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Trinity
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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");
use File::Basename;
use Time::localtime;
use Cwd;
use Carp;
use COMMON;
use Getopt::Long qw(:config posix_default no_ignore_case pass_through);
use Pipeliner;
use Fasta_reader;
my $VERSION = "v2.0.6";
BEGIN {
$ENV{TRINITY_HOME} = "$FindBin::Bin";
}
use HPC::GridRunner;
open (STDERR, ">&STDOUT"); ## capturing stderr and stdout in a single stdout stream
#directory defnintions
my $ROOTDIR = "$FindBin::RealBin";
my $UTILDIR = "$ROOTDIR/util";
my $MISCDIR = "$UTILDIR/misc";
my $INCHWORM_DIR = "$ROOTDIR/Inchworm/bin/";
my $CHRYSALIS_DIR = "$ROOTDIR/Chrysalis";
my $BUTTERFLY_DIR = "$ROOTDIR/Butterfly";
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";
my $PARAFLY = "$ROOTDIR/trinity-plugins/parafly/bin/ParaFly";
my $TRIMMOMATIC = "$ROOTDIR/trinity-plugins/Trimmomatic/trimmomatic.jar";
my $TRIMMOMATIC_DIR = "$ROOTDIR/trinity-plugins/Trimmomatic";
my $SAMBAMBA_BIN = '';
# Check architecture and assign sambamba
my $sys_arch = `uname -a 2>&1`;
if ($sys_arch =~ m/darwin.*?x86_64/i) {
$SAMBAMBA_BIN = "$UTILDIR/darwin_sambamba";
}
elsif ($sys_arch =~ m/linux.*?x86_64/i) {
$SAMBAMBA_BIN = "$UTILDIR/linux_sambamba";
}
# Site specific setup
my $KMER_SIZE = 25;
my $MAX_KMER_SIZE = 32;
my $INCHWORM_CUSTOM_PARAMS;
# option list:
my ($seqType, @left_files, @right_files, @single_files, $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 => '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 = &create_full_path("trinity_out_dir", 0);
# butterfly opts
$min_contig_length = 200;
$group_pairs_distance = 500;
my $path_reinforcement_distance;
my $PE_path_reinforcement_distance = 75;
my $SE_path_reinforcement_distance = 25;
my $bfly_opts = "";
my $bflyHeapSpaceMax = "4G";
my $bflyHeapSpaceInit = "1G";
my $BFLY_JAR = "";
# butterfly path merging criteria
my $NO_PATH_MERGING = 0;
my $MIN_PER_ID_SAME_PATH; # leave these at the butterfy defaults
my $MAX_DIFFS_SAME_PATH;
my $MAX_INTERNAL_GAP_SAME_PATH;
# misc opts
my $min_kmer_cov = 1;
my $meryl_opts = "";
my $inchworm_cpu = 6;
my $min_percent_read_iworm_kmers = -1; # experimental, off
my $CPU = 2;
my $np = 1;
my $mpiexec = "mpiexec";
my $bflyCPU;
my $bflyCalculateCPU = 0;
my $bflyGCThreads = 2;
my $long_reads = "";
## ADVANCED OPTIONS:
## Chrysalis opts
my $min_glue = 2;
my $min_iso_ratio = 0.05;
my $glue_factor = 0.05;
my $max_reads_per_graph = 200000;
my $max_reads_per_loop = 10000000;
my $min_pct_read_mapping = 0;
my $chrysalis_output_dir = "chrysalis";
my $component_directory;
my $NO_RUN_CHRYSALIS_FLAG = 0;
my $NO_DISTRIBUTED_TRINITY_EXEC = 0;
my $help_flag;
my $advanced_help_flag;
my $SHOW_CITATION_FLAG = 0;
my $show_version_flag = 0;
## Kmer methods
my $kmer_method = "";
## Jellyfish
my $max_memory;
## Grid computing options:
my $grid_conf_file;
## 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;
# flush each second, record procs+rest every 5 secs, use only process subsystem
my $collectl_param = "-F1 -i5:5 -sZ";
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 $PARALLEL_IWORM_FLAG = 1;
