diff --git a/Run_GeVarLi.sh b/Run_GeVarLi.sh
index 95a7c631a046d4f1ca1806bab50f8eb0b113e16d..c550985e0ae1514d4c880870b1e58c97b69ee522 100755
--- a/Run_GeVarLi.sh
+++ b/Run_GeVarLi.sh
@@ -10,13 +10,13 @@
### ###
###I###R###D######U###2###3###3#######T###R###A###N###S###V###I###H###M###I####
# Name ___________________ Run_GeVarLi.sh
-# Version ________________ v.2024.10
+# Version ________________ v.2025.01
# Author _________________ Nicolas Fernandez
# Affiliation ____________ IRD_U233_TransVIHMI
# Aim ____________________ Bash script running gevarli.smk snakefile
# Date ___________________ 2021.10.12
-# Latest modifications ___ 2024.11.15 (Update Snakemake)
-# Use ____________________ 'bash ./Run_GeVarLi.sh'
+# Latest modifications ___ 2025.01.08 (Prepare for Snakedeploy)
+# Use ____________________ 'bash Run_GeVarLi.sh'
###############################################################################
### COLORS ###
@@ -31,7 +31,7 @@ nc="\033[0m" # no color
###############################################################################
### ABOUT ###
#############
-version="2024.11" # Version
+version="2025.01" # Version
workdir=$(cd "$(dirname "${BASH_SOURCE[0]}" )" && pwd) # Get working directory
sample_test="SARS-CoV-2_Omicron-BA1_Covid-Seq-Lib-on-MiSeq_250000-reads"
@@ -47,8 +47,8 @@ ${blue}Author${nc} _________________ Nicolas Fernandez
${blue}Affiliation${nc} ____________ IRD_U233_TransVIHMI
${blue}Aim${nc} ____________________ Bash script for ${red}Ge${nc}nome assembling, ${red}Var${nc}iant calling and ${red}Li${nc}neage assignation
${blue}Date${nc} ___________________ 2021.10.12
-${blue}Latest modifications${nc} ___ 2024.10.01 (Dedicated installation scripts creation)
-${blue}Use${nc} ____________________ '${ylo}./Run_GeVarLi.sh${nc}'
+${blue}Latest modifications${nc} ___ 2025.01.08 (Prepare for Snakedeploy)
+${blue}Use${nc} ____________________ '${ylo}bash Run_GeVarLi.sh${nc}'
"
@@ -158,9 +158,9 @@ ${green}#####${nc} ${red}CONDA ACTIVATION${nc} ${green}#####${nc}
${green}----------------------------${nc}
"
-echo -e "conda activate ${ylo}workflow-base_v.${version}${nc}"
-
-conda activate workflow-base_v.${version}
+workflowbase_version="2024.11"
+echo -e "conda activate ${ylo}workflow-base_v.${workflowbase_version}${nc}"
+conda activate workflow-base_v.${workflowbase_version}
###############################################################################
@@ -179,6 +179,7 @@ yq_version=$(yq --version | sed 's/yq //') # Yq version
rename_version="1.601" # Rename version (ver. 1.601 from 2024-02)
graphviz_version="11.0.0" # GraphViz version (ver. 11.0.0 from 2024-02)
#graphviz_version=$#(dot -V | sed 's/dot - graphviz version //') # GraphViz version (ver. 11.0.0 from 2024-02)
+nextclade_version=""
fastq=$(expr $(ls -l ${workdir}/resources/reads/*.fastq.gz 2> /dev/null | wc -l)) # Get fastq.gz files count
if [[ "${fastq}" == "0" ]] # If no sample,
@@ -241,9 +242,7 @@ ${blue}Samples processed${nc} ______ ${red}${samples}${nc} samples (${ylo}${fast
${blue}Snakemake version${nc} ______ ${ylo}${snakemake_version}${nc}
${blue}Conda version${nc} __________ ${ylo}${conda_version}${nc}
-${blue}Conda frontend${nc} _________ ${ylo}${conda_frontend}${nc}
${blue}Mamba version${nc} __________ ${ylo}${mamba_version}${nc}
-${blue}Nextclade version${nc} ______ ${ylo}${nextclade_version}${nc}
${blue}Quality Ccontrol${nc} _______ [ ${red}${quality}${nc} ]
${blue}Trimming${nc} _______________ [ ${red}${trimming}${nc} ]
@@ -258,6 +257,7 @@ ${blue}Reference genome${nc} _______ ${ylo}${reference}${nc}
${blue}Aligner${nc} ________________ ${ylo}${aligner}${nc}
${blue}Min coverage${nc} ___________ ${red}${min_cov}${nc} X
${blue}Min allele frequency${nc} ___ ${red}${min_freq}${nc}
+
${blue}Nextclade dataset${nc} ______ ${red}${nextclade_dataset}${nc}
"
@@ -324,22 +324,26 @@ fi
###############################################################################
-### RENAME SAMPLES ###
-######################
+### RENAME SYMLINKS ###
+#######################
echo -e "
${green}------------------------------------------------------------------------${nc}
-${green}#####${nc} ${red}RENAME FASTQ FILES${nc} ${green}#####${nc}
-${green}------------------------------${nc}
+${green}#####${nc} ${red}RENAME FASTQ SYMLINKS${nc} ${green}#####${nc}
+${green}---------------------------------${nc}
"
-# Rename fastq files to remove "_001" Illumina pattern (mandatory)
-## De/comment line (#) if you want keep Illumina barcode-ID and/or Illumina line-ID
-echo -e "Removing ${red}'_S'${nc} index tag ID"
-rename "s/_S\d+_/_/" ${workdir}/resources/reads/*.fastq.gz 2> /dev/null # Remove barcode-ID like {_S001_}
-echo -e "Removing ${red}'_L'${nc} line tag ID"
-rename "s/_L\d+_/_/" ${workdir}/resources/reads/*.fastq.gz 2> /dev/null # Remove line-ID ID like {_L001_}
-echo -e "Removing ${red}'_001'${nc} illumina tag ID"
-rename "s/_001.fastq.gz/.fastq.gz/" ${workdir}/resources/reads/*.fastq.gz 2> /dev/null # Remove end-name ID like {_001}.fastq.gz
+# Remove tags from symlinks:
+## barcode-ID like {_S001_}
+## line-ID ID like {_L001_}
+## end-name ID like {_001}.fastq.gz
+mkdir -p ${workdir}/resources/symlinks/
+for fastq in ${workdir}/resources/reads/*.fastq.gz; do
+ symlinks=$(echo $(basename "${fastq}") | \
+ sed -E "s/_S\d+_//" | \
+ sed -E "s/_L\d+_//" | \
+ sed -E "s/_001.fastq.gz/.fastq.gz/")
+ ln -s "${fastq}" "${workdir}/resources/symlinks/${symlinks}"
+done
echo -e "
If you want to keep Illumina ${blue}barcode-ID${nc} and/or Illumina ${blue}line-ID${nc}, please edit ${ylo}Run_GeVarLi.sh${nc} script (l.335).
@@ -355,9 +359,6 @@ ${green}#####${nc} ${red}SNAKEMAKE PIPELINES${nc} ${green}#####${nc}
${green}-------------------------------${nc}
"
-# MODULES
-snakefiles_list="indexing_genomes gevarli"
-
echo -e "
${blue}## Unlocking Working Directory ##${nc}
${blue}---------------------------------${nc}
@@ -367,14 +368,11 @@ ${blue}---------------------------------${nc}
# Re-run all jobs the output of which is recognized as incomplete.
# Remove a lock on the working directory.
-for snakefile in ${snakefiles_list} ; do
- echo -e "${blue}-- ${snakefile} --${nc}" ;
- snakemake \
- --directory ${workdir}/ \
- --snakefile ${workdir}/workflow/snakefiles/${snakefile}.smk \
- --rerun-incomplete \
- --unlock ;
-done
+snakemake \
+ --directory ${workdir}/ \
+ --snakefile ${workdir}/workflow/Snakefile \
+ --rerun-incomplete \
+ --unlock
echo -e "
${blue}## Let's Run! ##${nc}
@@ -390,22 +388,17 @@ ${blue}----------------${nc}
# If defined in the rule, run job in a conda environment.
# Print out the shell commands that will be executed.
-for snakefile in ${snakefiles_list} ; do
- echo -e "${blue}-- ${snakefile} --${nc}" ;
- snakemake \
- --directory ${workdir}/ \
- --snakefile ${workdir}/workflow/snakefiles/${snakefile}.smk \
- --cores ${max_threads} \
- --max-threads ${max_threads} \
- --resources mem_gb=${max_memory} \
- --rerun-incomplete \
- --keep-going \
- --use-conda ;
-# --quiet host \
-# --quiet progress \
-# --quiet rules ;
-done
-
+snakemake \
+ --directory ${workdir}/ \
+ --snakefile ${workdir}/workflow/Snakefile\
+ --cores ${max_threads} \
+ --max-threads ${max_threads} \
+ --resources mem_gb=${max_memory} \
+ --rerun-incomplete \
+ --keep-going \
+ --use-conda \
+ --quiet host
+# Possible choices: all, host, progress, rules
###############################################################################
### CONCATENATE RESULTS ###
@@ -477,27 +470,23 @@ mkdir -p ${workdir}/results/10_Reports/files-summaries/ 2> /dev/null
graph_list="dag rulegraph filegraph"
extention_list="pdf png"
-for snakefile in ${snakefiles_list} ; do
- for graph in ${graph_list} ; do
- for extention in ${extention_list} ; do
- snakemake \
- --directory ${workdir}/ \
- --snakefile ${workdir}/workflow/snakefiles/${snakefile}.smk \
- --${graph} \
- | dot -T${extention} \
- 2> /dev/null \
- 1> ${workdir}/results/10_Reports/graphs/${snakefile}_${graph}.${extention} ;
- done ;
+for graph in ${graph_list} ; do
+ for extention in ${extention_list} ; do
+ snakemake \
+ --directory ${workdir}/ \
+ --snakefile ${workdir}/workflow/Snakefile \
+ --${graph} \
+ | dot -T${extention} \
+ 2> /dev/null \
+ 1> ${workdir}/results/10_Reports/graphs/${graph}.${extention} ;
done ;
done
-for snakefile in ${snakefiles_list} ; do
- snakemake \
- --directory ${workdir} \
- --snakefile ${workdir}/workflow/snakefiles/${snakefile}.smk \
- --summary > ${workdir}/results/10_Reports/files-summaries/${snakefile}_files-summary.txt \
- 2> /dev/null ;
-done
+snakemake \
+ --directory ${workdir} \
+ --snakefile ${workdir}/workflow/Snakefile \
+ --summary > ${workdir}/results/10_Reports/files-summaries/files-summary.txt \
+ 2> /dev/null
cp ${config_file} ${workdir}/results/10_Reports/config.log 2> /dev/null
@@ -512,14 +501,15 @@ ${green}------------------------${nc}
"
# Save and deactive environments
-mkdir -p ${workdir}/results/10_Reports/conda_env/ 2> /dev/null
-cp ${workdir}/workflow/environments/*.yaml ${workdir}/results/10_Reports/conda_env/
+mkdir -p ${workdir}/results/10_Reports/conda_envs/ 2> /dev/null
+cp ${workdir}/workflow/envs/*.yaml ${workdir}/results/10_Reports/conda_envs/
conda deactivate
# Cleanup
find ${workdir}/results/ -type f -empty -delete # Remove empty file (like empty log)
find ${workdir}/results/ -type d -empty -delete # Remove empty directory
rm -f ${workdir}/resources/reads/${sample_test}_R*.fastq.gz 2> /dev/null
+rm -rf ${workdir}/resources/symlinks/ 2> /dev/null
# Timer
time_stamp_end=$(date +"%Y-%m-%d %H:%M") # Get date / hour ending analyzes
@@ -542,8 +532,8 @@ Author _________________ Nicolas Fernandez
Affiliation ____________ IRD_U233_TransVIHMI
Aim ____________________ Bash script for GeVarLi
Date ___________________ 2021.10.12
-Latest modifications ___ 2024.10.01 (Dedicated installation scripts creation)
-Run ____________________ ./Run_GeVarLi.sh
+Latest modifications ___ 2025.01.08 (Prepare for Snakedeploy)
+Run ____________________ 'bash Run_GeVarLi.sh'
Operating System _______ ${os}
Shell __________________ ${shell}
@@ -567,9 +557,8 @@ Samples processed _______ ${samples} samples (${ylo}${fastq} fastq files)
Snakemake version _______ ${snakemake_version}
Conda version ___________ ${conda_version}
-Conda frontend __________ ${conda_frontend}
Mamba version ___________ ${mamba_version}
-Nextclade version _______ ${nextclade_version}
+
Quality Ccontrol ________ [ ${quality} ]
Trimming ________________ [ ${trimming} ]
@@ -584,16 +573,17 @@ Reference genome ________ ${reference}
Aligner _________________ ${aligner}
Min coverage ____________ ${min_cov} X
Min allele frequency ____ ${min_freq}
+
Nextclade dataset _______ ${nextclade_dataset}
" > ${workdir}/results/10_Reports/settings.log
# Gzip reports directory
cd ${workdir}/results/
tar -zcf 10_Reports_archive.tar.gz 10_Reports
+cd ${workdir}
# Gzip results directory
#mkdir -p ${workdir}/archives/ 2> /dev/null
-#cd ${workdir}/
#tar -zcf archives/Results_${time_stamp_archive}_${reference}_${aligner}-${min_cov}X_${samples}sp_archive.tar.gz results/
echo -e "
diff --git a/configuration/config.yaml b/configuration/config.yaml
index c636b9de6e4c6510e0ddc2c0f228aa076cae5ac4..6b1b4c69752d555ef9a78c8da9819b2444579f74 100755
--- a/configuration/config.yaml
+++ b/configuration/config.yaml
@@ -36,12 +36,12 @@ resources:
###############################################################################
modules: # GeVarLi modules analysis
# Available options, set at least one:
- - 'quality' # Perform reads quality control (FastQC, Fastq-Screen, MultiQC) default: ON
+ #- 'quality' # Perform reads quality control (FastQC, Fastq-Screen, MultiQC) default: ON
#- 'trimming' # Keep trimmed reads files after alignment (Cutadapt / Sickle-trim) default: OFF
#- 'cleapping' # Perform reads clipping (Bamclipper) default: OFF
- 'consensus' # Perform reads mapping to reference (BWA, Bowtie2, Minimap2) default: ON
- - 'covstats' # Perform coverage statistics (Fagstat, Covstats) default: ON
- - 'nextclade' # Perform nextclade clade assignation (Nexclade) default: ON
+ #- 'covstats' # Perform coverage statistics (Fagstat, Covstats) default: ON
+ #- 'nextclade' # Perform nextclade clade assignation (Nexclade) default: ON
#- 'pangolin' # Perform pangolin lineage assignation (Pangolin) default: OFF
###- 'gisaid' # Perform gisaid submission (GisAid : TODO) default: OFF
@@ -51,7 +51,7 @@ consensus:
reference: # Your reference, in fasta format (default: "SARS-CoV-2_Wuhan_MN-908947-3")
# Available options (not exhaustive), set one:
- 'SARS-CoV-2_Wuhan_MN-908947-3' # SARS-CoV-2 (Nextclade and Pangolin)
- #- 'Monkeypox-virus_Zaire_AF-380138-1' # Monkeypox (Nextclade and Pangolin)
+ - 'Monkeypox-virus_Zaire_AF-380138-1' # Monkeypox (Nextclade and Pangolin)
#- 'Monkeypox-virus_UK_MT-903345-1' # Monkeypox (Nextclade and Pangolin)
#- 'Swinepox-virus_India_MW-036632-1' # Swinepox (Nextclade)
#- 'Ebola-virus_Zaire_AF-272001-1' # Ebola (na)
@@ -96,8 +96,8 @@ consensus:
aligner: # Select your favorite aligner (default: 'bwa')
# Available options, set one:
- 'bwa' # Better, Faster, (Stronger, Harder...)
