Bam post analysis (#4)

* preseq and picard collectalignmentsummarymetrics added

* changed PICARD COLLECTALIGNMENTSUMMARYMETRICS to PICARD COLLECTMULTIPLEMETRICS and integrated the new wrapper as a temporary script

* integration of next steps with temporary use of future wrappers as scripts

* wrapper integration for collectmultiplemetrics and genomecov, rule to create an igv-file from bigWig files

* deeptools and phantompeakqualtools integration

* phantompeakqualtools, headers and multiqc integration

* draft for integration of additional plots from phantompeakqualtools data into multiqc

* Changes according view #4

* Cross-correlation plots are grouped now. Changes in the description of the plots.

* change to newer wrapper versions n all rules and code cleanup

* Changes according to view #4, temporary matplotlib dependency to multiqc added, github actions added

* github actions

* test config and test data

* changes according to PR #4

* update config

* more logs added

* lint: Mixed rules and functions in same snakefile -> moved a part of the rule_all input to common.smk, input functions for rule_all added

* undo the changes of the last commit

* moved all function from Snakefile to common.smk

* --cache flag added for github actions

* --cache flag added for github actions

* snakemake_output_cache location added

* test snakemake_output_cache location

* another test snakemake_output_cache location

* another test snakemake_output_cache location

* set cache in github actions

* fix: dependencies in pysam resulted in ContextualVersionConflict in multiqc

* test: set cache location in github actions

* removed config files in .test from gitignore

* pysam depenencies and changes for github actions

* directory for ngs-test-data added

* gitmodules

* config

* test submodules

* test submodules

* config added

* directory for snakemake output cache changed

* cache location removed

* creating directory for snakemake output cache in github actions

* test cache directory with mkdir and chmod

* code cleanup github actions

* code cleanup github actions

* conda-forge channel added to pysam env

* conda-forge channel added to pysam env

* rule phantompeak added in a script instead of a shell command via Rscript

* testing on saccharomyces cerevisiae data set with deactivated preseq_lc_extrap rule

* r-base environment added to rule phantompeak_correlation

* changed genome data back to human, added rule for downloading single chromosomes from the reference genome (to generate smaller test data)

* rule preseq_lc_extrap activated again, changed genome data back to human, added rule for downloading single chromosomes from the reference genome (to generate smaller test data)

* Add some TODOs for later

* reflect that compute_matrix requests two threads in the extra params

* also cache bwa_index, as this can take quite some time

Co-authored-by: David Laehnemann <david.laehnemann@hhu.de>

.github/workflows/main.yaml

+name: Tests
+
+on:
+  push:
+    branches:
+      - master
+  pull_request:
+    branches: [ master ]
+    branches_ignore: []
+
+jobs:
+  Linting:
+    runs-on: ubuntu-latest
+    steps:
+    - uses: actions/checkout@v1
+    - name: Lint workflow
+      uses: snakemake/snakemake-github-action@v1.9.0
+      with:
+        directory: .
+        snakefile: workflow/Snakefile
+        args: "--lint"
+        stagein: |
+          export TMPDIR=/tmp
+
+  Testing:
+    runs-on: ubuntu-latest
+    needs: Linting
+    steps:
+    - uses: actions/checkout@v1
+    - name: Checkout submodules
+      uses: textbook/git-checkout-submodule-action@2.0.0
+
+    - name: Test workflow (local test data)
+      uses: snakemake/snakemake-github-action@v1.9.0
+      with:
+        directory: .test
+        snakefile: workflow/Snakefile
+        args: "--use-conda --cache --show-failed-logs -j 10 --conda-cleanup-pkgs cache --conda-frontend mamba"
+        stagein: |
+          export TMPDIR=/tmp
+          rm -rf .test/resources .test/results
+          export SNAKEMAKE_OUTPUT_CACHE=/snakemake-cache
+          mkdir -p -m a+rw $SNAKEMAKE_OUTPUT_CACHE
+
+    - name: Test report
+      uses: snakemake/snakemake-github-action@v1.9.0
+      with:
+        directory: .test
+        snakefile: workflow/Snakefile
+        args: "--report report.zip"
+        stagein: |
+          export TMPDIR=/tmp

