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Trinity.pl
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Trinity.pl
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#!/usr/bin/env perl
use strict;
use warnings;
use threads;
no strict qw(subs refs);
use FindBin;
use lib ("$FindBin::Bin/PerlLib", "$FindBin::Bin/PerlLibAdaptors");
use File::Basename;
use Cwd;
use Carp;
use Getopt::Long qw(:config no_ignore_case bundling pass_through);
open (STDERR, ">&STDOUT"); ## capturing stderr and stdout in a single stdout stream
# Site specific setup
my $CPU_MAX = 64; # Set this to a high number if you really do want to use more processors. I suspect it may only cause trouble, though.
my $IWORM_KMER_SIZE = 25;
my $MIN_IWORM_LEN = 25;
# option list:
my ($seqType, $left_file, $right_file, $single_file, $SS_lib_type, $min_contig_length,
$group_pairs_distance, $jaccard_clip, $show_advanced_options,
$output_directory, $prep_only
);
# What is allowed for the options. Put string to be displayed in '%allowed'; this
# will be showed to the user via help and on error. Keys are the variable names.
# Actual hash to be used for checking is auto-generated. Fancy regex inside map
# is just to get rid of the syntaxical sugar 'or' in the display string.
my %allowed =
( seqType => 'cfa, cfq, fa, or fq'
, kmer_method => 'jellyfish, meryl, or inchworm'
);
my %allowed_check;
foreach my $all (keys %allowed) {
my %h = map { (my $s = $_) =~ s/^or //; $s => 1 } split ', ', $allowed{$all};
$allowed_check{$all} = \%h;
}
# defaults:
$output_directory = "trinity_out_dir";
$min_contig_length = 200;
$group_pairs_distance = 500;
my $path_reinforcement_distance = 75;
my $NO_RUN_BUTTERFLY_FLAG = 0;
my $RERUN_BUTTERFLY_FLAG = 0;
my $bfly_opts = "";
my $bflyHeapSpaceMax = "20G";
my $bflyHeapSpaceInit = "1G";
my $min_kmer_cov = 1;
my $meryl_opts = "";
my $inchworm_cpu = 6;
my $min_percent_read_iworm_kmers = -1;
my $CPU = 2;
my $bflyCPU;
my $bflyCalculateCPU = 0;
my $bflyGCThreads;
my $long_reads = "";
my $max_number_of_paths_per_node = 10;
my $lenient_path_extension = 0;
## ADVANCED OPTIONS:
# todo add some.
my $no_meryl_flag = 0;
## Chrysalis opts
my $min_glue = 2;
my $min_iso_ratio = 0.05;
my $glue_factor = 0.05;
my $max_reads_per_graph = 20000000;
my $max_reads_per_loop = 1000000;
my $min_pct_read_mapping = 0;
my $NO_RUN_QUANTIFYGRAPH_FLAG = 0;
my $NO_RUN_CHRYSALIS_FLAG = 0;
my $help_flag;
my $SHOW_CITATION_FLAG = 0;
my $VERSION = "BLEEDING_EDGE";
my $show_version_flag = 0;
## Kmer methods
my $kmer_method = "";
## Jellyfish
my $max_memory;
## Grid computing options:
my $grid_computing_module;
## Performance monitoring options
my $pm_logfile = "Trinity.timing";
my $pm_trinity_startstring;
my $pm_trinity_endstring;
my $pm_trinity_start=0;
my $pm_trinity_end=0;
my $pm_inchworm_start=0;
my $pm_inchworm_end=0;
my $pm_chrysalis_start=0;
my $pm_chrysalis_end=0;
my $pm_butterfly_start=0;
my $pm_butterfly_end=0;
my $pm_left_fa_size=0;
my $pm_right_fa_size=0;
my $pm_single_fa_size=0;
my $pm_trinity_fa_size=0;
my $pm_trinity_arguments="";
my $pm_inchworm_kmers=0;
my $pm_read_count=0;
my $run_with_collectl = 0;
my $collectl_output_directory = "collectl";
my $collectl_pid = 0;
my $collectl_out = "";
my $collectl_titlename = "";
my $start_dir = cwd();
## misc other opts, mostly for testing purposes
my $run_as_paired_flag = 0; ## in case we have paired reads in single fasta file, already oriented.