- #- 'bowtie2' # Depreciated (slower),"sensitivity" requiried (see below "Bowtie2" options)
- #- 'minimap2' # A versatile sequence alignment program
+ - 'bowtie2' # Depreciated (slower),"sensitivity" requiried (see below "Bowtie2" options)
+ - 'minimap2' # A versatile sequence alignment program
caller: 'ivar'
###############################################################################
@@ -275,24 +275,24 @@ fastq_screen:
###############################################################################
conda:
- yaml: # Conda environement yaml files:
- bamclipper: '../environments/bamclipper_v.1.0.0.yaml' # Bamclipper ver. 1.0.0
- bcftools: '../environments/bcftools_v.1.20.yaml' # Bcftools ver. 1.20
- bedtools: '../environments/bedtools_v.2.31.1.yaml' # Bedtools ver. 2.31.1
- bowtie2: '../environments/bowtie2_v.2.5.4.yaml' # Bowtie2 ver. 2.5.4
- bwa: '../environments/bwa_v.0.7.18.yaml' # BWA ver. 0.7.18
- cutadapt: '../environments/cutadapt_v.4.9.yaml' # Cutadapt ver. 4.9
- fastq_screen: '../environments/fastq-screen_v.0.15.3.yaml' # Fastq-Screen ver. 0.15.3 (with BWA ver. 0.7.18)
- fastqc: '../environments/fastqc_v.0.12.1.yaml' # FastQC ver. 0.12.1
- gawk: '../environments/gawk_v.5.3.0.yaml' # Awk (GNU) ver. 5.3.0
- ivar: '../environments/ivar_v.1.4.3.yaml' # iVar ver. 1.4.3 (with Samtools ver. 1.20)
- minimap2: '../environments/minimap2_v.2.28.yaml' # Minimap2 ver. 2.28
- multiqc: '../environments/multiqc_v.1.23.yaml' # MultiQC ver. 1.23 (with Pandoc ver. 3.3)
- nextclade: '../environments/nextclade_v.3.9.1.yaml' # Nextclade ver. 3.9.1
- pangolin: '../environments/pangolin_v.4.3.yaml' # Pangolin ver. 4.3
- samtools: '../environments/samtools_v.1.20.yaml' # Samtools ver. 1.20
- sickle_trim: '../environments/sickle-trim_v.1.33.yaml' # Sickle-trim ver. 1.33
- tsv2vcf: '../environments/tsv2vcf_v.1.1.yaml' # tsv2vcf ver. 1.1 (bcftools biopython numpy)
- workflow_base: '../environments/workflow-base_v.2024.11.yaml' # Workflow-Base ver. 2024.11
+ envs: # Conda environement yaml files:
+ bamclipper: 'envs/bamclipper_v.1.0.0.yaml' # Bamclipper ver. 1.0.0
+ bcftools: 'envs/bcftools_v.1.20.yaml' # Bcftools ver. 1.20
+ bedtools: 'envs/bedtools_v.2.31.1.yaml' # Bedtools ver. 2.31.1
+ bowtie2: 'envs/bowtie2_v.2.5.4.yaml' # Bowtie2 ver. 2.5.4
+ bwa: 'envs/bwa_v.0.7.18.yaml' # BWA ver. 0.7.18
+ cutadapt: 'envs/cutadapt_v.4.9.yaml' # Cutadapt ver. 4.9
+ fastq_screen: 'envs/fastq-screen_v.0.15.3.yaml' # Fastq-Screen ver. 0.15.3 (with BWA ver. 0.7.18)
+ fastqc: 'envs/fastqc_v.0.12.1.yaml' # FastQC ver. 0.12.1
+ gawk: 'envs/gawk_v.5.3.0.yaml' # Awk (GNU) ver. 5.3.0
+ ivar: 'envs/ivar_v.1.4.3.yaml' # iVar ver. 1.4.3 (with Samtools ver. 1.20)
+ minimap2: 'envs/minimap2_v.2.28.yaml' # Minimap2 ver. 2.28
+ multiqc: 'envs/multiqc_v.1.23.yaml' # MultiQC ver. 1.23 (with Pandoc ver. 3.3)
+ nextclade: 'envs/nextclade_v.3.9.1.yaml' # Nextclade ver. 3.9.1
+ pangolin: 'envs/pangolin_v.4.3.yaml' # Pangolin ver. 4.3
+ samtools: 'envs/samtools_v.1.20.yaml' # Samtools ver. 1.20
+ sickle_trim: 'envs/sickle-trim_v.1.33.yaml' # Sickle-trim ver. 1.33
+ tsv2vcf: 'envs/tsv2vcf_v.1.1.yaml' # tsv2vcf ver. 1.1 (bcftools biopython numpy)
+ workflow_base: 'envs/workflow-base_v.2024.11.yaml' # Workflow-Base ver. 2024.11
###############################################################################
diff --git a/version.txt b/version.txt
index 424805bac262e0487a8e9a7c654536c0a5e7e922..eb11b6f54b840a21cb459d580529691f99ac6dcd 100644
--- a/version.txt
+++ b/version.txt
@@ -1 +1 @@
-2024.11
+2025.01
diff --git a/workflow/Snakefile b/workflow/Snakefile
index 118850c0b068c9c4e289dc417375bad465b8965f..e58983186c74103324d3ba8252b639ce988a2208 100644
--- a/workflow/Snakefile
+++ b/workflow/Snakefile
@@ -1,4 +1,1454 @@
-include: "workflow/snakefiles/quality_control.smk"
-include: "workflow/snakefiles/indexing_genomes.smk"
-include: "workflow/snakefiles/gevarli.smk"
-conda_prefix: "workflow/environments/"
\ No newline at end of file
+###I###R###D######U###2###3###3#######T###R###A###N###S###V###I###H###M###I####
+### ###
+### /\ ______ ___ ____ _ _ __ ____ __ ____ ______ /\ ###
+### || \ \ \ \ / __( ___( \/ )__\ ( _ ( ) (_ _) / / / / || ###
+### || > > > > ( (_-.)__) \ /(__)\ ) /)(__ _)(_ < < < < || ###
+### || /_/_/_/ \___(____) \(__)(__(_)\_(____(____) \_\_\_\ || ###
+### \/ \/ ###
+### ###
+###I###R###D######U###2###3###3#######T###R###A###N###S###V###I###H###M###I####
+# Name ___________________ gevarli.smk
+# Version ________________ v.2025.01
+# Author _________________ Nicolas Fernandez
+# Affiliation ____________ IRD_U233_TransVIHMI
+# Aim ____________________ Snakefile with GeVarLi rules
+# Date ___________________ 2021.10.12
+# Latest modifications ___ 2025.01.08 (Prepare for Snakedeploy)
+# Use ____________________ snakemake -s Snakemake --use-conda
+
+###############################################################################
+### CONFIGURATION ###
+#####################
+
+configfile: "configuration/config.yaml"
+
+###############################################################################
+### FUNCTIONS ###
+#################
+
+def get_memory_per_thread(wildcards):
+ memory_per_thread = int(RAM) // int(CPUS)
+ if memory_per_thread < 1:
+ memory_per_thread = 1
+ return memory_per_thread
+
+def get_quality_input(wildcards):
+ quality_list = []
+ if "quality" in MODULES:
+ quality_list = "results/00_Quality_Control/multiqc/"
+ return quality_list
+
+def get_trimming_input(wildcards):
+ trimming_list = []
+ if "trimming" in MODULES:
+ trimming_list = expand("results/01_Trimming/sickle/{sample}_sickle-trimmed_SE.fastq.gz",
+ sample = SAMPLE)
+ return trimming_list
+
+def stash_or_trash(path):
+ if "trimming" in MODULES:
+ return path
+ else:
+ return temp(path)
+
+def get_cleapping_input(wildcards):
+ cleapping_list = []
+ if "cleapping" in MODULES:
+ cleapping_list = ""
+ return cleapping_list
+
+def get_bam_input(wildcards):
+ markdup = "results/02_Mapping/{reference}/{sample}_{aligner}_mark-dup.bam"
+ if "cleapping" in MODULES:
+ markdup = "results/02_Mapping/{reference}/{sample}_{aligner}_mark-dup.primerclipped.bam"
+ return markdup
+
+def get_bai_input(wildcards):
+ index = "results/02_Mapping/{reference}/{sample}_{aligner}_mark-dup.bam.bai"
+ if "cleapping" in MODULES:
+ index = "results/02_Mapping/{reference}/{sample}_{aligner}_mark-dup.primerclipped.bam.bai"
+ return index
+
+def get_flagstat_input(wildcards):
+ flagstat_list = []
+ if "covstats" in MODULES:
+ flagstat_list = expand(
+ "results/03_Coverage/{reference}/flagstat/{sample}_{aligner}_flagstat.{ext}",
+ reference = REFERENCE,
+ sample = SAMPLE,
+ aligner = ALIGNER,
+ ext = ["txt", "tsv", "json"]
+ )
+ return flagstat_list
+
+def get_covstats_input(wildcards):
+ covstats_list = []
+ if "covstats" in MODULES:
+ covstats_list = expand(
+ "results/03_Coverage/{reference}/{sample}_{aligner}_{min_cov}X_coverage-stats.tsv",
+ reference = REFERENCE,
+ sample = SAMPLE,
+ aligner = ALIGNER,
+ min_cov = MIN_COV
+ )
+ return covstats_list
+
+def get_consensus_input(wildcards):
+ consensus_list = []
+ if "consensus" in MODULES:
+ consensus_list = expand(
+ "results/05_Consensus/{reference}/{sample}_{aligner}_{min_cov}X_{caller}_consensus.fasta",
+ reference = REFERENCE,
+ sample = SAMPLE,
+ aligner = ALIGNER,
+ min_cov = MIN_COV,
+ caller = CALLER
+ )
+ return consensus_list
+
+def get_vcf_input(wildcards):
+ vcf_list = []
+ if "consensus" in MODULES:
+ vcf_list = expand(
+ "results/04_Variants/{reference}/{sample}_{aligner}_{min_cov}X_{caller}_variant-filt.vcf",
+ reference = REFERENCE,
+ sample = SAMPLE,
+ aligner = ALIGNER,
+ min_cov = MIN_COV,
+ caller = CALLER
+ )
+ return vcf_list
+
+def get_pangolin_input(wildcards):
+ pangolin_list = []
+ if "pangolin" in MODULES:
+ pangolin_list = expand(
+ "results/06_Lineages/{reference}/{sample}_{aligner}_{min_cov}X_{caller}_pangolin-report.csv",
+ reference = REFERENCE,
+ sample = SAMPLE,
+ aligner = ALIGNER,
+ min_cov = MIN_COV,
+ caller = CALLER
+ )
+ return pangolin_list
+
+def get_nextclade_input(wildcards):
+ nextclade_list = []
+ if "nextclade" in MODULES:
+ nextclade_list = expand(
+ "results/06_Lineages/{reference}/{sample}_{aligner}_{min_cov}X_{caller}_nextclade-report.tsv",
+ reference = REFERENCE,
+ sample = SAMPLE,
+ aligner = ALIGNER,
+ min_cov = MIN_COV,
+ caller = CALLER
+ )
+ return nextclade_list
+
+def get_gisaid_input(wildcards):
+ gisaid_list = []
+ if "gisaid" in MODULES:
+ gisaid_list = expand(
+ ""
+ )
+ return gisaid_list
+
+###############################################################################
+### WILDCARDS ###
+#################
+
+FASTQ, = glob_wildcards("resources/symlinks/{fastq}.fastq.gz")
+SAMPLE, = glob_wildcards("resources/symlinks/{sample}_R1.fastq.gz")
+
+###############################################################################
+### RESOURCES ###
+#################
+
+CPUS = config["resources"]["cpus"] # Threads (maximum)
+RAM = config["resources"]["ram"] # Memory (RAM) in Gb (maximum)
+MEM_GB = get_memory_per_thread # Memory per thread in GB (maximum)
+TMP_DIR = config["resources"]["tmp_dir"] # Temporary directory
+
+###############################################################################
+### ENVIRONMENTS ###
+####################
+
+MULTIQC = config["conda"]["envs"]["multiqc"] # Multi-QC conda env
+FASTQ_SCREEN = config["conda"]["envs"]["fastq_screen"] # Fastq-Screen conda env
+FASTQC= config["conda"]["envs"]["fastqc"] # FastQC conda env
+CUTADAPT = config["conda"]["envs"]["cutadapt"] # Cutadapt conda environment
+SICKLE_TRIM = config["conda"]["envs"]["sickle_trim"] # Sickle-Trim conda environment
+MINIMAP2 = config["conda"]["envs"]["minimap2"] # BWA conda environment
+BWA = config["conda"]["envs"]["bwa"] # BWA conda environment
+BOWTIE2 = config["conda"]["envs"]["bowtie2"] # Bowtie2 conda environment
+SAMTOOLS = config["conda"]["envs"]["samtools"] # SamTools conda environment
+BEDTOOLS = config["conda"]["envs"]["bedtools"] # BedTools conda environment
+BAMCLIPPER = config["conda"]["envs"]["bamclipper"] # BAMClipper
+GAWK = config["conda"]["envs"]["gawk"] # Awk (GNU) conda environment
+IVAR = config["conda"]["envs"]["ivar"] # iVar conda environment
+TSV2VCF = config["conda"]["envs"]["tsv2vcf"] # tsv2vcf conda environment
+BCFTOOLS = config["conda"]["envs"]["bcftools"] # BcfTools conda environment
+PANGOLIN = config["conda"]["envs"]["pangolin"] # Pangolin conda environment
+NEXTCLADE = config["conda"]["envs"]["nextclade"] # Nextclade conda environment
+
+###############################################################################
+### PARAMETERS ###
