moveing

EXAMPLE/getExp.sh

+#!/bin/bash
+#
+wget http://vm-lux.embl.de/~rmuench/files/samples.bz2
+tar xfvj samples.bz2
+rm samples.bz2
+exit

LICENSE

+    
+    metaSNV --- A metagenomic SNV calling pipeline
+    Copyright (C) 2017  P. I. Costea & R. Munch
+
+    This program is free software: you can redistribute it and/or modify
+    it under the terms of the GNU General Public License as published by
+    the Free Software Foundation, either version 3 of the License, or
+    (at your option) any later version.
+
+    This program is distributed in the hope that it will be useful,
+    but WITHOUT ANY WARRANTY; without even the implied warranty of
+    MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.  See the
+    GNU General Public License for more details.
+
+    You should have received a copy of the GNU General Public License
+    along with this program.  If not, see <http://www.gnu.org/licenses/>.

Makefile

+all: qaTools snpCaller
+clean:
+	cd src/qaTools && $(MAKE) clean
+	cd src/snpCaller && $(MAKE) clean
+
+qaTools:
+	cd src/$@ && $(MAKE)
+snpCaller:
+	cd src/$@ && $(MAKE)
+
+.PHONY: all clean

README.md

+# MetaSNV, a metagenomic SNV calling pipeline
+
+
+The metaSNV pipeline performs variant calling on aligned metagenomic samples.
+
+
+Download
+========
+
+Via Git:
+
+    git clone git@git.embl.de:rmuench/metaSNP.git
+    
+or [download](https://git.embl.de/rmuench/metaSNP/repository/archive.zip?ref=master) a zip file of the repository.
+
+Dependencies
+============
+
+* [Boost-1.53.0 or above](http://www.boost.org/users/download/)
+
+* [htslib](http://www.htslib.org/)
+ 
+* Python-2.7 or above
+
+#### Installing dependencies on Ubuntu/debian
+
+On an Ubuntu/debian system, the following sequence of commands will install all
+required packages (the first two are only necessary if you have not enabled the
+universe repository before):
+
+
+    sudo add-apt-repository "deb http://archive.ubuntu.com/ubuntu $(lsb_release -sc) universe"
+    sudo apt-get update
+
+    sudo apt-get install libhts-dev libboost-dev
+
+### Installing dependencies using anaconda
+
+If you use [anaconda](https://www.continuum.io/downloads), you can create an
+environment with all necessary dependencies using the following commands:
+
+    conda create --name metaSNV boost htslib pkg-config
+    source activate metaSNV
+    export CFLAGS=-I$CONDA_ENV_PATH/include
+    export LD_LIBRARY_PATH=$CONDA_ENV_PATH/lib:$LD_LIBRARY_PATH
+
+If you do not have a C++ compiler, anaconda can also install G++:
+
+    conda create --name metaSNV boost htslib pkg-config
+    source activate metaSNV
+
+    # Add this command:
+    conda install gcc
+
+    export CFLAGS=-I$CONDA_ENV_PATH/include
+    export LD_LIBRARY_PATH=$CONDA_ENV_PATH/lib:$LD_LIBRARY_PATH
+
+Setup & Compilation
+===================
+
+    make
+
+Workflow:
+=========
+## Required Files:
+
+* **'all\_samples'**    = a list of all BAM files, one /path/2/sample.bam per line (no duplicates)
+* **'ref\_db'**  = the reference database in fasta format (f.i. multi-sequence fasta)
+
+## Optional Files:
+* **'db\_ann'** = a gene annotation file for the reference database.
+
+## To use one of the provided reference databases:
+
+    ./getRefDB.sh
+    
+## 2. Run metaSNV
+
+    metaSNV.py project_dir/ all_samples ref_db ref_fasta [options]
+
+## 3. Part II: Post-Processing (Filtering & Analysis)
+
+### a) Filtering:
+Note: requires SNP calling (Part II) to be done!
+Caution: Perform this step seperately for individual SNPs and population SNPs.
+
+    usage: metaSNP_filtering.py
+
+        positional arguments:
