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Snakefile
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Snakefile
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from snakemake.utils import R
from snakemake.utils import min_version
import os
min_version("5.3.0") # New location of script executoin for R Markdown report
#report: "report/workflow.rst"
if os.path.isfile("config.yaml"):
configfile: "config.yaml"
# For a bash shell, needed on docker images to activate conda environments with source
# http://snakemake.readthedocs.io/en/stable/project_info/faq.html#i-want-to-configure-the-behavior-of-my-shell-for-all-rules-how-can-that-be-achieved-with-snakemake
shell.executable("/bin/bash")
PROJECT = config["project"] + "/"
rule final:
input: expand("{project}/stats/contaminants.txt \
{project}/{prog}/clst/{ds}.minsize{minsize}.{clmethod}.fasta \
{project}/{prog}/{ds}.minsize{minsize}.{clmethod}.taxonomy.biom \
{project}/report/report.html".split(),data=config["data"],project=config['project'],prog=["vsearch"],ds=config['project'],minsize=config['minsize'],clmethod=config['clustering'])
from snakemake.remote.FTP import RemoteProvider as FTPRemoteProvider
FTP = FTPRemoteProvider()
rule rename:
input:
forward = lambda wildcards: FTP.remote(config["data"][wildcards.data]["path"][0], keep_local=True, immediate_close=True) if config["remote"] else \
config["data"][wildcards.data]["path"][0],
reverse = lambda wildcards: FTP.remote(config["data"][wildcards.data]["path"][1], keep_local=True, immediate_close=True) if config["remote"] else \
config["data"][wildcards.data]["path"][1]
output:
forward=protected("{project}/gunzip/{data}_R1.fastq.gz"),
forward_md5=protected("{project}/gunzip/{data}_R1.fastq.gz.md5"),
reverse=protected("{project}/gunzip/{data}_R2.fastq.gz"),
reverse_md5=protected("{project}/gunzip/{data}_R2.fastq.gz.md5")
params:
prefix="{data}"
threads: 8
run:
if config["convert_to_casava1.8"]:
# BUGFIX: For baseclear data, convert ti casava 1.8 format and add 0 as tag
shell("zcat {input.forward} | awk '{{print (NR%4 == 1) ? \"@{params.prefix}_\" gsub(\"/1$\",\" 1:N:0:0\") substr($0,2) : $0}}' | gzip -c > {output.forward}")
shell("md5sum {output.forward} > {output.forward_md5}")
shell("zcat {input.reverse} | awk '{{print (NR%4 == 1) ? \"@{params.prefix}_\" gsub(\"/2$\",\" 2:N:0:0\") substr($0,2) : $0}}' | gzip -c > {output.reverse}")
shell("md5sum {output.reverse} > {output.reverse_md5}")
else:
shell("zcat {input.forward} | awk '{{print (NR%4 == 1) ? \"@{params.prefix}_\" substr($0,2) : $0}}' | gzip -c > {output.forward}")
shell("md5sum {output.forward} > {output.forward_md5}")
shell("zcat {input.reverse} | awk '{{print (NR%4 == 1) ? \"@{params.prefix}_\" substr($0,2) : $0}}' | gzip -c > {output.reverse}")
shell("md5sum {output.reverse} > {output.reverse_md5}")
rule readstat_raw:
input:
"{project}/gunzip/{data}_R1.fastq.gz",
output:
readstats = temporary("{project}/stats/raw/readstat.{data}.csv"),
readcount = temporary("{project}/stats/raw/readcount.{data}.csv")
params:
sample="{data}"
log:
"{project}/stats/raw/readstat.{data}.log"
conda:
"envs/khmer.yaml"
threads: 1
shell: """
readstats.py {input} --csv -o {output.readstats} 2> {log}
printf "%s\t" {params.sample} > {output.readcount}
tail -n +2 {output.readstats} | cut -d, -f 2 >> {output.readcount}
"""
rule readstat_raw_merge:
input:
expand("{project}/stats/raw/readcount.{data}.csv", project=config['project'], data=config["data"])
output:
protected("{project}/stats/readstat_raw.csv")
shell: """ echo "#SampleID\traw" > {output}
cat {input} >> {output}"""
rule filter_primers:
input:
forward="{project}/gunzip/{data}_R1.fastq.gz",
reverse="{project}/gunzip/{data}_R2.fastq.gz"
output:
forward="{project}/primers/{data}_R1.fastq",
reverse="{project}/primers/{data}_R2.fastq"
params:
forward_primer=config["forward_primer"],
reverse_primer=config["reverse_primer"]
log: "{project}/primers/cutadapt_{data}.log"
conda: "envs/cutadapt.yaml"
shell: """
FW_RC=$(python -c "from Bio.Seq import Seq; primer = Seq('{params.forward_primer}'); print(primer.reverse_complement())")
