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_processing_MB_0.2a.sh
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_processing_MB_0.2a.sh
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#!/bin/bash
###################################################################
# Bioinformatic data processing for metabarcoding
# for 16S V4 data, with amplicons generated by following Kozich et al. 2013 AEM
# by Alexander Keller (LMU München)
# email: [email protected]
# if you use this script, please kindly cite this article:
# https://doi.org/10.1098/rstb.2021.0171
###################################################################
if [ -z "$1" ]; then
echo 'No directory supplied' >&2
exit 1
fi
project=$1
now=$(date)
nowformat=$(date +"%y-%m-%d")
echo "-- create output folder $project.$nowformat; add first files"
mkdir -p $project.$nowformat
echo "-- moving in data dir"
cd $project
echo "-- loading config"
source config.txt
echo "-- creating directories"
mkdir -p logs
mkdir -p raw
mkdir -p tmp
if [ $classificationOnly -ne 1 ]
then
if [ $skip_preprocessing -ne 1 ]
then
echo "-- decompressing raw files"
#extracting files
find . -name '*.gz' -print0 | xargs -0 -I {} -P $threads gunzip {}
# looping through all files for merging
echo "-- starting merging and filter "
for f in *_R1_*.fastq; do
r=$(sed -e "s/_R1_/_R2_/" <<< "$f")
s=$(echo $f | sed "s/_L001.*//g")
total=$(grep "^@M" $f | wc -l)
echo " "
echo "===================================="
echo "Processing sample $s"
echo "(F: $f R: $r)"
echo "===================================="
$vsearch --fastq_mergepairs $f \
--reverse $r \
--fastq_minovlen $merge_minovlen \
--fastq_maxdiffs $merge_maxdiffs \
--fastqout $s.merge.fq \
--fastq_eeout \
--relabel R1+2-${s}_ \
--threads $threads 2> logs/vsearch.m.$s.log
var="$(grep "Merged" logs/vsearch.m.$s.log)"
echo "$s : $var" | tee -a logs/_merging.log
$vsearch --fastq_filter $s.merge.fq \
--fastq_stripleft $stripleft \
--fastq_stripright $stripright \
--fastq_maxee $fastq_maxee \
--fastq_minlen $fastq_minlen \
--fastq_maxlen $fastq_maxlen \
--fastq_maxns $fastq_maxns \
--fastaout $s.mergefiltered.fa \
--fasta_width 0 \
--threads $threads 2> logs/vsearch.mf.$s.log
var="$(grep "sequences kept" logs/vsearch.mf.$s.log)"
echo "$s : $var" | tee -a logs/_filter.log
$vsearch --fastq_truncee $fastq_truncee \
--fastq_filter $f \
--fastq_minlen $fastq_minlen \
--fastaout $s.trunc.fa \
--relabel R1-${s}_ \
--threads $threads 2> logs/vsearch.tf.$s.log
var="$(grep "sequences kept" logs/vsearch.tf.$s.log)"
echo "$s : $var" | tee -a logs/_truncfilter.log
done
cat *mergefiltered.fa > all.merge.fasta
cat *trunc.fa > all.trunc.fasta
if [ $use_fw_only -eq 1 ]
then
cp all.merge.fasta all.merge.fasta.bak
cp all.trunc.fasta all.merge.fasta
echo " "
echo "!!! INFO: using forward reads only"
echo " "
fi #end useFWonly
# cleanup
echo "-- cleanup"
mv *.fastq raw/
mv *.fq tmp/
mv *.fa tmp/
fi #end skippp
echo " "
echo "===================================="
echo "ASV generation and mapping"
echo "===================================="
echo "-- derep"
$vsearch --derep_fulllength all.merge.fasta \
--minuniquesize 2 \
--sizein \
--sizeout \
--fasta_width 0 \
--uc all.merge.derep.uc \
--output all.merge.derep.fa 2> logs/_derep.log
echo "-- denoise"
$vsearch --cluster_unoise all.merge.derep.fa \
--sizein --sizeout \
--relabel ASV \
--unoise_alpha $unoisealpha \
--centroids zotus.merge_chim.fa \
--threads $threads 2> logs/_unoise.log
grep "Clusters" logs/_unoise.log
grep "Singletons" logs/_unoise.log
echo "-- sorting"
$vsearch --sortbysize zotus.merge_chim.fa \
--output zotus.merge_sorted.fa \
--threads $threads 2> logs/_sort.log
echo "-- chimera removal"
$vsearch --uchime3_denovo zotus.merge_sorted.fa \
--abskew 16 \
--nonchimeras zotus.merge.fa \
--threads $threads 2> logs/_uchime.log
grep "Found" logs/_uchime.log
### create community table
echo "-- add barcodelabel"
cat all.merge.fasta | sed "s/^>R1+2-\(.*\)\_\([0-9]*\);/>R1+2-\1_\2;barcodelabel=\1;/g" | sed "s/^>R1-\([a-zA-Z0-9-]*\)\_\([0-9]*\)/>R1-\1_\2;barcodelabel=\1;/g" > all.merge.bc.fasta
echo "-- map data against ASVs"
$vsearch --usearch_global all.merge.bc.fasta --db zotus.merge.fa --strand plus --id 0.97 --uc map.merge.uc --otutabout asv.tab-csv --sizeout --threads $threads 2> logs/_mapping.log
grep "Matching" logs/_mapping.log
#python2.7 $p/uc2otutab.py map.trunc.uc > zotu_table.trunc.txt
# not necessary anymore
#echo "-- convert to table"
#python2.7 $pythonscripts/uc2otutab.py map.merge.uc > asv_table.merge.txt
echo "-- create output folder $project.$nowformat; add first files"
# copy final files into folde
mkdir -p ../$project.$nowformat
cp config.txt ../$project.$nowformat/config.txt
cp zotus.merge.fa ../$project.$nowformat/asvs.merge.fa
# cp asv_table.merge.txt ../$project.$nowformat/asv_table.merge.txt
cp asv.tab-csv ../$project.$nowformat/asv_table.merge.txt
# prepare final project information file
echo "name;id;own;year;marker;description;participants;doi;repository;accession;ignore" > ../$project.$nowformat/project.csv
echo "$project;$project;1;$nowformat;$marker;;;;;;" >> ../$project.$nowformat/project.csv
# prepare final sample information file
echo "project;name;host;collectionDate;location;country;bioregion;latitude;longitude;tissue;treatment;sampletype;notes" > ../$project.$nowformat/samples.csv
head -n 1 asv.tab-csv | sed -e "s/#OTU ID[[:space:]]//g" | tr "\t" "\n" | sed "s/^/$project;/" >> ../$project.$nowformat/samples.csv
fi #end classificationOnly
##### create taxonomy
echo " "
echo "===================================="
echo "Taxonomic classification"
echo "===================================="
# $vsearch
# old script version: keep to troubleshoot
# refDBs=($(grep "refdb" config.txt | cut -f2 -d"=" | sed 's/\"//g'))
# hieDBs=($(grep "hiedb" config.txt | cut -f2 -d"=" | sed 's/\"//g'))
threshold=$tax_threshold
echo "Classification threshold: $threshold"
# switch here for marker
rm taxonomy.vsearch
if [ "$marker" = "ITS2" ]
then
echo ",kingdom,phylum,order,family,genus,species" > taxonomy.vsearch
echo ",kingdom,phylum,order,family,genus,species" > taxonomy.blast
fi
if [ "$marker" = "COI-5P" ]
then
echo ",kingdom,phylum,class,order,family,genus,species" > taxonomy.vsearch
echo ",kingdom,phylum,class,order,family,subfamily,genus,species, subspecies" > taxonomy.blast
fi
if [ "$marker" = "16S" ]
then
echo ",kingdom,phylum,order,family,genus" > taxonomy.vsearch
echo ",kingdom,phylum,order,family,genus" > taxonomy.blast
fi
countdb=0
cp zotus.merge.fa zotus.direct.$countdb.uc.nohit.fasta
prevDB=$countdb
for db in "${refDBs[@]}"
do :
countdb=$((countdb+1))
echo "\n\n#### Direct vsearch Classification level: $countdb";
echo "DB: $db";
$vsearch --usearch_global zotus.direct.$prevDB.uc.nohit.fasta \
--db $db \
--id 0.$threshold \
--uc zotus.direct.$countdb.uc \
--fastapairs zotus.direct.$countdb.fasta \
--strand both \
--threads $threads 2> logs/_direct.$countdb.log
grep "^N[[:space:]]" zotus.direct.$countdb.uc | cut -f 9 > zotus.direct.$countdb.uc.nohit
$seqfilter zotus.merge.fa --ids zotus.direct.$countdb.uc.nohit --out zotus.direct.$countdb.uc.nohit.fasta
cut -f 9,10 zotus.direct.$countdb.uc | grep -v "*" | sed "s/[A-Za-z0-9_-]*;tax=//" >> taxonomy.vsearch
prevDB=$countdb
done
echo "\n\n#### Hierarchical vsearch classification";
echo "DB: $hieDBs";
$vsearch --sintax zotus.direct.$countdb.uc.nohit.fasta \
--db $hieDBs \
--tabbedout zotus.uc.merge.nohit.sintax \
--strand plus \
--sintax_cutoff $sintax_cutoff \