+##################
+
+MODULES = config["modules"] # Modules
+
+SUBSET = config["fastq_screen"]["subset"] # Fastq-Screen --subset
+FQC_CONFIG = config["fastq_screen"]["config"] # Fastq-Screen --conf
+#MQC_PATH = config["multiqc"]["path"] # MultiQC --conf
+#MQC_CONF = config["multiqc"]["config"] # MultiQC --conf
+#TAG = config["multiqc"]["tag"] # MultiQC --tag
+
+KMER_SIZE = config["minimap2"]["algorithm"]["k-mer_size"] # MM2 k-mer size
+MINIMIZER_SIZE = config["minimap2"]["algorithm"]["minimizer_size"] # MM2 minimizer window size
+SPLIT_SIZE = config["minimap2"]["algorithm"]["split_size"] # MM2 split index
+#HOMOPOLYMER = config["minimap2"]["algorithm"]["homopolymer"] # MM2 if PacBio
+BWA_ALGO = config["bwa"]["algorithm"] # BWA indexing algorithm
+BT2_ALGO = config["bowtie2"]["algorithm"] # BT2 indexing algorithm
+
+REFERENCE = config["consensus"]["reference"] # Genome reference sequence, in fasta format
+REF_PATH = config["consensus"]["path"] # Path to genomes references
+MIN_COV = config["consensus"]["min_cov"] # Minimum coverage, mask lower regions with 'N'
+IUPAC = config["consensus"]["iupac"] # Output variants in the form of IUPAC ambiguity codes
+ALIGNER = config["consensus"]["aligner"] # Aligner ('minimap2', 'bwa' or 'bowtie2')
+CALLER = config["consensus"]["caller"] # Variant Caller ('lofreq' or 'ivar')
+
+CUT_LENGTH = config["cutadapt"]["length"] # Cutadapt --minimum-length
+CUT_TRUSEQ = config["cutadapt"]["kits"]["truseq"] # Cutadapt --adapter Illumina TruSeq
+CUT_NEXTERA = config["cutadapt"]["kits"]["nextera"] # Cutadapt --adapter Illumina Nextera
+CUT_SMALL = config["cutadapt"]["kits"]["small"] # Cutadapt --adapter Illumina Small
+CUT_CLIPPING = config["cutadapt"]["clipping"] # Cutadapt --cut
+
+SIC_COMMAND = config["sickle_trim"]["command"] # Sickle-trim command
+SIC_ENCODING = config["sickle_trim"]["encoding"] # Sickle-trim --qual-type
+SIC_QUALITY = config["sickle_trim"]["quality"] # Sickle-trim --qual-threshold
+SIC_LENGTH = config["sickle_trim"]["length"] # Sickle-trim --length-treshold
+
+MM2_PATH = config["minimap2"]["path"] # Minimpa2 path to indexes
+MM2_PRESET = config["minimap2"]["preset"] # Minimap2 preset
+#MM2_LENGTH = config["minimap2"]["length"] # Minimap2 length
+#MM2_KMER_SIZE = config["minimap2"]["algorithm"]["k-mer_size"] # Minimap2 k-mer size
+#MM2_MINIMIZER_SIZE = config["minimap2"]["algorithm"]["minimizer_size"] # Minimap2 minimizer window size
+#MM2_SPLIT_SIZE = config["minimap2"]["algorithm"]["split_size"] # Minimap2 split index
+#MM2_HOMOPOLYMER = config["minimap2"]["algorithm"]["homopolymer"] # Minimap2 for PacBio
+
+BWA_PATH = config["bwa"]["path"] # BWA path to indexes
+BWA_ALGO = config["bwa"]["algorithm"] # BWA indexing algorithm
+
+BT2_PATH = config["bowtie2"]["path"] # Bowtie2 path to indexes
+BT2_ALGO = config["bowtie2"]["algorithm"] # Bowtie2 indexing algorithm
+BT2_SENSITIVITY = config["bowtie2"]["sensitivity"] # Bowtie2 sensitivity preset
+
+CLIPPATH = config["bamclipper"]["path"] # Bamclipper path to primers
+PRIMERS = config["bamclipper"]["primers"] # Bamclipper primers bed files
+UPSTREAM = config["bamclipper"]["upstream"] # Bamclipper upstream nucleotides
+DOWNSTREAM = config["bamclipper"]["downstream"] # Bamclipper downstream nucleotides
+
+IVAR_MIN_DEPTH = config["consensus"]["min_cov"] # iVar
+IVAR_MIN_FREQ = config["consensus"]["min_freq"] # iVar minimum allele frequency allowed
+IVAR_MIN_INSERT = config["consensus"]["min_freq"] # iVar minimum insertion frequency allowed
+#IVAR_MIN_DEPTH = config["ivar"]["min_depth"] # iVar
+#IVAR_MIN_FREQ = config["ivar"]["min_freq"] # iVar minimum allele frequency allowed
+#IVAR_MIN_INSERT = config["ivar"]["min_insert"] # iVar minimum insertion frequency allowed
+IVAR_MAX_DEPTH = config["ivar"]["max_depth"] # iVar
+IVAR_MIN_BQ = config["ivar"]["min_bq"] # iVar
+IVAR_MIN_QUAL = config["ivar"]["min_qual"] # iVar
+IVAR_MAP_QUAL = config["ivar"]["map_qual"] # iVar mapping quality
+
+NEXT_PATH = config["nextclade"]["path"] # Path to nextclade dataset
+NEXT_DATASET = config["nextclade"]["dataset"] # Nextclade dataset
+
+###############################################################################
+### RULES ###
+#############
+rule all:
+ input:
+ multiqc = get_quality_input,
+ trimming = get_trimming_input,
+ consensus = get_consensus_input,
+ flagstat = get_flagstat_input,
+ covstats = get_covstats_input,
+ vcf = get_vcf_input,
+ pangolin = get_pangolin_input,
+ nextclade = get_nextclade_input,
+ gisaid = get_gisaid_input
+
+
+###############################################################################
+################################## LINEAGES ###################################
+###############################################################################
+
+###############################################################################
+rule nextclade_lineage:
+ # Aim: nextclade lineage assignation
+ # Use: nextclade [QUERY.fasta] -t [THREADS] --outfile [NAME.csv]
+ message:
+ """
+ ~ Nextclade ∞ Assign Lineage ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ {wildcards.aligner}
+ Min. cov.: _______ {wildcards.min_cov}X
+ Variant caller: __ {wildcards.caller}
+ """
+ conda:
+ NEXTCLADE
+ resources:
+ cpus = CPUS
+ params:
+ path = NEXT_PATH,
+ dataset = NEXT_DATASET
+ input:
+ consensus = "results/05_Consensus/{reference}/{sample}_{aligner}_{min_cov}X_{caller}_consensus.fasta"
+ output:
+ lineage = "results/06_Lineages/{reference}/{sample}_{aligner}_{min_cov}X_{caller}_nextclade-report.tsv",
+ alignment = directory("results/06_Lineages/{reference}/{sample}_{aligner}_{min_cov}X_{caller}_nextclade-all/")
+ log:
+ "results/10_Reports/tools-log/nextclade/{reference}/{sample}_{aligner}_{min_cov}X_{caller}_lineage.log"
+ shell:
+ "nextclade " # Nextclade, assign queries sequences to clades and reports potential quality issues
+ "run " # Run analyzis
+ "--jobs {resources.cpus} " # -j: Number of CPU threads used by the algorithm (default: all available threads)
+ "--input-dataset {params.path}{params.dataset} " # -raq: Path to a directory containing a dataset (root-seq, tree and qc-config required)
+ "--output-tsv {output.lineage} " # -t: Path to output TSV results file
+ "--output-all {output.alignment} " # -O: Produce all of the output files into this directory, using default basename
+ "{input.consensus} " # Path to a .fasta file with input sequences
+ "&> {log}" # Log redirection
+
+###############################################################################
+rule pangolin_lineage:
+ # Aim: lineage mapping
+ # Use: pangolin [QUERY.fasta] -t [THREADS] --outfile [NAME.csv]
+ message:
+ """
+ ~ Pangolin ∞ Assign Lineage ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ {wildcards.aligner}
+ Min. cov.: _______ {wildcards.min_cov}X
+ Variant caller: __ {wildcards.caller}
+ """
+ conda:
+ PANGOLIN
+ resources:
+ cpus = CPUS,
+ tmp_dir = TMP_DIR
+ input:
+ consensus = "results/05_Consensus/{reference}/{sample}_{aligner}_{min_cov}X_{caller}_consensus.fasta"
+ output:
+ lineage = "results/06_Lineages/{reference}/{sample}_{aligner}_{min_cov}X_{caller}_pangolin-report.csv"
+ log:
+ "results/10_Reports/tools-log/pangolin/{reference}/{sample}_{aligner}_{min_cov}X_{caller}_lineage.log"
+ shell:
+ "pangolin " # Pangolinn, Phylogenetic Assignment of Named Global Outbreak LINeages
+ "{input.consensus} " # Query fasta file of sequences to analyse
+ "--threads {resources.cpus} " # -t: Number of threads
+ "--tempdir {resources.tmp_dir} " # Specify where you want the temp stuff to go (default: $TMPDIR)
+ "--outfile {output.lineage} " # Optional output file name (default: lineage_report.csv)
+ "&> {log}" # Log redirection
+
+###############################################################################
+################################## CONSENSUS ##################################
+###############################################################################
+
+###############################################################################
+rule sed_rename_headers:
+ # Aim: rename all fasta header with sample name
+ # Use: sed 's/[OLD]/[NEW]/' [IN] > [OUT]
+ message:
+ """
+ ~ Sed ∞ Rename Fasta Header ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ {wildcards.aligner}
+ Min. cov.: _______ {wildcards.min_cov}X
+ Variant caller: __ {wildcards.caller}
+ """
+ conda:
+ GAWK
+ input:
+ cons_tmp = "results/05_Consensus/{reference}/{sample}_{aligner}_{min_cov}X_{caller}_consensus.fasta.tmp"
+ output:
+ consensus = "results/05_Consensus/{reference}/{sample}_{aligner}_{min_cov}X_{caller}_consensus.fasta"
+ log:
+ "results/10_Reports/tools-log/sed/{reference}/{sample}_{aligner}_{min_cov}X_{caller}_fasta-header.log"
+ shell:
+ "sed " # Sed, a Stream EDitor used to perform basic text transformations on an input stream
+ "'s/^>.*$/>{wildcards.sample}_{wildcards.aligner}_{wildcards.min_cov}X_{wildcards.caller}/' "
+ "{input.cons_tmp} " # Input file
+ "1> {output.consensus} " # Output file
+ "2> {log}" # Log redirection
+
+###############################################################################
+########################### VARIANTS CALLING - IVAR ###########################
+###############################################################################
+
+###############################################################################
+rule convert_tsv2vcf:
+ message:
+ """
+ ~ iVar ∞ Convert TSV to VCF file ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ {wildcards.aligner}
+ Min. cov.: _______ {wildcards.min_cov}X
+ Variant caller: __ iVar
+ """
+ conda:
+ TSV2VCF
+ input:
+ tsv = "results/04_Variants/{reference}/{sample}_{aligner}_{min_cov}X_ivar_variant-call.tsv"
+ output:
+ vcf = "results/04_Variants/{reference}/{sample}_{aligner}_{min_cov}X_ivar_variant-filt.vcf"
+ log:
+ "results/10_Reports/tools-log/tsv2vcf/{reference}/{sample}_{aligner}_{min_cov}X_ivar_tsv2vcf.log"
+ shell:
+ "python3 " # Python 3
+ "workflow/scripts/ivar_variants_to_vcf.py " # Script (from viralrecon)
+ "{input.tsv} " # TSV input
+ "{output.vcf} " # VCF output
+ "&> {log}" # Log redirection
+
+###############################################################################
+rule ivar_consensus:
+ # Aim:
+ # Use:
+ message:
+ """
+ ~ iVar ∞ Call Consensus ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ {wildcards.aligner}
+ Min. cov.: _______ {wildcards.min_cov}X
+ Variant caller: __ iVar
+ """
+ conda:
+ IVAR
+ params:
+ min_depth = IVAR_MIN_DEPTH,
+ min_freq = IVAR_MIN_FREQ,
+ min_insert = IVAR_MIN_INSERT,
+ max_depth = IVAR_MAX_DEPTH,
+ min_bq = IVAR_MIN_BQ,
+ min_qual = IVAR_MIN_QUAL,
+ baq = IVAR_MAP_QUAL