RV_RC=$(python -c "from Bio.Seq import Seq; primer = Seq('{params.reverse_primer}'); print(primer.reverse_complement())")
cutadapt -g {params.forward_primer} -a "${{RV_RC}}" -G {params.reverse_primer} -A "${{FW_RC}}" -n 2 -o {output.forward} -p {output.reverse} {input} 2>&1 > {log}
"""
rule filter_contaminants:
input:
forward="{project}/primers/{data}_R1.fastq",
reverse="{project}/primers/{data}_R2.fastq"
output:
forward=temporary("{project}/filter/{data}_R1.fastq"),
reverse=temporary("{project}/filter/{data}_R2.fastq"),
stats="{project}/stats/{data}_contaminants_stats.txt"
params:
phix="refs/phix.fasta",
adapters="refs/illumina_scriptseq_and_truseq_adapters.fa",
quality=config["quality_control"]["trimming"]["quality"]
log: "{project}/filter/{data}.log"
conda: "envs/bbmap.yaml"
threads: 16
shell:"""bbduk.sh -Xmx8g in={input.forward} in2={input.reverse} out={output.forward} out2={output.reverse} \
ref={params.adapters},{params.phix} qtrim="rl" trimq={params.quality} threads={threads} stats={output.stats} 2> {log}"""
rule contaminants_stats:
input: expand("{project}/stats/{data}_contaminants_stats.txt", project=config['project'], data=config["data"])
output:
"{project}/stats/contaminants.txt"
shell: "grep '#' -v {input} | tr ':' '\t' > {output} || true"
if config["barcode_in_header"]:
rule remove_barcodes:
input:
forward="{project}/primers/{data}_R1.fastq",
reverse="{project}/primers/{data}_R2.fastq",
output:
barcodes=temp("{project}/barcode/{data}/barcodes.fastq"),
barcodes_fasta=temp("{project}/barcode/{data}/barcodes.fasta"),
forward="{project}/barcode/{data}_R1.fastq",
reverse="{project}/barcode/{data}_R2.fastq",
forward_unpaired="{project}/barcode/{data}_R1_unpaired.fastq",
reverse_unpaired="{project}/barcode/{data}_R2_unpaired.fastq",
params:
outdir="{project}/barcode/{data}/",
threshold=config['quality_control']['barcode']['threshold'],
length=config['quality_control']['barcode']['length'],
sep=config['quality_control']['barcode']['seperator']
log: "{project}/barcode/{data}.log"
conda: "envs/barcode.yaml"
threads: 8
shell: """extract_barcodes.py -f {input.forward} -s{params.sep} -l {params.length} -o {params.outdir} -c barcode_in_label && fastq_to_fasta < {output.barcodes} > {output.barcodes_fasta} && \
trimmomatic PE -threads {threads} -phred33 {input.forward} {input.reverse} {output.forward} {output.forward_unpaired} {output.reverse} {output.reverse_unpaired} ILLUMINACLIP:{output.barcodes_fasta}:0:0:{params.threshold} 2> {log}"""
rule readstat_filter:
input:
"{project}/filter/{data}_R1.fastq",
output:
readstats = temporary("{project}/stats/filter/readstat.{data}_R1.csv"),
readcount = temporary("{project}/stats/filter/readcount.{data}_R1.csv")
params:
sample="{data}"
log:
"{project}/stats/filter/readstat.{data}_R1.log"
conda:
"envs/khmer.yaml"
threads: 1
shell: """
readstats.py {input} --csv -o {output.readstats} 2> {log}
printf "%s\t" {params.sample} > {output.readcount}
tail -n +2 {output.readstats} | cut -d, -f 2 >> {output.readcount}
"""
rule readstat_filter_merge:
input:
expand("{project}/stats/filter/readcount.{data}_R1.csv", project=config['project'], data=config["data"])
output:
protected("{project}/stats/readstat_filter_R1.csv")
shell: """ echo "#SampleID\tfilter" > {output}
cat {input} >> {output}"""
rule readstat_filter_reverse:
input:
"{project}/filter/{data}_R2.fastq",
output:
temporary("{project}/stats/filter/readstat.{data}_R2.csv")
log:
"{project}/stats/filter/readstat.{data}_R2.log"
conda:
"envs/khmer.yaml"
threads: 1
shell: "readstats.py {input} --csv -o {output} 2> {log}"
rule readstat_reverse_merge:
input:
expand("{project}/stats/filter/readstat.{data}_R2.csv", project=config['project'], data=config["data"])
output:
protected("{project}/stats/readstat_filter_R2.csv")
shell: "cat {input[0]} | head -n 1 > {output} && for file in {input}; do tail -n +2 $file >> {output}; done;"
rule fastqc:
input:
forward="{project}/gunzip/{data}_R1.fastq",
reverse="{project}/gunzip/{data}_R2.fastq"