my $weldmer_size = 48;
my $FORCE_INCHWORM_KMER_METHOD = 0;
my $NO_TRIPLET_LOCK = 0;
# Note: For the Trinity logo below the backslashes are quoted in order to keep
# them from quoting the character than follows them. "\\" keeps "\ " from occuring.
my $usage = <<_EOUSAGE_;
###############################################################################
#
# ______ ____ ____ ____ ____ ______ __ __
# | || \\ | || \\ | || || | |
# | || D ) | | | _ | | | | || | |
# |_| |_|| / | | | | | | | |_| |_|| ~ |
# | | | \\ | | | | | | | | | |___, |
# | | | . \\ | | | | | | | | | | |
# |__| |__|\\_||____||__|__||____| |__| |____/
#
###############################################################################
#
# Required:
#
# --seqType <string> :type of reads: ( $allowed{seqType} )
# --JM <string> :(Jellyfish Memory) number of GB of system memory to use for
# k-mer counting by jellyfish (eg. 10G) *include the 'G' char
#
# If paired reads:
# --left <string> :left reads
# --right <string> :right reads
#
# Or, if unpaired reads:
# --single <string> :single reads (note, if single file contains pairs, can use flag: --run_as_paired )
#
####################################
## Misc: #########################
#
# --SS_lib_type <string> :Strand-specific RNA-Seq read orientation.
# if paired: RF or FR,
# if single: F or R. (dUTP method = RF)
# See web documentation.
# --long_reads <string> :fasta file containing corrected pac bio reads
#
#
# --output <string> :name of directory for output (will be
# created if it doesn't already exist)
# default( "$output_directory" )
# --CPU <int> :number of CPUs to use, default: $CPU
# --min_contig_length <int> :minimum assembled contig length to report
# (def=$min_contig_length)
# --jaccard_clip :option, set if you have paired reads and
# you expect high gene density with UTR
# overlap (use FASTQ input file format
# for reads).
# (note: jaccard_clip is an expensive
# operation, so avoid using it unless
# necessary due to finding excessive fusion
# transcripts w/o it.)
#
# --prep :Only prepare files (high I/O usage) and stop before kmer counting.
#
# --no_cleanup :retain all intermediate input files.
# --full_cleanup :only retain the Trinity fasta file, rename as \${output_dir}.Trinity.fasta
#
# --cite :get the Trinity literature citation and those of tools leveraged within.
# --monitoring :use collectl to monitor all steps of Trinity
#
# --version :reports Trinity version ($VERSION) and exits.
#
####################################################
# Inchworm and K-mer counting-related options: #####
#
# --min_kmer_cov <int> :min count for K-mers to be assembled by
# Inchworm (default: $min_kmer_cov)
# --inchworm_cpu <int> :number of CPUs to use for Inchworm, default is min(6, --CPU option)
#
###################################
# Chrysalis-related options: ######
#
# --min_glue <int> :min number of reads needed to glue two inchworm contigs
# together. (default: $min_glue)
# --min_iso_ratio <float> :min fraction of average kmer coverage between two iworm contigs
# required for gluing. (default: $min_iso_ratio)
# --glue_factor <float> :fraction of max (iworm pair coverage) for read glue support (default: $glue_factor)
# --max_reads_per_graph <int> :maximum number of reads to anchor within
# a single graph (default: $max_reads_per_graph)
# --max_reads_per_loop <int> :maximum number of reads to read into
# memory at once (default: $max_reads_per_loop)
# --min_pct_read_mapping <int> :minimum percent of a reads kmers that must map to an
# inchworm bundle (aka. component) default: 0
#
# --no_run_chrysalis :stop Trinity after Inchworm and before
# running Chrysalis
# --no_run_quantifygraph :stop Trinity just before running the
# parallel QuantifyGraph computes, to
# leverage a compute farm and massively
# parallel execution..
#
#####################################