.gitignore

+*
+results
+results/*
+!analysis/README.md
+!config
+!config/*
+!resources
+!resources/*
+!workflow
+!workflow/**
+!.gitignore
+!.gitattributes
+!.editorconfig
+!.travis.yml
+!.test
+!.test/config/*
+!.test/ngs-test-data
+!LICENSE
+!README.md

.gitmodules

+ 
+[submodule ".test/ngs-test-data"]
+	path = .test/ngs-test-data
+	url = https://github.com/snakemake-workflows/ngs-test-data

.test/config/bamtools_filtering_rules.json

+{
+  "filters" : [
+      {
+          "id" : "paired_end",
+          "isPaired" : "true"
+      },
+
+      {
+          "id" : "mismatch",
+          "tag" : "NM:<=4"
+      },
+
+      {
+          "id" : "min_size",
+          "insertSize" : ">=-2000"
+      },
+
+      {
+          "id" : "max_size",
+          "insertSize" : "<=2000"
+      }
+  ],
+
+    "rule" : " (paired_end & mismatch & min_size & max_size) | (!paired_end & mismatch) "
+
+}

.test/config/config.yaml

+# This file should contain everything to configure the workflow on a global scale.
+# In case of sample based data, it should be complemented by a samples.tsv file that contains
+# one row per sample. It can be parsed easily via pandas.
+samples: "config/samples.tsv"
+units: "config/units.tsv"
+
+resources:
+  ref:
+    # Number of chromosomes to consider for calling.
+    # The first n entries of the FASTA will be considered.
+    n_chromosomes: 25
+    # Ensembl species name
+    species: homo_sapiens
+    # Ensembl release
+    release: 101
+    # Genome build
+    build: GRCh38
+
+params:
+  lc_extrap: True
+  # TODO: move adapter parameters into a `adapter` column in units.tsv and check for its presence via the units.schema.yaml -- this enables unit-specific adapters, e.g. when integrating multiple datasets
+  # these cutadapt parameters need to contain the required flag(s) for
+  # the type of adapter(s) to trim, i.e.:
+  # * https://cutadapt.readthedocs.io/en/stable/guide.html#adapter-types
+  #   * `-a` for 3' adapter in the forward reads
+  #   * `-g` for 5' adapter in the forward reads
+  #   * `-b` for adapters anywhere in the forward reads
+  # also, separate capitalised letter flags are required for adapters in
+  # the reverse reads of paired end sequencing:
+  # * https://cutadapt.readthedocs.io/en/stable/guide.html#trimming-paired-end-reads
+  cutadapt-se: "-g AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT"
+   # reasoning behind parameters:
+  #   * `-e 0.005`: the default cutadapt maximum error rate of `0.2` is far too high, for Illumina
+  #     data the error rate is more in the range of `0.005` and setting it accordingly should avoid
+  #     false positive adapter matches
+  #   * `--minimum-overlap 7`: the cutadapt default minimum overlap of `5` did trimming on the level
+  #     of expected adapter matches by chance
+  cutadapt-pe: "-a AGATCGGAAGAGCACACGTCTGAACTCCAGTCA -g AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT -A AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT -G AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT"
+  cutadapt-others: "-e 0.005 --overlap 7"

.test/config/samples.tsv

+sample	condition	batch_effect	control	antibody
+A	treated	batch1	D	BCATENIN
+B	untreated	batch2	D	BCATENIN
+C	treated	batch1	D	TCF4
+D	untreated	batch2		