output:
zip="{project}/fastqc_raw/{data}_R1_fastqc.zip"
params:
dir="{project}/fastqc_raw",
adapters = config["adapters_fasta"]
log: "{project}/fastqc_raw/{data}.log"
threads: 2
conda: "envs/fastqc.yaml"
shell: "fastqc -q -t {threads} --contaminants {params.adapters} --outdir {params.dir} {input.forward} > {params.dir}/{log} && fastqc -q -t {threads} --contaminants {params.adapters} --outdir {params.dir} {input.reverse} > {params.dir}/{log}"
if config['mergepairs'] == 'none':
rule copy_forward:
input:
forward="{project}/barcode/{data}_R1.fastq" if config["barcode_in_header"] else\
"{project}/filter/{data}_R1.fastq",
output:
fasta = temporary("{project}/mergepairs/{data}.fasta")
conda: "envs/barcode.yaml"
shell: "fastq_to_fasta < {input} > {output}"
if config['mergepairs'] == 'pandaseq':
rule pandaseq:
input:
forward="{project}/barcode/{data}_R1.fastq" if config["barcode_in_header"] else\
"{project}/filter/{data}_R1.fastq",
reverse="{project}/barcode/{data}_R2.fastq" if config["barcode_in_header"] else\
"{project}/filter/{data}_R2.fastq",
output:
fasta = temporary("{project}/mergepairs/{data}.fasta")
params:
overlap = config['pandaseq_overlap'],
quality = config['pandaseq_quality'],
minlength = config['pandaseq_minlength'],
maxlength = config['pandaseq_maxlength'],
forward_primer = config['forward_primer'],
reverse_primer = config['reverse_primer']
log: "{project}/mergepairs/{data}_pandaseq.stdout"
threads: 1
conda: "envs/pandaseq.yaml"
shell: "pandaseq -N -o {params.overlap} -l {params.minlength} -L {params.maxlength} -f {input.forward} -r {input.reverse} -T {threads} -w {output.fasta} -g {log}"
rule fastqc_pandaseq:
input:
fastq = "{project}/mergepairs/{data}.fastq"
output:
dir="{project}/mergepairs/{data}_fastqc/",zip="{project}/fastqc_pandaseq/{data}_fastqc.zip"
params:
dir="{project}/fastqc_pandaseq",
adapters = config["adapters_fasta"]
log: "{project}/mergepairs/fastqc_{data}.log"
threads: 8
conda: "envs/fastqc.yaml"
shell: "fastqc -q -t {threads} --contaminants {params.adapters} --outdir {params.dir} {input.fastq} > {params.dir}/{log}"
rule readstat_mergepairs:
input:
fasta = "{project}/mergepairs/{data}.fasta"
output:
readstats = temporary("{project}/stats/mergepairs/readstat_mergepairs.{data}.csv"),
readcount = temporary("{project}/stats/mergepairs/readcount_mergepairs.{data}.csv")
params:
sample="{data}"
log:
"{project}/stats/readstat_mergepairs.{data}.log"
conda:
"envs/khmer.yaml"
threads: 1
shell: """
readstats.py {input} --csv -o {output.readstats} 2> {log}
printf "%s\t" {params.sample} > {output.readcount}
tail -n +2 {output.readstats} | cut -d, -f 2 >> {output.readcount}
"""
rule readstat_mergepairs_merge:
input:
expand("{project}/stats/mergepairs/readcount_mergepairs.{data}.csv", project=config['project'], data=config["data"])
output:
protected("{project}/stats/readstat_mergepairs.csv")
shell: """ echo "#SampleID\tmerged" > {output}
cat {input} >> {output}"""
if config['mergepairs'] == 'vsearch':
rule vsearch_merge:
input:
forward="{project}/barcode/{data}_R1.fastq" if config["barcode_in_header"] else\
"{project}/filter/{data}_R1.fastq",
reverse="{project}/barcode/{data}_R2.fastq" if config["barcode_in_header"] else\
"{project}/filter/{data}_R2.fastq",
output:
fasta = temporary("{project}/mergepairs/{data}.fasta")
params:
minmergelen=config['vsearch_minmergelen']
log: "{project}/mergepairs/{data}.log"
threads: 1
conda: "envs/vsearch.yaml"
shell: "vsearch --threads {threads} --fastq_mergepairs {input.forward} --reverse {input.reverse} --fastq_allowmergestagger --fastq_minmergelen {params.minmergelen} --fastaout {output} 2>&1 > {log}"
if config['its'] == True:
rule extract_its:
input:
fasta="{project}/mergepairs/{data}.fasta"
output:
fasta="{project}/itsx/{data}.ITS1.fasta" if config['its_region'] == 'ITS1' else "{project}/itsx/{data}.ITS2.fasta" if config['its_region'] == 'ITS2' else "{project}/itsx/{data}.full.fasta"
params:
basename="{project}/itsx/{data}",
dir="{project}/itsx"
log: "{project}/itsx/itsx{data}.log"
threads: 32
conda: "envs/itsx.yaml"