+            perc_FILE               input file with horizontal genome (taxon) coverage (breadth) per sample (percentage covered)
+            cov_FILE                input file with average genome (taxon) coverage
+                                    (depth) per sample (average number reads per site)
+            snp_FILE                input files from SNP calling
+            all_samples             list of input BAM files, one per line
+            output_dir/             output folder
+
+        optional arguments:
+            -h, --help              show this help message and exit
+            -p PERC, --perc PERC    Coverage breadth: Horizontal coverage cutoff
+                                    (percentage taxon covered) per sample (default: 40.0)
+            -c COV, --cov COV       Coverage depth: Average vertical coverage cutoff per
+                                    taxon, per sample (default: 5.0)
+            -m MINSAMPLES, --minsamples MINSAMPLES
+                                    Minimum number of sample required to pass the coverage
+                                    cutoffs per genome (default: 2)
+            -s SNPC, --snpc SNPC    FILTERING STEP II: SNP coverage cutoff (default: 5.0)
+            -i SNPI, --snpi SNPI    FILTERING STEP II: SNP occurence (incidence) cutoff
+                                    within samples_of_interest (default: 0.5)
+
+### b) Analysis:
+>   TODO: include R scripts for computing pairwise distances and visualization
+
+
+Example Tutorial
+================
+
+## 1. Run the setup & compilation steps and download the provided reference database.
+
+    ./getRefDB.sh
+
+## 2. Go to the EXAMPLE directory and download the samples with the getSamplesScript.sh
+
+    $ cd EXAMPLE
+    $ ./getSamplesScript.sh
+
+## 3. Initiate a new project in the parent directory
+
+    $ metaSNV_New tutorial
+
+## 4. Generate the 'all_samples' file
+
+    $ find `pwd`/EXAMPLE/samples -name “*.bam” > tutorial/all_samples
+
+## 5. Prepare and run the coverage estimation
+
+    $ metaSNV_COV tutorial/ tutorial/all_samples > runCoverage
+    $ bash runCoverage
+
+## 6. Perform a work load balancing step for run time optimization.
+
+    $ metaSNV_OPT tutorial/ db/Genomev9_definitions 5
+    $ bash runCoverage
+
+## 7. Prepare and run the SNV calling step
+
+    $ metaSNV_SNP tutorial/ tutorial/all_samples db/RepGenomesv9.fna -a db/RefOrganismDB_v9_gene.clean -l tutorial/bestsplits/ > runSNPcall
+    $ bash runSNPcall
+
+## 8. Run the post processing / filtering steps
+### a) Compute allele frequencies for each position that pass the given thresholds.
+
+    $ metaSNV_filtering.py tutorial/tutorial.all_perc.tab tutorial/tutorial.all_cov.tab tutorial/snpCaller/called_SNVs.best_split_* tutorial/all_samples tutorial/filtered/pop/
+
+### b) Compute pair-wise distances between samples on their SNP profiles and create a PCoA plot.
+    
+
+
+
+Advanced usage (tools and scripts)
+==================================
+
+If you are interested in using the pipeline in a more manual way (for example
+the metaSNV caller stand alone), you will find the executables for the
+individual steps in the `src/` directory.
+
+metaSNV caller
+--------------
+Calls SNVs from samtools pileup format and generates two outputs.
+
+    usage: ./snpCall [options] < stdin.mpileup > std.out.popSNPs
+
+    Options:
+        -f,     faidx indexed reference genome.
+        -g,     gene annotation file.
+        -i,     individual SNVs.
+
+Note: Expecting samtools mpileup as standard input
+
+### __Output__
+1. Population SNVs (pSNVs): 
