new modifications

config.json

 {
-	"THREADS": 5,
-	"SAMPLES": ["SRR5625858", "SRR5625859", "SRR5625860", "SRR5625864", "SRR5625861" ],
-	"FIVE_PRIMER": {"SRR5625858": "GTGYCAGCMGCCGCGGTA", "SRR5625859": "GTGYCAGCMGCCGCGGTA", "SRR5625860": "GTGYCAGCMGCCGCGGTA", "SRR5625864": "GTGYCAGCMGCCGCGGTA", "SRR5625861": "GTGYCAGCMGCCGCGGTA" },
-	"THREE_PRIMER": {"SRR5625858": "ACTYAAAKGAATTGRCGGGG", "SRR5625859": "ACTYAAAKGAATTGRCGGGG", "SRR5625860": "ACTYAAAKGAATTGRCGGGG", "SRR5625864": "ACTYAAAKGAATTGRCGGGG", "SRR5625861": "ACTYAAAKGAATTGRCGGGG"},
+	"THREADS": 20,
+	"unit": ["R1", "R2"],
+	"primer": ["fwd", "rev"],
 	"DATABASE": "/db/frogs_databanks/assignation/16S/silva_132_16S_pintail100/silva_132_16S_pintail100.fasta"
 }
\ No newline at end of file

dada2.R

@@ -2,9 +2,9 @@ library(dada2)
 
 derepF <- derepFastq(snakemake@input[[1]], verbose = TRUE)
 derepR <- derepFastq(snakemake@input[[2]], verbose = TRUE)
-errF <- learnErrors(snakemake@input[[1]], multithread = snakemake@threads, verbose = TRUE)
-errR <- learnErrors(snakemake@input[[2]], multithread = snakemake@threads, verbose = TRUE)
+errF <- learnErrors(snakemake@input[[1]], multithread = snakemake@threads, randomize = TRUE, verbose = TRUE)
+errR <- learnErrors(snakemake@input[[2]], multithread = snakemake@threads, randomize = TRUE, verbose = TRUE)
 dadaFs <- dada(derepF, err = errF, multithread = snakemake@threads, verbose = TRUE)
 dadaRs <- dada(derepR, err = errR, multithread = snakemake@threads, verbose = TRUE)
 mergers <- mergePairs(dadaFs, derepF, dadaRs, derepR, verbose=TRUE)
-saveRDS(mergers, snakemake@output[[1]])
+saveRDS(mergers, snakemake@output[[1]])
\ No newline at end of file

dada2.smk

 rule dada2:
 	input:
-		filt = "work/filter/{sample}_1.fastq.gz",
-		filtrev = "work/filter/{sample}_2.fastq.gz"
+		filt = "work/filter/{sample}_R1.fastq.gz",
+		filtrev = "work/filter/{sample}_R2.fastq.gz"
 	output:
 		"work/dada/{sample}.rds"
 	threads:

filterAndTrim.R

 library(dada2)
-
-filterAndTrim(snakemake@input[[1]],snakemake@output[[1]], snakemake@input[[2]], snakemake@output[[2]], maxN=0, rm.phix=TRUE, compress=TRUE, verbose = TRUE)
\ No newline at end of file
+library("ShortRead")
+library(Matrix)
+filterAndTrim(snakemake@input[[1]],snakemake@output[[1]], snakemake@input[[2]], snakemake@output[[2]], truncQ=2, truncLen=0, maxN=0, maxEE=Inf, rm.phix=TRUE, compress=TRUE, verbose = TRUE)
\ No newline at end of file

global.smk

@@ -3,14 +3,26 @@ shell.prefix("source /usr/local/genome/Anaconda2-5.1.0/etc/profile.d/conda.sh;")
 
 configfile: "./config.json"
 
-SAMPLES=config["SAMPLES"]
+#SAMPLES=config["SAMPLES"]
 unit=config["unit"]
+primer=config["primer"]
 
+import os
+import pandas as pd
+sample_table_file=config.get('sampletable','samples.tsv')
+SampleTable = pd.read_table(sample_table_file,index_col=0)
+SAMPLES = list(SampleTable.index)
+
+JAVA_MEM_FRACTION=0.85
+CONDAENV ='envs'
+PAIRED_END= ('R2' in SampleTable.columns)
+FRACTIONS= ['R1']
+if PAIRED_END: FRACTIONS+= ['R2']
 rule all:
 	input:#expand("DATA/{sample}.fastq.gz", sample=SAMPLES),
-		"report/multiqc_report.html",
-		expand("work/filter/{sample}_1.fastq.gz", sample=SAMPLES),
-		expand("work/filter/{sample}_2.fastq.gz", sample=SAMPLES),
+		#"report/multiqc_report.html",
+		#expand("work/filter/{sample}_R1.fastq.gz", sample=SAMPLES),
+		#expand("work/filter/{sample}_R2.fastq.gz", sample=SAMPLES),
 		expand("work/dada/{sample}.rds", sample=SAMPLES),
 		"work/dada/seqtab.fasta",
 		"work/dada/seqtab.tsv",
@@ -23,7 +35,7 @@ rule all:
 		#"report/tree.nwk"
 