### Butterfly-related options: ####
#
# --bfly_opts <string> :additional parameters to pass through to butterfly
# (see butterfly documentation).
# --max_number_of_paths_per_node <int> :only most supported (N) paths are extended from node A->B,
# mitigating combinatoric path explorations. (default: $max_number_of_paths_per_node)
# --group_pairs_distance <int> :maximum length expected between fragment pairs (default: $group_pairs_distance)
#
# --path_reinforcement_distance <int> :minimum overlap of reads with growing transcript
# path (default: $path_reinforcement_distance)
#
# --lenient_path_extension :require minimal read overlap to allow for path extensions.
# (equivalent to --path_reinforcement_distance=1)
#
# --no_triplet_lock : do not lock triplet-supported nodes
#
# --bflyHeapSpaceMax <string> :java max heap space setting for butterfly
# (default: $bflyHeapSpaceMax) => yields command
# 'java -Xmx$bflyHeapSpaceMax -jar Butterfly.jar ... \$bfly_opts'
# --bflyHeapSpaceInit <string> :java initial hap space settings for
# butterfly (default: $bflyHeapSpaceInit) => yields command
# 'java -Xms$bflyHeapSpaceInit -jar Butterfly.jar ... \$bfly_opts'
# --bflyGCThreads <int> :threads for garbage collection
# (default, not specified, so java decides)
# --bflyCPU <int> :CPUs to use (default will be normal
# number of CPUs; e.g., $CPU)
# --bflyCalculateCPU :Calculate CPUs based on 80% of max_memory
# divided by maxbflyHeapSpaceMax
# --no_run_butterfly :stops after the Chrysalis stage. You'll
# need to run the Butterfly computes
# separately, such as on a computing grid.
# Then, concatenate all the Butterfly assemblies by running:
# 'find trinity_out_dir/ -name "\*allProbPaths.fasta" \
# -exec cat {} + > trinity_out_dir/Trinity.fasta'
#
# --rerun_butterfly :will reexecute butterfly commands if composed differently
# from the previous execution.
#
#################################
# Grid-computing options: #######
#
# --grid_computing_module <string> : Perl module in $FindBin::RealBin/PerlLibAdaptors/
# that implements 'run_on_grid()'
# for naively parallel cmds. (eg. 'BroadInstGridRunner')
#
#
###############################################################################
#
# *Note, a typical Trinity command might be:
# Trinity.pl --seqType fq --JM 100G --left reads_1.fq --right reads_2.fq --CPU 6
#
# see: $FindBin::RealBin/sample_data/test_Trinity_Assembly/
# for sample data and 'runMe.sh' for example Trinity execution
# For more details, visit: http://trinityrnaseq.sf.net
#
###############################################################################
_EOUSAGE_
;
=EXPERIMENTAL_OPTIONS
## DO NOT USE! Not ready for prime-time yet, and doesn't seem to help.
# Chyrsalis-related options:
#
# --min_pcnt_read_iworm_kmers <int> :min percentage of a read sequence that must be composed of inchworm kmers to be pursued
# by chrysalis (default: $min_percent_read_iworm_kmers) note: off if < 0
#
# --FORCE_INCHWORM_KMER_METHOD :uses inchworm built-in kmer cataloger instead of jellyfish (not recommended)
=cut
my $ROOTDIR = "$FindBin::RealBin";
my $UTILDIR = "$ROOTDIR/util";
my $INCHWORM_DIR = "$ROOTDIR/Inchworm";
my $CHRYSALIS_DIR = "$ROOTDIR/Chrysalis";
my $BUTTERFLY_DIR = "$ROOTDIR/Butterfly";
my $MERYL_DIR = "$ROOTDIR/trinity-plugins/kmer/meryl";
my $JELLYFISH_DIR = "$ROOTDIR/trinity-plugins/jellyfish";
my $FASTOOL_DIR = "$ROOTDIR/trinity-plugins/fastool";
my $COLLECTL_DIR = "$ROOTDIR/trinity-plugins/collectl/bin";
my $COREUTILS_DIR = "$ROOTDIR/trinity-plugins/coreutils/bin";
unless (@ARGV) {
die "$usage\n";
}
# Log command line parameters for performance monitoring
foreach (@ARGV) {
$pm_trinity_arguments = $pm_trinity_arguments . " " . $_;
};
my $NO_FASTOOL = 0;
my $NO_CLEANUP = 0;
my $FULL_CLEANUP = 0;
&GetOptions(
'h|help' => \$help_flag,
## general opts
"seqType=s" => \$seqType,
"left=s" => \$left_file,
"right=s" => \$right_file,
"single=s" => \$single_file,
"SS_lib_type=s" => \$SS_lib_type,
"long_reads=s" => \$long_reads,
"output=s" => \$output_directory,
"min_contig_length=i" => \$min_contig_length,
"jaccard_clip" => \$jaccard_clip,
"cite" => \$SHOW_CITATION_FLAG,
'CPU=i' => \$CPU,
'prep' => \$prep_only,
# Butterfly opts
'no_run_butterfly' => \$NO_RUN_BUTTERFLY_FLAG,
'no_triplet_lock' => \$NO_TRIPLET_LOCK,
"group_pairs_distance=i" => \$group_pairs_distance,