+Population wide variants that occur with a frequency of 1 % at positions with at least 4x coverage.
+
+2. Individual specific SNVs (iSNVs):
+Non population variants, that occur with a frequency of 10 % at positions with at least 10x coverage.
+
+
+[qaCompute](https://github.com/CosteaPaul/qaTools)
+-------------------------------------------------
+Computes normal and span coverage from a bam/sam file. 
+Also counts unmapped and sub-par quality reads.
+
+### __Parameters:__
+   m	    -	Compute median coverage for each contig/chromosome. 
+   		Will make running a bit slower. Off by default.
+   
+   q [INT]  -   Quality threshold. Any read with a mapping quality under
+                INT will be ignored when computing the coverage.
+                
+		NOTE: bwa outputs mapping quality 0 for reads that map with
+		equal quality in multiple places. If you want to condier this,
+		set q to 0.
+		
+   d        -   Print coverage histrogram over each individual contig/chromosome.
+   	        These details will be printed in file <output>.detail
+   	        
+   p [INT]  -   Print coverage profile to bed file, averaged over given window size.  
+   
+   i        -   Silent run. Will not print running info to stdout.
+    
+   s [INT]  -   Compute span coverage. (Use for mate pair libs)
+                Instead of actual read coverage, using the options will consider
+                the entire span of the insert as a read, if insert size is
+		lower than INT. 
+ 		For an accurate estimation of span coverage, I recommend
+		setting an insert size limit INT around 3*std_dev of your lib's 
+		insert size distribution.
+    
+   c [INT]  -   Maximum X coverage to consider in histogram.
+    
+   h [STR]  -   Use different header. 
+                Because mappers sometimes break the headers or simply don't output them, 
+		this is provieded as a non-kosher way around it. Use with care!
+    
+   For more info on the parameteres try ./qaCompute
+   
+
+metaSNV_filtering.py
+--------------------   
+usage: metaSNV filtering [-h] [-p PERC] [-c COV] [-m MINSAMPLES] [-s SNPC]
+                         [-i SNPI] 
+                         perc_FILE cov_FILE snp_FILE [snp_FILE ...]
+                         all_samples output_dir/
+
+metaSNV filtering
+
+positional arguments:
+  perc_FILE             input file with horizontal genome (taxon) coverage
+                        (breadth) per sample (percentage covered)
+  cov_FILE              input file with average genome (taxon) coverage
+                        (depth) per sample (average number reads per site)
+  snp_FILE              input files from SNP calling
+  all_samples           list of input BAM files, one per line
+  output_dir/           output folder
+
+optional arguments:
+  -h, --help            show this help message and exit
+  -p PERC, --perc PERC  Coverage breadth: Horizontal coverage cutoff
+                        (percentage taxon covered) per sample (default: 40.0)
+  -c COV, --cov COV     Coverage depth: Average vertical coverage cutoff per
+                        taxon, per sample (default: 5.0)
+  -m MINSAMPLES, --minsamples MINSAMPLES
+                        Minimum number of sample that have to pass the
+                        filtering criteria in order to write an output for the
+                        representative Genome (default: 2)
+  -s SNPC, --snpc SNPC  FILTERING STEP II: SNP coverage cutoff (default: 5.0)
+  -i SNPI, --snpi SNPI  FILTERING STEP II: SNP occurence (incidence) cutoff
+                        within samples_of_interest (default: 0.5)
+
+