+ input:
+ mark_dup = get_bam_input,
+ variant_call = "results/04_Variants/{reference}/{sample}_{aligner}_{min_cov}X_ivar_variant-call.tsv"
+ output:
+ prefix = temp("results/05_Consensus/{reference}/{sample}_{aligner}_{min_cov}X_ivar_consensus"),
+ cons_tmp = temp("results/05_Consensus/{reference}/{sample}_{aligner}_{min_cov}X_ivar_consensus.fasta.tmp"),
+ qual_txt = "results/05_Consensus/{reference}/ivar_consensus-quality/{sample}_{aligner}_{min_cov}X_ivar_consensus.qual.txt",
+ log:
+ "results/10_Reports/tools-log/ivar/{reference}/{sample}_{aligner}_{min_cov}X_ivar_consensus.log"
+ shell:
+ "samtools mpileup " # Samtools mpileup, tools for alignments in the SAM format with command multi-way pileup
+ "--verbosity 0 " # Set level of verbosity [INT]
+ "-a " # -a: output all positions (including zero depth)
+ "-a " # -a -a / -aa: output absolutely all positions, including unused ref. sequences
+ "--count-orphans " # -A: do not discard anomalous read pairs
+ "--max-depth {params.max_depth} " # -d: max per-file depth; avoids excessive memory usage [INT] (default: 8000)
+ "{params.baq} " # --no-BAQ / -B: disable BAQ (per-Base Alignment Quality)
+ "--min-BQ {params.min_bq} " # -Q: skip bases with baseQ/BAQ smaller than [INT] (default: 13)
+ #"--reference {input.masked_ref} " # Reference sequence FASTA FILE
+ "{input.mark_dup} " # Markdup BAM input
+ "2>&1 | grep -v '[mpileup] 1 samples in 1 input files' " # Remove this stdout
+ "| " ### PIPE to iVar
+ "ivar consensus " # iVar, with command 'consensus': Call consensus from aligned BAM file
+ "-p {output.prefix} " # -p: prefix
+ "-q {params.min_qual} " # -q: Minimum quality score threshold to count base [INT] (Default: 20)
+ "-t {params.min_freq} " # -t: Minimum frequency threshold to call variants [FLOAT] (Default: 0.03)
+ "-c {params.min_insert} " # -c: Minimum insertion frequency threshold to call consensus [FLOAT] (Default: 0.8)
+ "-m {params.min_depth} " # -m: Minimum read depth to call variants [INT] (Default: 0)
+ "-n N " # -n: Character to print in regions with less than minimum coverage (Default: N)
+ #"-i {wildcards.sample} " # -i: Name of fasta header (default: Consensus_<prefix>_threshold_<min_freq>_quality_<min_qual>_<min_insert>
+ "&> {log} " # Log redirection
+ "&& mv {output.prefix}.fa {output.cons_tmp} " # copy consensus.fa (temp) to consensus.fasta.tmp (tmp)
+ "&& mv {output.prefix}.qual.txt {output.qual_txt} " # cppty consensus.qual.txt (tmp) to ivar_consensus-quality/ directory
+ "&& touch {output.prefix}" # Touch done
+
+###############################################################################
+rule ivar_variant_calling:
+ # Aim: SNVs and Indels calling
+ # Use: samtools mpileup -aa -A -d 0 -B -Q 0 --reference [<reference-fasta] <input.bam> | ivar variants -p <prefix> [-q <min-quality>] [-t <min-frequency-threshold>] [-m <minimum depth>] [-r <reference-fasta>] [-g GFF file]
+ # Note: samtools mpileup output must be piped into ivar variants
+ message:
+ """
+ ~ iVar ∞ Call Variants ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ {wildcards.aligner}
+ Min. cov.: _______ {wildcards.min_cov}X
+ Variant caller: __ iVar
+ """
+ conda:
+ IVAR
+ params:
+ min_depth = IVAR_MIN_DEPTH,
+ min_freq = IVAR_MIN_FREQ,
+ min_insert = IVAR_MIN_INSERT,
+ max_depth = IVAR_MAX_DEPTH,
+ min_bq = IVAR_MIN_BQ,
+ min_qual = IVAR_MIN_QUAL,
+ baq = IVAR_MAP_QUAL
+ input:
+ masked_ref = "results/04_Variants/{reference}/{sample}_{aligner}_{min_cov}X_masked-ref.fasta",
+ mark_dup = get_bam_input
+ output:
+ variant_call = "results/04_Variants/{reference}/{sample}_{aligner}_{min_cov}X_ivar_variant-call.tsv"
+ log:
+ "results/10_Reports/tools-log/ivar/{reference}/{sample}_{aligner}_{min_cov}X_ivar_variant-call.log"
+ shell:
+ "samtools mpileup " # Samtools mpileup, tools for alignments in the SAM format with command multi-way pileup
+ "--verbosity 0 " # Set level of verbosity [INT]
+ "-a " # -a: output all positions (including zero depth)
+ "-a " # -a -a / -aa: output absolutely all positions, including unused ref. sequences
+ "--count-orphans " # -A: do not discard anomalous read pairs
+ "--max-depth {params.max_depth} " # -d: max per-file depth; avoids excessive memory usage (default: 8000) [INT]
+ "{params.baq} " # --no-BAQ / -B: disable BAQ (per-Base Alignment Quality)
+ "--min-BQ {params.min_bq} " # -Q: skip bases with baseQ/BAQ smaller than (default: 13) [INT]
+ "--reference {input.masked_ref} " # Reference sequence FASTA FILE
+ "{input.mark_dup} " # Markdup BAM input
+ "2>&1 | grep -v '[mpileup] 1 samples in 1 input files' " # Remove this stdout
+ "| " ### pipe to iVar
+ "ivar variants " # iVar, with command 'variants': Call variants from aligned BAM file
+ "-p {output.variant_call} " # -p: prefix
+ "-q {params.min_qual} " # -q: Minimum quality score threshold to count base (Default: 20) [INT]
+ "-t {params.min_freq} " # -t: Minimum frequency threshold to call variants (Default: 0.03) [FLOAT]
+ "-m {params.min_depth} " # -m: Minimum read depth to call variants (Default: 0) [INT]
+ "-r {input.masked_ref} " # -r: Reference file used for alignment (translate the nuc. sequences and identify intra host single nuc. variants)
+ #"-g " # -g: A GFF file in the GFF3 format can be supplied to specify coordinates of open reading frames (ORFs)
+ "&> {log}" # Log redirection
+
+
+###############################################################################
+############################ MASKING LOW COVERAGE #############################
+###############################################################################
+
+###############################################################################
+rule bedtools_masking:
+ # Aim: masking low coverage regions
+ # Use: bedtools maskfasta [OPTIONS] -fi [REFERENCE.fasta] -bed [RANGE.bed] -fo [MASKEDREF.fasta]
+ message:
+ """
+ ~ BedTools ∞ Mask Low Coverage Regions ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ {wildcards.aligner}
+ Min. cov.: _______ {wildcards.min_cov}X
+ """
+ conda:
+ BEDTOOLS
+ params:
+ path = REF_PATH
+ input:
+ low_cov_mask = "results/03_Coverage/{reference}/bed/{sample}_{aligner}_{min_cov}X_low-cov-mask.bed"
+ output:
+ masked_ref = "results/04_Variants/{reference}/{sample}_{aligner}_{min_cov}X_masked-ref.fasta"
+ log:
+ "results/10_Reports/tools-log/bedtools/{reference}/{sample}_{aligner}_{min_cov}X_masking.log"
+ shell:
+ "bedtools maskfasta " # Bedtools maskfasta, mask a fasta file based on feature coordinates
+ "-fi {params.path}{wildcards.reference}.fasta " # Input FASTA file
+ "-bed {input.low_cov_mask} " # BED/GFF/VCF file of ranges to mask in -fi
+ "-fo {output.masked_ref} " # Output masked FASTA file
+ "&> {log}" # Log redirection
+
+###############################################################################
+rule bedtools_merged_mask:
+ # Aim: merging overlaps
+ # Use: bedtools merge [OPTIONS] -i [FILTERED.bed] -g [GENOME.fasta]
+ message:
+ """
+ ~ BedTools ∞ Merge Overlaps ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ {wildcards.aligner}
+ Min. cov.: _______ {wildcards.min_cov}X
+ """
+ conda:
+ BEDTOOLS
+ input:
+ min_cov_filt = "results/03_Coverage/{reference}/bed/{sample}_{aligner}_{min_cov}X_min-cov-filt.bed"
+ output:
+ low_cov_mask = temp("results/03_Coverage/{reference}/bed/{sample}_{aligner}_{min_cov}X_low-cov-mask.bed")
+ log:
+ "results/10_Reports/tools-log/bedtools/{reference}/{sample}_{aligner}_{min_cov}X_merging.log"
+ shell:
+ "bedtools merge " # Bedtools merge, merges overlapping BED/GFF/VCF entries into a single interval
+ "-i {input.min_cov_filt} " # -i: BED/GFF/VCF input to merge
+ "1> {output.low_cov_mask} " # merged output
+ "2> {log}" # Log redirection
+
+###############################################################################
+rule awk_min_covfilt:
+ # Aim: minimum coverage filtration
+ # Use: awk '$4 < [MIN_COV]' [BEDGRAPH.bed] 1> [FILTERED.bed]
+ message:
+ """
+ ~ Awk ∞ Minimum Coverage Filtration ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ {wildcards.aligner}
+ Min. cov.: _______ {wildcards.min_cov}X
+ """
+ conda:
+ GAWK
+ input:
+ genome_cov = "results/02_Mapping/{reference}/{sample}_{aligner}_genome-cov.bed"
+ output:
+ min_cov_filt = temp("results/03_Coverage/{reference}/bed/{sample}_{aligner}_{min_cov}X_min-cov-filt.bed")
+ log:
+ "results/10_Reports/tools-log/awk/{reference}/{sample}_{aligner}_{min_cov}X_min-cov-filt.log"
+ shell:
+ "awk " # Awk, a program that you can use to select particular records in a file and perform operations upon them
+ "'$4 < {wildcards.min_cov}' " # Minimum coverage for masking regions in consensus sequence
+ "{input.genome_cov} " # BedGraph coverage input
+ "1> {output.min_cov_filt} " # Minimum coverage filtered bed output
+ "2> {log} " # Log redirection
+
+###############################################################################
+################################## STATISTICS #################################
+###############################################################################
+
+###############################################################################
+rule awk_coverage_statistics:
+ # Aim: computing genomme coverage stats
+ # Use: awk {FORMULA} END {{print [RESULTS.tsv] [BEDGRAPH.bed]
+ message:
+ """
+ ~ Awk ∞ Compute Genome Coverage Statistics from BED file ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ {wildcards.aligner}
+ """
+ conda:
+ GAWK
+ input:
+ cutadapt = "results/10_Reports/tools-log/cutadapt/{sample}.log",
+ sickle = "results/10_Reports/tools-log/sickle-trim/{sample}.log",
+ samtools = "results/10_Reports/tools-log/samtools/{reference}/{sample}_{aligner}_mark-dup.log",
+ flagstat = "results/03_Coverage/{reference}/flagstat/{sample}_{aligner}_flagstat.json",
+ histogram = "results/03_Coverage/{reference}/histogram/{sample}_{aligner}_coverage-histogram.txt",
+ genome_cov = "results/02_Mapping/{reference}/{sample}_{aligner}_genome-cov.bed"
+ output:
+ cov_stats = "results/03_Coverage/{reference}/{sample}_{aligner}_{min_cov}X_coverage-stats.tsv"
+ log:
+ "results/10_Reports/tools-log/awk/{reference}/{sample}_{aligner}_{min_cov}X_coverage-stats.log"
+ shell:
+ r""" rawReads=$(grep -o -E """ # Get raw reads
+ r""" 'Total read pairs processed:.+' {input.cutadapt} """ #
+ r""" | sed -E 's/Total read pairs processed:\ +//' """ #
+ r""" | sed 's/,//g') ; """ #
+ #
+ r""" cutadaptPF=$(grep -o -E """ # Get cutadapt Passing Filtes reads
+ r""" 'Pairs written \(passing filters\):.+' {input.cutadapt} """ #
+ r""" | sed -E 's/Pairs written \(passing filters\):\ +//' """ #
+ r""" | sed 's/,//g') ; """ #
+ #