my $NO_PARALLEL_IWORM = 0;
## Quality trimming params
my $RUN_TRIMMOMATIC_FLAG = 0;
my $trimmomatic_quality_trim_params = "ILLUMINACLIP:$TRIMMOMATIC_DIR/adapters/TruSeq3-PE.fa:2:30:10 SLIDINGWINDOW:4:5 LEADING:5 TRAILING:5 MINLEN:25";
## Normalize reads
my $NORMALIZE_READS_FLAG = 0;
my $normalize_max_read_cov = 50;
my $NORMALIZE_BY_READ_SET = 0;
my $grid_node_CPU = 1;
my $grid_node_max_memory = "1G";
# 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 $basic_usage = qq^
###############################################################################
#
# ______ ____ ____ ____ ____ ______ __ __
# | || \\ | || \\ | || || | |
# | || D ) | | | _ | | | | || | |
# |_| |_|| / | | | | | | | |_| |_|| ~ |
# | | | \\ | | | | | | | | | |___, |
# | | | . \\ | | | | | | | | | | |
# |__| |__|\\_||____||__|__||____| |__| |____/
#
###############################################################################
#
# Required:
#
# --seqType <string> :type of reads: ( $allowed{seqType} )
#
# --max_memory <string> :suggested max memory to use by Trinity where limiting can be enabled. (jellyfish, sorting, etc)
# provied in Gb of RAM, ie. '--max_memory 10G'
#
# If paired reads:
# --left <string> :left reads, one or more file names (separated by commas, no spaces)
# --right <string> :right reads, one or more file names (separated by commas, no spaces)
#
# Or, if unpaired reads:
# --single <string> :single reads, one or more file names, comma-delimited (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.
#
# --CPU <int> :number of CPUs to use, default: $CPU
# --min_contig_length <int> :minimum assembled contig length to report
# (def=$min_contig_length)
#
# --long_reads <string> :fasta file containing error-corrected or circular consensus (CCS) pac bio reads
#
# --genome_guided_bam <string> :genome guided mode, provide path to coordinate-sorted bam file.
# (see genome-guided param section under --show_full_usage_info)
#
# --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.)
#
# --trimmomatic :run Trimmomatic to quality trim reads
# see '--quality_trimming_params' under full usage info for tailored settings.
#
#
# --normalize_reads :run in silico normalization of reads. Defaults to max. read coverage of $normalize_max_read_cov.
# see '--normalize_max_read_cov' under full usage info for tailored settings.
#
# --no_distributed_trinity_exec :do not run Trinity phase 2 (assembly of partitioned reads), and stop after generating command list.
#
#
# --output <string> :name of directory for output (will be
# created if it doesn't already exist)
# default( your current working directory: "$output_directory"
# note: must include 'trinity' in the name as a safety precaution! )
#
# --full_cleanup :only retain the Trinity fasta file, rename as \${output_dir}.Trinity.fasta
#
# --cite :show the Trinity literature citation
#
# --verbose :provide additional job status info during the run.
#
# --version :reports Trinity version ($VERSION) and exits.
#
# --show_full_usage_info :show the many many more options available for running Trinity (expert usage).
^;