--threads $threads 2> logs/_sintax.log
cut -f1,4 zotus.uc.merge.nohit.sintax | sed -E -e "s/\_[0-9]+//g" -e "s/,s:.*$//" >> taxonomy.vsearch
# v3 idea [TODO]: phylo + spc estimation on sintax results
#cut -f4 zotus.uc.merge.nohit.sintax | head |
#cut -f1,4 zotus.uc.merge.nohit.sintax | head | sed "s/^\(.*\)[[:space:]]\(.*\)$/\2/"
#mkdir -p unresolved
#cut -f4 zotus.uc.merge.nohit.sintax | head | sort | uniq | sed "s/^.*,//" | sort > unresolved/tmp.taxa
#grep "k:Viridiplantae,p:Streptophyta,c:Magnoliopsida,o:Rosales,f:Moraceae,g:Sorocea" taxonomy.vsearch
## get those with unresolved phylos
# #BLAST local DBs
# countdb=0
# cp zotus.merge.fa zotus.blast.$countdb.uc.nohit.fasta
# prevDB=$countdb
# touch zotus.blast.hits
# touch taxonomy.blast
#
# for db in "${refDBs[@]}"
# do :
# countdb=$((countdb+1))
# echo "\n\n#### Direct BLAST Classification level: $countdb";
# echo "DB: $db";
# #makeblastdb -in $db -parse_seqids -blastdb_version 5 -dbtype nucl
# blastn -outfmt '6 qseqid sseqid length pident qcovs' -max_target_seqs 1 -query zotus.blast.$prevDB.uc.nohit.fasta -subject $db -perc_identity $threshold -qcov_hsp_perc 90 -num_threads $threads > zotus.blast.$countdb.out
# cut -f1 -d" " zotus.blast.$countdb.out | cut -f1 >> zotus.blast.hits
# $sf zotus.merge.fa --ids-exclude --ids zotus.blast.hits --out zotus.blast.$countdb.uc.nohit.fasta
# prevDB=$countdb
# cut -f 1,2 zotus.blast.$countdb.out >> taxonomy.blast
#
# done
#BLAST web nt instead of sintax here?
echo "-- polishing and copying output files"
python ../_resources/python/fix_output_files.py --tax taxonomy.vsearch --asv asv.tab-csv
cp taxonomy.vsearch ../$project.$nowformat/taxonomy.vsearch
cp asv.tab-csv ../$project.$nowformat/asv_table.merge.txt
#sed -i .bak -e "s/c:.*,o:/o:/g" -e "s/,[A-Za-z0-9_-]*;tax=//" -e "s/ /,/" -e "s/,.*k:/,k:/" taxonomy.vsearch
# sed -i .bak -e "s/c:.*,o:/o:/g" -e "s/[A-Za-z0-9]*;tax=//" -e "s/ /,/" taxonomy.blast
# if [ "$classificationOnly" = "16S" ]
# then
# sed -e "s/,.*d:/,d:/" -e "s/p:Actinobacteria/p:Actinobacteria_phylum/" -e "s/_\d,/,/" taxonomy.vsearch > ../$project.$nowformat/taxonomy.vsearch
# else
# sed -e "s/,.*d:/,d:/" taxonomy.vsearch > ../$project.$nowformat/taxonomy.vsearch
# fi
#sed -i .bak "s/OTUId//" ../$project.$nowformat/asv_table.merge.txt
# without python scripts
# sed -i .bak "s/^\#OTU ID//" ../$project.$nowformat/asv_table.merge.txt
# sed -i .bak "s/;size=[0-9]*//" ../$project.$nowformat/taxonomy.vsearch
#cp config.txt ../$project.$nowformat/
echo "-- create R script"
# CREATE R SCRIPT
cat ../_resources/R_template_header.R > ../$project.$nowformat/R_$project.v0.R
echo "# Created: $now" >>../$project.$nowformat/R_$project.v0.R
echo "# Project: $project" >>../$project.$nowformat/R_$project.v0.R
echo "# Marker: $marker" >>../$project.$nowformat/R_$project.v0.R
echo "# For: $datasupplier" >>../$project.$nowformat/R_$project.v0.R
cat ../_resources/R_template_libraries.R >> ../$project.$nowformat/R_$project.v0.R
echo "\n\n# Setting working directory (check path)" >>../$project.$nowformat/R_$project.v0.R
echo "setwd('$(pwd)/../$project.$nowformat')" >>../$project.$nowformat/R_$project.v0.R
echo "\n\n# Custom functions inclusion" >>../$project.$nowformat/R_$project.v0.R
echo "marker=\"$marker\"" >>../$project.$nowformat/R_$project.v0.R
echo "source('$(pwd)/../_resources/metabarcoding_tools_0-1a.R')" >>../$project.$nowformat/R_$project.v0.R
cat ../_resources/R_template_ITS2.R >> ../$project.$nowformat/R_$project.v0.R
cp ../_resources/metabarcoding_tools_0-1a.R ../$project.$nowformat/
mkdir -p ../$project.$nowformat/plots
$vsearch -v > logs/software.version
cd ..
# cleanup if wanted
if [ $compressionCleanup -eq 1 ]
then
echo "-- compressing large files"
sh _compression_cleanup_1.sh $project
fi