+ r""" sicklePF=$(grep -o -E """ # Get sickle Passing Filtes reads
+ r""" 'FastQ paired records kept:.+' {input.sickle} """ #
+ r""" | sed -E 's/FastQ paired records kept:\ +//') ; """ #
+ #
+ r""" totalDuplicate=$(grep -o -E """ # Get total duplicated reads
+ r""" 'DUPLICATE TOTAL:.+' {input.samtools} """ #
+ r""" | sed -E 's/DUPLICATE TOTAL:\ +//') ; """ #
+ #
+ r""" estimatedLibrarySize=$(grep -o -E """ # Get estimated library size
+ r""" 'ESTIMATED_LIBRARY_SIZE:.+' {input.samtools} """ #
+ r""" | sed -E 's/ESTIMATED_LIBRARY_SIZE:\ +//') ; """ #
+ #
+ r""" samtoolsPF=$(grep -o -E """ # Get samtool Passing Filter reads
+ r""" 'WRITTEN: .+' {input.samtools} """ #
+ r""" | sed -E 's/WRITTEN:\ +//') ; """ #
+ #
+ r""" mappedReads=$(grep -o -E -m 1 """ # Get mapped reads
+ r""" '"mapped": .+' {input.flagstat} """ #
+ r""" | sed -E 's/"mapped":\ +//' """ #
+ r""" | sed 's/,//g') ; """ #
+ #
+ r""" mappedPercentReads=$(grep -o -E -m 1 """ # Get mapped precent reads
+ r""" '"mapped %": .+' {input.flagstat} """ #
+ r""" | sed -E 's/"mapped %":\ +//' """ #
+ r""" | sed 's/,//g') ; """ #
+ #
+ r""" covPercentAt1X=$(grep -o -E """ # Get coverage percent @1X
+ r""" 'Percent covered:.+' {input.histogram} """ #
+ r""" | sed -E 's/Percent covered:\ +//') ; """ #
+ #
+ r""" awk """ # Awk, a program to select particular records in a file and perform operations upon them
+ r""" -v rawReads="${{rawReads}}" """ # Define external variable
+ r""" -v cutadaptPF="${{cutadaptPF}}" """ # Define external variable
+ r""" -v sicklePF="${{sicklePF}}" """ # Define external variable
+ r""" -v totalDuplicate="${{totalDuplicate}}" """ # Define external variable
+ r""" -v estimatedLibrarySize="${{estimatedLibrarySize}}" """ # Define external variable
+ r""" -v samtoolsPF="${{samtoolsPF}}" """ # Define external variable
+ r""" -v mappedReads="${{mappedReads}}" """ # Define external variable
+ r""" -v mappedPercentReads="${{mappedPercentReads}}" """ # Define external variable
+ r""" -v covPercentAt1X="${{covPercentAt1X}}" """ # Define external variable
+ r""" '$4 >= {wildcards.min_cov} {{supMin_Cov+=$3-$2}} ; """ # Genome size (>= min_cov @X)
+ r""" {{genomeSize+=$3-$2}} ; """ # Genome size (total)
+ r""" {{totalBases+=($3-$2)*$4}} ; """ # Total bases @1X
+ r""" {{totalBasesSq+=(($3-$2)*$4)**2}} """ # Total bases square @1X
+ r""" END """ # END
+ r""" {{print """ # Print
+ r""" "sample_id", "\t", """ # header: Sample ID
+ r""" "raw_paired_reads", "\t", """ # header: Raw paired reads
+ r""" "cutadapt_pairs_PF", "\t", """ # header: Cutadapt Passing Filters
+ r""" "sickle_reads_PF", "\t", """ # header: Sickle-trim Passing Filters
+ r""" "duplicated_reads", "\t", """ # header:
+ r""" "duplicated_percent_%","\t", """ # header:
+ r""" "estimated_library_size*", "\t", """ # header:
+ r""" "samtools_pairs_PF", "\t", """ # header:
+ r""" "mapped_with", "\t", """ # header: aligner
+ r""" "mapped_on", "\t", """ # header: reference
+ r""" "mapped_reads", "\t", """ # header:
+ r""" "mapped_percent_%", "\t", """ # header:
+ r""" "mean_depth", "\t", """ # header: mean depth
+ r""" "standard_deviation", "\t", """ # header: standard deviation
+ r""" "cov_percent_%_@1X", "\t", """ # header: coverage percentage @1X
+ r""" "cov_percent_%" "\t", """ # header: coverage percentage
+ r""" "@_min_cov" """ # header: @_[min_cov]_X
+ r""" ORS """ # \n newline
+ r""" "{wildcards.sample}", "\t", """ # value: Sample ID
+ r""" rawReads, "\t", """ # value: Raw sequences
+ r""" cutadaptPF, "\t", """ # value: Cutadapt Passing Filter
+ r""" sicklePF, "\t", """ # value: Sickle Passing Filter
+ r""" totalDuplicate, "\t", """ # value:
+ r""" int(((totalDuplicate)/(rawReads*2))*100), "%", "\t", """ # value: (divided by 2 to estimated pairs)
+ r""" estimatedLibrarySize, "\t", """ # value:
+ r""" samtoolsPF, "\t", """ # value:
+ r""" "{wildcards.aligner}", "\t", """ # value: aligner
+ r""" "{wildcards.reference}", "\t", """ # value: reference
+ r""" mappedReads, "\t", """ # value:
+ r""" mappedPercentReads, "%", "\t", """ # value:
+ r""" int(totalBases/genomeSize), "\t", """ # value: mean depth
+ r""" int(sqrt((totalBasesSq/genomeSize)-(totalBases/genomeSize)**2)), "\t", """ # Standard deviation value
+ r""" covPercentAt1X, "\t", """ # value
+ r""" supMin_Cov/genomeSize*100, "%", "\t", """ # Coverage percent (@ min_cov X) value
+ r""" "@{wildcards.min_cov}X" """ # @ min_cov X value
+ r""" }}' """ # Close print
+ r""" {input.genome_cov} """ # BedGraph coverage input
+ r""" 1> {output.cov_stats} """ # Mean depth output
+ r""" 2> {log}""" # Log redirection
+
+###############################################################################
+rule bedtools_genome_coverage:
+ # Aim: computing genome coverage sequencing
+ # Use: bedtools genomecov [OPTIONS] -ibam [MARK-DUP.bam] 1> [BEDGRAPH.bed]
+ message:
+ """
+ ~ BedTools ∞ Compute Genome Coverage ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ {wildcards.aligner}
+ """
+ conda:
+ BEDTOOLS
+ input:
+ mark_dup = get_bam_input,
+ index = get_bai_input
+ output:
+ genome_cov = "results/02_Mapping/{reference}/{sample}_{aligner}_genome-cov.bed"
+ log:
+ "results/10_Reports/tools-log/bedtools/{reference}/{sample}_{aligner}_genome-cov.log"
+ shell:
+ "bedtools genomecov " # Bedtools genomecov, compute the coverage of a feature file among a genome
+ "-bga " # Report depth in BedGraph format, regions with zero coverage are also reported
+ "-ibam {input.mark_dup} " # The input file is in BAM format, must be sorted by position
+ "1> {output.genome_cov} " # BedGraph output
+ "2> {log} " # Log redirection
+
+###############################################################################
+rule samtools_coverage_histogram:
+ # Aim: alignment depth and percent coverage histogram
+ # Use: samtools coverage --histogram [INPUT.bam]
+ message:
+ """
+ ~ SamTools ∞ Calcul Depth and Coverage from BAM file ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ {wildcards.aligner}
+ """
+ conda:
+ SAMTOOLS
+ resources:
+ cpus = CPUS
+ #bins = BINS,
+ #depth = DEPTH
+ input:
+ mark_dup = get_bam_input
+ output:
+ histogram = "results/03_Coverage/{reference}/histogram/{sample}_{aligner}_coverage-histogram.txt"
+ log:
+ "results/10_Reports/tools-log/samtools/{reference}/{sample}_{aligner}_coverage-histogram.log"
+ shell:
+ "samtools coverage " # Samtools coverage, tools for alignments in the SAM format with command to alignment depth and percent coverage
+ "--histogram " # -m: show histogram instead of tabular output
+ "--verbosity 4 " # Set level of verbosity [INT] (default: 3)
+ "--n-bins 149 " # -w: number of bins in histogram (default: terminal width - 40) (todo: {params.bins})
+ "--depth 0 " # -d maximum allowed coverage depth [INT] (default: 1000000 ; 0 removing any depth limit) (todo: {params.depth})
+ "--output {output.histogram} " # write output to FILE (default: stdout)
+ "{input.mark_dup} ; " # Mark_dup bam input
+ "echo >> {output.histogram} ; " # Newline
+ "samtools coverage " # Samtools coverage, tools for alignments in the SAM format with command to alignment depth and percent coverage
+ "--verbosity 4 " # Set level of verbosity [INT] (default: 3)
+ "{input.mark_dup} " # Mark_dup bam input
+ ">> {output.histogram} " # write output to FILE (default: stdout)
+ "2> {log}" # Log redirection
+
+###############################################################################
+rule samtools_flagstat_ext:
+ # Aim: simple stats
+ # Use: samtools flagstat -@ [THREADS] [INPUT.bam]
+ message:
+ """
+ ~ SamTools ∞ Calcul simple Stats from BAM file ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ {wildcards.aligner}
+ """
+ conda:
+ SAMTOOLS
+ resources:
+ cpus = CPUS
+ input:
+ mark_dup = get_bam_input
+ output:
+ flagstat = "results/03_Coverage/{reference}/flagstat/{sample}_{aligner}_flagstat.{ext}"
+ log:
+ "results/10_Reports/tools-log/samtools/{reference}/{sample}_{aligner}_flagstat-{ext}.log"
+ shell:
+ "samtools flagstat " # Samtools flagstat, tools for alignments in the SAM format with command to simple stat
+ "--threads {resources.cpus} " # -@: Number of additional threads to use (default: 1)
+ "--verbosity 4 " # Set level of verbosity [INT] (default: 3)
+ "--output-fmt {wildcards.ext} " # -O Specify output format (none, tsv and json)
+ "{input.mark_dup} " # Mark_dup bam input
+ "1> {output.flagstat} " # Mark_dup index output
+ "2> {log}" # Log redirection
+
+###############################################################################
+############################## PRIMER CLEAPING ###############################
+###############################################################################
+
+###############################################################################
+rule bamclipper_amplicon_primers:
+ # Aim: soft-clip amplicon PCR primers from BAM alignments
+ # Use: bamclipper.sh -n [THREADS] -b [MARKDUP.bam] -p [PRIMER.bed] -u [UPSTREAM] -d [DOWNSTREAM]
+ message:
+ """
+ ~ BAMClipper ∞ soft-clipping amplicon PCR primers from BAM alignments ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ {wildcards.aligner}
+ """
+ conda:
+ BAMCLIPPER
+ resources:
+ cpus = CPUS
+ params:
+ path = CLIPPATH,
+ primers = PRIMERS,
+ upstream = UPSTREAM,
+ downstream = DOWNSTREAM
+ input:
+ markdup = "results/02_Mapping/{reference}/{sample}_{aligner}_mark-dup.bam",
+ index = "results/02_Mapping/{reference}/{sample}_{aligner}_mark-dup.bam.bai"
+ output:
+ bamclip = "results/02_Mapping/{reference}/{sample}_{aligner}_mark-dup.primerclipped.bam",
+ baiclip = "results/02_Mapping/{reference}/{sample}_{aligner}_mark-dup.primerclipped.bam.bai"
+ log:
+ "results/10_Reports/tools-log/bamclipper/{reference}/{sample}_{aligner}_primers-clip.log"
+ shell:
+ "bamclipper.sh " # BAMClipper, remove primer sequences from BAM alignments of PCR amplicons by soft-clipping
+ "-b {input.markdup} " # Indexed BAM alignment file
+ "-p {params.path}{params.primers}.bedpe " # BEDPE of primer pair locations
+ "-n {resources.cpus} " # Number of threads (default: 1)
+ "-u {params.upstream} " # Number of nuc. upstream for assigning alignments to primers (default: 5)
+ "-d {params.downstream} " # Number of nuc. downstream for assigning alignments to primers (default: 5)
+ #"-o results/02_Mapping/ " # Path to write output (if BamClipper v.1.1.2) (todo)
+ "&> {log} " # Log redirection
+ "&& mv {wildcards.sample}_{wildcards.aligner}_mark-dup.primerclipped.bam {output.bamclip} " # because BamClipper v.1 default output system...
+ "&& mv {wildcards.sample}_{wildcards.aligner}_mark-dup.primerclipped.bam.bai {output.baiclip}" # because BamClipper v.1 default output system...