# TODO: Filter on specific list of organisms?
# Only ITS2 region?
shell: "ITSx --cpu {threads} --preserve TRUE -i {input.fasta} -o {params.basename} 2> {log}"
# Combine per sample files to a single project file
rule mergefiles:
input:
fasta = expand(rules.extract_its.output, data=config["data"], project=config['project']) if config['its'] \
else expand(PROJECT + "mergepairs/{data}.fasta", data=config["data"]),
output:
fasta=temporary("{project}/mergefiles/{project}.fasta")
params:
samples=config["data"]
shell: """cat {input} > {output}"""
rule length:
input:
"{project}/mergefiles/{project}.fasta"
output:
protected("{project}/stats/readlength.csv")
shell: "awk -f ../src/hydra/seqlen.awk {input} > {output}"
# Dereplication
rule derep:
input:
"{project}/mergefiles/{ds}.fasta"
output:
temporary("{project}/{prog}/{ds}.derep.fasta")
threads: 8
conda: "envs/vsearch.yaml"
shell: "vsearch -derep_fulllength {input} -output {output} -sizeout -threads {threads}"
# Abundance sort and discard singletons
rule sortbysize:
input:
"{project}/{prog}/{ds}.derep.fasta"
output:
temporary("{project}/{prog}/{ds}.sorted.minsize{minsize}.fasta")
params:
minsize="{minsize}"
threads: 8
conda: "envs/vsearch.yaml"
shell: "vsearch -sortbysize {input} -output {output} -minsize {params.minsize}"
if config['clustering'] == "usearch_smallmem":
# Uclust clustering
rule smallmem:
input:
"{project}/{prog}/{ds}.sorted.minsize{minsize}.fasta"
output:
otus=protected("{project}/{prog}/clst/{ds}.minsize{minsize}.usearch_smallmem.fasta")
threads: 8
conda: "envs/vsearch.yaml"
shell: "vsearch --cluster_smallmem {input} --usersort -centroids {output.otus} --id 0.97 -sizeout"
#
# Swarm
#
if config['clustering'] == "swarm":
# Swarm
rule swarm:
input:
"{project}/{prog}/{ds}.sorted.minsize{minsize}.fasta"
output:
swarms="{project}/{prog}/{ds}.minsize{minsize}.swarm.swarms",
stats="{project}/{prog}/{ds}.minsize{minsize}.swarm.stats"
params: d="1"
threads: 16
conda: "envs/swarm.yaml"
shell: "swarm -d {params.d} -t {threads} -z -u uclust.out -s {output.stats} < {input} > {output.swarms}"
rule swarm_get_seed:
input:
swarms="{project}/{prog}/{ds}.minsize{minsize}.swarm.swarms",
amplicons="{project}/{prog}/{ds}.sorted.minsize{minsize}.fasta"
output:
seeds="{project}/{prog}/clst/{ds}.minsize{minsize}.swarm.fasta"
shell: "SEEDS=$(mktemp); cut -d ' ' -f 1 {input.swarms} | sed -e 's/^/>/' > '${{SEEDS}}'; grep -A 1 -F -f '${{SEEDS}}' {input.amplicons} | sed -e '/^--$/d' > {output.seeds}"
#
# Chimera checking
#
rule uchime:
input:
"{project}/{prog}/clst/{ds}.minsize{minsize}.{clmethod}.fasta"
output:
chimeras="{project}/{prog}/uchime/{ds}.minsize{minsize}.{clmethod}.chimeras",
nonchimeras="{project}/{prog}/uchime/{ds}.minsize{minsize}.{clmethod}.fasta"
log: "{project}/{prog}/uchime/{ds}.minsize{minsize}.{clmethod}.uchime.log"
conda: "envs/vsearch.yaml"
shell: "vsearch --uchime_denovo {input} --nonchimeras {output.nonchimeras} --chimeras {output.chimeras} > {log}"
#
# Mapping
#
rule make_otu_names:
input:
"{project}/{prog}/uchime/{ds}.minsize{minsize}.{clmethod}.fasta"
output:
"{project}/{prog}/otus/{ds}.minsize{minsize}.{clmethod}.fasta"
conda: "envs/qiime.yaml"
shell: "python2.7 uparse_scripts/fasta_number.py {input} OTU_ > {output}"
rule mapping:
input:
otus="{project}/{prog}/otus/{ds}.minsize{minsize}.{clmethod}.fasta",
reads="{project}/mergefiles/{ds}.fasta"
output:
"{project}/{prog}/otus/{ds}.minsize{minsize}.{clmethod}.uc"
conda: "envs/vsearch.yaml"
threads: 32
shell: "vsearch -usearch_global {input.reads} -threads {threads} -db {input.otus} -strand plus -id 0.97 -uc {output}"
rule create_otutable:
input:
"{project}/{prog}/otus/{ds}.minsize{minsize}.{clmethod}.uc"
output:
"{project}/{prog}/otus/{ds}.minsize{minsize}.{clmethod}.otutable.txt"
conda: "envs/qiime.yaml"
shell: "python2.7 uparse_scripts/uc2otutab.py {input} > {output}"
# convert to biom file
rule biom_otu:
