🔀 Merge pull request #74 from cnr-ibba/issue-73

⚡ Optimize `samtools/depth` step
cnr-ibba · Apr 11, 2024 · d41676e · d41676e
2 parents 055c09d + 0b84168
commit d41676e
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diff --git a/.vscode/settings.json b/.vscode/settings.json
@@ -10,6 +10,7 @@
         "coverage",
         "cpus",
         "demultiplexing",
+        "depthfile",
         "downsampling",
         "fasta",
         "fastq",

diff --git a/CHANGELOG.md b/CHANGELOG.md
@@ -3,6 +3,15 @@
 The format is based on [Keep a Changelog](https://keepachangelog.com/en/1.0.0/)
 and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0.html).
 
+## 0.6.1 - [2024-04-11]
+
+- Calculate `samtools/depth` on each chromosomes ([#73](https://github.com/cnr-ibba/nf-resequencing-mem/issues/73))
+
+### `Fixed`
+
+- Passing args to `modules/local/freebayes_splitcram`
+- Calculate `samtools/depth` without _0 coverage_ regions
+
 ## 0.6.0 - [2024-04-04]
 
 - Replace `*.bam` file format with `*.cram` ([#9](https://github.com/cnr-ibba/nf-resequencing-mem/issues/9))

diff --git a/assets/multiqc_config.yml b/assets/multiqc_config.yml
@@ -1,7 +1,7 @@
 report_comment: >
-  This report has been generated by the <a href="https://github.com/cnr-ibba/nf-resequencing-mem/releases/tag/v0.6.0" target="_blank">nf-resequencing-mem</a>
+  This report has been generated by the <a href="https://github.com/cnr-ibba/nf-resequencing-mem/releases/tag/v0.6.1" target="_blank">nf-resequencing-mem</a>
   analysis pipeline. For information about how to interpret these results, please see the
-  <a href="https://github.com/cnr-ibba/nf-resequencing-mem/blob/v0.6.0/README.md" target="_blank">documentation</a>.
+  <a href="https://github.com/cnr-ibba/nf-resequencing-mem/blob/v0.6.1/README.md" target="_blank">documentation</a>.
 report_section_order:
   "nf-resequencing-mem-methods-description":
     order: -1000

diff --git a/bin/split_ref_by_depth.py b/bin/split_ref_by_depth.py
@@ -2,14 +2,19 @@
 # -*- coding: utf-8 -*-
 
 """
+
 This script will write a list of region from a reference file relying on
-coverage depth
+coverage depth. Is supposed to work on a single chromosome at a time, it
+requires chromosome length in order to work on sample depth with no 0 regions
 
 Author: Paolo Cozzi
-Date: 2024/03/21
+Date: 2024/04/11
 
 Usage:
-    python split_ref_by_depth.py --depth_file <depth_file>
+    python split_ref_by_depth.py --depth_file <depth_file> \
+      --chromosome <chromosome> --chromosome_length <chromosome_length> \
+      [--min_length <min_length>] [--max_coverage <max_coverage>] \
+      [--overlap_size <overlap_size>] [--verbose <verbose>] [--quiet <quiet>]
 
 Arguments:
     depth_file: The path of samtools depth output file
@@ -126,49 +131,40 @@ def append_or_extend_region(
 
 
 def split_ref_by_coverage(
-        depthfile: str, max_coverage: int, min_length: int, overlap_size: int):
+        depthfile: str, chromosome: str, chromosome_length: int,
+        max_coverage: int, min_length: int, overlap_size: int):
     with gzip.open(depthfile, "rt") as handle:
         reader = csv.reader(handle, delimiter="\t", lineterminator="\n")
         header = next(reader)
 
         # header: ["#CHROM", "POS", "Sample_1.bam", "Sample_2.bam", ...]
         logging.debug(f"Got header: {header}")
 
-        # Inizialize some variables
-        line = next(reader)
-        start_chrom = line[0]
-        start_pos = int(line[1])
-        old_pos = start_pos
-        cumulative_coverage = 0
+        # test if I have reads aligned in this file
+        try:
+            line = next(reader)
+
+        except StopIteration:
+            logging.error(
+                f"File '{depthfile}' has no coverage data"
+            )
+            return [[chromosome, 0, chromosome_length]]
 
+        # Initialize some variables
         regions = []
+        start_chrom = chromosome
+        start_pos = 1
+        old_pos = start_pos
+        cumulative_coverage = 0
 
         logging.debug(f"Starting from chrom: '{start_chrom}'")
 
         for i, line in enumerate(reader):
             chrom, pos = line[0], int(line[1])
 
             if chrom != start_chrom:
-                logging.debug(f"{i}: Got a new chromosome '{chrom}'")
-                logging.debug(
-                    f"{i}: Test for a new region with: "
-                    f"{[start_chrom, start_pos, old_pos]}"
-                    f" ({old_pos-start_pos+1} bp; "
-                    f"{cumulative_coverage:.2e} cumulative coverage)"
-                )
-
-                # add and open a new region
-                regions = append_or_extend_region(
-                    regions,
-                    [start_chrom, start_pos, old_pos],
-                    min_length,
-                    overlap_size
-                )
-
-                # reset variables
-                start_chrom = chrom
-                start_pos = pos
-                cumulative_coverage = 0
+                logging.critical(f"{i}: Got a new chromosome '{chrom}'")
+                raise Exception("This script works on a single chromosome at a time")
 
             # determine region size
             length = pos - start_pos
@@ -219,6 +215,14 @@ def split_ref_by_coverage(
                 overlap_size
             )
 