 #include: "prefetech.smk"
-include: "quality.smk"
-include: "preprocess.smk"
-include: "dada2.smk"
-#include: "affiliation.smk"
+#include: "quality.smk"
+#include: "revcomp_primer.smk"
+#include: "preprocess.smk"
+include: "dada2.smk"
\ No newline at end of file

preprocess.smk

-rule cutadapt:
-	input:
-		fwd = "DATA/{sample}_1.fastq.gz",
-		rev = "DATA/{sample}_2.fastq.gz"
-	output:
-		cut = "work/cutadapt/{sample}_1.fastq.gz",
-		cutrev = "work/cutadapt/{sample}_2.fastq.gz"
-	params:
-		five = lambda wildcards: config["FIVE_PRIMER"][wildcards.sample],
-		three = lambda wildcards: config["THREE_PRIMER"][wildcards.sample]
-	shell:
-		"conda activate cutadapt-2.5 "
-		"&& "
-		"cutadapt "
-		"-g {params.five} "
-		"-a {params.three} "
-		"-G {params.five} "
-		"-A {params.three} "
-		"--error-rate 0.1 "
-		"--discard-untrimmed "
-		"--match-read-wildcards "
-		"-o {output.cut} "
-		"-p {output.cutrev} "
-		"{input} "
-		"&& "
-		"conda deactivate"
+rule cutadapt_pe:
+    input:
+        fwd='DATA/{sample}_R1.fastq.gz',
+        rev='DATA/{sample}_R2.fastq.gz',
+        fwd_prime='Primers/fwd_primers.fasta',
+        rev_prime='Primers/rev_primers.fasta',
+        fwd_revcomp_prime='Primers/fwd_revcomp_primers.fasta',
+        rev_revcomp_prime='Primers/rev_revcomp_primers.fasta'
+    output:
+        fwd_trim='work/cutadapt/{sample}_R1.fastq.gz',
+        rev_trim='work/cutadapt/{sample}_R2.fastq.gz'
+    log:
+        'report/cutadapt/cutadapt_{sample}'
+    params:
+        discard = '--discard-untrimmed ',
+        indels = '--no-indels ',
+        trim_n='--trim-n ' 
+    shell:
+        '''
+         cutadapt -g file:{input.fwd_prime} -G file:{input.rev_prime} -a file:{input.rev_revcomp_prime} -A file:{input.fwd_revcomp_prime} -o {output.fwd_trim} -p {output.rev_trim} --max-n=0 -m 50 --error-rate 0.1 {params.discard}{params.indels}{params.trim_n}--pair-filter any {input.fwd} {input.rev} > {log} 
+        ''' 
 
 rule filter:
 	input:
-		cut = "work/cutadapt/{sample}_1.fastq.gz",
-		cutrev = "work/cutadapt/{sample}_2.fastq.gz"
+		cut = "work/cutadapt/{sample}_R1.fastq.gz",
+		cutrev = "work/cutadapt/{sample}_R2.fastq.gz"
 	output:
-		filt = "work/filter/{sample}_1.fastq.gz",
-		filtrev = "work/filter/{sample}_2.fastq.gz"
+		filt = "work/filter/{sample}_R1.fastq.gz",
+		filtrev = "work/filter/{sample}_R2.fastq.gz"
 	script:
 		"filterAndTrim.R"

quality.smk

 rule fastqc:
     input:
-        fwd = "DATA/{sample}_1.fastq.gz",
-        rev = "DATA/{sample}_2.fastq.gz"
+        fwd = "DATA/{sample}_R1.fastq.gz",
+        rev = "DATA/{sample}_R2.fastq.gz"
     output:
         zip = "work/fastqc/{sample}_{unit}_fastqc.zip",
         html = "work/fastqc/{sample}_{unit}_fastqc.html"
@@ -32,4 +32,4 @@ rule multiqc:
         "--outdir {params.output} "
         "{input} "
         "&& "
-        "conda deactivate "
+        "conda deactivate "
\ No newline at end of file

revcomp_primer.smk

+import os
+def yield_fasta(fasta, gz=False, rev_comp=False):
+    '''Make a generator that yields (seq_header, seq) for each entry)'''
+    complement = {'A':'T', 'C':'G', 'G':'C', 'T':'A', 'N':'N', 'X':'X', 'M':'K', 'R':'Y', 'W':'W', 'S':'S', 'Y':'R', 'K':'M','V':'B', 'H':'D', 'D':'H', 'B':'V'}
+    if gz==False:
+        fhs=open(fasta, 'r')
+    else:
+        fhs=gzip.open(fasta, 'rt')
+    l=fhs.readline().strip()
+    while l != '':
+        if l.startswith('>'):   #if header
+            header=l  #save header
+            if rev_comp is False:
+                seq=fhs.readline().strip().upper()
+            else:
+                seq=''
+                for base in fhs.readline().strip().upper()[::-1]:
+                    seq+=complement[base]
+            yield header, seq# yield data
+        l=fhs.readline().strip()		
+rule revcomp_primer:
+    input:
+        primers = "Primers/{primer}_primers.fasta"
+    output:
+        primers = temporary("Primers/{primer}_revcomp_primers.fasta")
+    run:
+        with open(output[0], 'w') as fho:
+                fho.writelines(['{}\n{}\n'.format(x[0],x[1]) for x in yield_fasta(input[0], rev_comp=True)])
\ No newline at end of file