input:
"{project}/{prog}/otus/{ds}.minsize{minsize}.{clmethod}.otutable.txt"
output:
"{project}/{prog}/otus/{ds}.minsize{minsize}.{clmethod}.biom"
conda: "envs/biom-format.yaml"
shell: "biom convert -i {input} --to-json -o {output} --table-type='OTU table'"
#
# Taxonomy
#
if config["classification"] == "sina":
rule download_silva_arb:
output: temporary("SILVA_132_SSURef_NR99_13_12_17_opt.arb")
shell: """
RELEASE=132
URL="https://www.arb-silva.de/fileadmin/silva_databases/release_${{RELEASE}}/ARB_files"
FILE="SILVA_${{RELEASE}}_SSURef_NR99_13_12_17_opt.arb.gz"
# Download and check
wget -c ${{URL}}/${{FILE}}{{,.md5}} && md5sum -c ${{FILE}}.md5
gunzip ${{FILE}}
"""
rule create_index_sina:
input:
"SILVA_132_SSURef_NR99_13_12_17_opt.arb"
output:
temporary("SILVA_132_SSURef_NR99_13_12_17_opt.arb.index.arb"),
temporary("SILVA_132_SSURef_NR99_13_12_17_opt.arb.index.arb.pt"),
temporary("SILVA_132_SSURef_NR99_13_12_17_opt.arb.index.ARM"),
temporary("SILVA_132_SSURef_NR99_13_12_17_opt.arb.index.ARF")
conda: "envs/sina.yaml"
shell: """
cp SILVA_132_SSURef_NR99_13_12_17_opt.arb SILVA_132_SSURef_NR99_13_12_17_opt.arb.index.arb
arb_pt_server -build_clean -DSILVA_132_SSURef_NR99_13_12_17_opt.arb.index.arb
arb_pt_server -build -DSILVA_132_SSURef_NR99_13_12_17_opt.arb.index.arb
"""
rule sina:
input:
fasta="{project}/{prog}/otus/{ds}.minsize{minsize}.{clmethod}.fasta",
arb="SILVA_132_SSURef_NR99_13_12_17_opt.arb",
index="SILVA_132_SSURef_NR99_13_12_17_opt.arb.index.arb",
pt="SILVA_132_SSURef_NR99_13_12_17_opt.arb.index.arb.pt",
arm="SILVA_132_SSURef_NR99_13_12_17_opt.arb.index.ARM",
arf="SILVA_132_SSURef_NR99_13_12_17_opt.arb.index.ARF"
output:
csv="{project}/{prog}/sina/{ds}.minsize{minsize}.{clmethod}.sina.csv",
align="{project}/{prog}/sina/{ds}.minsize{minsize}.{clmethod}.sina.align",
priority: -1
threads: 24
# TODO: turn is set to all to get classification. Reverse the reads in earlier stage!
log: "{project}/{prog}/sina/{ds}.minsize{minsize}.{clmethod}.sina.log"
conda: "envs/sina.yaml"
shell: "sina --log-file {log} -i {input.fasta} --intype fasta -o {output.align} --outtype fasta --meta-fmt csv --db {input.arb} --overhang remove --turn all --search --search-db {input.arb} --search-min-sim 0.95 --search-no-fast --search-kmer-len 10 --lca-fields tax_slv --num-pts {threads} || true"
rule sina_get_taxonomy:
input:
csv="{project}/{prog}/sina/{ds}.minsize{minsize}.{clmethod}.sina.csv"
output:
taxonomy="{project}/{prog}/sina/{ds}.minsize{minsize}.{clmethod}.sina.taxonomy.txt"
shell: "tail -n +2 {input.csv} | cut -d, -f 1,10 | tr , '\\t' > {output.taxonomy}"
# Tree
rule filter_alignment:
input:
align="{project}/{prog}/sina/{ds}.minsize{minsize}.{clmethod}.sina.align"
output:
filtered="{project}/{prog}/sina/{ds}.minsize{minsize}.{clmethod}.sina_pfiltered.fasta"
params:
outdir="{project}/{prog}/sina/"
conda: "envs/qiime.yaml"
shell: "filter_alignment.py -i {input.align} -o {params.outdir} --suppress_lane_mask_filter --entropy_threshold 0.10"
rule make_tree:
input:
align="{project}/{prog}/sina/{ds}.minsize{minsize}.{clmethod}.sina_pfiltered.fasta"
output:
tree="{project}/{prog}/{ds}.minsize{minsize}.{clmethod}.tre"
conda: "envs/qiime.yaml"
shell: "make_phylogeny.py -i {input.align} -t fasttree -o {output.tree}"
rule sina_convert_tax:
input:
taxonomy="{project}/{prog}/sina/{ds}.minsize{minsize}.{clmethod}.sina.taxonomy.txt",
output:
taxonomy="{project}/{prog}/sina/{ds}.minsize{minsize}.{clmethod}.sina.qiimeformat.taxonomy",
run:
if config["use_full_lineage"]:
shell("""awk -F"[;\t]" '{{printf $1"\t"; for(i=2;i<NF;i++){{printf i-1"__%s;", $i}}; printf "\\n"}}' {input.taxonomy} > {output.taxonomy}""")
else:
shell("""cat {input.taxonomy} | awk -F"[;\t]" 'BEGIN{{print "OTUs,Domain,Phylum,Class,Order,Family,Genus"}}{{print $1"\\tk__"$2"; p__"$3"; c__"$4"; o__"$5"; f__"$6"; g__"$7"; s__"$8}}' > {output.taxonomy}""")