SETUPFILE

+# Required Variables for metaSNP compilation
+
+HTSLIB_CFLAGS := $(shell pkg-config htslib --cflags)
+HTSLIB_LIBS := $(shell pkg-config htslib --libs)
+
+# For boost, only includes are necessary:
+BOOST_CFLAGS :=

all_samples

+/net/mraid08/export/jafar/Microbiome/Analyses/Costea/metaSNP/EXAMPLE/samples/s5.sorted.bam
+/net/mraid08/export/jafar/Microbiome/Analyses/Costea/metaSNP/EXAMPLE/samples/s2.sorted.bam
+/net/mraid08/export/jafar/Microbiome/Analyses/Costea/metaSNP/EXAMPLE/samples/s1.sorted.bam
+/net/mraid08/export/jafar/Microbiome/Analyses/Costea/metaSNP/EXAMPLE/samples/s6.sorted.bam
+/net/mraid08/export/jafar/Microbiome/Analyses/Costea/metaSNP/EXAMPLE/samples/s3.sorted.bam
+/net/mraid08/export/jafar/Microbiome/Analyses/Costea/metaSNP/EXAMPLE/samples/s4.sorted.bam

getRefDB.sh

+#!/bin/bash
+
+download_9() {
+    [ -d "db" ] || mkdir -p db
+    cd db
+    echo "Downloading the archive."
+    wget vm-lux.embl.de/~rmuench/files/db_f9.tar.bz2
+    echo "This might take up to 10 minutes."
+    echo "Extracting files!"
+    echo "Patience please..."
+    tar xjvf db_f9.tar.bz2
+    rm db_f9.tar.bz2
+    exit	
+}
+
+download_11() {
+    [ -d "db" ] || mkdir -p db
+    cd db
+    echo "Downloading the archive."
+    wget vm-lux.embl.de/~rmuench/files/db_f11.tar.bz2
+	echo "This might take up to 20 minutes."
+    echo "Extracting files!"
+    echo "Patience please..."
+    tar xjvf db_f11.tar.bz2
+    rm db_f11.tar.bz2
+    exit
+}
+
+
+echo "You are about to download the metaSNP database"
+echo ""
+echo "Requirements:"
+echo "  - Freeze9:  12G available disc space (1753 genomes)"
+echo "  - Freeze11: 33G available disc space (5278 genomes)"
+echo ""
+echo "Please make a selection by entering the right number:"
+
+select yn in "f9" "f11" "cancel"; do
+    case $yn in
+        f9 ) download_9 ; break;;
+        f11 ) download_11; break;;
+        cancel ) exit;;
+    esac
+done