'bfly_opts=s' => \$bfly_opts,
'bflyHeapSpaceMax=s' => \$bflyHeapSpaceMax,
'bflyHeapSpaceInit=s' => \$bflyHeapSpaceInit,
'bflyGCThreads=i' => \$bflyGCThreads,
'bflyCPU=i' => \$bflyCPU,
'bflyCalculateCPU' => \$bflyCalculateCPU,
'max_number_of_paths_per_node=i' => \$max_number_of_paths_per_node,
'lenient_path_extension' => \$lenient_path_extension,
'path_reinforcement_distance=i' => \$path_reinforcement_distance,
'rerun_butterfly' => \$RERUN_BUTTERFLY_FLAG,
# Inchworm & kmer catalog opts
'min_kmer_cov=i' => \$min_kmer_cov,
'inchworm_cpu=i' => \$inchworm_cpu,
'FORCE_INCHWORM_KMER_METHOD' => \$FORCE_INCHWORM_KMER_METHOD,
# Jellyfish
'JM=s' => \$max_memory, # in GB
# Chrysalis -related opts
'min_glue=i' => \$min_glue,
'glue_factor=f' => \$glue_factor,
'min_iso_ratio=f' => \$min_iso_ratio,
'min_pcnt_read_iworm_kmers=i' => \$min_percent_read_iworm_kmers,
'no_run_quantifygraph' => \$NO_RUN_QUANTIFYGRAPH_FLAG,
'max_reads_per_graph=i' => \$max_reads_per_graph,
'max_reads_per_loop=i' => \$max_reads_per_loop,
'no_run_chrysalis' => \$NO_RUN_CHRYSALIS_FLAG,
'min_pct_read_mapping=i' => \$min_pct_read_mapping,
'weldmer_size=i' => \$weldmer_size,
# Grid computing options
'grid_computing_module=s' => \$grid_computing_module,
"show_advanced_options" => \$show_advanced_options,
# misc
'run_as_paired' => \$run_as_paired_flag,
'no_fastool' => \$NO_FASTOOL,
'no_cleanup' => \$NO_CLEANUP,
'full_cleanup' => \$FULL_CLEANUP,
'version' => \$show_version_flag,
'monitoring' => \$run_with_collectl,
);
if ($SHOW_CITATION_FLAG) {
&show_lit_citation();
exit(0);
}
if ($help_flag) {
die "$usage\n";
}
if ($show_version_flag) {
print "Trinity version: $VERSION\n";
exit(1);
}
if ($NO_CLEANUP && $FULL_CLEANUP) {
die "cannot set --no_cleanup and --full_cleanup as they contradict";
}
if (@ARGV) {
die "Error, do not understand options: @ARGV\n";
}
## Check options set:
# Subroutine takes variable *reference* plus name of variable. Lower-cases
# variable value and checks to see if it one of the allowed ones.
# 'die' has new-line in order to keep line number from being shown to user.
sub check_option {
my ($option, $name) = @_;
$$option = lc $$option;
if ($$option eq '') {
die "Error, option '--$name' is required.\n";
}
if (!defined $allowed_check{$name}{$$option}) {
die "Error, option '--$name' ($$option) not one of $allowed{$name}\n";
}
}
check_option( \$seqType, 'seqType' );
## load adaptors
my $grid_computing_method = "";
if ($grid_computing_module) {
my $perl_lib_repo = "$FindBin::Bin/../PerlLibAdaptors";
print STDERR "-importing module: $grid_computing_module\n";
require "$grid_computing_module.pm" or die "Error, could not import perl module at run-time: $grid_computing_module";
$grid_computing_method = $grid_computing_module . "::run_on_grid";
}
my $USE_FASTOOL = 1; # by default, using fastool for fastq to fasta conversion
if ($NO_FASTOOL) {
$USE_FASTOOL = 0;
}
if ($SS_lib_type) {
unless ($SS_lib_type =~ /^(R|F|RF|FR)$/) {
die "Error, unrecognized SS_lib_type value of $SS_lib_type. Should be: F, R, RF, or FR\n";
}
}
unless ( ($left_file && $right_file) || $single_file ) {
die "Error, need either options 'left' and 'right' or option 'single'\n";
}
if ($min_iso_ratio > 1) {
die "Error, --min_iso_ratio should be <= 1 \n";
}
## keep the original 'xG' format string for the --JM option, then calculate the numerical value for max_memory
my $JM_string = $max_memory; ## this one is used in the Chrysalis exec string
if ($max_memory) {
$max_memory =~ /^([\d\.]+)G$/ or die "Error, cannot parse max_memory value of $max_memory. Set it to 'xG' where x is a numerical value\n";
$max_memory = $1;
$max_memory *= 1024**3; # convert to from gig to bytes
}
else {
die "Error, must specify max memory for jellyfish to use, eg. --JM 10G \n";
}
## Try to remove stack limits
if ($^O eq "linux") { # cannot set stacksize on newer macs for some reason...
# &try_unlimit_stacksize();
}
my $curr_limit_settings = `/bin/sh -c 'ulimit -a' `;
unless ($curr_limit_settings && $curr_limit_settings =~ /\w/) {
$curr_limit_settings = `/bin/csh -c limit`; # backup, probably not needed.