+
+###############################################################################
+############################## REMOVE DUPLICATS ###############################
+###############################################################################
+
+###############################################################################
+rule samtools_index_markdup:
+ # Aim: indexing marked as duplicate BAM file
+ # Use: samtools index -@ [THREADS] -b [MARK-DUP.bam] [INDEX.bai]
+ message:
+ """
+ ~ SamTools ∞ Index 'Marked as Duplicate' BAM file ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ {wildcards.aligner}
+ """
+ conda:
+ SAMTOOLS
+ resources:
+ cpus = CPUS
+ input:
+ mark_dup = "results/02_Mapping/{reference}/{sample}_{aligner}_mark-dup.bam"
+ output:
+ index = "results/02_Mapping/{reference}/{sample}_{aligner}_mark-dup.bam.bai"
+ log:
+ "results/10_Reports/tools-log/samtools/{reference}/{sample}_{aligner}_mark-dup-index.log"
+ shell:
+ "samtools index " # Samtools index, tools for alignments in the SAM format with command to index alignment
+ "-@ {resources.cpus} " # --threads: Number of additional threads to use (default: 1)(NB, --threads form dose'nt work)
+ "-b " # -b: Generate BAI-format index for BAM files (default)
+ "{input.mark_dup} " # Mark_dup bam input
+ "{output.index} " # Mark_dup index output
+ "&> {log}" # Log redirection
+
+###############################################################################
+rule samtools_markdup:
+ # Aim: marking duplicate alignments
+ # Use: samtools markdup -@ [THREADS] -r -s -O BAM [SORTED.bam] [MARK-DUP.bam]
+ message:
+ """
+ ~ SamTools ∞ Mark Duplicate Alignments ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ {wildcards.aligner}
+ """
+ conda:
+ SAMTOOLS
+ resources:
+ cpus = CPUS
+ input:
+ sorted = "results/02_Mapping/{reference}/{sample}_{aligner}_sorted.bam"
+ output:
+ mark_dup = "results/02_Mapping/{reference}/{sample}_{aligner}_mark-dup.bam"
+ log:
+ "results/10_Reports/tools-log/samtools/{reference}/{sample}_{aligner}_mark-dup.log"
+ shell:
+ "samtools markdup " # Samtools markdup, tools for alignments in the SAM format with command mark duplicates
+ "--threads {resources.cpus} " # -@: Number of additional threads to use (default: 1)
+ "-r " # -r: Remove duplicate reads
+ "-s " # -s: Report stats
+ "--output-fmt BAM " # -O: Specify output format: SAM, BAM, CRAM (here, BAM format)
+ "{input.sorted} " # Sorted bam input
+ "{output.mark_dup} " # Mark_dup bam output
+ "&> {log}" # Log redirection
+
+###############################################################################
+rule samtools_sorting:
+ # Aim: sorting
+ # Use: samtools sort -@ [THREADS] -m [MEM_GB] -T [TMP_DIR] -O BAM -o [SORTED.bam] [FIX-MATE.bam]
+ message:
+ """
+ ~ SamTools ∞ Sort BAM file ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ {wildcards.aligner}
+ """
+ conda:
+ SAMTOOLS
+ resources:
+ cpus = CPUS,
+ mem_gb = MEM_GB,
+ tmp_dir = TMP_DIR
+ input:
+ fix_mate = "results/02_Mapping/{reference}/{sample}_{aligner}_fix-mate.bam"
+ output:
+ sorted = temp("results/02_Mapping/{reference}/{sample}_{aligner}_sorted.bam")
+ log:
+ "results/10_Reports/tools-log/samtools/{reference}/{sample}_{aligner}_sorted.log"
+ shell:
+ "samtools sort " # Samtools sort, tools for alignments in the SAM format with command to sort alignment file
+ "--threads {resources.cpus} " # -@: Number of additional threads to use (default: 1)
+ "-m {resources.mem_gb}G " # -m: Set maximum memory per thread, suffix K/M/G recognized (default: 768M)
+ "-T {resources.tmp_dir} " # -T: Write temporary files to PREFIX.nnnn.bam
+ "--output-fmt BAM " # -O: Specify output format: SAM, BAM, CRAM (here, BAM format)
+ "-o {output.sorted} " # Sorted bam output
+ "{input.fix_mate} " # Fixmate bam input
+ "&> {log}" # Log redirection
+
+###############################################################################
+rule samtools_fixmate:
+ # Aim: filling in mate coordinates
+ # Use: samtools fixmate -@ [THREADS] -m -O BAM [SORT-BY-NAMES.bam] [FIX-MATE.bam]
+ message:
+ """
+ ~ SamTools ∞ Fil Mate Coordinates ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ {wildcards.aligner}
+ """
+ conda:
+ SAMTOOLS
+ resources:
+ cpus = CPUS
+ input:
+ sort_by_names = "results/02_Mapping/{reference}/{sample}_{aligner}_sort-by-names.bam"
+ output:
+ fix_mate = temp("results/02_Mapping/{reference}/{sample}_{aligner}_fix-mate.bam")
+ log:
+ "results/10_Reports/tools-log/samtools/{reference}/{sample}_{aligner}_fix-mate.log"
+ shell:
+ "samtools fixmate " # Samtools fixmate, tools for alignments in the SAM format with command to fix mate information
+ "--threads {resources.cpus} " # -@: Number of additional threads to use (default: 1)
+ "-m " # -m: Add mate score tag
+ "--output-fmt BAM " # -O: Specify output format: SAM, BAM, CRAM (here, BAM format)
+ "{input.sort_by_names} " # Sort_by_names bam input
+ "{output.fix_mate} " # Fix_mate bam output
+ "&> {log}" # Log redirection
+
+###############################################################################
+rule samtools_sortbynames:
+ # Aim: sorting by names
+ # Use: samtools sort -t [THREADS] -m [MEM_GB] -n -O BAM -o [SORT-BY-NAMES.bam] [MAPPED.sam]
+ message:
+ """
+ ~ SamTools ∞ Sort by Names BAM file ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ {wildcards.aligner}
+ """
+ conda:
+ SAMTOOLS
+ resources:
+ cpus = CPUS,
+ mem_gb = MEM_GB
+ input:
+ mapped = "results/02_Mapping/{reference}/{sample}_{aligner}-mapped.sam"
+ output:
+ sort_by_names = temp("results/02_Mapping/{reference}/{sample}_{aligner}_sort-by-names.bam")
+ log:
+ "results/10_Reports/tools-log/samtools/{reference}/{sample}_{aligner}_sort-by-names.log"
+ shell:
+ "samtools sort " # Samtools sort, tools for alignments in the SAM format with command to sort alignment file
+ "--threads {resources.cpus} " # -@: Number of additional threads to use (default: 1)
+ "-m {resources.mem_gb}G " # -m: Set maximum memory per thread, suffix K/M/G recognized (default: 768M)
+ "-n " # -n: Sort by read name (not compatible with samtools index command)
+ "--output-fmt BAM " # -O: Specify output format: SAM, BAM, CRAM (here, BAM format)
+ "-o {output.sort_by_names} " # -o: Write final output to FILE rather than standard output
+ "{input.mapped} " # Mapped reads input
+ "&> {log}" # Log redirection
+
+
+###############################################################################
+################################### MAPPING ###################################
+###############################################################################
+
+###############################################################################
+rule minimap2_mapping:
+ # Aim: reads mapping against reference sequence
+ # Use: minimap2
+ message:
+ """
+ ~ Minimap2 ∞ Map Reads ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ MiniMap2
+ """
+ conda:
+ MINIMAP2
+ resources:
+ cpus = CPUS
+ params:
+ preset = MM2_PRESET
+ #length = LENGTH
+ input:
+ mm2_indexes = "resources/indexes/minimap2/{reference}.mmi",
+ fwd_reads = "results/01_Trimming/sickle/{sample}_sickle-trimmed_R1.fastq.gz",
+ rev_reads = "results/01_Trimming/sickle/{sample}_sickle-trimmed_R2.fastq.gz"
+ output:
+ mapped = temp("results/02_Mapping/{reference}/{sample}_minimap2-mapped.sam")
+ log:
+ "results/10_Reports/tools-log/minimap2/{sample}_{reference}.log"
+ shell:
+ "minimap2 " # Minimap2, a versatile sequence alignment program
+ "-x {params.preset} " # -x: presets (always applied before other options)
+ "-t {resources.cpus} " # -t: Number of threads (default: 3)
+ "-a " # -a: output in the SAM format (PAF by default)
+ #"-F {params.length} " # -F: max fragment length, effective with -x sr mode (default: 800)
+ "{input.mm2_indexes} " # Reference index filename prefix.mmi
+ # (-k, -w, -I and -H can't be changed during mapping)
+ #"resources/genomes/{wildcards.reference}.fasta " # Reference genome fasta format (for custom -kwIH)
+ #"-k {params.kmer_size} " # -k: k-mer size (default: "21", no larger than "28") [INT]
+ #"-w {params.minimizer_size} " # -w: minimizer window size (default: "11") [INT]
+ #"-I {params.split_size} " # -I: split index for every {NUM} input bases (default: "8G") [INT]
+ #"{params.homopolymer} " # -H: use homopolymer-compressed k-mer (preferrable for PacBio)
+ "{input.fwd_reads} " # Forward input reads
+ "{input.rev_reads} " # Reverse input reads
+ "1> {output.mapped} " # SAM output
+ "2> {log}" # Log redirection
+
+###############################################################################
+rule bwa_mapping:
+ # Aim: reads mapping against reference sequence
+ # Use: bwa mem -t [THREADS] -x [REFERENCE] [FWD_R1.fq] [REV_R2.fq] 1> [MAPPED.sam]
+ message:
+ """
+ ~ BWA-MEM ∞ Map Reads ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ BWA
+ """
+ conda:
+ BWA
+ resources:
+ cpus = CPUS
+ input:
+ bwa_indexes = "resources/indexes/bwa/{reference}",
+ fwd_reads = "results/01_Trimming/sickle/{sample}_sickle-trimmed_R1.fastq.gz",
+ rev_reads = "results/01_Trimming/sickle/{sample}_sickle-trimmed_R2.fastq.gz"
+ output:
+ mapped = temp("results/02_Mapping/{reference}/{sample}_bwa-mapped.sam")
+ log:
+ "results/10_Reports/tools-log/bwa/{sample}_{reference}.log"
+ shell:
+ "bwa mem " # BWA-MEM algorithm, performs local alignment
+ "-t {resources.cpus} " # -t: Number of threads (default: 12)
+ "-v 1 " # -v: Verbosity level: 1=error, 2=warning, 3=message, 4+=debugging
+ "{input.bwa_indexes} " # Reference index filename prefix
+ "{input.fwd_reads} " # Forward input reads
+ "{input.rev_reads} " # Reverse input reads
+ "1> {output.mapped} " # SAM output
+ "2> {log}" # Log redirection
+
+###############################################################################
+rule bowtie2_mapping:
+ # Aim: reads mapping against reference sequence
+ # Use: bowtie2 -p [THREADS] -x [REFERENCE] -1 [FWD_R1.fq] -2 [REV_R2.fq] -S [MAPPED.sam]
+ message:
+ """
+ ~ Bowtie2 ∞ Map Reads ~
+ Sample: __________ {wildcards.sample}
+ Reference: _______ {wildcards.reference}
+ Aligner: _________ Bowtie2
+ """
+ conda:
+ BOWTIE2
+ resources:
+ cpus = CPUS
+ params:
+ sensitivity = BT2_SENSITIVITY
+ input:
+ bt2_indexes = "resources/indexes/bowtie2/{reference}",
+ fwd_reads = "results/01_Trimming/sickle/{sample}_sickle-trimmed_R1.fastq.gz",
+ rev_reads = "results/01_Trimming/sickle/{sample}_sickle-trimmed_R2.fastq.gz"
+ output:
+ mapped = temp("results/02_Mapping/{reference}/{sample}_bowtie2-mapped.sam")
+ log:
+ "results/10_Reports/tools-log/bowtie2/{sample}_{reference}.log"
+ shell:
+ "bowtie2 " # Bowtie2, an ultrafast and memory-efficient tool for aligning sequencing reads to long reference sequences
+ "--threads {resources.cpus} " # -p: Number of alignment threads to launch (default: 1)
+ "--reorder " # Keep the original read order (if multi-processor option -p is used)
+ "-x {input.bt2_indexes} " # -x: Reference index filename prefix (minus trailing .X.bt2)
+ "{params.sensitivity} " # Preset (default: "--sensitive")
+ # sensitive: same as [-D 15 -R 2 -N 0 -L 22 -i S,1,1.15]
+ "-q " # -q: Query input files are FASTQ .fq/.fastq (default)
+ "-1 {input.fwd_reads} " # Forward input reads
+ "-2 {input.rev_reads} " # Reverse input reads
+ "1> {output.mapped} " # -S: File for SAM output (default: stdout)
+ "2> {log}" # Log redirection
+
+###############################################################################
+################################## INDEXING ###################################
+###############################################################################
+
+###############################################################################
+rule minimap2_genome_indexing:
+ # Aim: index sequences in the FASTA format
+ # Use: minimap2 [OPTIONS] -d [INDEX.mmi] <query.fasta>
+ message:
+ """
+ ~ Minimap2 ∞ Index Genome ~
+ Reference: _______ {wildcards.reference}
+ """
+ conda:
+ MINIMAP2
+ params:
+ kmer_size = KMER_SIZE,
+ minimizer_size = MINIMIZER_SIZE,
+ split_size = SPLIT_SIZE
+ #homopolymer = HOMOPOLYMER
+ input:
+ fasta = "resources/genomes/{reference}.fasta"
+ output:
+ mm2_indexes = multiext("resources/indexes/minimap2/{reference}",
+ ".mmi")
+ log:
+ "results/10_Reports/tools-log/minimap2-indexes/{reference}.log"
+ shell:
+ "minimap2 " # Minimap2, index sequences
+ "-k {params.kmer_size} " # -k: k-mer size (default: "21", no larger than "28") [INT]
+ "-w {params.minimizer_size} " # -w: minimizer window size (default: "11") [INT]
+ "-I {params.split_size} " # -I: split index for every {NUM} input bases (default: "8G") [INT]
+ #"{params.homopolymer} " # use homopolymer-compressed k-mer (preferrable for PacBio)
+ "-d {output.mm2_indexes} " # -d: dump index to FILE []
+ "{input.fasta} " # Reference sequences in the FASTA format
+ "&> {log}" # Log redirection
+
+###############################################################################
+rule bwa_genome_indexing:
+ # Aim: index sequences in the FASTA format
+ # Use: bwa index -a [ALGO] -p [PREFIX] <genome.fasta>
+ message:
+ """
+ ~ BWA-SW ∞ Index Genome ~