rule biom_tax_sina:
input:
taxonomy="{project}/{prog}/sina/{ds}.minsize{minsize}.{clmethod}.sina.qiimeformat.taxonomy",
biom="{project}/{prog}/otus/{ds}.minsize{minsize}.{clmethod}.biom",
meta=config["metadata"]
output:
biom=protected("{project}/{prog}/{ds}.minsize{minsize}.{clmethod}.taxonomy.biom"),
otutable=protected("{project}/{prog}/{ds}.minsize{minsize}.{clmethod}.taxonomy.otutable.txt")
conda: "envs/biom-format.yaml"
shell: """
biom add-metadata -i {input.biom} -o {output.biom} --observation-metadata-fp {input.taxonomy} --observation-header OTUID,taxonomy --sc-separated taxonomy --float-fields confidence --sample-metadata-fp {input.meta} --output-as-json && \
biom convert --to-tsv --header-key=taxonomy -i {output.biom} -o {output.otutable}
"""
if config["classification"] == "stampa":
rule download_silva:
output:
"SILVA_128_SSURef_tax_silva_trimmed.fasta"
params:
forward_primer=config["forward_primer"],
reverse_primer=config["reverse_primer"]
conda: "envs/cutadapt.yaml"
# Download script adapted from https://github.com/frederic-mahe/stampa
shell: """
RELEASE=128
URL="https://www.arb-silva.de/fileadmin/silva_databases/release_${{RELEASE}}/Exports"
FILE="SILVA_${{RELEASE}}_SSURef_tax_silva.fasta.gz"
# Download and check
wget -c ${{URL}}/${{FILE}}{{,.md5}} && md5sum -c ${{FILE}}.md5
# Define variables and output files
INPUT="SILVA_${{RELEASE}}_SSURef_tax_silva.fasta.gz"
OUTPUT="${{INPUT/.fasta.gz/_trimmed.fasta}}"
LOG="${{INPUT/.fasta.gz/_trimmed.log}}"
PRIMER_F={params.forward_primer}
PRIMER_R={params.reverse_primer}
MIN_LENGTH=32
MIN_F=$(( ${{#PRIMER_F}} * 2 / 3 ))
MIN_R=$(( ${{#PRIMER_R}} * 2 / 3 ))
CUTADAPT="cutadapt --discard-untrimmed --minimum-length ${{MIN_LENGTH}}"
# Trim forward & reverse primers, format
zcat "${{INPUT}}" | sed '/^>/ ! s/U/T/g' | \
${{CUTADAPT}} -g "${{PRIMER_F}}" -O "${{MIN_F}}" - 2> "${{LOG}}" | \
${{CUTADAPT}} -a "${{PRIMER_R}}" -O "${{MIN_F}}" - 2>> "${{LOG}}" | \
sed '/^>/ s/;/|/g ; /^>/ s/ /_/g ; /^>/ s/_/ /1' > "${{OUTPUT}}"
"""
rule download_unite:
output:
"UNITE-7.2-28.06.2017.fasta"
params:
forward_primer=config["forward_primer"],
reverse_primer=config["reverse_primer"]
conda: "envs/cutadapt.yaml"
shell:"""
RELEASE=7.2
DATE=28.06.2017
URL="https://unite.ut.ee/sh_files/"
FILE="UNITE_public_${{DATE}}.fasta.zip"
# Download and check
wget -c ${{URL}}/${{FILE}}
unzip ${{FILE}}
# Define variables and output files
PRIMER_F={params.forward_primer}
PRIMER_R={params.reverse_primer}
FNAME=ITS9
RNAME=ITS4
INPUT="UNITE_public_${{DATE}}.fasta"
OUTPUT="UNITE-${{RELEASE}}-${{DATE}}.fasta"
LOG="UNITE-${{RELEASE}}_${{DATE}}.log"
MIN_LENGTH=32
MIN_F=$(( ${{#PRIMER_F}} * 2 / 3 ))
MIN_R=$(( ${{#PRIMER_R}} * 2 / 3 ))
CUTADAPT="cutadapt --discard-untrimmed --minimum-length ${{MIN_LENGTH}}"
# Trim forward & reverse primers, format
cat "${{INPUT}}" | sed '/^>/ ! s/U/T/g' | \
${{CUTADAPT}} -g "${{PRIMER_F}}" -O "${{MIN_F}}" - 2> "${{LOG}}" | \
${{CUTADAPT}} -a "${{PRIMER_R}}" -O "${{MIN_F}}" - 2>> "${{LOG}}" | \
sed 's/\ /+/'g | sed 's/|/\ /'g | sed 's/;/|/g' | \
awk '/^>/ {{env=index($0,"s__Fungi_sp");}} {{if (!env) print;}}' | \
awk '/^>/ {{env=index($0,"k__Fungi");}} {{if (env) print;}}' > "${{OUTPUT}}"
"""
rule stampa:
input:
fasta="{project}/{prog}/otus/{ds}.minsize{minsize}.{clmethod}.fasta",
db = "SILVA_128_SSURef_tax_silva_trimmed.fasta" if config["reference_db"] == "silva" else "UNITE_public_28.06.2017.fasta" if config["reference_db"] == "unite" else None,
output:
swarm="{project}/{prog}/stampa/{ds}.minsize{minsize}.{clmethod}.fasta",
hits="{project}/{prog}/stampa/hits.{ds}.minsize{minsize}.{clmethod}_usearch_global",
results="{project}/{prog}/stampa/results.{ds}.minsize{minsize}.{clmethod}_usearch_global",
taxonomy="{project}/{prog}/stampa/{ds}.minsize{minsize}.{clmethod}.taxonomy.txt",
params:
stampadir="{project}/{prog}/stampa/",
conda: "envs/vsearch.yaml"
threads: 32
