Pacbio align

.github/workflows/ci.yml

@@ -41,3 +41,7 @@ jobs:
         # Remember that you can parallelise this by using strategy.matrix
         run: |
           nextflow run ${GITHUB_WORKSPACE} -profile test,docker --outdir ./results
+
+      - name: Run pipeline with unaligned test data
+        run: |
+          nextflow run ${GITHUB_WORKSPACE} -profile test_align,docker --outdir ./results --align

.github/workflows/sanger_test.yml

@@ -19,8 +19,9 @@ jobs:
           parameters: |
             {
               "outdir": "${{ secrets.TOWER_WORKDIR_PARENT }}/results/${{ github.repository }}/results-${{ github.sha }}",
+              "align":  true
             }
-          profiles: test,sanger,singularity,cleanup
+          profiles: test_align,sanger,singularity,cleanup
 
       - uses: actions/upload-artifact@v3
         with:

.github/workflows/sanger_test_full.yml

@@ -32,8 +32,9 @@ jobs:
           parameters: |
             {
               "outdir": "${{ secrets.TOWER_WORKDIR_PARENT }}/results/${{ github.repository }}/results-${{ env.REVISION }}",
+              "align":  true,
             }
-          profiles: test_full,sanger,singularity,cleanup
+          profiles: test_full_align,sanger,singularity,cleanup
 
       - uses: actions/upload-artifact@v3
         with:

assets/samplesheet_test_align.csv

@@ -0,0 +1,4 @@
+sample,datatype,datafile
+icCanRufa1,pacbio,https://tolit.cog.sanger.ac.uk/test-data/Cantharis_rufa/genomic_data/icCanRufa1/pacbio/m64094_200730_174533.ccs.bc1010_BAK8A_OA--bc1010_BAK8A_OA_0_03.bam
+icCanRufa1,pacbio,https://tolit.cog.sanger.ac.uk/test-data/Cantharis_rufa/genomic_data/icCanRufa1/pacbio/m64094_200730_174533.ccs.bc1010_BAK8A_OA--bc1010_BAK8A_OA_0_02.bam
+icCanRufa1XXXXX,pacbio,https://tolit.cog.sanger.ac.uk/test-data/Cantharis_rufa/genomic_data/icCanRufa1/pacbio/m64094_200730_174533.ccs.bc1010_BAK8A_OA--bc1010_BAK8A_OA_0_02.bam

assets/samplesheet_test_full_align.csv

@@ -0,0 +1,2 @@
+sample,datatype,datafile
+ilPolIcar1,pacbio,/lustre/scratch124/tol/projects/darwin/data/insects/Polyommatus_icarus/genomic_data/ilPolIcar1/pacbio/m64016_191206_183623.ccs.bc1019_BAK8B_OA--bc1019_BAK8B_OA.bam

bin/check_samplesheet.py

@@ -108,6 +108,11 @@ def validate_unique_samples(self):
         """
         if len(self._seen) != len(self.validated):
             raise AssertionError("The combination of sample name and data file must be unique.")
+        seen = Counter()
+        for row in self.validated:
+            sample = row[self._sample_col]
+            seen[sample] += 1
+            row[self._sample_col] = f"{sample}_T{seen[sample]}"
 
 
 def read_head(handle, num_lines=10):

bin/pacbio_filter.sh

@@ -0,0 +1,6 @@
+#!/bin/bash
+
+input=$1
+output=$2
+
+grep -v 'MG551957' $input | awk -v OFS='\t' '{if (($2 ~ /NGB00972/ && $3 >= 97 && $4 >= 44) || ($2 ~ /NGB00973/ && $3 >= 97 && $4 >= 34) || ($2 ~ /^bc/ && $3 >= 99 && $4 >= 16)) print $1}' | sort -u > $output

conf/base.config

@@ -16,7 +16,7 @@ process {
     time   = { check_max( 4.h  * task.attempt, 'time'   ) }
 
     errorStrategy = { task.exitStatus in ((130..145) + 104) ? 'retry' : 'finish' }
-    maxRetries    = 1
+    maxRetries    = 5
     maxErrors     = '-1'
 