metaSNV.py

+#!/usr/bin/env python
+# This code is part of the metagenomic SNV calling pipeline (metaSNV)
+# Helper script to initiate a new project file structure.
+import argparse
+from sys import stderr, exit
+from os import path
+from glob import glob
+import os
+import shutil
+import subprocess
+import multiprocessing
+
+basedir = os.path.dirname(os.path.abspath(__file__))
+
+
+def mkdir_p(dirname):
+    '''Equivalent to 'mkdir -p' on the command line'''
+    from os import makedirs
+    try:
+        makedirs(dirname)
+    except OSError:
+        pass
+
+
+def create_directories(basedir):
+    mkdir_p(basedir)
+    for sub in ['cov',
+                'bestsplits',
+                'snpCaller',
+                'filtered',
+                'filtered/pop',
+                'filtered/ind',
+                'distances']:
+        mkdir_p(path.join(basedir, sub))
+
+
+
+def exit_worker(signum, frame):
+    raise RuntimeError("Keyboard Interrupt")
+def init_worker():
+    import signal
+    signal.signal(signal.SIGINT, exit_worker)
+
+def run_sample(sample, command):
+    '''Simply wraps subprocess.call and returns contextual information in order
+    to provide a good error message (if necessary)'''
+
+    ret = subprocess.call(command)
+    return sample, command, ret
+
+
+def compute_opt(args):
+    out_dir = path.join(args.project_dir, 'cov')
+    try:
+        os.makedirs(out_dir)
+    except:
+        pass
+    p = multiprocessing.Pool(args.threads, init_worker)
+    results = []
+    for line in open(args.all_samples):
+        line = line.rstrip()
+        name = path.basename(line)
+        cmd = ['{}/src/qaTools/qaCompute'.format(basedir)
+                ,'-c' ,'10', '-d',
+                '-i', line, '{}/{}.cov'.format(out_dir, name)]
+        if args.print_commands:
+            print(" ".join(cmd))
+        else:
+            results.append(p.apply_async(run_sample, (name, cmd)))
+
+    p.close()
+    p.join()
+    for r in results:
+        sample, command, ret = r.get()
+        if ret:
+            stderr.write("Failure in sample {}".format(sample))
+            stderr.write("Call to {} failed.".format(' '.join(cmd)))
+            exit(1)
+def split_opt(args):
+
+    if args.n_splits > 100:
+        stderr.write("Maximum number of splits is 100.\n")
+        exit(1)
+
+    older_files = glob(args.project_dir + '/bestsplits/*')
+    if older_files:
+        stderr.write("\nremoving old splits.\n")
+        for f in older_files:
+            os.unlink(f)
+
+    cov_dir = path.join(args.project_dir, 'cov')
+    cov_files = glob(cov_dir + '/*.cov')
+    project_name = path.basename(args.project_dir)
+    if not cov_files:
+	if not args.print_commands:
+        	stderr.write("Coverage files not found.\n")
+	else:
+		stderr.write("Coverage files not found.\nFinish running the commands prinnted above and then run this command again.\n")
+        exit(1)
+    for f in cov_files:
+        cmd = ['python',
+	            path.join(basedir, 'src/computeGenomeCoverage.py'),
+                f,
+                f + '.detail',
+                f + '.summary']
+        subprocess.call(cmd)
+    print("\nCoverage summary here: {}".format(args.project_dir))
+    print("	Average vertical genome coverage: '{}/{}.all_cov.tab'".format(args.project_dir, project_name))
+    print("	Horizontal genome coverage (1X): '{}/{}.all_perc.tab'".format(args.project_dir, project_name))
+    cmd = ['python',
+            '{}/src/collapse_coverages.py'.format(basedir),
+            args.project_dir]
+    subprocess.call(cmd)
+
+
+    print("\nCalculating best database split:")
+    # usage createOptimumSplit.sh <all_cov.tab> <all_perc.tab> <geneDefinitions> <INT_NrSplits> <.outfile>
+    cmd = ['python',
+            '{}/src/createOptimumSplit.py'.format(basedir),
+            "{}/{}.all_cov.tab".format(args.project_dir, project_name),
+            "{}/{}.all_perc.tab".format(args.project_dir, project_name),
+            args.ctg_len,
+            str(args.n_splits),
+            path.join(args.project_dir, "bestsplits", "best_split")]
+    subprocess.call(cmd)
+
+
+def execute_snp_call(args, snpCaller, ifile, ofile, split):
+    db_ann_args = []
+    if args.db_ann != '':
+        db_ann_args = ['-g', args.db_ann]
+    split_args = []
+    if split:
+        split_args = ['-l', str(split)]
+    samtools_cmd = ['samtools',
+                'mpileup',
+                '-f', args.ref_db
+                ] + split_args + [
+                '-B',
+                '-b', args.all_samples]
+    snpcaller_cmd = [
+                snpCaller, '-f', args.ref_db] + db_ann_args + [
+                '-i', ifile]
+    if args.print_commands:
+        print(" ".join(samtools_cmd + ['|'] + snpcaller_cmd + ['>', ofile]))