}
print "Current settings:\n$curr_limit_settings\n\n";
## Check Java version:
my $java_version = `java -version 2>&1 `;
unless ($java_version =~ /(java|openjdk) version \"1\.[7]\./) {
die "Error, Trinity requires access to Java version 1.6. Currently installed version is: $java_version";
}
# Give the variable with memory size and a user-oriented name
sub bfly_check {
my ($mem, $name) = @_;
my ($num, $type) = $mem =~ /^(\d+)([MG])$/;
if (!defined $mem || !defined $type) {
die "Error, $name must be set to a value of format: \\d+G or \\d+M (eg. 1G or 1000M)\n Currently: $mem\n";
}
return $type eq 'G' ? $num * 1024**3 : $num * 1024**2;
}
my $bflyHeapSpaceMaxBytes = bfly_check($bflyHeapSpaceMax , 'bflyHeapSpaceMax' );
my $bflyHeapSpaceInitBytes = bfly_check($bflyHeapSpaceInit, 'bflyHeapSpaceInit');
if ($bflyHeapSpaceInitBytes > $bflyHeapSpaceMaxBytes) {
die "Error, bflyHeapSpaceInit ($bflyHeapSpaceInit) must be less or equal to bflyHeapSpaceMax ($bflyHeapSpaceMax).\n";
}
if ($CPU > $CPU_MAX) {
print STDERR "Warning, --CPU $CPU might be excessive. Limiting it to $CPU_MAX for now.\n";
$CPU = $CPU_MAX;
}
if ($inchworm_cpu > $CPU) {
$inchworm_cpu = $CPU;
}
if ($bflyCalculateCPU && $max_memory) {
$bflyCPU = int ($max_memory * 0.80 / $bflyHeapSpaceMaxBytes);
}
$bflyCPU = $CPU if !defined $bflyCPU;
if ($bflyCPU > $CPU_MAX) {
print STDERR "Warning, --bflyCPU $bflyCPU might be excessive. Limiting it to $CPU_MAX for now.\n";
$bflyCPU = $CPU_MAX;
}
if (defined($bflyGCThreads) && $bflyGCThreads > 32) {
die "Error, you probably want fewer than $bflyGCThreads java garbage collection threads. Try a number less than 32.";
}
$ENV{OMP_NUM_THREADS} = $CPU; ## for Inchworm and Chrysalis
my $PAIRED_MODE = ( ($left_file && $right_file) || $run_as_paired_flag) ? 1:0;
if ($PAIRED_MODE) {
## be sure we can find 'bowtie', since we use it as part of the iworm pair scaffolding step
my $bowtie_path = `which bowtie`;
if ($bowtie_path =~ /\w/) {
print "Paired mode requires bowtie. Found bowtie at: $bowtie_path\n";
}
else {
die "Error, cannot find path to bowtie, which is now needed as part of Chrysalis' read scaffolding step";
}
}
sub perfmon_start {
open (FILE, ">", "$output_directory/$pm_logfile") or die;
print FILE "Statistics:\n";
print FILE "===========\n";
print FILE "Trinity Version: $VERSION\n";
my $tempp="";
$tempp=`ldd $INCHWORM_DIR/bin/inchworm | grep "libgomp"`;
if ($tempp eq "") {
print FILE "Compiler: Intel\n";
} else {
print FILE "Compiler: GCC\n";
}
print FILE "Trinity Parameters: $pm_trinity_arguments\n";
$pm_trinity_startstring = `date`;
$pm_trinity_start = `date +%s`;
close (FILE);
}
sub perfmon_end {
$pm_trinity_endstring = `date`;
$pm_trinity_end = `date +%s`;
my $timestamp = `date +%s`;
if ( -e "$output_directory/$pm_logfile" ) {
open (FILE, '>>', "$output_directory/$pm_logfile") or die;
if ($PAIRED_MODE) {
print FILE "Paired mode\n";
print FILE " Input data\n";
if ($left_file && $right_file) {
print FILE " Left.fasta $pm_left_fa_size MByte\n";
print FILE " Right.fasta $pm_right_fa_size MByte\n";
} else {
print FILE " Single.fasta $pm_single_fa_size MByte\n";
}
} else {
print FILE "Unpaired read mode\n";