my $full_usage = qq^
#
# --KMER_SIZE <int> :kmer length to use (default: 25) max=32
#
# --prep :Only prepare files (high I/O usage) and stop before kmer counting.
#
# --no_cleanup :retain all intermediate input files.
#
# --no_version_check :dont run a network check to determine if software updates are available.
#
####################################################
# 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)
#
# --no_run_inchworm :stop after running jellyfish, before inchworm. (phase 1, read clustering only)
#
###################################
# Chrysalis-related options: ######
#
# --max_reads_per_graph <int> :maximum number of reads to anchor within
# a single graph (default: $max_reads_per_graph)
# --min_glue <int> :min number of reads needed to glue two inchworm contigs
# together. (default: $min_glue)
#
# --no_bowtie :dont run bowtie to use pair info in chrysalis clustering.
#
# --no_run_chrysalis :stop after running inchworm, before chrysalis. (phase 1, read clustering only)
#
#####################################
### Butterfly-related options: ####
#
# --bfly_opts <string> :additional parameters to pass through to butterfly
# (see butterfly options: java -jar Butterfly.jar ).
# (note: only for expert or experimental use. Commonly used parameters are exposed through this Trinity menu here).
#
# //////////////////////////////////
# Alternative reconstruction modes:
# Default mode is the 'regular' Butterfly transcript reconstruction by graph node extension.
#
# --PasaFly PASA-like algorithm for maximally-supported isoforms
# or
# --CuffFly Cufflinks-like algorithm to report minimum transcripts
#
#
# Butterfly read-pair grouping settings (used for all reconstruction modes to define 'pair paths'):
#
# --group_pairs_distance <int> :maximum length expected between fragment pairs (default: $group_pairs_distance)
# (reads outside this distance are treated as single-end)
#
# ///////////////////////////////////////////////
# Butterfly default reconstruction mode settings. (no CuffFly or PasaFly custom settings are currently available).
#
# --path_reinforcement_distance <int> :minimum overlap of reads with growing transcript
# path (default: PE: $PE_path_reinforcement_distance, SE: $SE_path_reinforcement_distance)
# Set to 1 for the most lenient path extension requirements.
#
#
# /////////////////////////////////////////
# Butterfly transcript reduction settings:
#
# --no_path_merging : all final transcript candidates are output (including SNP variations, however, some SNPs may be unphased)
#
# By default, alternative transcript candidates are merged (in reality, discarded) if they are found to be too similar, according to the following logic:
#
# (identity=(numberOfMatches/shorterLen) > 95.0% or if we have <= 2 mismatches) and if we have internal gap lengths <= 10
#
# with parameters as:
#
# --min_per_id_same_path <int> default: 98 min percent identity for two paths to be merged into single paths
# --max_diffs_same_path <int> default: 2 max allowed differences encountered between path sequences to combine them
# --max_internal_gap_same_path <int> default: 10 maximum number of internal consecutive gap characters allowed for paths to be merged into single paths.
#
# If, in a comparison between two alternative transcripts, they are found too similar, the transcript with the greatest cumulative
# compatible read (pair-path) support is retained, and the other is discarded.
#
#
# //////////////////////////////////////////////
# Butterfly Java and parallel execution settings.
#
# --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: $bflyGCThreads))
# --bflyCPU <int> :CPUs to use (default will be normal
# number of CPUs; e.g., $CPU)
# --bflyCalculateCPU :Calculate CPUs based on 80% of max_memory
# divided by maxbflyHeapSpaceMax
#
# --bfly_jar <string> : /path/to/Butterfly.jar, otherwise default
# Trinity-installed version is used.
#
#
#
################################################################################
#### Quality Trimming Options ####
#
# --quality_trimming_params <string> defaults to: "$trimmomatic_quality_trim_params"
#
################################################################################
#### In silico Read Normalization Options ###
#
# --normalize_max_read_cov <int> defaults to 50
# --normalize_by_read_set run normalization separate for each pair of fastq files,
# then one final normalization that combines the individual normalized reads.
# Consider using this if RAM limitations are a consideration.
#
################################################################################