+    else:
+        with open(ofile, 'wt') as ofile:
+            samtools_call = subprocess.Popen(samtools_cmd, stdout=subprocess.PIPE)
+            snpcaller_call = subprocess.Popen(snpcaller_cmd, stdin=samtools_call.stdout, stdout=ofile)
+            samtools_call.stdout.close()
+            return snpcaller_call.wait()
+
+
+def snp_call(args):
+    out_dir = path.join(args.project_dir, 'snpCaller')
+    try:
+        os.makedirs(out_dir)
+    except:
+        pass
+
+    shutil.copy(args.all_samples,args.project_dir+'/all_samples')
+
+    snpCaller = basedir + "/src/snpCaller/snpCall"
+
+    indiv_out = path.join(out_dir, "indiv_called")
+    called_SNP = path.join(out_dir, "called_SNPs")
+
+
+## ACTUAL COMMANDLINE
+# 	Note: Due to a bug in samtools v0.1.18, -Q 20 might be erroneous to use.
+#	Note: Different phred score scales might be disregarded.
+#	Note: If samtools > v0.1.18 is used -Q 20 filtering is highly recommended.
+
+    threads = (args.threads if not args.print_commands else 1)
+    p = multiprocessing.Pool(threads, init_worker)
+    results = []
+    if args.n_splits > 1:
+        splits = glob('{}/bestsplits/best_split_*'.format(args.project_dir))
+        for split in splits:
+            results.append(p.apply_async(execute_snp_call,
+                                    (args,
+                                    snpCaller,
+                                    '{}.{}'.format(indiv_out, path.basename(split)),
+                                    '{}.{}'.format(called_SNP, path.basename(split)),
+                                    split)))
+        p.close()
+        p.join()
+        for r in results:
+            v = r.wait()
+            if v > 0:
+                stderr.write("SNV calling failed")
+                exit(1)
+    else:
+        v = execute_snp_call(args, snpCaller, indiv_out, called_SNP, None)
+        if v > 0:
+            stderr.write("SNV calling failed")
+            exit(1)
+
+
+
+def main():
+    parser = argparse.ArgumentParser(description='Compute SNV profiles')
+    parser.add_argument('project_dir', metavar='DIR',
+                        help='A metaSNP initialized project directory')
+    parser.add_argument('all_samples', metavar='FILE',
+                        help='File with an input list of bam files, one file per line')
+    parser.add_argument("ref_db", metavar='REF_DB_FILE',
+                        help='reference multi-sequence FASTA file used for the alignments.')
+    parser.add_argument('--db_ann', metavar='BED_FILE',default='',
+                        help='Database gene annotation.')
+    parser.add_argument('--print-commands', default=False, action='store_true',
+                        help='Instead of executing the commands, simply print them out')
+    parser.add_argument('--threads', metavar='INT', default=1,type=int,
+                        help='Number of jobs to run simmultaneously. Will create same number of splits, unless n_splits set differently.')
+    parser.add_argument('--n_splits', metavar='INT', default=1,type=int,
+                        help='Number of bins to split ref into')
+    parser.add_argument('--ctg_len',metavar='BED_FILE',default='',
+			help='Per contig lengths, required for splitting.')
+    args = parser.parse_args()
+    if not path.isfile(args.ref_db):
+        stderr.write('''
+ERROR:	No reference database or annotation file found!"
+ERROR:	'{}' is not a file."
+
+SOLUTION: run getRefDB.sh or set up a custom database before running metaSNP caller
+        '''.format(args.ref_db))
+        parser.print_help()
+        exit(1)
+
+    if not path.isfile(basedir+"/src/qaTools/qaCompute") or not path.isfile(basedir+"/src/snpCaller/snpCall"):
+	stderr.write('''
+ERROR:  No binaries found
+
+SOLUTION: make\n\n'''.format(basedir))	
+	exit(1)
+
+    if (args.n_splits > 1 or args.threads > 1) and args.ctg_len=='':
+	stderr.write('''
+ERROR:  No contig information supplied. Cannot compute splits
+
+SOLUTION: Supply contig information through --ctg_len\n\n'''.format(basedir))  
+        exit(1)	
+
+    if args.threads > 1 and args.n_splits==1:
+	args.n_splits=args.threads
+
+    if path.exists(args.project_dir) and not args.print_commands:
+    	stderr.write("Project directory '{}' already exists\n\n\n".format(args.project_dir))
+        exit(1)
+    create_directories(args.project_dir)
+    compute_opt(args)
+    if args.n_splits > 1:
+	split_opt(args)
+    snp_call(args)
+
+if __name__ == '__main__':
+    main()
+

metaSNV_post.py

+#!/usr/bin/env python
+import os               # interacting with operating system
+import sys		# interact with system commandline 
+import time		# print current date/time
+import argparse		# manage command line options and arguments
+import glob
+
+