# Create STAMPA compatible input
# Replace underscore in otu names and add fake abundance information
shell:"""
sed 's/_/:/' {input.fasta} | awk '/^>/ {{$0=\">\" substr($0,2) \"_1\"}}1' > {output.swarm} && \
vsearch --usearch_global {output.swarm} --threads {threads} --dbmask none --qmask none --rowlen 0 --notrunclabels --userfields query+id1+target --maxaccepts 0 --maxrejects 32 --top_hits_only --output_no_hits --db {input.db} --id 0.5 --iddef 1 --userout {output.hits} && \
python2.7 stampa_merge.py {params.stampadir} && \
sed 's/:/_/' {output.results} | sed 's/|/;/g' | cut -f 1,4 > {output.taxonomy}
"""
rule biom_tax_stampa:
input:
taxonomy="{project}/{prog}/stampa/{ds}.minsize{minsize}.{clmethod}.taxonomy.txt",
biom="{project}/{prog}/otus/{ds}.minsize{minsize}.{clmethod}.biom",
meta=config["metadata"]
output:
taxonomy="{project}/{prog}/stampa/{ds}.minsize{minsize}.{clmethod}.taxonomy.qiimeformat.txt",
biom="{project}/{prog}/{ds}.minsize{minsize}.{clmethod}.taxonomy.biom",
otutable="{project}/{prog}/{ds}.minsize{minsize}.{clmethod}.taxonomy.otutable.txt"
conda: "envs/biom-format.yaml"
shell: """cat {input.taxonomy} | awk -F"[;\t]" 'BEGIN{{print "OTUs,Domain,Phylum,Class,Order,Family,Genus"}}{{print $1"\\tk__"$2"; p__"$3"; c__"$4"; o__"$5"; f__"$6"; g__"$7"; s__"$8}}' > {output.taxonomy} && \
biom add-metadata -i {input.biom} -o {output.biom} --observation-metadata-fp {output.taxonomy} --observation-header OTUID,taxonomy --sc-separated taxonomy --float-fields confidence --sample-metadata-fp {input.meta} --output-as-json && \
biom convert --to-tsv --header-key=taxonomy -i {output.biom} -o {output.otutable}
"""
if config["classification"] == "blast":
rule blastn:
input:
"{project}/{prog}/otus/{ds}.minsize{minsize}.{clmethod}.fasta"
output:
hits="{project}/{prog}/blast/{ds}.minsize{minsize}.{clmethod}.blastout.txt",
params:
db = config["blast_db"],
max_hits = config["blast_max_hits"]
threads: 32
conda: "envs/blast.yaml"
shell: """blastn -query {input} -db {params.db} -evalue 1e-5 -outfmt "6 qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore" -out {output.hits} -num_threads {threads} -max_target_seqs {params.max_hits}"""
rule lca:
input:
hits="{project}/{prog}/blast/{ds}.minsize{minsize}.{clmethod}.blastout.txt",
fasta = "{project}/{prog}/otus/{ds}.minsize{minsize}.{clmethod}.fasta"
output:
lca="{project}/{prog}/blast/{ds}.minsize{minsize}.{clmethod}.lca.txt",
taxonomy="{project}/{prog}/blast/{ds}.minsize{minsize}.{clmethod}.taxonomy.txt"
params:
taxref = "/data/db/pr2/gb203/lotus_lca.tax",
run:
shell("./LCA -i {input.hits} -r {params.taxref} -o {output.lca} -id '99,96,93,91,88,78,0'")
shell("""cat {output.lca} | awk -F"\t" 'BEGIN{{}}{{gsub(" ","_",$0);gsub("\\"","",$0);print $1"\\td__"$2";p__"$3";c__"$4";o__"$5";f_"$6";g__"$7";s__"$8}}' > {output.taxonomy}""")
# Detect OTUs without blast hit
hits = []
with open(output.lca) as fh:
for line in fh:
line = line.strip().split("\t")
hits.append(line[0])
with open(input.fasta) as fasta_file, open(output.taxonomy, "a") as outfile:
for line in fasta_file:
line = line.strip()
if line.startswith(">"):
if line[1:] not in hits:
outfile.write("%s\tk__?;p__?;c__?;o__?;f__?;g__?;s__?\n" % line[1:])
rule biom_tax_blast:
input:
taxonomy="{project}/{prog}/blast/{ds}.minsize{minsize}.{clmethod}.taxonomy.txt",
biom="{project}/{prog}/otus/{ds}.minsize{minsize}.{clmethod}.biom",
meta=config["metadata"]
output:
biom="{project}/{prog}/{ds}.minsize{minsize}.{clmethod}.taxonomy.biom",
otutable="{project}/{prog}/{ds}.minsize{minsize}.{clmethod}.taxonomy.otutable.txt"
conda: "envs/biom-format.yaml"
shell: """biom add-metadata -i {input.biom} -o {output.biom} --observation-metadata-fp {input.taxonomy} --observation-header OTUID,taxonomy --sc-separated taxonomy --float-fields confidence --sample-metadata-fp {input.meta} --output-as-json && \
biom convert --to-tsv --header-key=taxonomy -i {output.biom} -o {output.otutable}