#### Genome-guided de novo assembly
#
# * required:
#
# --genome_guided_max_intron <int> :maximum allowed intron length (also maximum fragment span on genome)
#
# * optional:
#
# --genome_guided_min_coverage <int> :minimum read coverage for identifying and expressed region of the genome. (default: 1)
#
# --genome_guided_min_reads_per_partition <int> :default min of 10 reads per partition
#
#
#################################
# Grid-computing options: #######
#
# --grid_conf <string> :configuration file for supported compute farms
# ex. TRINITY_HOME/htc_conf/BroadInst_LSF.conf
# currently supported computing gris: LSF, SGE
#
# --grid_node_CPU <int> number of threads for each parallel process to leverage. (default: $grid_node_CPU)
#
# --grid_node_max_memory <string> max memory targeted for each grid node. (default: $grid_node_max_memory)
#
#
^;
my $usage_synopsis = qq^#
#
###############################################################################
#
# *Note, a typical Trinity command might be:
#
# Trinity --seqType fq --max_memory 50G --left reads_1.fq --right reads_2.fq --CPU 6
#
#
# and for Genome-guided Trinity:
#
# Trinity --genome_guided_bam rnaseq_alignments.csorted.bam --max_memory 50G\
# --genome_guided_max_intron 10000 --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.github.io
#
###############################################################################
^;
my $advanced_usage = <<_ADVANCEDUSAGE_;
###################################################################################
## Not intended for users, instead for experimentation by developers ##
###################################################################################
#
#
# Inchworm-related options:
#
# --INCHWORM_CUSTOM_PARAMS <string> :additional parameters to be passed on to Inchworm
# --FORCE_INCHWORM_KMER_METHOD :uses inchworm built-in kmer cataloger instead of jellyfish (not recommended)
# --NO_PARALLEL_IWORM : turn off parallel iworm assembly
# --iworm_opts <string> : options for inchworm
#
#
# 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
#
# --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_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
#
# --bowtie_components :use bowtie2 to generate readsToTranscripts mappings
#
#
# Other:
# --monitoring :use collectl to monitor all steps of Trinity
#
# --bypass_java_version_check : skip check for required java version 1.7
#
_ADVANCEDUSAGE_
;
my $usage = $basic_usage . $usage_synopsis;
unless (@ARGV) {
die "$usage\n";
}
# Log command line parameters for performance monitoring
foreach (@ARGV) {
$pm_trinity_arguments = $pm_trinity_arguments . " " . $_;
};
my $sort_exec = &COMMON::get_sort_exec($CPU);
my $NO_FASTOOL = 0;
my $NO_CLEANUP = 0;
my $FULL_CLEANUP = 0;
my $NO_BOWTIE = 0;
my $BOWTIE_COMP = 0;
my $NO_RUN_INCHWORM_FLAG = 0;
my $JELLY_S;
my $PASAFLY_MODE = 0;
my $CUFFFLY_MODE = 0;
my $full_usage_info_flag;
my $NO_TRIPLET_LOCK;
## Genome-guided params:
my $genome_guided_max_intron;
my $genome_guided_bam;
my $genome_guided_min_coverage = 1;
my $genome_guided_min_reads_per_partition = 10;
my $genome_guided_just_prep_flag = 0;
## trinity complete flag
my $TRINITY_COMPLETE_FLAG = 0;
my @ORIG_ARGS = @ARGV;
my $CHRYSALIS_DEBUG_WELD_ALL = 0;
my $iworm_opts = "";
my $bypass_java_version_check = 0;
my $SEE = 0;
my $ANANAS_DIR = "";
my $NO_VERSION_CHECK = 0;
&GetOptions(
'h|help' => \$help_flag,
'advanced_help' => \$advanced_help_flag,
'show_full_usage_info' => \$full_usage_info_flag,
'verbose' => \$SEE,
'no_version_check' => \$NO_VERSION_CHECK,
## general opts
"seqType=s" => \$seqType,
"left=s{,}" => \@left_files,
"right=s{,}" => \@right_files,
"single=s{,}" => \@single_files,
"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,
'np=i' => \$np,
'mpiexec=s' => \$mpiexec,
'prep' => \$prep_only,
'KMER_SIZE=i' => \$KMER_SIZE,
# Quality trimming:
'trimmomatic' => \$RUN_TRIMMOMATIC_FLAG,
'quality_trimming_params=s' => \$trimmomatic_quality_trim_params,
# In silico read normalization
'normalize_reads' => \$NORMALIZE_READS_FLAG,
'normalize_max_read_cov=i' => \$normalize_max_read_cov,
'normalize_by_read_set' => \$NORMALIZE_BY_READ_SET,
# Butterfly opts
"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,
'bfly_jar=s' => \$BFLY_JAR,
'path_reinforcement_distance=i' => \$path_reinforcement_distance,