+ Reference: _______ {wildcards.reference}
+ """
+ conda:
+ BWA
+ params:
+ algorithm = BWA_ALGO
+ input:
+ fasta = "resources/genomes/{reference}.fasta"
+ output:
+ prefix = "resources/indexes/bwa/{reference}",
+ bwa_indexes = multiext("resources/indexes/bwa/{reference}",
+ ".amb", ".ann", ".bwt", ".pac", ".sa")
+ log:
+ "results/10_Reports/tools-log/bwa-indexes/{reference}.log"
+ shell:
+ "bwa index " # BWA-SW algorithm, index sequences
+ "{params.algorithm} " # -a: Algorithm for constructing BWT index (default: auto)
+ "-p {output.prefix} " # -p: Prefix of the output database
+ "{input.fasta} " # Reference sequences in the FASTA format
+ "&> {log} " # Log redirection
+ "&& touch {output.prefix}" # Touch done
+
+###############################################################################
+rule bowtie2_genome_indexing:
+ # Aim: index sequences in the FASTA format
+ # Use: bowtie2-build [OPTIONS] <reference_in> <bt2_index_base>
+ message:
+ """
+ ~ Bowtie2-build ∞ Index Genome ~
+ Reference: _______ {wildcards.reference}
+ """
+ conda:
+ BOWTIE2
+ resources:
+ cpus = CPUS
+ params:
+ algorithm = BT2_ALGO
+ input:
+ fasta = "resources/genomes/{reference}.fasta"
+ output:
+ prefix = "resources/indexes/bowtie2/{reference}",
+ bt2_indexes = multiext("resources/indexes/bowtie2/{reference}",
+ ".1.bt2", ".2.bt2", ".3.bt2", ".4.bt2",
+ ".rev.1.bt2", ".rev.2.bt2")
+ log:
+ "results/10_Reports/tools-log/bowtie2-indexes/{reference}.log"
+ shell:
+ "bowtie2-build " # Bowtie2-build, index sequences
+ "--quiet " # -q: quiet
+ "--threads {resources.cpus} " # Number of threads
+ "{params.algorithm} " # Force (or no by default) generated index to be 'large',
+ # even if ref has fewer than 4 billion nucleotides
+ "-f " # Reference files are FASTA (default)
+ "{input.fasta} " # Reference sequences files (comma-separated list) in the FASTA format
+ "{output.prefix} " # Write bt2 data to files with this dir/basename
+ "&> {log} " # Log redirection
+ "&& touch {output.prefix}" # Touch done
+
+
+###############################################################################
+################################## TRIMMING ###################################
+###############################################################################
+
+###############################################################################
+rule sickle_trim_quality:
+ # Aim: windowed adaptive trimming tool for FASTQ files using quality
+ # Use: sickle [COMMAND] [OPTIONS]
+ message:
+ """
+ ~ Sickle-trim ∞ Trim Low Quality Sequences ~
+ Sample: __________ {wildcards.sample}
+ """
+ conda:
+ SICKLE_TRIM
+ params:
+ command = SIC_COMMAND,
+ encoding = SIC_ENCODING,
+ quality = SIC_QUALITY,
+ length = SIC_LENGTH
+ input:
+ fwd_reads = "results/01_Trimming/cutadapt/{sample}_cutadapt-removed_R1.fastq.gz",
+ rev_reads = "results/01_Trimming/cutadapt/{sample}_cutadapt-removed_R2.fastq.gz"
+ output:
+ fwd_reads = stash_or_trash("results/01_Trimming/sickle/{sample}_sickle-trimmed_R1.fastq.gz"),
+ rev_reads = stash_or_trash("results/01_Trimming/sickle/{sample}_sickle-trimmed_R2.fastq.gz"),
+ single = stash_or_trash("results/01_Trimming/sickle/{sample}_sickle-trimmed_SE.fastq.gz")
+ log:
+ "results/10_Reports/tools-log/sickle-trim/{sample}.log"
+ shell:
+ "sickle " # Sickle, a windowed adaptive trimming tool for FASTQ files using quality
+ "{params.command} " # Paired-end or single-end sequence trimming
+ "-t {params.encoding} " # --qual-type: Type of quality values, solexa ; illumina ; sanger ; CASAVA, < 1.3 ; 1.3 to 1.7 ; >= 1.8
+ "-q {params.quality} " # --qual-threshold: Threshold for trimming based on average quality in a window (default: 20)
+ "-l {params.length} " # --length-threshold: Threshold to keep a read based on length after trimming (default: 20)
+ "-f {input.fwd_reads} " # --pe-file1: Input paired-end forward fastq file
+ "-r {input.rev_reads} " # --pe-file2: Input paired-end reverse fastq file
+ "-g " # --gzip-output: Output gzipped files
+ "-o {output.fwd_reads} " # --output-pe1: Output trimmed forward fastq file
+ "-p {output.rev_reads} " # --output-pe2: Output trimmed reverse fastq file (must use -s option)
+ "-s {output.single} " # --output-single: Output trimmed singles fastq file
+ "&> {log} " # Log redirection
+
+###############################################################################
+rule cutadapt_adapters_removing:
+ # Aim: finds and removes adapter sequences, primers, poly-A tails and other types of unwanted sequence from your high-throughput sequencing reads
+ # Use: cutadapt [OPTIONS] -a/-A [ADAPTER] -o [OUT_FWD.fastq.gz] -p [OUT_REV.fastq.gz] [IN_FWD.fastq.gz] [IN_REV.fastq.gz]
+ # Rmq: multiple adapter sequences can be given using further -a options, but only the best-matching adapter will be removed
+ message:
+ """
+ ~ Cutadapt ∞ Remove Adapters ~
+ Sample: __________ {wildcards.sample}
+ """
+ conda:
+ CUTADAPT
+ resources:
+ cpus = CPUS
+ params:
+ length = CUT_LENGTH,
+ truseq = CUT_TRUSEQ,
+ nextera = CUT_NEXTERA,
+ small = CUT_SMALL,
+ cut = CUT_CLIPPING
+ input:
+ fwd_reads = "resources/reads/{sample}_R1.fastq.gz",
+ rev_reads = "resources/reads/{sample}_R2.fastq.gz"
+ #fwd_reads = "/users/illumina/local/data/run_1/FATSQ/{sample}_R1.fastq.gz",
+ #rev_reads = "/users/illumina/local/data/run_1/FATSQ/{sample}_R2.fastq.gz"
+ output:
+ fwd_reads = temp("results/01_Trimming/cutadapt/{sample}_cutadapt-removed_R1.fastq.gz"),
+ rev_reads = temp("results/01_Trimming/cutadapt/{sample}_cutadapt-removed_R2.fastq.gz")
+ log:
+ "results/10_Reports/tools-log/cutadapt/{sample}.log"
+ shell:
+ "cutadapt " # Cutadapt, finds and removes unwanted sequence from your HT-seq reads
+ "--cores {resources.cpus} " # -j: Number of CPU cores to use. Use 0 to auto-detect (default: 1)
+ "--cut {params.cut} " # -u: Remove 'n' first bases (5') from forward R1 (hard-clipping, default: 0)
+ "-U {params.cut} " # -U: Remove 'n' first bases (5') from reverse R2 (hard-clipping, default: 0)
+ "--trim-n " # --trim-n: Trim N's on ends (3') of reads
+ "--minimum-length {params.length} " # -m: Discard reads shorter than length
+ "--adapter {params.truseq} " # -a: Sequence of an adapter ligated to the 3' end of the first read
+ "-A {params.truseq} " # -A: 3' adapter to be removed from second read in a pair
+ "--adapter {params.nextera} " # -a: Sequence of an adapter ligated to the 3' end of the first read
+ "-A {params.nextera} " # -A: 3' adapter to be removed from second read in a pair
+ "--adapter {params.small} " # -a: Sequence of an adapter ligated to the 3' end of the first read
+ "-A {params.small} " # -A: 3' adapter to be removed from second read in a pair
+ "--output {output.fwd_reads} " # -o: Write trimmed reads to FILE
+ "--paired-output {output.rev_reads} " # -p: Write second read in a pair to FILE
+ "{input.fwd_reads} " # Input forward reads R1.fastq
+ "{input.rev_reads} " # Input reverse reads R2.fastq
+ "&> {log} " # Log redirection
+
+###############################################################################
+############################### QUALITY CONTROL ###############################
+###############################################################################
+
+###############################################################################
+rule multiqc_reports_aggregation:
+ # Aim: aggregates bioinformatics analyses results into a single report
+ # Use: multiqc [OPTIONS] --output [MULTIQC/] [FASTQC/] [MULTIQC/]
+ priority: 999 # Explicit high priority
+ message:
+ """
+ ~ MultiQC ∞ Aggregat HTML Qualities Reports ~
+ """
+ conda:
+ MULTIQC
+ params:
+ #config = MQC_CONFIG,
+ #tag = TAG
+ input:
+ fastqc = expand("results/00_Quality_Control/fastqc/{fastq}/",
+ fastq = FASTQ),
+ fastq_screen = expand("results/00_Quality_Control/fastq-screen/{fastq}/",
+ fastq = FASTQ)
+ output:
+ multiqc = directory("results/00_Quality_Control/multiqc/")
+ log:
+ "results/10_Reports/tools-log/multiqc.log"
+ shell:
+ "multiqc " # Multiqc, searches in given directories for analysis & compiles a HTML report
+ "--quiet " # -q: Only show log warning
+ "--no-ansi " # Disable coloured log
+ #"--config {params.config} " # Specific config file to load
+ #"--tag {params.tag} " # Use only modules which tagged with this keyword
+ #"--pdf " # Creates PDF report with 'simple' template (require xelatex)
+ "--export " # Export plots as static images in addition to the report
+ "--outdir {output.multiqc} " # -o: Create report in the specified output directory
+ "{input.fastqc} " # Input FastQC files
+ "{input.fastq_screen} " # Input Fastq-Screen
+ "&> {log}" # Log redirection
+
+###############################################################################
+rule fastqscreen_contamination_checking:
+ # Aim: screen if the composition of the library matches with what you expect
+ # Use fastq_screen [OPTIONS] --outdir [DIR/] [FASTQ.GZ]
+ message:
+ """
+ ~ Fasts-Screen ∞ Screen Contamination ~
+ Fastq: __________ {wildcards.fastq}
+ """
+ conda:
+ FASTQ_SCREEN
+ resources:
+ cpus = CPUS
+ params:
+ config = FQC_CONFIG,
+ subset = SUBSET
+ input:
+ fastq = "resources/reads/{fastq}.fastq.gz"
+ output:
+ fastq_screen = directory("results/00_Quality_Control/fastq-screen/{fastq}/")
+ log:
+ "results/10_Reports/tools-log/fastq-screen/{fastq}.log"
+ shell:
+ "fastq_screen " # FastqScreen, what did you expect ?
+ "-q " # --quiet: Only show log warning
+ "--threads {resources.cpus} " # --threads: Specifies across how many aligner will be allowed to run
+ "--aligner 'bwa' " # -a: choose aligner 'bowtie', 'bowtie2', 'bwa'
+ "--conf {params.config} " # path to configuration file
+ "--subset {params.subset} " # Don't use the whole sequence file, but create a subset of specified size
+ "--outdir {output.fastq_screen} " # Output directory
+ "{input.fastq} " # Input file.fastq
+ "&> {log}" # Log redirection
+
+###############################################################################
+rule fastqc_quality_control:
+ # Aim: reads sequence files and produces a quality control report
+ # Use: fastqc [OPTIONS] --output [DIR/] [FASTQ.GZ]
+ message:
+ """
+ ~ FastQC ∞ Quality Control ~
+ Fastq: __________ {wildcards.fastq}
+ """
+ conda:
+ FASTQC
+ resources:
+ cpus = CPUS
+ input:
+ fastq = "resources/reads/{fastq}.fastq.gz"
+ output:
+ fastqc = directory("results/00_Quality_Control/fastqc/{fastq}/")
+ log:
+ "results/10_Reports/tools-log/fastqc/{fastq}.log"
+ shell:
+ "mkdir -p {output.fastqc} " # (*) this directory must exist as the program will not create it
+ "2> /dev/null && " # in silence and then...
+ "fastqc " # FastQC, a high throughput sequence QC analysis tool
+ "--quiet " # -q: Supress all progress messages on stdout and only report errors
+ "--threads {resources.cpus} " # -t: Specifies files number which can be processed simultaneously
+ "--outdir {output.fastqc} " # -o: Create all output files in the specified output directory (*)
+ "{input.fastq} " # Input file.fastq
+ "&> {log}" # Log redirection
+
+###############################################################################
diff --git a/workflow/environments/bamclipper_v.1.0.0.yaml b/workflow/envs/bamclipper_v.1.0.0.yaml
similarity index 100%
rename from workflow/environments/bamclipper_v.1.0.0.yaml
rename to workflow/envs/bamclipper_v.1.0.0.yaml
diff --git a/workflow/environments/bcftools_v.1.20.yaml b/workflow/envs/bcftools_v.1.20.yaml
similarity index 100%
rename from workflow/environments/bcftools_v.1.20.yaml
rename to workflow/envs/bcftools_v.1.20.yaml
diff --git a/workflow/environments/bedtools_v.2.31.1.yaml b/workflow/envs/bedtools_v.2.31.1.yaml
similarity index 100%
rename from workflow/environments/bedtools_v.2.31.1.yaml
rename to workflow/envs/bedtools_v.2.31.1.yaml
diff --git a/workflow/environments/bowtie2_v.2.5.4.yaml b/workflow/envs/bowtie2_v.2.5.4.yaml
similarity index 100%
rename from workflow/environments/bowtie2_v.2.5.4.yaml
rename to workflow/envs/bowtie2_v.2.5.4.yaml
diff --git a/workflow/environments/bwa_v.0.7.18.yaml b/workflow/envs/bwa_v.0.7.18.yaml
similarity index 100%
rename from workflow/environments/bwa_v.0.7.18.yaml
rename to workflow/envs/bwa_v.0.7.18.yaml
diff --git a/workflow/environments/cutadapt_v.4.9.yaml b/workflow/envs/cutadapt_v.4.9.yaml
similarity index 100%
rename from workflow/environments/cutadapt_v.4.9.yaml
rename to workflow/envs/cutadapt_v.4.9.yaml
diff --git a/workflow/environments/fastq-screen_v.0.15.3.yaml b/workflow/envs/fastq-screen_v.0.15.3.yaml
similarity index 100%
rename from workflow/environments/fastq-screen_v.0.15.3.yaml
rename to workflow/envs/fastq-screen_v.0.15.3.yaml
diff --git a/workflow/environments/fastqc_v.0.12.1.yaml b/workflow/envs/fastqc_v.0.12.1.yaml
similarity index 100%
rename from workflow/environments/fastqc_v.0.12.1.yaml
rename to workflow/envs/fastqc_v.0.12.1.yaml
diff --git a/workflow/environments/gawk_v.5.3.0.yaml b/workflow/envs/gawk_v.5.3.0.yaml
similarity index 100%
rename from workflow/environments/gawk_v.5.3.0.yaml
rename to workflow/envs/gawk_v.5.3.0.yaml
diff --git a/workflow/environments/ivar_v.1.4.3.yaml b/workflow/envs/ivar_v.1.4.3.yaml