print FILE " Input data\n";
print FILE " Single.fasta $pm_single_fa_size MByte\n";
}
}
$pm_inchworm_kmers = `cat $output_directory/inchworm.kmer_count`;
print FILE " Number of unique KMERs: $pm_inchworm_kmers";
print FILE " Number of reads: $pm_read_count";
print FILE " Output data\n";
my $pm_temp = -s "$output_directory/Trinity.fasta" || 0;
$pm_temp = $pm_temp / 1024 / 1024;
my $pm_trinity_fa_size = sprintf('%.0f', $pm_temp);
print FILE " Trinity.fasta $pm_trinity_fa_size MByte\n\n";
print FILE "Runtime\n";
print FILE "=======\n";
print FILE "Start: $pm_trinity_startstring";
print FILE "End: $pm_trinity_endstring";
my $pm_trinity_time = $pm_trinity_end - $pm_trinity_start;
print FILE "Trinity.pl $pm_trinity_time seconds\n";
my $pm_inchworm_time = $pm_inchworm_end - $pm_inchworm_start;
print FILE " Inchworm $pm_inchworm_time seconds\n";
my $pm_chrysalis_time = $pm_chrysalis_end - $pm_chrysalis_start;
print FILE " Chrysalis $pm_chrysalis_time seconds\n";
my $pm_butterfly_time = $pm_butterfly_end - $pm_butterfly_start;
print FILE " Butterfly $pm_butterfly_time seconds\n";
my $pm_rest_time = $pm_trinity_time - $pm_butterfly_time - $pm_chrysalis_time - $pm_inchworm_time;
print FILE " Rest $pm_rest_time seconds\n";
close (FILE);
}
main: {
$ENV{OMP_NUM_THREADS} = $inchworm_cpu;
unless ($NO_RUN_BUTTERFLY_FLAG) {
print STDERR "-since butterfly will eventually be run, lets test for proper execution of java\n";
&test_java_failure_capture();
}
## create complete paths for input files:
$left_file = &create_full_path($left_file) if $left_file;
$right_file = &create_full_path($right_file) if $right_file;
$single_file = &create_full_path($single_file) if $single_file;
$output_directory = &create_full_path($output_directory);
$long_reads = &create_full_path($long_reads) if $long_reads;
unless (-d $output_directory) {
mkdir $output_directory or die "Error, cannot mkdir $output_directory";
}
chdir ($output_directory) or die "Error, cannot cd to $output_directory";
if ($run_with_collectl){
$collectl_output_directory = "$start_dir/collectl";
`rm -rf $collectl_output_directory `;
$collectl_output_directory = &create_full_path($collectl_output_directory);
unless (-d $collectl_output_directory) {
mkdir $collectl_output_directory or die "Error, cannot mkdir $collectl_output_directory";
}
my $user = $ENV{USER};
my $cmd = "cd $collectl_output_directory && ${COLLECTL_DIR}/collectl -i5:5 -sZ --procfilt U$user -F0 -f $collectl_output_directory/y &";
&process_cmd($cmd);
#&process_cmd("sync; sleep 1"); #try to make sure that collectl has started
print STDERR "CHECKING FOR COLLECTL PID\n";
while (! $collectl_pid) {
$collectl_pid = `ps h -C collectl -o pid | head -n1`;
chomp $collectl_pid;
print STDERR "COLLECTL PID = $collectl_pid\n";
sleep(1);
}
}
&perfmon_start();
## create inchworm file name
my $inchworm_file = "inchworm.K$IWORM_KMER_SIZE.L$MIN_IWORM_LEN";
unless ($SS_lib_type) {
$inchworm_file .= ".DS";
}
$inchworm_file .= ".fa";
$inchworm_file = &create_full_path($inchworm_file);
my $trinity_target_fa = ($single_file) ? "single.fa" : "both.fa";
my $inchworm_target_fa = $trinity_target_fa; # change this later if we have long_reads
## Don't prep the inputs if Inchworm already exists.... Resuming earlier operations.
my $inchworm_finished_checkpoint_file = "$inchworm_file.finished";
if (-s $inchworm_file && -e $inchworm_finished_checkpoint_file) {
print "\n\n#######################################################################\n"
. "Inchworm file: $inchworm_file detected.\n"
. "Skipping Inchworm Step, Using Previous Inchworm Assembly\n"
. "#######################################################################\n\n";
sleep(2);
}
else {
## Prep data for Inchworm
if ($left_file && $right_file) {
unless (-s $trinity_target_fa && !-e "left.fa" && !-e "right.fa") {
my ($left_SS_type, $right_SS_type);
if ($SS_lib_type) {
($left_SS_type, $right_SS_type) = split(//, $SS_lib_type);
}
print("Converting input files. (in parallel)");
my $thr1;
my $thr2;
if (!(-s "left.fa")) {
$thr1 = threads->create('prep_seqs', $left_file, $seqType, "left", $left_SS_type);
} else {
$thr1 = threads->create(sub { print ("left file exists, nothing to do");});
}
if (!(-s "right.fa")) {
$thr2 = threads->create('prep_seqs', $right_file, $seqType, "right", $right_SS_type);
} else {
$thr2 = threads->create(sub { print ("right file exists, nothing to do");});
}
$thr1->join();
$thr2->join();
print("Done converting input files.");
## Calculate input file sizes for performance monitoring
my $pm_temp = -s "$left_file";
$pm_temp = $pm_temp / 1024 / 1024;
$pm_left_fa_size = sprintf('%.0f', $pm_temp);
$pm_temp = -s "$right_file";
$pm_temp = $pm_temp / 1024 / 1024;
$pm_right_fa_size = sprintf('%.0f', $pm_temp);
&process_cmd("cat left.fa right.fa > $trinity_target_fa") unless (-s $trinity_target_fa && (-s $trinity_target_fa == ((-s "left.fa") + (-s "right.fa"))));
unless (-s $trinity_target_fa == ((-s "left.fa") + (-s "right.fa"))){
die "$trinity_target_fa is smaller (".(-s $trinity_target_fa)." bytes) than the combined size of left.fa and right.fa (".((-s "left.fa") + (-s "right.fa"))." bytes)\n";
}
unlink ("left.fa", "right.fa"); # no longer needed now that we have 'both.fa', which is needed by chryaslis
}
}
elsif ($single_file) {
&prep_seqs($single_file, $seqType, "single", $SS_lib_type) unless (-s "single.fa");
## Calculate input file sizes for performance monitoring
my $pm_temp = -s "$single_file";
$pm_temp = $pm_temp / 1024 / 1024;
$pm_single_fa_size = sprintf('%.0f', $pm_temp);
}
else {
die "not sure what to do. "; # should never get here.
}
if ($long_reads) {
$inchworm_target_fa .= ".wLongReads.fa";
&process_cmd("cat $long_reads $trinity_target_fa > $inchworm_target_fa");
}
}
if ($prep_only){
print "Data has been prepared. Exiting now as per user request\n";
exit();
}
#################
## Inchworm step:
$pm_inchworm_start = `date +%s`;
unless (-s $inchworm_file && -e $inchworm_finished_checkpoint_file) {
&run_inchworm($inchworm_file, $inchworm_target_fa, $SS_lib_type, $kmer_method);
&process_cmd("touch $inchworm_finished_checkpoint_file");
}
$pm_inchworm_end = `date +%s`;
unless (-s $inchworm_file) {
if ($FULL_CLEANUP && -e $inchworm_file && -e $inchworm_finished_checkpoint_file) {
## GG-trinity mode, clean-up gracefully
&process_cmd("rm -rf $output_directory");
exit(0);
}
else {
die "Error, no Inchworm output is detected at: $inchworm_file";
}
}
if ($jaccard_clip) {
eval {
if ($jaccard_clip && $left_file && $right_file) {
$inchworm_file = &run_jaccard_clip_left_right($inchworm_file, $left_file, $right_file, $seqType, $SS_lib_type);
}
elsif ($jaccard_clip && $single_file) {
$inchworm_file = &run_jaccard_clip_single_but_really_paired($inchworm_file, $single_file, $seqType, $SS_lib_type);
}
};
if ($@) {
if ($FULL_CLEANUP) {
## GG-trinity mode, clean up gracefully
&process_cmd("rm -rf $output_directory");
exit(0);
}
else {
die "Error, jaccard-clip failed: $@";
}
}
}
if ($NO_RUN_CHRYSALIS_FLAG) {
print "\n\n\n";
print "#########################################################################\n";
print "Inchworm is complete. --no_run_chrysalis was specified, so stopping here.\n";
print "#########################################################################\n\n\n";
exit(0);
}
$ENV{OMP_NUM_THREADS} = $CPU;
##################
## Chrysalis step:
if ($min_percent_read_iworm_kmers > 0) {
### EXPERIMENTAL: DO NOT USE!
$trinity_target_fa = &extract_reads_with_iworm_kmers($trinity_target_fa, $inchworm_file, $min_percent_read_iworm_kmers, $SS_lib_type);