     // Process-specific resource requirements

conf/modules.config

@@ -20,15 +20,73 @@ process {
         ]
     }
 
+    if( params.align ) {
+
+        withName: '.*:ALIGN_PACBIO:FILTER_PACBIO:SAMTOOLS_CONVERT' {
+            ext.args = "-e '[rq]>=0.99' -x fi -x fp -x ri -x rp --write-index --output-fmt bam"
+        }
+
+        withName: '.*:ALIGN_PACBIO:FILTER_PACBIO:SAMTOOLS_COLLATE' {
+            ext.prefix = { "${meta.id}.collate" }
+        }
+
+        withName: '.*:ALIGN_PACBIO:FILTER_PACBIO:BLAST_BLASTN' {
+            ext.args = '-task blastn -reward 1 -penalty -5 -gapopen 3 -gapextend 3 -dust yes -soft_masking true -evalue .01 -searchsp 1750000000000 -outfmt 6'
+        }
+
+        withName: '.*:ALIGN_PACBIO:FILTER_PACBIO:SAMTOOLS_FILTER' {
+            ext.prefix = { "${meta.id}.filter" }
+        }
+
+        withName: '.*:ALIGN_PACBIO:FILTER_PACBIO:SAMTOOLS_FASTQ' {
+            ext.args = '-F 0x200 -nt'
+        }
+
+        withName: '.*:.*:ALIGN_PACBIO:MINIMAP2_ALIGN' {
+            ext.args = { "-ax map-hifi --cs=short -R ${meta.read_group}" }
+        }
+
+        withName: '.*:.*:ALIGN_PACBIO:SAMTOOLS_MERGE' {
+            ext.args = { "-c -p" }
+            ext.prefix = { "${meta.id}.merge" }
+        }
+
+        withName: '.*:CONVERT_STATS:SAMTOOLS_VIEW' {
+            ext.prefix = { "${meta2.id}.${meta.datatype}.${meta.id}" }
+            ext.args   = '--output-fmt cram --write-index'
+        }
+
+        withName: '.*:CONVERT_STATS:SAMTOOLS_STATS' {
+            ext.prefix = { "${input.baseName}" }
+        }
+
+        withName: '.*:CONVERT_STATS:SAMTOOLS_FLAGSTAT' {
+            ext.prefix = { "${bam.baseName}" }
+        }
+
+        withName: '.*:CONVERT_STATS:SAMTOOLS_IDXSTATS' {
+            ext.prefix = { "${bam.baseName}" }
+        }
+
+        withName: '.*:ALIGN_PACBIO:CONVERT_STATS:.*' {
+            publishDir = [
+                path: { "${params.outdir}/variant_calling" },
+                mode: params.publish_dir_mode,
+                saveAs: { filename -> filename.equals('versions.yml') ? null : filename }
+            ]
+        }
+    } else{
+
+        withName: '.*:INPUT_MERGE:SAMTOOLS_MERGE' {
+            ext.args   = '--write-index'
+        }
+    }
+
     withName: '.*:INPUT_FILTER_SPLIT:SAMTOOLS_VIEW' {
         ext.args   = '--output-fmt cram --write-index -F 0x900'
         ext.prefix = { "${meta.id}_filtered" }
     }
 
-    withName: '.*:INPUT_MERGE:SAMTOOLS_MERGE' {
-        ext.args   = '--write-index'
-    }
-
     withName: '.*:DEEPVARIANT_CALLER:DEEPVARIANT' {
         ext.args = '--model_type=PACBIO'
     }

conf/test_align.config

@@ -0,0 +1,27 @@
+/*
+~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
+    Nextflow config file for running minimal tests
+~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
+    Defines input files and everything required to run a fast and simple pipeline test.
+
+    Use as follows:
+        nextflow run sanger-tol/variantcalling -profile test_align,<docker/singularity> --outdir <OUTDIR> --align
+
+----------------------------------------------------------------------------------------
+*/
+
+params {
+    config_profile_name        = 'Test profile with alignment'
+    config_profile_description = 'Minimal unaligned test dataset to check pipeline function'
+
+    // Limit resources so that this can run on GitHub Actions
+    max_cpus   = 2
+    max_memory = '6.GB'
+    max_time   = '6.h'
+
+    // Input data
+    input  = "${projectDir}/assets/samplesheet_test_align.csv"
+
+    // Fasta references
+    fasta = "https://tolit.cog.sanger.ac.uk/test-data/Cantharis_rufa/assembly/GCA_947369205.1_OX376310.1_CANBKR010000003.1.fasta.gz"
+}

conf/test_full_align.config

@@ -0,0 +1,25 @@
+/*
+~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
+    Nextflow config file for running full-size tests with alignment
+~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
+    Defines input files and everything required to run a full size pipeline test.
+
+    Use as follows:
+        nextflow run sanger-tol/variantcalling -profile test_full_align,<docker/singularity> --outdir <OUTDIR> --align
+
+----------------------------------------------------------------------------------------
+*/
+
+cleanup = true
+
+params {
+    config_profile_name        = 'Full test profile with alignment'
+    config_profile_description = 'Full non-aligned test dataset to check pipeline function'
+
+    // Input data for full size test
+    input = "${projectDir}/assets/samplesheet_test_full_align.csv"
+
+    // Fasta references
+    fasta = "/lustre/scratch124/tol/projects/darwin/data/insects/Polyommatus_icarus/assembly/release/ilPolIcar1.1/insdc/GCA_937595015.1.fasta.gz"
+
+}

