of 1000 nucleotides per second is assumed. +Actual figures vary from 2 nucleotides/second to 30000 nucleotides/second. + ## build.nf The [`build.nf`](#buildnf) script is used to build reference needed for smallGRCh37. diff --git a/docs/use_cases.md b/docs/use_cases.md new file mode 100644 index 0000000000..894343f37c --- /dev/null +++ b/docs/use_cases.md @@ -0,0 +1,122 @@ +# Use cases + +The workflow has two pre-processing options: `mapping` and `recalibrate`. +Using the `mapping` directive one will have a pair of mapped, deduplicated and recalibrated BAM files in the `Preprocessing/Recalibrated/` directory. +This is the usual option you have to give when you are starting from raw FASTQ data: + +```bash +nextflow run nf-core/sarek/main.nf --sample mysample.tsv --tools +``` + +`mapping` will start by default, you do not have to give any additional parameters, only the TSV file describing the sample (see below). + +During the execution of the workflow a `execution_trace.txt`, a `execution_timeline.html` and a `execution_report.html` files are generated automatically. +These files contain statistics about resources used and processes finished. +If you start a new workflow or restart/resume a sample, the previous version will be renamed as `execution_trace.txt.1`, `execution_timeline.html.1` and `execution_report.html.1` respectively. +Also, older version are renamed with incremented numbers. + +## Starting from raw FASTQ - pair of FASTQ files + +The workflow should be started in this case with the smallest set of options as written above: + +```bash +nextflow run nf-core/sarek/main.nf --sample mysample.tsv --tools +``` + +The TSV file should look like: +``` +SUBJECT_ID XX 0 SAMPLE_ID 1 /samples/normal_1.fastq.gz /samples/normal_2.fastq.gz +``` + +See the [input files documentation](docs/input.md) for more information. + +## Starting from raw FASTQ - a directory with normal sample only + +The `--sample` option can be also used to point Sarek to a directory with FASTQ files: +```bash +nextflow run nf-core/sarek/main.nf --sample path/to/FASTQ/files --tools +``` +The given directory is searched recursively for FASTQ files that are named `*_R1_*.fastq.gz`, and a matching pair with the same name except `_R2_` instead of `_R1_` is expected to exist alongside. +All of the found FASTQ files are considered to belong to the sample. +Each FASTQ file pair gets its own read group (`@RG`) in the resulting BAM file. + +### Metadata when using `--sample` with a directory + +When using `--sample` with a directory, the metadata about the sample that are written to the BAM header in the `@RG` tag are determined in the following way. + +- The sample name (`SM`) is derived from the the last component of the path given to `--sample`. +That is, you should make sure that that directory has a meaningful name! For example, with `--sample=/my/fastqs/sample123`, the sample name will be `sample123`. +- The read group id is set to *flowcell.samplename.lane*. +The flowcell id and lane number are auto-detected from the name of the first read in the FASTQ file. + +## Starting from raw FASTQ - pair of FASTQ files for tumor/normal samples + +The workflow command line is just the same as before, but the TSV contains extra lines. +You can see the second column is used to distinguish normal and tumor samples. +You can add as many relapse samples as many you have, providing their name in the third column is different. +Each will be compared to the normal one-by-one. +Usually there are more read groups - sequencing lanes - for a single sequencing run, and in a flowcell different lanes have to be recalibrated separately. +This is captured in the TSV file only in the following manner, adding read group numbers or IDs in the fourth column. +All lanes belonging to the same Sample will be merged together after the FASTQ pairs are mapped to the reference genome. +Obviously, if you do not have relapse samples, you can leave out the two last lines. + +``` +SUBJECT_ID XX 0 SAMPLE_ID_N 1 /samples/normal1_1.fastq.gz /samples/normal1_2.fastq.gz +SUBJECT_ID XX 0 SAMPLE_ID_N 2 /samples/normal2_1.fastq.gz /samples/normal2_2.fastq.gz +SUBJECT_ID XX 1 SAMPLE_ID_T 3 /samples/tumor3_1.fastq.gz /samples/tumor3_2.fastq.gz +SUBJECT_ID XX 1 SAMPLE_ID_T 4 /samples/tumor4_1.fastq.gz /samples/tumor4_2.fastq.gz +SUBJECT_ID XX 1 SAMPLE_ID_T 5 /samples/tumor5_1.fastq.gz /samples/tumor5_2.fastq.gz +SUBJECT_ID XX 1 SAMPLE_ID_R 7 /samples/relapse7_1.fastq.gz /samples/relapse7_2.fastq.gz +SUBJECT_ID XX 1 SAMPLE_ID_R 9 /samples/relapse9_1.fastq.gz /samples/relapse9_2.fastq.gz +``` + +See the [input files documentation](docs/input.md) for more information. + +## Starting from recalibration + +```bash +nextflow run nf-core/sarek/main.nf --sample mysample.tsv --step recalibrate --tools +``` + +And the corresponding TSV file should be like: +Obviously, if you do not have tumor or relapse samples, you can leave out the two last lines. + +``` +SUBJECT_ID XX 0 SAMPLE_ID_N /samples/SAMPLE_ID_N.bam /samples/SAMPLE_ID_N.bai /samples/SAMPLE_ID_N.recal.table +SUBJECT_ID XX 1 SAMPLE_ID_T /samples/SAMPLE_ID_T.bam /samples/SAMPLE_ID_T.bai /samples/SAMPLE_ID_T.recal.table +SUBJECT_ID XX 1 SAMPLE_ID_R /samples/SAMPLE_ID_R.bam /samples/SAMPLE_ID_R.bai /samples/SAMPLE_ID_R.recal.table +``` + +See the [input files documentation](docs/input.md) for more information. + +## Starting from a recalibrated BAM file + +At this step we are assuming that all the required preprocessing is over, we only want to run variant callers or other tools using recalibrated BAM files. + +```bash +nextflow run nf-core/sarek/main.nf --step variantcalling --tools +``` + +And the corresponding TSV file should be like: + +``` +SUBJECT_ID XX 0 SAMPLE_ID_N /samples/SAMPLE_ID_N.bam /samples/SAMPLE_ID_N.bai +SUBJECT_ID XX 1 SAMPLE_ID_T /samples/SAMPLE_ID_T.bam /samples/SAMPLE_ID_T.bai +SUBJECT_ID XX 1 SAMPLE_ID_R /samples/SAMPLE_ID_R.bam /samples/SAMPLE_ID_R.bai +``` + +See the [input files documentation](docs/input.md) for more information. + +If you want to restart a previous run of the pipeline, you may not have a recalibrated BAM file. +In this case, you need to start with `--step=recalibrate` (see previous section). + +## Using Sarek with targeted (whole exome or panel) sequencing data + +The recommended flow for targeted sequencing data is to use the workflow as it is, but also provide a BED file containing targets for all steps using the `--targetBED` option. +The workflow will pick up these intervals, and activate the `--exome` flag to process deeper coverage. +It is adviced to pad the variant calling regions (exons or the target) to some extent before submitting to the workflow. +To add the target BED file configure the flow like: + +```bash +nextflow run nf-core/sarek/main.nf --tools haplotypecaller,strelka,mutect2 --targetBED targets.bed --sample my_panel.tsv +``` From 3cee83514f869a53a67c2aab6dbe1d6373c77a13 Mon Sep 17 00:00:00 2001 From: MaxUlysse Date: Tue, 28 May 2019 15:20:26 +0200 Subject: [PATCH 22/22] update docs --- main.nf | 5 ++--- 1 file changed, 2 insertions(+), 3 deletions(-) diff --git a/main.nf b/main.nf index b51f47b62c..7bff7070ea 100644 --- a/main.nf +++ b/main.nf @@ -53,7 +53,6 @@ def helpMessage() { --tools Specify tools to use for variant calling Available: ASCAT, ControlFREEC, FreeBayes, HaplotypeCaller Manta, mpileup, MuTect2, Strelka - Default: HaplotypeCaller, Manta, Strelka --annotateTools Specify from which tools Sarek will annotate VCF, only for step annotate Available: HaplotypeCaller, Manta, MuTect2, Strelka @@ -118,7 +117,7 @@ params.sequencing_center = null params.snpEff_cache = null params.step = 'mapping' params.targetBED = null -params.tools = "HaplotypeCaller,Manta,Strelka" +params.tools = null params.vep_cache = null stepList = defineStepList() @@ -1855,7 +1854,7 @@ multiQCReport = Channel.empty() Channel.fromPath(params.multiqc_config) -process RunMultiQC { +process MultiQC { publishDir "${params.outdir}/Reports/MultiQC", mode: params.publishDirMode input: