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MG-TK.pl
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#!/usr/bin/env perl
#The Metagenomic Assembly, Genomic Recovery and Assembly Independent Mapping Tool (formerly MATAFILER, now mg-tk)
#main mg-tk routine
# (c) Falk Hildebrand, 2016-2024
#examples
#./mg-tk.pl map2tar test/refCtg.fasta,test/refCtg.fasta test1,test2
#./mg-tk.pl map2tar test/TEC2/v5/TEC2.MM4.BEE.GF.rn.fa TEC2
#./mg-tk.pl -map dir/map
use warnings;
use strict;
use File::Basename;
use Cwd 'abs_path';
use POSIX;
use Getopt::Long qw( GetOptions );
use vars qw($CONFIG_FILE);
#load MF specific modules
use Mods::GenoMetaAss qw(readMap getDirsPerAssmblGrp lcp readFastHD prefixFAhd prefix_find gzipopen
readFasta writeFasta systemW getAssemblPath filsizeMB
iniCleanSeqSetHR checkSeqTech is3rdGenSeqTech hasSuppRds
getRawSeqsAssmGrp getCleanSeqsAssmGrp addFileLocs2AssmGrp);
use Mods::IO_Tamoc_progs qw(getProgPaths setConfigFile jgi_depth_cmd inputFmtSpades inputFmtMegahit createGapFillopt
buildMapperIdx mapperDBbuilt decideMapper checkMapsDoneSH greaterComputeSpace);
use Mods::SNP qw(SNPconsensus_vcf SNPconsensus_vcf2 );
use Mods::TamocFunc qw (cram2bsam getSpecificDBpaths getFileStr displayPOTUS bam2cram checkMF checkMFFInstall);
use Mods::phyloTools qw(fixHDs4Phylo);
#use Mods::Binning qw (runMetaBat runCheckM runSemiBin runMetaDecoder );
use Mods::Subm qw (qsubSystemWaitMaxJobs qsubSystem emptyQsubOpt findQsubSys qsubSystemJobAlive MFnext add2SampleDeps numUserJobs);
#some useful HPC commands..
#bjobs | awk '$3=="CDDB" {print $1}' |xargs bkill
#bjobs | grep 'dWXmOT' | cut -f11 -d' ' | xargs -t -i bkill {}
#squ | grep 'r' | cut -f11 -d' ' | xargs -t -i scontrol update TimeLimit=84:00:00 jobid={}
#squ | grep 'dencyNev' | cut -f11 -d' ' | xargs -t -i scancel {}
#bhosts | cut -f1 -d' ' | grep -v HOST_NAME | xargs -t -i ssh {} 'killall -u hildebra'
#hosts=`bhosts | grep ok | cut -d" " -f 1 | grep compute | tr "\\n" ","`; pdsh -w $hosts "rm -rf /tmp/hildebra"
#local subs
sub announce_MGTK;
sub smplStats; sub checkDrives;
sub isLastSampleInAssembly;
sub uploadRawFilePrep; sub unploadRawFilePostprocess;
sub seedUnzip2tmp; sub cleanInput; #unzipping reads; removing these at later stages ; remove tmp dirs
sub manageFiles;sub clean_tmp;
sub sdmClean; sub sdmOptSet; #qual filter reads
sub mergeReads; #merge reads via flash
sub krakHSap; sub krakenTaxEst;sub prepKraken;
sub metagAssemblyRun;
sub createPsAssLongReads; #pseudo assembler
sub prepPreAssmbl; #hybrid pacbio/ill assemblies
sub genePredictions; sub run_prodigal; #gene prediction
sub mapReadsToRef; sub scndMap2Genos;
sub runContigStats;#sub bam2cram;
sub mocat_reorder; sub postSubmQsub;
sub detectRibo; sub RiboMeta;sub riboSummary;
sub runOrthoPlacement;
sub runDiamond; sub DiaPostProcess; sub IsDiaRunFinished;
sub nopareil; sub calcCoverage;sub d2metaDist;
sub metphlanMapping; sub mergeMP2Table;
sub mOTU2Mapping; sub mergeMotu2Table; sub prepMOTU2;
sub genoSize; sub check_map_done; sub check_depth_done;
sub postprocess;
sub setDefaultMFconfig;
sub getCmdLineOptions;
sub setupHPC;
#------- version history MATAFILER --------
#.22: fixing of assembly.txt paths, if output folders were later copied around
#.23: upgrade to metaphlan3 & motus3 & general update to workflow that these no longer need sdm filtered reads
#.24: new sdm version, better HDD space usage control, reworked EBIupload routines, sdm cores back to 4, bug fixes..
#.25: added SemiBin and MetaDecoder
#.26: added pprodigal, MUSCLE5, addapted cc.bin resource usage, semibin integration in MGS.pl, checkM2 by default used
#.27: added "loopTillComplete" functionality
#.28: extended loopTillComplete, better sq control
#.29: added more control of tmp node space, better reporting on scratch usage & paired samples
#.30: changed installer massively, now split into 3 packs: matafiler.yml, gtdbtk.yml, Rbase.yml. motus.yml is only for motus, as massive dep problems with this one..
#.31 further cleanup to installer, sdm does now trimmomatic job (saves 1 step), sdm v2.07, tmp_space refinements, SNP calling performance improved, flow improved
#.32: more automatation to ssd & RAM usage
#.33: fixes metagStats, phylotools, cc.bin and strainWithin.pl
#.34: cut5PR1 and cut5PR2 mapping headers added. sdm 2.10
#.35: bugfixes to flow control (should be less iteration MF now), replaced bedtools with mosdetph
#.36: rm mosdepth, replaced with samtools depth; integrated kma, but bowtie2 remains default aligner
#.37: bug fix rmDup correctly activated
#.38: first implementation of automatic sample lock; furthermore bam->fastq functionality (single end) implemented
#.39: smplLock improvements, genecat improvements, prevent rm bwt idx
#.40: refactor how internally file paths are transferred and kept up-to-date between different subfunctions
#.41: 31.1.24: bugfixes related to refactor. full implementation of eggNOGmapper. bugfixes in MGS and genecat workflows
#.42: 2.3.24: reworked how nodeTmpSpace & Ram are denoted internally; starting binner now in same round as other steps (one less round of starting MF)
#.43: 17.3.24: first version of hybrid ill-PB assemblies working! also reworked getProgPaths to be faster
#.44: 20.3.24: hybrid ill-PB implemented for assembly groups. PB assembler implemented for assembly group. added proper subfunction to manage assembly mode
#.45: 13.4.24: reactivated ribofinder module
#.46: 23.4.24: hybrid assembly reworked. refactor code. less false subm dependencies.
#.47: 24.4.24: gene coverage by default gz'd, more safety checks that related files are gz'd. semiBin2 by default used now. SemiBin2 can use support read coverage profiles in addition to primary reads (increases MAG recovery).
#.48: stabilized hybrid assm. Contigstats uses .gz as input.
#.49: 17.5.24: tentative strobealign added
#.50: 18.5.24: bugfixes workflow, autodetect to rm locks, code cleanups
#.51: code refactor
#.52: 6.6.24: start implementation (.3di). more checks on correct pipe excecution.
#,53: 27.7.24: routine to check if some essential programs have been correctly installed and are avaialble. added flag -checkInstall. Moved geneCat.pl and MGS.pl to secScripts/ dir to avoid confusion on where to start..
#.54: 15.8.24: renamed MATAFILER to mg-tk
my $MATFILER_ver = 0.54;
#operation mode?
my $ARGV0 = "";$ARGV0 = $ARGV[0] if (@ARGV > 0);
#----------------- defaults -----------------
my %MFconfig; #config is more overall configuration for MATAFILER
my $rawFileSrchStr1 = '.*1\.f[^\.]*q\.gz$';
my $rawFileSrchStr2 = '.*2\.f[^\.]*q\.gz$';
my $rawFileBamSrchSing = ""; #inputBAMregex
my $rawFileSrchStrSingl = "";
my $rawFileSrchStrXtra1= '.*1_sequence\.f[^\.]*q\.gz$';
my $rawFileSrchStrXtra2= '.*2_sequence\.f[^\.]*q\.gz$';
my $submSytem = ""; #user supplied submission system flag.. default is empty and autodetect
my $mateInsertLength = 20000; #controls expected mate insert size , import for bowtie2 mappings
my %jmp=();
my $logDir = ""; #this is the local logdir
my $sharedTmpDirP = ""; #e.g. /scratch/MG-TK/
#$sharedTmpDirP = "/g/scb/bork/hildebra/tmp/" if (`hostname` !~ m/submaster/);
my $nodeTmpDirBase = "";#/tmp/MG-TK/
my $nodeHDDspace = 30; #30 Gb; default value
my $baseDir = ""; my $baseOut = "";
my $mapFile = ""; my $baseID = "";
my $configFile = "";
#more specific control: unfiniRew=rewrite unfinished sample dir; $redoCS = redo ContigStats completely;
#removeInputAgain=remove unzipped files from scratch, after sdm; remove_reads_tmpDir = leave cleaned reads on scratch after everything finishes
my $unfiniRew=0; my $redoCS=0; my $removeInputAgain=1; my $remove_reads_tmpDir=1;
my $OKtoRWassGrps = 0;my $rmBinFailAssmbly = 0;
my $skipWrongPairedSmpls=1; #check in sdm output if wrong read pairs present
my $defaultContigSubs = "as"; #default subprts for contigstats..
my $silent = 0;
my $redoFails = 0;my $XfirstReads = -1;
my $killDepNever = 0; my $checkMaxNumJobs = 0; #slurm related.. $killDepNever=1 kills jobs in state "DependencyNeverFinished" (happens a lot), while $checkMaxNumJobs=X halts the pipeline if more than X jobs are already queued up
my $excludeNodes = ""; #excluding certain nodes..
my $readsRpairs=-1; #are reads given in pairs? default: -1 = no clue
#my $useTrimomatic=0; #trimmomatic step now replaced by sdm solution -> $MFopt{trimAdapters}
my $abortOnEmptyInput = 0;
#my $relaxedSmplNames = 0;#don't abort when SMPLID in map contains strange chars -> this is now handled by adding to map "#RelaxSMPLID TRUE"
my $ignoreSmpl = "";
my $splitFastaInput = 0; #assembly as input..
my $importMocat = 0; my $mocatFiltPath = "reads.screened.screened.adapter.on.hg19.solexaqa/";
my $alwaysDoStats = 1;
my $rmScratchTmp=1;#Default; extremely important option as this adds a lot of overhead and scratch usage space, but reduces later overhead a lot and makes IO more stable
my $unpackZip = 0; #only goes through with read filtering, needed to get files for luis
my $filterFromSource=1; #powerful option that skips the unzip step.. use careful
my $doDateFileCheck = 0; #very specific option for Moh's reads that were of different dates..
$MFconfig{DoFreeGlbTmp} = 0;
$MFconfig{defaultReadLength} = 150; $MFconfig{defaultReadLengthX} = 5000;
$MFconfig{oldStylFolders} =0; #0=smpl name as out folder; 1=inputdir as out foler (legacy)
$MFconfig{mocatLinkDir} = "";
$MFconfig{wcKeysForJob} = ""; #EI specific system to register jobs under certain flag
$MFconfig{prefSinglFQgreps} = 0; #if grep of files (rawSrchString) has multi assignments, which grep to trust more?
$MFconfig{rmSmplLocks} = 0;
$MFconfig{uploadRawRds} = ""; #prepare raw input fastq's for upload 2 EBI? Clean reads will be stored in this dir
#control broad flow
my $FROM1=0; my $TO1=999999999999;
#counter on where MF is in map
my $JNUM=0;
my $doSubmit=1;
my $rewrite=0;
my $loop2completion = "0" ; #
my $loop2c_winsize=0;
my $loop2completion_ini=0;
#MFcontstants: object to store essential paths/file endings
my %MFcontstants;
#MFopt: global object with options for MG-TK. Added in MF v0.5, slowly rebuild MF around this system
my %MFopt;
#keep track of DBs that the metagenome will be filtered against..
my @filterHostDB = ();
#track secondary mapping and ref DBs
my %map2ndTogRefDB;my %make2ndMapDecoy;
my @bwt2outD =(); my @DBbtRefX = (); my @DBbtRefGFF=(); my @bwt2ndMapNmds;
my @scaffTarExternalOLib1; my @scaffTarExternalOLib2;
my @EBIjobs = (); #keeps track of $MFconfig{uploadRawRds} jobs, submits postprocessing (md5)
#----------- map all reads to a specific reference - options ---------
#progStats: object to track progress of programs/submissions
my %progStats;#count up progress of submitted jobs in current run
#HDDspace: object to handle HDD usage: Always format as "XXG" XX = space requirements in Gb. Excecption: "-1"
my %HDDspace;
my %inputFileSizeMB; #stores file size/sample
my %locStats; #keeps statistics of samples in hash (from already finished samples
#say hello to user
announce_MGTK();
setDefaultMFconfig();
getCmdLineOptions;
checkMF(1);
#set up further dependencies for MF
if ($loop2completion =~ m/(\d+):(\d+)/){
$loop2c_winsize = int($2);$loop2completion=$1;$loop2completion_ini=$1;
print "Loop2completion=$loop2completion; Window size=$loop2c_winsize\n";
} elsif ($loop2completion ne "0") {
$loop2completion = 6 ;$loop2completion_ini=6; #set to std number of iterations..
}
#dirs from config file--------------------------
#can be overwritten by $map{opt}{GlbTmpD} $map{opt}{NodeTmpD}
$sharedTmpDirP = getProgPaths("globalTmpDir",0) unless ($sharedTmpDirP ne "");
$nodeTmpDirBase = getProgPaths("nodeTmpDir",0) unless ($nodeTmpDirBase ne "");
#programs of global (pun) importance --------------------------
my $smtBin = getProgPaths("samtools");#
my $pigzBin = getProgPaths("pigz");
my $avx2Constr = getProgPaths("avx2_constraint",0);
#set up link to submission system on cluster
my $QSBoptHR = setupHPC();
# the map and some base parameters (base ID, in path, out path) can be (re)set
my %map; my %AsGrps; my %DOs;#DOs only required for metabat, to use all mappings within an assembly group
my ($hr,$hr2) = readMap($mapFile,0,\%map,\%AsGrps,$MFconfig{oldStylFolders});
%AsGrps = %{$hr2}; %map = %{$hr};
if ($MFopt{DoMetaBat2}){
my ($hrD,$hrM) = getDirsPerAssmblGrp(\%map,\%AsGrps);
%DOs = %{$hrD};
}
#$baseDir = $map{inDir} if (exists($map{inDir} ));
$baseOut = $map{opt}{outDir} if (exists($map{opt}{outDir} ) && $map{opt}{outDir} ne "");
$baseID = $map{opt}{baseID} if (exists($map{opt}{baseID} ) && $map{opt}{baseID} ne "");
die "provide an outdir in the mapping file\n" if ($baseOut eq "");
die "provide a baseID in the mapping file\n" if ($baseID eq "");
#overwrite tmp dirs??
if ($map{opt}{GlbTmpD} ne ""){print "Taking Global temp dir from map: $map{opt}{GlbTmpD} \n";$sharedTmpDirP = $map{opt}{GlbTmpD} ;}
if ($map{opt}{NodeTmpD} ne ""){print "Taking Node temp dir from map: $map{opt}{NodeTmpD} \n";$nodeTmpDirBase = $map{opt}{NodeTmpD} ;}
my $runTmpDirGlobal = "$sharedTmpDirP/$baseID/";
my $runTmpDBDirGlobal = "$runTmpDirGlobal/DB/";
unless (-d $runTmpDBDirGlobal){
system "mkdir -p $runTmpDBDirGlobal" ;
#and check that this dir exists...
sleep(1);
die "Can't create $runTmpDBDirGlobal\n" unless (-d $runTmpDBDirGlobal);
}
my $globaldDiaDBdir = $runTmpDBDirGlobal."DiamDB/";
system "mkdir -p $globaldDiaDBdir" unless (-d $globaldDiaDBdir);
#----------- map all reads to a specific reference, preparation ---------
my $map2ndMpde=0;#0=map2tar;2=map2DB;3=map2GC
#----------- scaffolding external contigs parameters
my $scaffTarExternal = "";my $scaffTarExternalName = "";
my $scaffTarExtLibTar = ""; my $bwt2ndMapDep = "";
map2ndPrep();
#some base stats kept in vars
my $sequencer = "hiSeq";#plattform the algos have to deal with
#my $DBpath="";
my $assDir="";
my $continue_JNUM = 0; #debug, set to 0 for full run
my $prevAssembly = ""; my $shortAssembly = "";#files with full length and short length assemblies
#my $mmpuOutTab = "";
#fixed stones
my $STOpreAssmblDone = "preassmblDone.sto"; #marks preAssebmly (hybrid assemblies etc) is done
my $STOassmbleDone = "ass.done.sto"; #marks assembly is done
#fixed dirs for specific set of samples
my $dir_MP2 = $baseOut."pseudoGC/Phylo/MP2/"; #metaphlan 2 dir
my $dir_mOTU2 = $baseOut."pseudoGC/Phylo/mOTU2/"; #mOUT 2 dir
my $dir_TaxTar = $baseOut."pseudoGC/Phylo/TaxaTarget/"; #taxaTar dir
my $dir_ContigStats = "/assemblies/metag/ContigStats/";
my $dir_RibFind = $baseOut."pseudoGC/Phylo/RiboFind/"; #ribofinder dir
my $dir_KrakFind = $baseOut."pseudoGC/Phylo/KrakenTax/$MFopt{globalKraTaxkDB}/"; #kraken dir
system("mkdir -p $baseOut") unless (-d $baseOut);
my $globalLogDir = $baseOut."LOGandSUB/"; #this is the gloabl logdir (across all samples in current run)
system("mkdir -p $globalLogDir/sdm") unless (-d "$globalLogDir/sdm");
open $QSBoptHR->{LOG},">",$globalLogDir."qsub.log";# unless ($doSubmit == 0);
print $globalLogDir."qsub.log\n";
my $collectFinished = $baseOut."runFinished.log\n";
my $globalNPD = $baseOut."NonPareil/";
system "mkdir -p $globalNPD" unless (-d "$globalNPD");
system "cp $mapFile $globalLogDir/inmap.txt";
my $presentAssemblies = 0; my $totalChecked=0;
my @samples = @{$map{opt}{smpl_order}}; my @allSmplNames;
my @allFilter1; my @allFilter2; my @inputRawFQs;
my @EmptySample;
my $statStr = ""; my $statStr5 = "";
my %sampleSDMs;
my $curSmpl = "";
#my %assmblGrpLog; #security that no assmblGrp exists twice.. #actually not needed..
my $baseSDMopt = getProgPaths("baseSDMopt_rel"); #"/g/bork3/home/hildebra/dev/Perl/reAssemble2Spec/data/sdm_opt_inifilter_relaxed.txt";
if ($MFopt{useSDM} ==2 ){$baseSDMopt = getProgPaths("baseSDMopt");}#"/g/bork3/home/hildebra/dev/Perl/reAssemble2Spec/data/sdm_opt_inifilter.txt";}
my $baseSDMoptMiSeq = getProgPaths("baseSDMoptMiSeq_rel");#"/g/bork3/home/hildebra/dev/Perl/reAssemble2Spec/data/sdm_opt_miSeq.txt";
if ($MFopt{useSDM} ==2 ){$baseSDMoptMiSeq = getProgPaths("baseSDMoptMiSeq");}#"/g/bork3/home/hildebra/dev/Perl/reAssemble2Spec/data/sdm_opt_miSeq_relaxed.txt"; }
my @unzipjobs; my $curUnzipDep = "";
my $sdmjNamesAll = "";
my $waitTime = 0;
my @grandDeps; #used for loop2completion , collects dependencies
my $emptCmd = "sleep 333";
#qsubSystem($globalLogDir."emptyrun.sh","sleep 333",1,"1G",0,"empty","","",1);
#set up kraken human filter
my $krakDeps = ""; my $krakenDBDirGlobal = $runTmpDirGlobal;
if ($MFopt{DoKraken} && $MFopt{globalKraTaxkDB} eq ""){die "Kraken tax specified, but no DB specified\n";}
$krakDeps = prepKraken() if ($MFopt{humanFilter} || ($MFopt{DoKraken}) || $MFopt{DoEukGenePred});
#profiling prep
my $mOTU2Deps = prepMOTU2();
#redo d2s intersample distance?
if ($MFopt{DoCalcD2s}) {$MFopt{DoCalcD2s} = !-e "$baseOut/d2StarComp/d2meta.stone";}
#same route as metaphlan2
prepMetaphlan();
my $from = $FROM1; my $to = $TO1;
if ($to > @samples){
print "Reset range of samples to ". @samples."\n";
$to = @samples;
}
if ($loop2c_winsize > 0){
$to = $from + $loop2c_winsize; $to = $TO1 if ($to > $TO1);
}
#--------------------------------------------------------------------------------
#--------------------------------------------------------------------------------
#--------------------------------------------------------------------------------
#--------------------------------------------------------------------------------
#die $to."\n";
#for loop that goes over every single sample in the .map
for ($JNUM=$from; $JNUM<$to;$JNUM++){
#local flow control
next if (exists($jmp{$JNUM}));
qsubSystemWaitMaxJobs($checkMaxNumJobs, $killDepNever);
#set up initial local paths for a given sample
my $dir2rd=""; my $curDir = "";my $curOutDir = "";
$curSmpl = $samples[$JNUM];
$dir2rd = $map{$curSmpl}{dir};
$dir2rd = $map{$curSmpl}{prefix} if ($dir2rd eq "");
#print "$curSmpl $map{$curSmpl}{dir} $map{$curSmpl}{rddir} $map{$curSmpl}{wrdir}\n"; next;
my $SmplName = $map{$curSmpl}{SmplID};
if ($dir2rd eq "" ){#very specific read dir..
if ($map{$curSmpl}{SupportReads} ne ""){
$curDir = ""; $curOutDir = "$baseOut$SmplName/";
} else {
die "Can;t find valid path for $SmplName\n";
}
$dir2rd = $SmplName;
} else {
$curDir = $map{$curSmpl}{rddir}; $curOutDir = $map{$curSmpl}{wrdir};
}
$logDir = "$curOutDir/LOGandSUB/";
#ignore samples .. for various reasons ------------------------------------------------------------------------------------
if ($ignoreSmpl ne ""){if ($ignoreSmpl =~ m/$SmplName/){print "\n ======= Ignoring sample $SmplName =======\n";next;}}
my $smplLockF = "$logDir/$MFcontstants{DefaultSampleLock}";
if (-e $smplLockF){
if ($MFconfig{rmSmplLocks}){ system "rm -f $smplLockF";
} else {print "\n >>>>>>>>>> Sample $SmplName is locked! <<<<<<<<<< \n";next;}
}
$QSBoptHR->{LOCKfile} = $smplLockF; #set lockfile to be created if any job is submitted
push (@allSmplNames,$SmplName);
#die "$curDir\n$curOutDir\n$baseOut\n";
my $samplReadLength = $MFconfig{defaultReadLength}; #some default value
if (exists $map{$curSmpl}{readLength} && $map{$curSmpl}{readLength} != 0){
$samplReadLength = $map{$curSmpl}{readLength};
}
my $samplReadLengthX = $MFconfig{defaultReadLengthX}; #for any supplementary reads (eg PacBio)
if (exists $map{$curSmpl}{readLengthX} && $map{$curSmpl}{readLengthX} != 0){
$samplReadLengthX = $map{$curSmpl}{readLengthX};
}
#key IDs for sample
my $cAssGrp = $curSmpl;
my $cMapGrp = $map{$curSmpl}{MapGroup};
if ($map{$curSmpl}{AssGroup} ne "-1"){ $cAssGrp = $map{$curSmpl}{AssGroup};}
my $contigStatsUsed = 0;
%locStats = ();
$locStats{hasPaired} = 0; $locStats{hasSingle} = 0;
$totalChecked++;
#die $SmplName." $samplReadLength\n";
print "\n======= $SmplName - $JNUM - $dir2rd =======\n" unless($silent);
#set up dirs ------------------------------------------------------------------------------------
my $smplTmpDir = "$runTmpDirGlobal$SmplName/"; #curTmpDir
my $nodeSpTmpD = "$nodeTmpDirBase/$SmplName";
my @checkLocs = ($smplTmpDir);
push(@checkLocs,$curDir) if ($curDir ne "");
my $mapOut = "$smplTmpDir/mapping/";
#$DBpath="$curOutDir/readDB/";
my $finalCommAssDir = "$curOutDir/assemblies/metag/";
my $finalCommAssDirSingle = $finalCommAssDir; #this is only used for checking..
my $finalMapDir = "$curOutDir/mapping/";
my $KrakenOD = $curOutDir."Tax/kraken/$MFopt{globalKraTaxkDB}/";
$AsGrps{$cAssGrp}{AssemblSmplDirs} .= $curOutDir."\n";
my $AssemblyGo=0; my $MappingGo=0; #controls if assemblies / mappings are done in respective groups
#complicated flow control for multi sample assemblies
die "cAssGrp eq \"$cAssGrp\" ".$baseID."\n" if ($cAssGrp eq "");
my $assmGrpTag = "AssmblGrp_$cAssGrp";
#die "AssemblGrp exists already!!: \"$assmGrpTag\"\n" if (exists($assmblGrpLog{$assmGrpTag}));
#$assmblGrpLog{$assmGrpTag} = 1;
$assDir="$runTmpDirGlobal/$assmGrpTag/";
$finalCommAssDir = "$baseOut/$assmGrpTag/metag/" if ( !exists($AsGrps{$cAssGrp}{CntAimAss}) || $AsGrps{$cAssGrp}{CntAimAss}>1);
#my $metaGpreAssmblDir = "$runTmpDirGlobal/pre$assmGrpTag/";
my $metaGpreAssmblDir = "$smplTmpDir/pre$assmGrpTag/"; #needs to be sample specific (to allow for different coverage)
#assign job name (dependency) only ONCE
if ( !exists($AsGrps{$cAssGrp}{CntAss}) || $AsGrps{$cAssGrp}{CntAss} == 0){
$AsGrps{$cAssGrp}{AssemblJobName} = "";#"_XXASpl$cAssGrp"."XX_" ;
$AsGrps{$cAssGrp}{CSfinJobName} = "";#"_XXCSpl$cAssGrp"."XX_" ;
}
my @sampleDeps = ();
$AsGrps{$cAssGrp}{CntAss} ++;
print "AssmblyGrp: " . $AsGrps{$cAssGrp}{CntAss} .":".$AsGrps{$cAssGrp}{CntAimAss}. "; " if ($AsGrps{$cAssGrp}{CntAimAss} > 1 && !$silent);
if ($AsGrps{$cAssGrp}{CntAss} >= $AsGrps{$cAssGrp}{CntAimAss} ){
#print "running assembluy\n";
$AssemblyGo = 1;
if ($AsGrps{$cAssGrp}{CntAimAss}<=1){
$assDir="$smplTmpDir/assemblies/";
}
}
#mapping groups?
$AsGrps{$cMapGrp}{CntMap} ++;
print "MapGroup: ".$AsGrps{$cMapGrp}{CntMap} .":".$AsGrps{$cMapGrp}{CntAimMap}.";\n" unless ($silent);
if (!exists($AsGrps{$cMapGrp}{CntMap})){ die "Can;t find CntMap for $cMapGrp";}
if ($AsGrps{$cMapGrp}{CntMap} >= $AsGrps{$cMapGrp}{CntAimMap} ) {
#print "running mapping";
$MappingGo = 1;
}
#die "MG: $MappingGo\n";
if ($MFopt{DoAssembly} ==0 ){$AssemblyGo=0;}
#mapping related
my $cramthebam = 1;
my $bamcramMap = "bam"; if ($cramthebam){$bamcramMap = "cram";}
#---------------------------- 2 ----------------------------------
#set up dirs for this sample
my $metagAssDir = $assDir."metag/";
my $geneDir = $metagAssDir."genePred/";
#system("mkdir -p $metagAssDir $geneDir");
my $metaGassembly=$metagAssDir."scaffolds.fasta.filt";
my $finalCommScaffDir = "$finalCommAssDir/scaffolds/";
my $metaGscaffDir = "$metagAssDir/scaffolds/";
my $STOfinScaff = "$finalCommScaffDir/scaffDone.sto";
my $pseudoAssFile = "$metagAssDir/longReads.fasta.filt";
my $pseudoAssFileFinal = "$finalCommAssDir/longReads.fasta.filt";
my $finAssLoc = "$finalCommAssDir/scaffolds.fasta.filt";
my $ContigStatsDir = "$curOutDir/$dir_ContigStats/";
my $coveragePerCtg = "$ContigStatsDir/Coverage.percontig.gz";
my $suppCoveragePerCtg = "$ContigStatsDir/Cov.sup.percontig.gz";
my $nonParDir = $curOutDir."nonpareil/";
#SNP calling on assembly related files
my $SNPdir = "$curOutDir/SNP/";
my $contigsSNP = "$SNPdir/contig.SNPc.$MFopt{SNPcallerFlag}.fna"; #keep without gz, although will be gz'd
my $genePredSNP = "$SNPdir/genes.shrtHD.SNPc.$MFopt{SNPcallerFlag}.fna.gz";
my $genePredAASNP = "$SNPdir/proteins.shrtHD.SNPc.$MFopt{SNPcallerFlag}.faa.gz";
my $vcfSNP = "$SNPdir/allSNP.$MFopt{SNPcallerFlag}.vcf"; $vcfSNP = "" if (!$MFopt{saveVCF});
my $CRAMmap = "$finalMapDir/$SmplName-smd.cram";
my $SupCRAMmap = "$finalMapDir/$SmplName.sup-smd.cram";
my $inputRawFile = "$curOutDir/input_raw.txt";
my $binningDir = "$finalCommAssDir/Binning/";
my @smplIDs = ("");#smpls in current assembly group
if ($MFopt{DoMetaBat2}){
@smplIDs = @{$DOs{$cAssGrp}{SmplID}};
}
my $BinningOut = "$binningDir/MB2/$smplIDs[-1]";
$BinningOut = "$binningDir/SB/$smplIDs[-1]" if ($MFopt{DoMetaBat2} == 2);
$BinningOut = "$binningDir/MD/$smplIDs[-1]" if ($MFopt{DoMetaBat2} == 3);
my $STOcram = "$CRAMmap.sto";
my $STOsupCram = "$SupCRAMmap.sto";
my $STOmapFinal="$finalMapDir/done.sto";
# collect stats on seq qual, assembly etc
if ($alwaysDoStats){
my ($statsHD,$curStats,$statsHD5,$curStats5) = smplStats($curOutDir,$assDir,$SmplName);
#add to global string that is later written
if ($statStr eq ""){
$statStr.="SMPLID\tDIR\t".$statsHD."\n".$curSmpl."\t$dir2rd\t".$curStats."\n";
$statStr5.="SMPLID\tDIR\t".$statsHD5."\n".$curSmpl."\t$dir2rd\t".$curStats5."\n";
} else {$statStr.=$curSmpl."\t$dir2rd\t".$curStats."\n"; $statStr5.=$curSmpl."\t$dir2rd\t".$curStats5."\n";}
}
#die "$doPreAssmFlag\n$finalCommAssDir/$STOpreAssmblDone\n";
#detect what already exists..
my $efinAssLoc = 0; $efinAssLoc = 1 if (-s $finAssLoc && -e "$finalCommAssDir/$STOassmbleDone");
#activate if two assemblies for single sample required, e.g. hybrid assemblies
my $ePreAssmbly = 0; $ePreAssmbly = 1 if (-s $finAssLoc && -e "$finalCommAssDir/$STOpreAssmblDone");
my $ePreAssmblPck = 0; $ePreAssmblPck = 1 if (-s $finAssLoc && -e "$metaGpreAssmblDir/moved.sto");
my $doPreAssmFlag = 0; my $postPreAssmblGo =0 ;
($doPreAssmFlag,$postPreAssmblGo,$ePreAssmblPck) = prepPreAssmbl($finalCommAssDir,$metaGpreAssmblDir,$finalMapDir, "$smplTmpDir/preAssmblData/",
$ContigStatsDir, $curSmpl, $cAssGrp, $efinAssLoc, $ePreAssmblPck);#moves files to new locations
my $eCovAsssembly = 0; $eCovAsssembly = 1 if (-e $coveragePerCtg);
my $eSuppCovAsssembly = 0; $eSuppCovAsssembly = 1 if (-e $suppCoveragePerCtg);
#will be created in contigstats step (not related to bowtie & sortbam)
my $emetaGassembly = 0; $emetaGassembly = 1 if (-e $metaGassembly && -e "$metagAssDir/$STOassmbleDone");
my $eFinMapCovGZ = 0; $eFinMapCovGZ = 1 if (-e $STOcram && -e "$finalMapDir/$SmplName-smd.bam.coverage.gz");#"$finalMapDir/$SmplName-smd.bam.coverage.gz";
#$MFopt{mapSupport2Assembly}
my $locMapSup2Assembly =0; $locMapSup2Assembly =1 if ($MFopt{mapSupport2Assembly} && $map{$curSmpl}{"SupportReads"} ne "");
my $eFinSupMapCovGZ = 0; $eFinSupMapCovGZ = 1 if ($locMapSup2Assembly && -e $STOsupCram && -e "$finalMapDir/$SmplName.sup-smd.bam.coverage.gz");
#die "$eFinSupMapCovGZ $finalMapDir\n";
my $dfinalCommAssDir = 0 ; $dfinalCommAssDir = 1 if (-d $finalCommAssDir);
my $eFinalMapDir = 0; $eFinalMapDir = 1 if (-s $STOmapFinal);
#upload2EBI
my $DoUploadRawReads = 0; $DoUploadRawReads = 1 if ($MFconfig{uploadRawRds} ne "");
#print "$emetaGassembly XX";
#die "$eFinMapCovGZ $STOcram && -e $finalMapDir/$SmplName-smd.bam.coverage.gz\n";
#first take care of moving finished assemblies..
if (!$efinAssLoc && $emetaGassembly){
print "moving finished assembly to final location\n";
#print "$metaGassembly\n";
systemW "mkdir -p $finalCommAssDir" unless ($dfinalCommAssDir);
systemW "rsync -r --remove-source-files $metagAssDir/* $finalCommAssDir/";
$efinAssLoc = 1; $emetaGassembly = 0;
}
my $locRedoSNPcalling =0;
my $locRedoAssMapping = $MFopt{redoAssMapping};
#check if current assembly group is the same as before!
my $locRewrite = 0; my $locRedoAssembl = 0;
if ($efinAssLoc && -e "$finalCommAssDir/smpls_used.txt"){
my $currAssmlCnt = 0;#`cat $finalCommAssDir/smpls_used.txt | grep -v '^\\\$' | wc -l`;
open I,"<$finalCommAssDir/smpls_used.txt " or die "Smple used: $!\n"; while (<I>){$currAssmlCnt++ unless m/^$/;} close I;
chomp $currAssmlCnt; $currAssmlCnt = int($currAssmlCnt);
if ($currAssmlCnt < $AsGrps{$cAssGrp}{CntAimAss}){
print "$cAssGrp assembl count has changed! (from $currAssmlCnt to $AsGrps{$cAssGrp}{CntAimAss})\n$finalCommAssDir\nRemoving assembly and all processed reads\n";
unless ($OKtoRWassGrps) {print "Stopping MATAFILER, human intervention needed.. use the flag \"-OKtoRWassGrps 1\" to allow MATAFILER to delete files\n"; die;}
$locRewrite=1;
}
}
if ($eFinMapCovGZ && (!$ePreAssmblPck && !$ePreAssmbly && !$efinAssLoc && !$emetaGassembly) ){#impossible, so reason must be severe!
print "Mapping exists, but no assembly, removing mapping..\n$finalMapDir\n$finAssLoc\n$metaGassembly\n";
die unless ($OKtoRWassGrps);
$locRewrite = 0; $locRedoAssembl = 0;
}
if ($efinAssLoc && $finAssLoc ne "$finalCommAssDirSingle/scaffolds.fasta.filt" && -s "$finalCommAssDirSingle/scaffolds.fasta.filt"){
print "Something wrong.. assembly group assembly and single assembly present:\n$finAssLoc\n$finalCommAssDirSingle/scaffolds.fasta.filt\n";
#die;
$locRedoAssMapping=1;$locRedoSNPcalling=1;
system "rm -fr $finalCommAssDirSingle; mkdir -p $finalCommAssDirSingle;\n";
$eCovAsssembly=0;$eFinMapCovGZ=0;$eFinalMapDir=0;$eFinSupMapCovGZ=0;$eSuppCovAsssembly=0;$eSuppCovAsssembly=0;
}
if ($locStats{totRds}==0 && !defined($locStats{uniqAlign}) && -e $inputRawFile && $eFinMapCovGZ){ #do a deeper look
my $line = getFileStr("$inputRawFile",0); #open I,"<$inputRawFile"; my $line = <I>; close I; chomp($line);
#my @spl = split /,/,$line; my $inFileSize = -s $spl[0];
print "weird empty: $line \nredoing..\n";
$locRewrite = 1;$locRedoAssembl = 1;
}
if ( ($skipWrongPairedSmpls || $OKtoRWassGrps) && -e "$logDir/sdmReadCleaner.sh.etxt" && `tail -n 70 $logDir/sdmReadCleaner.sh.etxt | grep 'invalid paired read' ` ne ""){
print "$logDir/sdmReadCleaner.sh.etxt problems! Delete outdir\n";
if ($OKtoRWassGrps){
$locRewrite=1 ;
}elsif ($skipWrongPairedSmpls){
next ;
}
}
#die;
#-------------------------- DELETION SECTION -----------------------------------
#DELETION SECTION
#redo run - or parts thereof
if ($OKtoRWassGrps && ($rewrite || $locRewrite)){
print "Deleting previous results.. rerun MATAFILER for sample\n";
system ("rm -r -f $assDir $finalCommAssDir");
system("rm -f -r $curOutDir $smplTmpDir $collectFinished ");
#next; #too deep, needs a complete new round over dir..
#$efinAssLoc = 0; $eFinMapCovGZ = 0; $emetaGassembly = 0;
$efinAssLoc =0 ;$eCovAsssembly = 0; $eSuppCovAsssembly=0; $eFinSupMapCovGZ=0; $emetaGassembly = 0; $eFinMapCovGZ = 0;$dfinalCommAssDir =0;$eFinalMapDir = 0;$locRewrite = 0; $locRedoAssembl = 0;$eSuppCovAsssembly=0;
}
#unzip and change ifastap & cfp1/cfp2
if (@unzipjobs > 12){#only run 8 jobs in parallel, lest the cluster breaks down..
#my @last_n = @unzipjobs[-8..-1];
$curUnzipDep = $unzipjobs[-12];#join(";",@last_n);
$waitTime=0;
}
#automatically delete mapping, if assembly no longer exists..
#print "locRedoAssMapping : $locRedoAssMapping\n";
if ($MFopt{map2Assembly} ){
if ($eFinMapCovGZ && !$eFinalMapDir){$locRedoAssMapping = 1 ; print "R0 ";}
if (!$efinAssLoc && !$ePreAssmbly && !$emetaGassembly ){$locRedoAssMapping = 1 ;}#print "R1";}
if (!$MappingGo && $eFinalMapDir){$locRedoAssMapping = 1 ;print "R2 ";}
if (-e $STOcram && !-e "$finalMapDir/$SmplName-smd.bam.coverage.gz" && !-e "$finalMapDir/$SmplName-smd.bam.coverage"){$locRedoAssMapping = 1 ;print "R3 ";}
if ($eFinMapCovGZ && (exists($locStats{uniqAlign}) && $locStats{uniqAlign} > 20) && -s $CRAMmap <300){$locRedoAssMapping = 1 ;print "R4";}
#print "$CRAMmap :: $locRedoAssMapping\n";
print "redo assem mapping!" . " -s $CRAMmap \n" if ($locRedoAssMapping && -e $CRAMmap); #. (-s $CRAMmap)
#die "$STOcram && !-e $finalMapDir/$SmplName-smd.bam.coverage.gz";
}
#die "locRedoAssMapping : $locRedoAssMapping $finalMapDir : !$efinAssLoc && !$emetaGassembly\n" if ($locRedoAssMapping);
my $sizemap = -s $CRAMmap;
#print "size map: " . $sizemap . "\n";
#delete assembly
if ($MFopt{redoAssembly} || $locRedoAssembl){
print "Removing assembly ... \n" if ($emetaGassembly);
system "rm -fr $finalCommAssDir";
$efinAssLoc = 0;
$locRedoAssMapping=1;
}
#delete mapping to assembly
if ($locRedoAssMapping){
#die "locDel\n";
print "Deleting previous assembly mapping, size map: ". (-s $CRAMmap) . "\n$CRAMmap\n" if ($MappingGo && $eFinalMapDir);
#die "$finAssLoc && !-e $metaGassembly\n";
system "rm -fr $finalMapDir $mapOut $ContigStatsDir/Coverage.* $ContigStatsDir/Cov.sup.*";
$eFinMapCovGZ = 0; $emetaGassembly = 0;
#are there SNPs called? remove as well..
$locRedoSNPcalling=1;
}
#Case: primary assembly mapping was done, support reads were not yet mapped.. need to redo binning
#print "$locMapSup2Assembly && !$eFinSupMapCovGZ) && ($MFopt{map2Assembly} && $eFinMapCovGZ \n";
if ( ($locMapSup2Assembly && !$eFinSupMapCovGZ) && ($MFopt{map2Assembly} && $eFinMapCovGZ ) ){
print "redoing binning due to support mapping not included..\n";
system("rm -rf $binningDir/");
}
#debug case: binning was empty
if ($MFopt{DoMetaBat2} && $MFopt{BinnerRedoEmpty} && -e $BinningOut && !-s $BinningOut){
#die;
print "redoing binning due to empty bins (flag -redoEmptyBins 1) ..\n";
system "rm -rf $binningDir";
}
if ((!$eFinMapCovGZ && $eCovAsssembly) #redo only contigstats related to coverage..
|| ($eSuppCovAsssembly && !$eFinSupMapCovGZ) || $redoCS){
print "redoing contig stats global..\n";
system("rm -rf $finalCommAssDir/ContigStats/ $ContigStatsDir $binningDir/");
$eCovAsssembly = 0; $eSuppCovAsssembly=0; #contigstats needs redoing..
}
system "rm -r $KrakenOD" if ($MFopt{RedoKraken} && -d $KrakenOD);
if ($MFopt{RedoRiboFind}){system "rm -rf $curOutDir/ribos";}
if ($MFopt{RedoRiboAssign}){system "rm -rf $curOutDir/ribos//ltsLCA";}
if ($MFopt{DoRibofind} && -e "$curOutDir/LOGandSUB/RiboLCA.sh.etxt"){
#my $LCAetxt = `cat $curOutDir/LOGandSUB/RiboLCA.sh.etxt`;
#if ($LCAetxt =~ m/ParseError thrown: Unexpected character .\@. found/){system "rm -rf $curOutDir/ribos";}
}
if ($locRedoSNPcalling){system "rm -fr $SNPdir";}
if ($MFopt{redoSNPcons}){ system "rm -rf $genePredSNP* $contigsSNP* $genePredAASNP* $smplTmpDir/SNP $logDir/SNP";
} elsif ($MFopt{redoSNPgene}){ system "rm -rf $genePredSNP* $genePredAASNP* ";
}
my $boolGenePredOK=0;
if ($MFopt{DoEukGenePred}){
$boolGenePredOK = 1 if (-s "$finalCommAssDir/genePred/proteins.bac.shrtHD.faa" || ($MFopt{pseudoAssembly} && -e "$finalCommAssDir/genePred/proteins.bac.shrtHD.faa"));
} else {
$boolGenePredOK = 1 if (-s "$finalCommAssDir/genePred/proteins.shrtHD.faa" || ($MFopt{pseudoAssembly} && -e "$finalCommAssDir/genePred/proteins.shrtHD.faa") );
}
#central flag
my $boolAssemblyOK=0;
$boolAssemblyOK=1 if ($boolGenePredOK && $efinAssLoc );#&& (!$MFopt{map2Assembly} || $eFinMapCovGZ ) );
#die "$boolGenePredOK && $efinAssLoc && (!$MFopt{map2Assembly} || $eFinMapCovGZ ) $locRedoAssMapping\n";
#&& (-s "$finalMapDir/$SmplName-smd.bam" || -s "$finalMapDir/$SmplName-smd.cram")
#die "$boolAssemblyOK\n$finalCommAssDir/genePred/proteins.shrtHD.faa\n$finalMapDir/$SmplName-smd.bam.coverage.gz\n";
if ( ( !$boolAssemblyOK && $unfiniRew==1 ) ){
die "Deleting previous results..\n";
system("rm -f -r $curOutDir $smplTmpDir $collectFinished");
$efinAssLoc = 0; $eFinMapCovGZ = 0; $emetaGassembly = 0;
} elsif ($boolAssemblyOK && $eCovAsssembly) {
#check that assembly path fits..
getAssemblPath($curOutDir,$finalCommAssDir);
}
#--------------------- secondary map deletions & flags --------------------------
my $boolScndMappingOK = 1; my $iix =0;
my $boolScndCoverageOK = 1;
if ($MFopt{MapRewrite2nd}){
print "rewriting secondary map\n";
foreach my $bwt2outDTT (@bwt2outD){
my $expectedMapCovGZ = "$bwt2outDTT/$bwt2ndMapNmds[$iix]"."_".$SmplName."-0-smd.bam.coverage.gz"; #$bamcramMap : 2nd map only has .bam output
system "rm -f $expectedMapCovGZ*";
$eFinMapCovGZ = 0;
}
}
#die "eFinMapCovGZ $eFinMapCovGZ\n";
foreach my $bwt2outDTT (@bwt2outD){
my $expectedMapCovGZ = "$bwt2outDTT/$bwt2ndMapNmds[$iix]"."_".$SmplName."-0-smd.bam.coverage.gz";
my $expectedMapBam = "$bwt2outDTT/$bwt2ndMapNmds[$iix]"."_".$SmplName."-0-smd.bam";
$iix++;
#print $expectedMapCovGZ."\n";
if ( -e "$expectedMapCovGZ" && -e $expectedMapBam && $MappingGo ){
$boolScndMappingOK=1;
}else{
$boolScndMappingOK=0; $boolScndCoverageOK=0;
#be clean
system "rm -f $expectedMapBam*";
last;
}
if ($MFopt{mapModeCovDo} && (!-e $expectedMapCovGZ.".median.percontig" || !-e $expectedMapCovGZ.".percontig"|| !-e $expectedMapCovGZ.".pergene")){
$boolScndCoverageOK=0;
system "rm -f $expectedMapCovGZ.*";
}
}
if (@bwt2outD == 0 ){$boolScndMappingOK = 1 ; $boolScndCoverageOK=1;}#|| !$MappingGo);
#die "$boolScndMappingOK\n";
#print $boolScndMappingOK."\n$boolAssemblyOK\n";
#--------------------- other flags --------------------------
#contamination flag: redo/do contaminant removal to eg make sure human contamination is correctly logged
my $calcContamination = 0;
$calcContamination = 1 if ($locStats{contamination} eq "?\t" && $efinAssLoc && $MFopt{completeContaStats});
#print "Conta: $calcContamination \"$locStats{contamination}\"\n";
#Kraken flag
my $calcKraken =0;
$calcKraken = 1 if ($MFopt{DoKraken} && (!-d $KrakenOD || !-e "$KrakenOD/krakDone.sto"));
if (!$calcKraken && $MFopt{DoKraken}){
opendir D, $KrakenOD; my @krkF = grep {/krak\./} readdir(D); closedir D;
foreach my $kf (@krkF){
$kf =~ m/krak\.(.*)\.cnt\.tax/; my $thr = $1;# die $thr." $kf\n";
system "mkdir -p $dir_KrakFind/$thr" unless (-d "$dir_KrakFind/$thr"); #system "mkdir -p $dir_RibFind/SSU/" unless (-d "$dir_RibFind/SSU/"); system "mkdir -p $dir_RibFind/LSU/" unless (-d "$dir_RibFind/LSU/");
system "cp $KrakenOD/$kf $dir_KrakFind/$thr/$SmplName.$thr.krak.txt";
}
} else {$progStats{KrakTaxFailCnts}++;}
#system "rm -f $curOutDir/ribos//ltsLCA/LSU_ass.sto $curOutDir/ribos//ltsLCA/Assigned.sto"; fix for new LSU assignments
my $calcGenoSize=0; $calcGenoSize=1 if ($MFopt{DoGenoSizeEst} && !-e "$curOutDir/MicroCens/MC.0.result");
my $calcRibofind = 0; my $calcRiboAssign = 0;
$calcRibofind = 1 if ($MFopt{DoRibofind} && (!-e "$curOutDir/ribos//SSU_pull.sto"|| !-e "$curOutDir/ribos//LSU_pull.sto" || ($MFopt{doRiboAssembl} && !-e "$curOutDir/ribos/Ass/allAss.sto" ))); #!-e "$curOutDir/ribos//ITS_pull.sto"||
$calcRiboAssign = 1 if ($MFopt{DoRibofind} && ( !-e "$curOutDir/ribos//ltsLCA/Assigned.sto" || !-e "$curOutDir/ribos//ltsLCA/SSU_ass.sto") ); #!-e "$curOutDir/ribos//ltsLCA/ITS_ass.sto"|| #ITS no longer required.. unreliable imo
#die "$calcRibofind\n\n";
#die "$calcRibofind $calcRiboAssign\n";
RiboMeta($calcRibofind,$calcRiboAssign,$curOutDir,$SmplName);
my ($calcDiamond,$calcDiaParse) = IsDiaRunFinished($curOutDir);
#die "XX $calcDiamond $calcDiaParse\n";
my $calcMetaPhlan=0;
#die $dir_MP2."$SmplName.MP2.sto";
if ($MFopt{DoMetaPhlan}){
if (!-e $dir_MP2."$SmplName.MP2.sto"){
$calcMetaPhlan=1 ;
$progStats{metaPhl2FailCnts}++;
} else { $progStats{metaPhl2ComplCnts}++;}
}
my $calcMOTU2=0;
if ($MFopt{DoMOTU2}){
if (!-e $dir_mOTU2."$SmplName.Motu2.sto"){
$calcMOTU2=1;
$progStats{mOTU2FailCnts}++;
} else { $progStats{mOTU2ComplCnts}++;}
}
my $calcTaxaTar = 0;
if ($MFopt{DoTaxaTarget}){
if (!-e $dir_TaxTar."$SmplName.TaxTar.sto"){
$calcTaxaTar=1 ;
$progStats{taxTarFailCnts}++;
} else { $progStats{taxTarComplCnts}++;}
}
#TODO
my $allMapDone =0;#used for SNP calling and Binning - but binning requires info if all maps are finished from all samples
$allMapDone = 1 if (-e "$finalMapDir/$SmplName-smd.$bamcramMap" && $eCovAsssembly && !$ePreAssmbly && ($eSuppCovAsssembly || !$locMapSup2Assembly) && $AsGrps{$cAssGrp}{MapDeps} !~ m/[^;]/ );
if ( ($boolAssemblyOK || ($doPreAssmFlag && $ePreAssmbly && !$ePreAssmblPck)) && !$locRedoAssMapping ){#causes a lot of overhead but mainly to avoid unpacking reads again..
print "present: $curOutDir \n"; $presentAssemblies ++;#= $AsGrps{$cAssGrp}{CntAimAss};
#base is present, but is the additions?
if ($MFopt{map2Assembly} && $MFopt{doBam2Cram} && #.cram.sto to check that everything went well
$AssemblyGo && $MappingGo && $eFinMapCovGZ && $allMapDone ){
#(-e "$finalCommAssDir/scaffolds.fasta.filt.$MFcontstants{mini2IdxFileSuffix}" || -e "$finalCommAssDir/scaffolds.fasta.filt${bwt2IdxFileSuffix}.1.bt2") &&
#delete bwt2 index to save some space..
#need to ensure that all mapping in mapping group are fine..
system "rm -f $finalCommAssDir/scaffolds.fasta.filt$MFcontstants{bwt2IdxFileSuffix}* $finalCommAssDir/scaffolds.fasta.filt.fai $finalCommAssDir/scaffolds.fasta.filt.$MFcontstants{mini2IdxFileSuffix} $finalCommAssDir/scaffolds.fasta.filt.$MFcontstants{kmaIdxFileSuffix}";
system "rm -f $finalMapDir/${SmplName}*smd.bam.bai $finalMapDir/${SmplName}*smd.bam.coverage.c* $finalMapDir/${SmplName}*smd.bam.coverage.gen* $finalMapDir/${SmplName}*smd.bam.coverage.m* $finalMapDir/${SmplName}*smd.bam.coverage.p*";
}
my $subprts = $defaultContigSubs; my $CSthre = 1;
if ($AssemblyGo ){
$subprts = $defaultContigSubs."gFG";$CSthre = 6; $subprts .= "m" if ($MFopt{DoBinning});
$subprts .= "4" if ($MFopt{kmerPerGene} ); $subprts .= "k" if ($MFopt{kmerAssembly} );
} #k
#call contigStats only from 1 (actually two) places: end of for loop
#my ($tmpCSj,$tmpCSc,$tmpCSd) = runContigStats($curOutDir ,"",$finalCommAssDir,$subprts,1,$samplReadLength,$nodeSpTmpD,$AssemblyGo,$CSthre, $curSmpl);
#add2SampleDeps(\@sampleDeps, [$tmpCSj]);
#$contigStatsUsed = $tmpCSd;
#print "SmplDps: @sampleDeps\n";
#die "$boolScndMappingOK && !$MFopt{DoCalcD2s} && !$calcRibofind && !$calcDiamond && !$calcMetaPhlan && !$calcKraken\n";
}
my $calcMetaBat2 = 0;
if ($MFopt{DoMetaBat2} && $boolAssemblyOK && $AssemblyGo && $AsGrps{$cAssGrp}{MapDeps} !~ m/[^;]/ && (!-e "$BinningOut.cm" && !-s "$BinningOut.cm2")) {
$calcMetaBat2=$MFopt{DoMetaBat2};
}
#die "Binner: $calcMetaBat2 :: $MFopt{DoMetaBat2} && $boolAssemblyOK && $AssemblyGo (!-e $BinningOut.cm && !-s $BinningOut.cm2)\n$BinningOut\n" ;
#not complete yet? Then delete..