}
## butterfly commands can be reparameterized for exploring different assembly requirements
## chrysalis will just run or resume depending on what's already been processed.
$pm_chrysalis_start = `date +%s`;
my $butterfly_cmds = &run_chrysalis($inchworm_file, $inchworm_target_fa,
$min_contig_length, $group_pairs_distance, $SS_lib_type, $trinity_target_fa);
$pm_chrysalis_end = `date +%s`;
print "Butterfly_cmds: $butterfly_cmds\n";
if ($butterfly_cmds && -s $butterfly_cmds) {
if ($NO_RUN_BUTTERFLY_FLAG) {
print "\n\nYou've opted to run butterfly commands independently from this script, such as on a computing grid.\n\n";
print "Butterfly commands to execute are available here:\n"
. "\t$butterfly_cmds\n\n";
print "After executing Butterfly commands, concatenate all Butterfly outputs by running:\n"
. "\t\tfind $output_directory/ -name \"\*allProbPaths.fasta\" -exec cat {} + > $output_directory/Trinity.fasta\n\n\n";
}
else {
## Run Butterfly
print "Inchworm and Chrysalis complete. Butterfly commands to execute are provided here:\n"
. $butterfly_cmds . "\n\n";
print STDERR "---------------------------------------------------------------\n"
. "-------------------- Butterfly --------------------------------\n"
. "-- (Reconstruct transcripts from reads and de Bruijn graphs) --\n"
. "---------------------------------------------------------------\n\n";
$pm_butterfly_start = `date +%s`;
if ($grid_computing_module) {
my @bfly_cmds = `cat $butterfly_cmds`;
chomp @bfly_cmds;
&$grid_computing_method(@bfly_cmds);
}
else {
my $cmd = "$INCHWORM_DIR/bin/ParaFly -c $butterfly_cmds -shuffle -CPU $bflyCPU -failed_cmds failed_butterfly_commands.$$.txt -v "; # shuffle them since the first ones are usually the longest-running ones.
&process_cmd($cmd);
}
$pm_butterfly_end = `date +%s`;
## capture results:
# my $cmd = 'find ./chrysalis -name "*allProbPaths.fasta" -exec cat {} + > Trinity.fasta.tmp';
# no longer scan the file system... we know which files should exist
my $cmd = "$UTILDIR/print_butterfly_assemblies.pl ./chrysalis/component_file_listing.txt > Trinity.fasta.tmp";
&process_cmd($cmd);
}
}
if ($FULL_CLEANUP) {
print "Fully cleaning up.\n";
$output_directory =~ s|/+$||g; # remove any trailing directory slash
if (-s "Trinity.fasta.tmp") {
rename("Trinity.fasta.tmp", "$output_directory.Trinity.fasta") or die "Error, cannot rename Trinity.fasta.tmp to $output_directory.Trinity.fasta";
}
&process_cmd("rm -rf $output_directory");
print "\n\n";
print "###################################################################\n";
print "Butterfly assemblies are written to $output_directory.Trinity.fasta\n";
print "###################################################################\n\n\n";
}
else {
if (-s "Trinity.fasta.tmp") {
rename("Trinity.fasta.tmp", "Trinity.fasta") or die "Error, cannot rename Trinity.fasta.tmp to Trinity.fasta"; # now that process has finished.
}
if (-s "Trinity.fasta") {
print "\n\n";
print "###################################################################\n";
print "Butterfly assemblies are written to $output_directory/Trinity.fasta\n";
print "###################################################################\n\n\n";
}
else {
die "ERROR, no butterfly assemblies written to $output_directory/Trinity.fasta";
}
}
&perfmon_end();
# finish monitoring and create collectl statistics
if ($run_with_collectl){
&process_cmd("kill -15 $collectl_pid");
#&process_cmd("sync");
#&process_cmd("sleep 3");
chdir ($collectl_output_directory) or die "Error, cannot cd to $collectl_output_directory";
&process_cmd("$COLLECTL_DIR/make_data_files.sh");
&process_cmd("$COLLECTL_DIR/timetable.sh");
$collectl_titlename = "${VERSION} ${CPU} ${left_file}${single_file}";
&process_cmd("$COLLECTL_DIR/plot.sh \"$collectl_titlename\" ${CPU}");
}
exit(0);
}
####
sub run_chrysalis {
my ($inchworm_file, $reads_file,
$min_contig_length, $group_pairs_distance, $SS_lib_type, $pairs_fa) = @_;
my $butterfly_cmds = &create_full_path("chrysalis/butterfly_commands");
my $quantify_graph_cmds = &create_full_path("chrysalis/quantifyGraph_commands");
my $adjusted_butterfly_cmds = "$butterfly_cmds.adj";
my $chrysalis_finished_checkpoint = "chrysalis/chrysalis.finished";
if (-s $butterfly_cmds && -e $chrysalis_finished_checkpoint) {
print "###################################################################\n";
print "#### Chrysalis results already exist. Not rerunning Chrysalis. ####\n";
print "###################################################################\n\n\n";
sleep(2);