modules.json

@@ -10,6 +10,11 @@
                         "git_sha": "911696ea0b62df80e900ef244d7867d177971f73",
                         "installed_by": ["modules"]
                     },
+                    "blast/blastn": {
+                        "branch": "master",
+                        "git_sha": "3f5420aa22e00bd030a2556dfdffc9e164ec0ec5",
+                        "installed_by": ["modules"]
+                    },
                     "cat/cat": {
                         "branch": "master",
                         "git_sha": "911696ea0b62df80e900ef244d7867d177971f73",
@@ -25,11 +30,46 @@
                         "git_sha": "516189e968feb4ebdd9921806988b4c12b4ac2dc",
                         "installed_by": ["modules"]
                     },
+                    "gunzip": {
+                        "branch": "master",
+                        "git_sha": "3f5420aa22e00bd030a2556dfdffc9e164ec0ec5",
+                        "installed_by": ["modules"]
+                    },
+                    "minimap2/align": {
+                        "branch": "master",
+                        "git_sha": "3f5420aa22e00bd030a2556dfdffc9e164ec0ec5",
+                        "installed_by": ["modules"]
+                    },
+                    "samtools/collate": {
+                        "branch": "master",
+                        "git_sha": "3f5420aa22e00bd030a2556dfdffc9e164ec0ec5",
+                        "installed_by": ["modules"]
+                    },
                     "samtools/faidx": {
                         "branch": "master",
                         "git_sha": "fd742419940e01ba1c5ecb172c3e32ec840662fe",
                         "installed_by": ["modules"]
                     },
+                    "samtools/fasta": {
+                        "branch": "master",
+                        "git_sha": "6f4299292ef2c5b66e6829527b2647c301b77cc9",
+                        "installed_by": ["modules"]
+                    },
+                    "samtools/fastq": {
+                        "branch": "master",
+                        "git_sha": "3f5420aa22e00bd030a2556dfdffc9e164ec0ec5",
+                        "installed_by": ["modules"]
+                    },
+                    "samtools/flagstat": {
+                        "branch": "master",
+                        "git_sha": "63e817de8c617131447192ab2c4e70b4ed4071f7",
+                        "installed_by": ["modules"]
+                    },
+                    "samtools/idxstats": {
+                        "branch": "master",
+                        "git_sha": "63e817de8c617131447192ab2c4e70b4ed4071f7",
+                        "installed_by": ["modules"]
+                    },
                     "samtools/merge": {
                         "branch": "master",
                         "git_sha": "e7ce60acc8a33fa17429e966364657a63016e870",
@@ -41,14 +81,25 @@
                         "git_sha": "a0f7be95788366c1923171e358da7d049eb440f9",
                         "installed_by": ["modules"]
                     },
+                    "samtools/stats": {
+                        "branch": "master",
+                        "git_sha": "3f5420aa22e00bd030a2556dfdffc9e164ec0ec5",
+                        "installed_by": ["modules"]
+                    },
                     "samtools/view": {
                         "branch": "master",
-                        "git_sha": "3ffae3598260a99e8db3207dead9f73f87f90d1f",
+                        "git_sha": "3f5420aa22e00bd030a2556dfdffc9e164ec0ec5",
+                        "installed_by": ["modules"],
+                        "patch": "modules/nf-core/samtools/view/samtools-view.diff"
+                    },
+                    "untar": {
+                        "branch": "master",
+                        "git_sha": "3f5420aa22e00bd030a2556dfdffc9e164ec0ec5",
                         "installed_by": ["modules"]
                     },
                     "vcftools": {
                         "branch": "master",
-                        "git_sha": "8fc1d24c710ebe1d5de0f2447ec9439fd3d9d66a",
+                        "git_sha": "485558b40040fc3ace093d9084210125d8ba4c97",
                         "installed_by": ["modules"],
                         "patch": "modules/nf-core/vcftools/vcftools.diff"
                     }

modules/local/pacbio_filter.nf

@@ -0,0 +1,30 @@
+process PACBIO_FILTER {
+    tag "$meta.id"
+    label 'process_single'
+
+    conda "conda-forge::gawk=5.1.0"
+    container "${ workflow.containerEngine == 'singularity' && !task.ext.singularity_pull_docker_container ?
+        'https://depot.galaxyproject.org/singularity/gawk:5.1.0' :
+        'quay.io/biocontainers/gawk:5.1.0' }"
+
+    input:
+    tuple val(meta), path(txt)
+
+    output:
+    path("*.blocklist"),  emit: list
+    path  "versions.yml", emit: versions
+
+    when:
+    task.ext.when == null || task.ext.when
+
+    script:
+    def prefix = task.ext.prefix ?: "${meta.id}"
+    """
+    pacbio_filter.sh $txt ${prefix}.blocklist
+
+    cat <<-END_VERSIONS > versions.yml
+    "${task.process}":
+        GNU Awk: \$(echo \$(awk --version 2>&1) | grep -i awk | sed 's/GNU Awk //; s/,.*//')
+    END_VERSIONS
+    """
+}