.test/config/units.tsv

+sample	unit	fragment_len_mean	fragment_len_sd	fq1	fq2	platform
+A	1			ngs-test-data/reads/a.chr21.1.fq	ngs-test-data/reads/a.chr21.2.fq	ILLUMINA
+B	1			ngs-test-data/reads/b.chr21.1.fq	ngs-test-data/reads/b.chr21.2.fq	ILLUMINA
+B	2	300	14	ngs-test-data/reads/b.chr21.1.fq		ILLUMINA
+C	1			ngs-test-data/reads/a.chr21.1.fq	ngs-test-data/reads/a.chr21.2.fq	ILLUMINA
+D	1			ngs-test-data/reads/b.chr21.1.fq	ngs-test-data/reads/b.chr21.2.fq	ILLUMINA

ngs-test-data @ 80837752

+Subproject commit 8083775249918d187e375ae9800d81842ec3c3e9

config/config.yaml

@@ -6,10 +6,18 @@ units: "config/units.tsv"
 
 resources:
   ref:
-    # path to referece genome file in FASTA format
-    genome: "resources/ref/genome.chr21.fa"
-    
+    # Number of chromosomes to consider for calling.
+    # The first n entries of the FASTA will be considered.
+    n_chromosomes: 25
+    # Ensembl species name
+    species: homo_sapiens
+    # Ensembl release
+    release: 101
+    # Genome build
+    build: GRCh38
+
 params:
+  lc_extrap: True
   # these cutadapt parameters need to contain the required flag(s) for
   # the type of adapter(s) to trim, i.e.:
   # * https://cutadapt.readthedocs.io/en/stable/guide.html#adapter-types

config/samples.tsv

-sample	condition	batch_effect
-A	treated	batch1
-B	untreated	batch1
-C	treated	batch2
-D	untreated	batch2
+sample	condition	batch_effect	control	antibody
+A	treated	batch1	D	BCATENIN
+B	untreated	batch2	D	BCATENIN
+C	treated	batch1	D	TCF4
+D	untreated	batch2		

config/units.tsv

 sample	unit	fragment_len_mean	fragment_len_sd	fq1	fq2	platform
-A	1			raw/a.chr21.1.fq	raw/a.chr21.2.fq	ILLUMINA
-B	1			raw/b.chr21.1.fq	raw/b.chr21.2.fq	ILLUMINA
-B	2	300	14	raw/b.chr21.1.fq		ILLUMINA
-C	1			raw/a.chr21.1.fq	raw/a.chr21.2.fq	ILLUMINA
-D	1			raw/b.chr21.1.fq	raw/b.chr21.2.fq	ILLUMINA
+A	1			resources/reads/a.chr21.1.fq	resources/reads/a.chr21.2.fq	ILLUMINA
+B	1			resources/reads/b.chr21.1.fq	resources/reads/b.chr21.2.fq	ILLUMINA
+B	2	300	14	resources/reads/b.chr21.1.fq		ILLUMINA
+C	1			resources/reads/a.chr21.1.fq	resources/reads/a.chr21.2.fq	ILLUMINA
+D	1			resources/reads/b.chr21.1.fq	resources/reads/b.chr21.2.fq	ILLUMINA

workflow/Snakefile

@@ -2,57 +2,14 @@
 # After configuring, running snakemake -n in a clone of this repository should successfully execute a dry-run of the workflow.
 
 include: "rules/common.smk"
+include: "rules/ref.smk"
 include: "rules/qc.smk"
 include: "rules/cutadapt.smk"
 include: "rules/mapping.smk"
 include: "rules/filtering.smk"
 include: "rules/stats.smk"
 include: "rules/utils.smk"
-
-def all_input(wildcards):
-
-    wanted_input = []
-
-    # QC with fastQC and multiQC
-    wanted_input.extend(["results/qc/multiqc/multiqc.html"])
-
-    # trimming reads
-    for (sample, unit) in units.index:
-        if is_single_end(sample, unit):
-            wanted_input.extend(expand(
-                    [
-                        "results/trimmed/{sample}-{unit}.fastq.gz",
-                        "results/trimmed/{sample}-{unit}.se.qc.txt"
-                    ],
-                    sample = sample,
-                    unit = unit
-                )
-            )
-        else:
-            wanted_input.extend(
-                expand (
-                    [
-                        "results/trimmed/{sample}-{unit}.1.fastq.gz",
-                        "results/trimmed/{sample}-{unit}.2.fastq.gz",
-                        "results/trimmed/{sample}-{unit}.pe.qc.txt"
-                    ],
-                    sample = sample,
-                    unit = unit
-            )
-        )
-
-    # mapping, merging and filtering bam-files
-    for sample in samples.index:
-        wanted_input.extend(
-            expand (
-                [
-                    "results/orphan_rm_sorted/{sample}.bam"
-                ],
-                sample = sample
-            )
-        )
-
-    return wanted_input
+include: "rules/post-analysis.smk"
 