similarity index 100%
rename from workflow/environments/ivar_v.1.4.3.yaml
rename to workflow/envs/ivar_v.1.4.3.yaml
diff --git a/workflow/environments/minimap2_v.2.28.yaml b/workflow/envs/minimap2_v.2.28.yaml
similarity index 100%
rename from workflow/environments/minimap2_v.2.28.yaml
rename to workflow/envs/minimap2_v.2.28.yaml
diff --git a/workflow/environments/multiqc_v.1.23.yaml b/workflow/envs/multiqc_v.1.23.yaml
similarity index 100%
rename from workflow/environments/multiqc_v.1.23.yaml
rename to workflow/envs/multiqc_v.1.23.yaml
diff --git a/workflow/environments/nextclade_v.3.9.1.yaml b/workflow/envs/nextclade_v.3.9.1.yaml
similarity index 100%
rename from workflow/environments/nextclade_v.3.9.1.yaml
rename to workflow/envs/nextclade_v.3.9.1.yaml
diff --git a/workflow/environments/pangolin_v.4.3.yaml b/workflow/envs/pangolin_v.4.3.yaml
similarity index 100%
rename from workflow/environments/pangolin_v.4.3.yaml
rename to workflow/envs/pangolin_v.4.3.yaml
diff --git a/workflow/environments/samtools_v.1.20.yaml b/workflow/envs/samtools_v.1.20.yaml
similarity index 100%
rename from workflow/environments/samtools_v.1.20.yaml
rename to workflow/envs/samtools_v.1.20.yaml
diff --git a/workflow/environments/sickle-trim_v.1.33.yaml b/workflow/envs/sickle-trim_v.1.33.yaml
similarity index 100%
rename from workflow/environments/sickle-trim_v.1.33.yaml
rename to workflow/envs/sickle-trim_v.1.33.yaml
diff --git a/workflow/environments/tsv2vcf_v.1.1.yaml b/workflow/envs/tsv2vcf_v.1.1.yaml
similarity index 100%
rename from workflow/environments/tsv2vcf_v.1.1.yaml
rename to workflow/envs/tsv2vcf_v.1.1.yaml
diff --git a/workflow/environments/workflow-base_v.2024.11.yaml b/workflow/envs/workflow-base_v.2024.11.yaml
similarity index 100%
rename from workflow/environments/workflow-base_v.2024.11.yaml
rename to workflow/envs/workflow-base_v.2024.11.yaml
diff --git a/workflow/snakefiles/gevarli.smk b/workflow/rules/gevarli.smk
similarity index 90%
rename from workflow/snakefiles/gevarli.smk
rename to workflow/rules/gevarli.smk
index 501a4228538694728a8ece1447882eb370dfd15a..ba9ad8c82356e3c043054d03171487c97a8a588e 100755
--- a/workflow/snakefiles/gevarli.smk
+++ b/workflow/rules/gevarli.smk
@@ -203,6 +203,13 @@ FQC_CONFIG = config["fastq_screen"]["config"] # Fastq-Screen --conf
#MQC_CONF = config["multiqc"]["config"] # MultiQC --conf
#TAG = config["multiqc"]["tag"] # MultiQC --tag
+KMER_SIZE = config["minimap2"]["algorithm"]["k-mer_size"] # MM2 k-mer size
+MINIMIZER_SIZE = config["minimap2"]["algorithm"]["minimizer_size"] # MM2 minimizer window size
+SPLIT_SIZE = config["minimap2"]["algorithm"]["split_size"] # MM2 split index
+#HOMOPOLYMER = config["minimap2"]["algorithm"]["homopolymer"] # MM2 if PacBio
+BWA_ALGO = config["bwa"]["algorithm"] # BWA indexing algorithm
+BT2_ALGO = config["bowtie2"]["algorithm"] # BT2 indexing algorithm
+
REFERENCE = config["consensus"]["reference"] # Genome reference sequence, in fasta format
REF_PATH = config["consensus"]["path"] # Path to genomes references
MIN_COV = config["consensus"]["min_cov"] # Minimum coverage, mask lower regions with 'N'
@@ -1046,10 +1053,10 @@ rule minimap2_mapping:
resources:
cpus = CPUS
params:
- mm2_path = MM2_PATH,
preset = MM2_PRESET
#length = LENGTH
input:
+ mm2_indexes = "resources/indexes/minimap2/{reference}.mmi"
fwd_reads = "results/01_Trimming/sickle/{sample}_sickle-trimmed_R1.fastq.gz",
rev_reads = "results/01_Trimming/sickle/{sample}_sickle-trimmed_R2.fastq.gz"
output:
@@ -1057,21 +1064,22 @@ rule minimap2_mapping:
log:
"results/10_Reports/tools-log/minimap2/{sample}_{reference}.log"
shell:
- "minimap2 " # Minimap2, a versatile sequence alignment program
- "-x {params.preset} " # -x: presets (always applied before other options)
- "-t {resources.cpus} " # -t: Number of threads (default: 3)
- "-a " # -a: output in the SAM format (PAF by default)
- #"-F {params.length} " # -F: max fragment length, effective with -x sr mode (default: 800)
- "{params.mm2_path}{wildcards.reference}.mmi " # Reference index filename prefix.mmi (-k, -w, -I and -H can't be changed during mapping)
+ "minimap2 " # Minimap2, a versatile sequence alignment program
+ "-x {params.preset} " # -x: presets (always applied before other options)
+ "-t {resources.cpus} " # -t: Number of threads (default: 3)
+ "-a " # -a: output in the SAM format (PAF by default)
+ #"-F {params.length} " # -F: max fragment length, effective with -x sr mode (default: 800)
+ "{input.mm2_indexes} " # Reference index filename prefix.mmi
+ # (-k, -w, -I and -H can't be changed during mapping)
#"resources/genomes/{wildcards.reference}.fasta " # Reference genome fasta format (for custom -kwIH)
- #"-k {params.kmer_size} " # -k: k-mer size (default: "21", no larger than "28") [INT]
- #"-w {params.minimizer_size} " # -w: minimizer window size (default: "11") [INT]
- #"-I {params.split_size} " # -I: split index for every {NUM} input bases (default: "8G") [INT]
- #"{params.homopolymer} " # -H: use homopolymer-compressed k-mer (preferrable for PacBio)
- "{input.fwd_reads} " # Forward input reads
- "{input.rev_reads} " # Reverse input reads
- "1> {output.mapped} " # SAM output
- "2> {log}" # Log redirection
+ #"-k {params.kmer_size} " # -k: k-mer size (default: "21", no larger than "28") [INT]
+ #"-w {params.minimizer_size} " # -w: minimizer window size (default: "11") [INT]
+ #"-I {params.split_size} " # -I: split index for every {NUM} input bases (default: "8G") [INT]
+ #"{params.homopolymer} " # -H: use homopolymer-compressed k-mer (preferrable for PacBio)
+ "{input.fwd_reads} " # Forward input reads
+ "{input.rev_reads} " # Reverse input reads
+ "1> {output.mapped} " # SAM output
+ "2> {log}" # Log redirection
###############################################################################
rule bwa_mapping:
@@ -1088,9 +1096,8 @@ rule bwa_mapping:
BWA
resources:
cpus = CPUS
- params:
- bwa_path = BWA_PATH
input:
+ bwa_indexes = "resources/indexes/bwa/{reference}"
fwd_reads = "results/01_Trimming/sickle/{sample}_sickle-trimmed_R1.fastq.gz",
rev_reads = "results/01_Trimming/sickle/{sample}_sickle-trimmed_R2.fastq.gz"
output:
@@ -1098,14 +1105,14 @@ rule bwa_mapping:
log:
"results/10_Reports/tools-log/bwa/{sample}_{reference}.log"
shell:
- "bwa mem " # BWA-MEM algorithm, performs local alignment
- "-t {resources.cpus} " # -t: Number of threads (default: 12)
- "-v 1 " # -v: Verbosity level: 1=error, 2=warning, 3=message, 4+=debugging
- "{params.bwa_path}{wildcards.reference} " # Reference index filename prefix
- "{input.fwd_reads} " # Forward input reads
- "{input.rev_reads} " # Reverse input reads
- "1> {output.mapped} " # SAM output
- "2> {log}" # Log redirection
+ "bwa mem " # BWA-MEM algorithm, performs local alignment
+ "-t {resources.cpus} " # -t: Number of threads (default: 12)
+ "-v 1 " # -v: Verbosity level: 1=error, 2=warning, 3=message, 4+=debugging
+ "{input.bwa_indexes} " # Reference index filename prefix
+ "{input.fwd_reads} " # Forward input reads
+ "{input.rev_reads} " # Reverse input reads
+ "1> {output.mapped} " # SAM output
+ "2> {log}" # Log redirection
###############################################################################
rule bowtie2_mapping:
@@ -1123,9 +1130,9 @@ rule bowtie2_mapping:
resources:
cpus = CPUS
params:
- bt2_path = BT2_PATH,
sensitivity = BT2_SENSITIVITY
input:
+ bt2_indexes = "resources/indexes/bowtie2/{reference}",
fwd_reads = "results/01_Trimming/sickle/{sample}_sickle-trimmed_R1.fastq.gz",
rev_reads = "results/01_Trimming/sickle/{sample}_sickle-trimmed_R2.fastq.gz"
output:
@@ -1136,14 +1143,118 @@ rule bowtie2_mapping:
"bowtie2 " # Bowtie2, an ultrafast and memory-efficient tool for aligning sequencing reads to long reference sequences
"--threads {resources.cpus} " # -p: Number of alignment threads to launch (default: 1)
"--reorder " # Keep the original read order (if multi-processor option -p is used)
- "-x {params.bt2_path}{wildcards.reference} " # -x: Reference index filename prefix (minus trailing .X.bt2) [Bowtie-1 indexes are not compatible]
- "{params.sensitivity} " # Preset (default: "--sensitive", same as [-D 15 -R 2 -N 0 -L 22 -i S,1,1.15])
+ "-x {input.indexes} " # -x: Reference index filename prefix (minus trailing .X.bt2)
+ "{params.sensitivity} " # Preset (default: "--sensitive")
+ # sensitive: same as [-D 15 -R 2 -N 0 -L 22 -i S,1,1.15]
"-q " # -q: Query input files are FASTQ .fq/.fastq (default)
"-1 {input.fwd_reads} " # Forward input reads
"-2 {input.rev_reads} " # Reverse input reads
"1> {output.mapped} " # -S: File for SAM output (default: stdout)
"2> {log}" # Log redirection
+###############################################################################
+################################## INDEXING ###################################
+###############################################################################
+
+###############################################################################
+rule minimap2_genome_indexing:
+ # Aim: index sequences in the FASTA format
+ # Use: minimap2 [OPTIONS] -d [INDEX.mmi] <query.fasta>
+ message:
+ """
+ ~ Minimap2 ∞ Index Genome ~
+ Reference: _______ {wildcards.reference}
+ """
+ conda:
+ MINIMAP2
+ params:
+ kmer_size = KMER_SIZE,
+ minimizer_size = MINIMIZER_SIZE,
+ split_size = SPLIT_SIZE
+ #homopolymer = HOMOPOLYMER
+ input:
+ fasta = "resources/genomes/{reference}.fasta"
+ output:
+ mm2_indexes = multiext("resources/indexes/minimap2/{reference}",
+ ".mmi")
+ log:
+ "results/10_Reports/tools-log/minimap2-indexes/{reference}.log"
+ shell:
+ "minimap2 " # Minimap2, index sequences
+ "-k {params.kmer_size} " # -k: k-mer size (default: "21", no larger than "28") [INT]
+ "-w {params.minimizer_size} " # -w: minimizer window size (default: "11") [INT]
+ "-I {params.split_size} " # -I: split index for every {NUM} input bases (default: "8G") [INT]
+ #"{params.homopolymer} " # use homopolymer-compressed k-mer (preferrable for PacBio)
+ "-d {output.indexes} " # -d: dump index to FILE []
+ "{input.fasta} " # Reference sequences in the FASTA format
+ "&> {log}" # Log redirection
+
+###############################################################################
+rule bwa_genome_indexing:
+ # Aim: index sequences in the FASTA format
+ # Use: bwa index -a [ALGO] -p [PREFIX] <genome.fasta>
+ message:
+ """
+ ~ BWA-SW ∞ Index Genome ~
+ Reference: _______ {wildcards.reference}
+ """
+ conda:
+ BWA
+ params:
+ algorithm = BWA_ALGO
+ input:
+ fasta = "resources/genomes/{reference}.fasta"
+ output:
+ prefix = temp("resources/indexes/bwa/{reference}"),
+ indexes = multiext("resources/indexes/bwa/{ref_seq}",
+ ".amb", ".ann", ".bwt", ".pac", ".sa")
+ log:
+ "results/10_Reports/tools-log/bwa-indexes/{reference}.log"
+ shell:
+ "bwa index " # BWA-SW algorithm, index sequences
+ "{params.algorithm} " # -a: Algorithm for constructing BWT index (default: auto)
+ "-p {output.prefix} " # -p: Prefix of the output database
+ "{input.fasta} " # Reference sequences in the FASTA format
+ "&> {log} " # Log redirection
+ "&& touch {output.prefix}" # Touch done
+
+###############################################################################
+rule bowtie2_genome_indexing:
+ # Aim: index sequences in the FASTA format
+ # Use: bowtie2-build [OPTIONS] <reference_in> <bt2_index_base>
+ message:
+ """
+ ~ Bowtie2-build ∞ Index Genome ~
+ Reference: _______ {wildcards.reference}
+ """
+ conda:
+ BOWTIE2
+ resources:
+ cpus = CPUS
+ params:
+ algorithm = BT2_ALGO
+ input:
+ fasta = "resources/genomes/{reference}.fasta"
+ output:
+ prefix = temp("resources/indexes/bowtie2/{reference}"),
+ indexes = multiext("resources/indexes/bowtie2/{ref_seq}",
+ ".1.bt2", ".2.bt2", ".3.bt2", ".4.bt2",
+ ".rev.1.bt2", ".rev.2.bt2")
+ log:
+ "results/10_Reports/tools-log/bowtie2-indexes/{reference}.log"
+ shell:
+ "bowtie2-build " # Bowtie2-build, index sequences
+ "--quiet " # -q: quiet
+ "--threads {resources.cpus} " # Number of threads
+ "{params.algorithm} " # Force (or no by default) generated index to be 'large',
+ # even if ref has fewer than 4 billion nucleotides
+ "-f " # Reference files are FASTA (default)
+ "{input.fasta} " # Reference sequences files (comma-separated list) in the FASTA format
+ "{output.prefix} " # Write bt2 data to files with this dir/basename
+ "&> {log} " # Log redirection
+ "&& touch {output.prefix}" # Touch done
+
+
###############################################################################
################################## TRIMMING ###################################
###############################################################################
diff --git a/workflow/snakefiles/indexing_genomes.smk b/workflow/rules/indexing_genomes.smk
similarity index 100%
rename from workflow/snakefiles/indexing_genomes.smk
rename to workflow/rules/indexing_genomes.smk
diff --git a/workflow/snakefiles/quality_control.smk b/workflow/rules/quality_control.smk
similarity index 100%
rename from workflow/snakefiles/quality_control.smk
rename to workflow/rules/quality_control.smk