"""
if config["classification"] == "rdp":
rule rdp:
input:
fasta = "{project}/{prog}/otus/{ds}.minsize{minsize}.{clmethod}.fasta"
output:
"{project}/{prog}/rdp/{ds}.minsize{minsize}.{clmethod}.rdp"
params:
traindir="/data/db/unite/UNITE_retrained/"
conda: "envs/rdp.yaml"
shell: "classifier classify -Xms512M -Xmx8g -t {params.traindir}/rRNAClassifier.properties -o {output} {input}"
rule rdp_filter:
input:
rdpout = "{project}/{prog}/rdp/{ds}.minsize{minsize}.{clmethod}.rdp",
fasta = "{project}/{prog}/otus/{ds}.minsize{minsize}.{clmethod}.fasta"
output:
"{project}/{prog}/rdp/{ds}.minsize{minsize}.{clmethod}.filtered.rdp"
params:
cutoff = config["rdp_confidence_cutoff"]
run:
hits = []
with open(input.rdpout) as rdpout, open(output[0], "w") as outfile:
for line in rdpout:
parts = line.strip().split("\t")
tax_list = []
for i in range(5, len(parts), 3):
confidence = float(parts[i+2])
taxonomy = parts[i].split("|")[-1].replace(" ", "_")
tax_level = parts[i+1]
if tax_level == "domain" or tax_level == "kingdom":
tax_prefix = "k__"
elif tax_level == "phylum":
tax_prefix = "p__"
elif tax_level == "class":
tax_prefix = "c__"
elif tax_level == "order":
tax_prefix = "o__"
elif tax_level == "family":
tax_prefix = "f__"
elif tax_level == "genus":
tax_prefix = "g__"
elif tax_level == "species":
tax_prefix = "s__"
if confidence > float(params.cutoff):
tax_list.append(tax_prefix + taxonomy)
else:
tax_list.append(tax_prefix + "unclassified")
tax_string = ";".join(tax_list)
outfile.write("%s\t%s\n" % (parts[0],tax_string))
# Keep track of OTUs with classification
hits.append(parts[0])
# Add OTUs without RDP classification
with open(input.fasta) as fasta_file, open(output[0], "a") as outfile:
for line in fasta_file:
line = line.strip()
if line.startswith(">"):
if line[1:] not in hits:
outfile.write("%s\td__unclassified;p__unclassified;c__unclassified;o__unclassified;f__unclassified;g__unclassified;s__unclassified\n" % line[1:])
rule biom_tax_rdp:
input:
biom="{project}/{prog}/otus/{ds}.minsize{minsize}.{clmethod}.biom",
taxonomy="{project}/{prog}/rdp/{ds}.minsize{minsize}.{clmethod}.filtered.rdp",
meta=config["metadata"]
output:
biom="{project}/{prog}/{ds}.minsize{minsize}.{clmethod}.taxonomy.biom",
otutable="{project}/{prog}/{ds}.minsize{minsize}.{clmethod}.taxonomy.otutable.txt"
conda: "envs/biom-format.yaml"
shell: """biom add-metadata -i {input.biom} -o {output.biom} --observation-metadata-fp {input.taxonomy} --observation-header OTUID,taxonomy --sc-separated taxonomy --float-fields confidence --sample-metadata-fp {input.meta} --output-as-json && \
biom convert --to-tsv --header-key=taxonomy -i {output.biom} -o {output.otutable}
"""
rule workflow_graph:
output: temporary("{project}/report/workflow.svg")
conda: "envs/rulegraph.yaml"
shell: "snakemake --rulegraph | dot -Tsvg > {output}"
rule combine_readcount:
input:
"{project}/stats/readstat_raw.csv",
"{project}/stats/readstat_filter_R1.csv",
"{project}/stats/readstat_mergepairs.csv"
output:
"{project}/stats/readcount.csv"
shell: "paste -d '\\t' {input} | cut -f 1,2,4,6 > {output}"
rule report:
input:
workflow = "{project}/report/workflow.svg",
readstat = "{project}/stats/readcount.csv",
biom = expand("{{project}}/{prog}/{ds}.minsize{minsize}.{clmethod}.taxonomy.biom", prog=["vsearch"],ds=config['project'],minsize=2,clmethod=config['clustering']),
otutable = expand("{{project}}/{prog}/{ds}.minsize{minsize}.{clmethod}.taxonomy.otutable.txt", prog=["vsearch"],ds=config['project'],minsize=2,clmethod=config['clustering']),
otus= expand("{{project}}/{prog}/otus/{ds}.minsize{minsize}.{clmethod}.fasta", prog=["vsearch"],ds=config['project'],minsize=2,clmethod=config['clustering']),
output:
"{project}/report/report.html"
params:
prefix="{project}/report/report",
mergemethod = config['mergepairs']
conda: "envs/report.yaml"
script:
"report.Rmd"