if ($redoFails && ($calcRibofind||$calcDiamond || $calcDiaParse ||$calcMOTU2 || $calcMetaPhlan || $calcTaxaTar)){
die "now recalc $curSmpl\n";
system ("rm -r -f $assDir $finalCommAssDir");
system("rm -f -r $curOutDir $smplTmpDir $collectFinished ");
}
system("mkdir -p $logDir") unless (-d $logDir);
$QSBoptHR->{LocationCheckStrg} = checkDrives(\@checkLocs);
#die "$metaGassembly\n$finAssLoc\n$nodeSpTmpD\n";
# #----------------------- FLAGS ------------------------
# #and some more flags for subprocesses
my $nonPareilFlag = !-s "$nonParDir/$SmplName.npo" && $MFopt{DoNonPareil} ;
my $scaffoldFlag = 0; if ( !-e $STOfinScaff && $map{$curSmpl}{"SupportReads"} =~ m/mate/i ){$scaffoldFlag = 1 ;}# print "SUPP:: $map{$curSmpl}{SupportReads}\n";}
my $assemblyFlag = 0; $assemblyFlag = 1 if ( $MFopt{DoAssembly} && !$boolAssemblyOK && !$efinAssLoc && !$emetaGassembly);
#die "$assemblyFlag = 1 if ( $MFopt{DoAssembly} && !$boolAssemblyOK && !$efinAssLoc && !-e $metaGassembly\n $doPreAssmFlag\n";
my $calcReadMerge = 0;
$calcReadMerge = 1 if ($MFopt{doReadMerge} && ($MFopt{calcOrthoPlacement} || $calcDiamond || $calcGenoSize));
my $mapAssFlag = 0; $mapAssFlag = 1 if ($MFopt{map2Assembly} && !$eFinMapCovGZ );
my $calcCoverage = 0; $calcCoverage =1 if (!$eCovAsssembly && $MFopt{map2Assembly});
#only for support reads (from hybrid assemblies)
my $mapSuppAssFlag =0;$mapSuppAssFlag = 1 if ($locMapSup2Assembly && !$eFinSupMapCovGZ && $efinAssLoc );#hasSuppRds(\%AsGrps,$cAssGrp,$curSmpl ) );
my $calcSuppCoverage = 0; $calcSuppCoverage =1 if ($MFopt{mapSupport2Assembly} && !$eSuppCovAsssembly && $map{$curSmpl}{"SupportReads"} ne "");
#die "$mapSuppAssFlag = 1 if ($locMapSup2Assembly && !$eFinSupMapCovGZ && $efinAssLoc \n";
my $assemblyBuildIndexFlag=0;
$assemblyBuildIndexFlag=1 if ($MFopt{DoAssembly} && !$assemblyFlag && $MFopt{map2Assembly} && ($mapAssFlag || $mapSuppAssFlag )
# && $AssemblyGo -> do already in first round, store dependency
&& ! mapperDBbuilt($finAssLoc,$MFopt{MapperProg}) );
#die "$assemblyBuildIndexFlag $MFopt{DoAssembly} && !$assemblyFlag && $MFopt{map2Assembly} && ($mapAssFlag || $mapSuppAssFlag ) \n". mapperDBbuilt($finAssLoc,$MFopt{MapperProg}) ."\n";
#print "build $assemblyBuildIndexFlag $MFopt{DoAssembly} && !$assemblyFlag && $MFopt{map2Assembly} && $mapAssFlag && $MFopt{MapperProg}\n";
#requires only bam/cram && assembly
my $calcConsSNP=0; $calcConsSNP =1 if ($MFopt{DoConsSNP} && (!-e $genePredSNP || -s $genePredSNP < 100 || ($MFopt{saveVCF} && !-e $vcfSNP)));
#die "genePredSNP $genePredSNP\n";
my $calc2ndMapSNP = 0; $calc2ndMapSNP = 1 if ($MFopt{Do2ndMapSNP});
my $pseudAssFlag = 0; $pseudAssFlag = 1 if ($MFopt{pseudoAssembly} && $map{$curSmpl}{ExcludeAssem} eq "0" && (!-e $pseudoAssFileFinal.".sto" || !$boolGenePredOK));
my $dowstreamAnalysisFlag = 0;
#$unpackZip simulates dowstreamAnalysisFlag, just to get sdm running..
$dowstreamAnalysisFlag=1 if ( $unpackZip || $calcContamination || $MFopt{calcOrthoPlacement} || $scaffTarExternal ne "" || $assemblyFlag
|| $pseudAssFlag || $scaffoldFlag || $nonPareilFlag || $calcGenoSize || $calcDiamond || $calcDiaParse
|| $MFopt{DoCalcD2s} || $calcKraken || $calcRibofind || $calcRiboAssign || $calcMOTU2 || $calcMetaPhlan || $calcTaxaTar );
my $requireRawReadsFlag = 0;#only list modules that really need raw reads
$requireRawReadsFlag = 1 if ( !$boolScndMappingOK || $calcContamination);
my $seqCleanFlag = 0; $seqCleanFlag =1 if ($dowstreamAnalysisFlag && !-e "$smplTmpDir/seqClean/filterDone.stone" );
my $porechopFlag = 0;
$porechopFlag = 1 if ($MFopt{usePorechop} && $dowstreamAnalysisFlag && !-e "$smplTmpDir/rawRds/poreChopped.stone");
#die "$assemblyFlag\t$seqCleanFlag\t$boolScndMappingOK\n";
my $calcUnzip=0;
$calcUnzip=1 if ($porechopFlag || $seqCleanFlag || $mapAssFlag || $mapSuppAssFlag || (!$MFopt{useUnmapped} && !$boolScndMappingOK));
#print "chk1 $mapSuppAssFlag $calcSuppCoverage $eSuppCovAsssembly\n" ;
# #----------------------- END FLAGS ------------------------
#some more flow control..
if (
!$DoUploadRawReads && $boolScndMappingOK && !$MFopt{DoCalcD2s} &&
!$calcConsSNP && !$calcMetaBat2 && !$calc2ndMapSNP && $boolAssemblyOK && $boolScndCoverageOK
&& !$calcCoverage && !$calcSuppCoverage && !$dowstreamAnalysisFlag
#&& !$calcRibofind && !$calcRiboAssign && !$MFopt{calcOrthoPlacement} && !$calcGenoSize && !$calcDiamond && !$calcDiaParse &&
#!$calcMetaPhlan && !$calcTaxaTar && !$calcMOTU2 && !$calcKraken && $scaffTarExternal eq ""
){
#free some scratch
system "rm -rf $smplTmpDir &" if ($rmScratchTmp );
#system "rm -f $smplLockF" if (-e $smplLockF);
print "next due to sample finished";
MFnext($smplLockF,\@sampleDeps,$JNUM ,$QSBoptHR); next;
}
#report for debugging:
#print "!$calcConsSNP && !$calcMetaBat2 && !$calc2ndMapSNP && $boolAssemblyOK && $boolScndCoverageOK \n && $boolScndMappingOK && !$MFopt{DoCalcD2s} && !$DoUploadRawReads \n && !$calcRibofind && !$calcRiboAssign && !$MFopt{calcOrthoPlacement} && !$calcGenoSize && !$calcDiamond && !$calcDiaParse && \n !$calcMetaPhlan && !$calcTaxaTar && !$calcMOTU2 && !$calcKraken && $scaffTarExternal eq \n $allMapDone\n $eFinMapCovGZ && $eCovAsssembly && $calcCoverage\n";
if (!$assemblyFlag && $AssemblyGo && !$efinAssLoc && $emetaGassembly){
die "assem copy\n"; #should actually never be here..
push(@{$AsGrps{$cAssGrp}{AssCopies}}, $assDir."/metag/*",$finalCommAssDir);
}
#die "$pseudAssFlag\n$boolGenePredOK\n$pseudoAssFileFinal\n";
if ($scaffTarExternal ne "" && $map{$curSmpl}{"SupportReads"} !~ /mate/i && $scaffTarExtLibTar ne $curSmpl ){print"scNxt\n";next;}
#has this sample extra reads (e.g. long reads?)
#die "$assemblyFlag || !$boolScndMappingOK || $nonPareilFlag || $calcDiamond || $MFopt{DoCalcD2s} || $calcKraken\n";
#die "$curDir\n";
#-----------------------------------------------------------------------------------------
# actual job submissions starts from here
#-----------------------------------------------------------------------------------------
#unzipping & sorting of files into scratch dir..
#also does either trimomatic or porechop..
#die "calcUnzip $calcUnzip\n" if ($calcUnzip);
# my ($jdep,$cfp1ar,$cfp2ar,$cfpsar,$WT,$rawFiles, $mmpuNum, $libInfoRef, $inputRawSize) =
#my %seqSet = (pa1 => \@pa1, pa2 => \@pa2, pas => \@pas, paX1 => \@paX1, paX2 => \@paX2, paXs => \@paXs, libInfo => \@libInfo, libInfoX => \@libInfoX,
# totalInputSizeMB => $totalInputSizeMB,rawReads => $rawReads,mmpu => $mmpu, WT => $WT);
my ($jdep,$hrefSeqSet) =
seedUnzip2tmp($curDir,$curSmpl,$curUnzipDep,$nodeSpTmpD,
$smplTmpDir,$waitTime,$importMocat,$AsGrps{$cMapGrp}{CntMap},$calcUnzip,$finalMapDir,