+        # check for last region end
+        last_region = regions[-1]
+        if last_region[2] < chromosome_length:
+            logging.debug(
+                f"Extending the last region: {last_region} to {chromosome_length}"
+            )
+            last_region[2] = chromosome_length
+
     return regions
 
 
@@ -232,6 +236,14 @@ def split_ref_by_coverage(
         '-d', '--depth_file', required=True,
         help="The output of samtools depth file for all samples"
     )
+    parser.add_argument(
+        '-c', '--chromosome', required=True, type=str,
+        help="The chromosome name"
+    )
+    parser.add_argument(
+        '-l', '--chromosome_length', required=True, type=int,
+        help="The length of the chromosome"
+    )
     parser.add_argument(
         "--min_length", default=100_000, type=int,
         help="minimum fragment length in bp"
@@ -263,7 +275,13 @@ def split_ref_by_coverage(
 
     # split reference by coverage depth
     regions = split_ref_by_coverage(
-        args.depth_file, args.max_coverage, args.min_length, args.overlap_size)
+        args.depth_file,
+        args.chromosome,
+        args.chromosome_length,
+        args.max_coverage,
+        args.min_length,
+        args.overlap_size
+    )
 
     logging.info(f"Number of regions: {len(regions)}")
 

diff --git a/conf/base.config b/conf/base.config
@@ -26,17 +26,17 @@ process {
     withLabel:process_single {
         cpus   = { check_max( 1                  , 'cpus'    ) }
         memory = { check_max( 4.GB * task.attempt, 'memory'  ) }
-        time   = { check_max( 4.h  * task.attempt, 'time'    ) }
+        time   = { check_max( 6.h  * task.attempt, 'time'    ) }
     }
     withLabel:process_low {
         cpus   = { check_max( 2     * task.attempt, 'cpus'    ) }
         memory = { check_max( 4.GB  * task.attempt, 'memory'  ) }
-        time   = { check_max( 4.h   * task.attempt, 'time'    ) }
+        time   = { check_max( 6.h   * task.attempt, 'time'    ) }
     }
     withLabel:process_medium {
         cpus   = { check_max( 6     * task.attempt, 'cpus'    ) }
         memory = { check_max( 12.GB * task.attempt, 'memory'  ) }
-        time   = { check_max( 8.h   * task.attempt, 'time'    ) }
+        time   = { check_max( 12.h   * task.attempt, 'time'    ) }
     }
     withLabel:process_high {
         cpus   = { check_max( 12    * task.attempt, 'cpus'    ) }

diff --git a/modules/local/freebayes_splitcram.nf b/modules/local/freebayes_splitcram.nf
@@ -23,7 +23,11 @@ process FREEBAYES_SPLITCRAM {
     def prefix = task.ext.prefix ?: "${meta.id}"
     """
     split_ref_by_depth.py \\
-        --depth_file ${depth} > ${prefix}.regions.txt
+        ${args} \\
+        --depth_file ${depth} \\
+        --chromosome ${meta.id} \\
+        --chromosome_length ${meta.length} \\
+        > ${prefix}.regions.txt
 
     cat <<-END_VERSIONS > versions.yml
     "${task.process}":

diff --git a/modules/nf-core/samtools/depth/main.nf b/modules/nf-core/samtools/depth/main.nf
diff --git a/modules/nf-core/samtools/depth/samtools-depth.diff b/modules/nf-core/samtools/depth/samtools-depth.diff
diff --git a/nextflow.config b/nextflow.config
@@ -162,7 +162,7 @@ manifest {
     description     = 'Nextflow Resequencing with BWA MEM'
     mainScript      = 'main.nf'
     nextflowVersion = '!>=23.04.0'
-    version         = '0.6.0'
+    version         = '0.6.1'
 }
 
 // Load modules.config for DSL2 module specific options

diff --git a/subworkflows/local/cram_freebayes_parallel/main.nf b/subworkflows/local/cram_freebayes_parallel/main.nf
@@ -18,8 +18,16 @@ workflow CRAM_FREEBAYES_PARALLEL {
     main:
     ch_versions = Channel.empty()
 
-    // calculate total coverage depth for all samples
-    SAMTOOLS_DEPTH( bam, [[], []] )
+    // read chromosome list from fasta index
+    chromosome_ch = fai.map{ it -> it[1] }
+        .splitCsv(header: false, sep: '\t')
+        .map{ row -> [[id: row[0], length: row[1]], row[0]] }
+        // .view()
+
+    // calculate total coverage depth for all samples by chromosome
+    // bam is a value channel; chromosome_ch is a queue channel
+    // for every chromosome
+    SAMTOOLS_DEPTH( bam, bai, chromosome_ch )
     ch_versions = ch_versions.mix(SAMTOOLS_DEPTH.out.versions)
 
     // split fasta in chunks relying BAM size
@@ -33,7 +41,15 @@ workflow CRAM_FREEBAYES_PARALLEL {
         .map{ it -> [[id: it.trim()], it.trim()]}
 
     // call freebayes on each region
-    FREEBAYES_CHUNK ( regions_ch, bam, bai, SAMTOOLS_DEPTH.out.bam_list, fasta, fai )
+    FREEBAYES_CHUNK (
+        regions_ch,
+        bam,
+        bai,
+        // converting to a value channel
+        SAMTOOLS_DEPTH.out.bam_list.first(),
+        fasta,
+        fai
+    )
     ch_versions = ch_versions.mix(FREEBAYES_CHUNK.out.versions)
 
     // merge freebayes chunks