 rule all:
     input: all_input

workflow/envs/curl.yaml

+channels:
+  - conda-forge
+dependencies:
+  - curl ==7.71

workflow/envs/gawk.yaml

+channels:
+  - conda-forge
+  - defaults
+dependencies:
+  - gawk ==5.1.0

workflow/envs/perl.yaml

+channels:
+  - bioconda
+  - defaults
+dependencies:
+  - perl-getopt-long ==2.50

workflow/envs/phantom_corr.yaml

+channels:
+  - conda-forge
+dependencies:
+  - r-base ==4.0

workflow/envs/phantompeakqualtools.yaml

+channels:
+  - conda-forge
+  - bioconda
+dependencies:
+  - phantompeakqualtools ==1.2.2
+  - samtools ==1.10
+  - r-snow ==0.4_3
+  - r-snowfall ==1.84_6.1
+  - r-bitops ==1.0_6
+  - r-catools ==1.18.0
+  - bioconductor-rsamtools ==2.4.0

workflow/envs/pysam.yaml

 channels:
+  - conda-forge
   - bioconda
+  - defaults
 dependencies:
-  - pysam ==0.15.4
+  - python ==3.7
+  - pysam ==0.16
+

workflow/header/nsc_header.txt

+# parent_id: spp
+# parent_name: 'ChIP-seq processing pipeline'
+# id: nsc_coefficient
+# section_name: 'Normalized strand cross-correlation coefficient (NSC)'
+# description: "The normalized strand cross-correlation coefficient is a quality control metric based on strand-shift cross-correlation plot.
+# The barplot is generated using run_spp.R script from
+# <a href='https://github.com/kundajelab/phantompeakqualtools' target='_blank'>phantompeakqualtools</a> and shows for each sample on y-axis the
+# normalized strand cross-correlation coefficient on x-axis.
+# The coefficient is defined by the ratio of maximal cross-correlation value divided by the background cross-correlation.
+# The higher NSC values are the better the enrichment.
+# For more information about calculation and interpretation of this value please see
+# <a href='https://hbctraining.github.io/In-depth-NGS-Data-Analysis-Course/sessionV/lessons/CC_metrics_extra.html' target='_blank'>Quality Metrics Based on Cross-Correlation</a>."
+# format: 'tsv'
+# plot_type: 'bargraph'
+# pconfig:
+#    title: 'Normalized strand cross-correlation coefficient'
+#    ylab: 'NSC coefficient'
+#    ymin: 1
+#    tt_decimals: 1

workflow/header/rsc_header.txt

+# parent_id: spp
+# parent_name: 'ChIP-seq processing pipeline'
+# id: rsc_coefficient
+# section_name: 'Relative strand cross-correlation coefficient (RSC)'
+# description: "The relative strand cross-correlation coefficient is a quality control metric based on strand-shift cross-correlation plot.
+# The barplot is generated using run_spp.R script from
+# <a href='https://github.com/kundajelab/phantompeakqualtools' target='_blank'>phantompeakqualtools</a> and shows for each sample on y-axis the
+# relative strand cross-correlation coefficient on x-axis.
+# The coefficient is defined by the ratio of maximal cross-correlation value divided by the phantom peak cross-correlation value after removal
+# of the background cross-correlation for both values.
+# RSC values >1 indicates a high enrichment.
+# For more information about calculation and interpretation of this value please see
+# <a href='https://hbctraining.github.io/In-depth-NGS-Data-Analysis-Course/sessionV/lessons/CC_metrics_extra.html' target='_blank'>Quality Metrics Based on Cross-Correlation</a>."
+# format: 'tsv'
+# plot_type: 'bargraph'
+# pconfig:
+#    title: 'Relative strand cross-correlation coefficient'
+#    ylab: 'RSC coefficient'
+#    ymin: 0
+#    tt_decimals: 1