'no_path_merging' => \$NO_PATH_MERGING,
'min_per_id_same_path=i' => \$MIN_PER_ID_SAME_PATH,
'max_diffs_same_path=i' => \$MAX_DIFFS_SAME_PATH,
'max_internal_gap_same_path=i' => \$MAX_INTERNAL_GAP_SAME_PATH,
'PasaFly' => \$PASAFLY_MODE,
'CuffFly' => \$CUFFFLY_MODE,
# Inchworm & kmer catalog opts
'min_kmer_cov=i' => \$min_kmer_cov,
'inchworm_cpu=i' => \$inchworm_cpu,
'FORCE_INCHWORM_KMER_METHOD' => \$FORCE_INCHWORM_KMER_METHOD,
'INCHWORM_CUSTOM_PARAMS=s' => \$INCHWORM_CUSTOM_PARAMS,
'no_run_inchworm' => \$NO_RUN_INCHWORM_FLAG,
'iworm_opts=s' => \$iworm_opts,
'max_memory|M=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,
'max_reads_per_graph=i' => \$max_reads_per_graph,
'max_reads_per_loop=i' => \$max_reads_per_loop,
'min_pct_read_mapping=i' => \$min_pct_read_mapping,
'weldmer_size=i' => \$weldmer_size,
"no_bowtie" => \$NO_BOWTIE,
"bowtie_comp" => \$BOWTIE_COMP,
"no_run_chrysalis" => \$NO_RUN_CHRYSALIS_FLAG,
# Grid computing options
'grid_conf=s' => \$grid_conf_file,
"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,
'no_distributed_trinity_exec' => \$NO_DISTRIBUTED_TRINITY_EXEC,
# hidden (don't look here! ;)
'KMER_SIZE=i' => \$KMER_SIZE,
'jelly_s=i' => \$JELLY_S,
'NO_PARALLEL_IWORM' => \$NO_PARALLEL_IWORM,
'chrysalis_debug_weld_all' => \$CHRYSALIS_DEBUG_WELD_ALL,
# genome guided
"genome_guided_bam=s" => \$genome_guided_bam,
"genome_guided_max_intron=i" => \$genome_guided_max_intron,
"genome_guided_min_coverage=i" => \$genome_guided_min_coverage,
"genome_guided_min_reads_per_partition=i" => \$genome_guided_min_reads_per_partition,
"genome_guided_just_prep" => \$genome_guided_just_prep_flag,
"trinity_complete" => \$TRINITY_COMPLETE_FLAG,
"grid_node_CPU=i" => \$grid_node_CPU,
"grid_node_max_memory=s" => \$grid_node_max_memory,
"bypass_java_version_check" => \$bypass_java_version_check,
"ananas_dir=s" => \$ANANAS_DIR,
);
if ($SHOW_CITATION_FLAG) {
&show_lit_citation();
exit(0);
}
if ($full_usage_info_flag) {
$usage = $basic_usage . $full_usage . $usage_synopsis;
die "$usage\n";
}
if ($advanced_help_flag) {
die "$advanced_usage\n";
}
if ($help_flag) {
die "$usage\n";
}
if ($show_version_flag) {
&version_check();
exit(1);
}
if ($NO_CLEANUP && $FULL_CLEANUP) {
die "cannot set --no_cleanup and --full_cleanup as they contradict";
}
if ($KMER_SIZE > $MAX_KMER_SIZE) {
die "Error, kmer size can be at most $MAX_KMER_SIZE ";
}
if ($NO_PARALLEL_IWORM) {
# turn it off.
$PARALLEL_IWORM_FLAG = 0;
}
my $MIN_IWORM_LEN = $KMER_SIZE;
if (@ARGV) {
die "Error, do not understand options: @ARGV\n";
}
if ($run_with_collectl && $^O !~ /linux/i) {
print STDERR "WARNING, --monitoring can only be used on linux. Turning it off.\n\n";
$run_with_collectl = 0;
}
unless ($BFLY_JAR) {
$BFLY_JAR = "$BUTTERFLY_DIR/Butterfly.jar";
}
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 $SS_lib_type. Should be: F, R, RF, or FR\n";
}
if (@single_files) {
if ($run_as_paired_flag) {
die "Error, if you have paired-end reads and want to run as strand-specific, then you must specify the --left and --right parameters";
}
if (length($SS_lib_type) != 1) {
die "Error, with --single reads, the --SS_lib_type can be 'F' or 'R' only.\n";
}
}
if (@left_files && @right_files) {
if (length($SS_lib_type) != 2) {
die "Error, with paired end reads, the --SS_lib_type can be 'RF' or 'FR' only.\n";
}
}
}
unless ($genome_guided_bam || (@left_files && @right_files) || @single_files ) {
die "Error, need either options 'left' and 'right' or option 'single' or 'genome_guided_bam'\n";
}
unless ($genome_guided_bam) {
unless ($seqType) {
die "Error, need --seqType specified\n";
}
}
if (@left_files) {
@left_files = split(",", join(",", @left_files));
}
if (@right_files) {
@right_files = split(",", join(",", @right_files));
}
if (@single_files) {
@single_files = split(",", join(",", @single_files));
}
if ($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 jellyfish_ram
my $jellyfish_ram = $max_memory; ## this one is used in the Chrysalis exec string
if ($jellyfish_ram) {
$jellyfish_ram =~ /^([\d\.]+)G$/ or die "Error, cannot parse jellyfish_ram value of $jellyfish_ram. Set it to 'xG' where x is a numerical value\n";
$jellyfish_ram = $1;
$jellyfish_ram *= 1024**3; # convert to from gig to bytes
}
else {
die "Error, must specify max memory for jellyfish to use, eg. --max_memory 10G \n";
}
## Check Java version:
unless ($NO_RUN_INCHWORM_FLAG || $NO_RUN_CHRYSALIS_FLAG || $bypass_java_version_check) {
my $java_version = `java -Xmx64m -version 2>&1 `;
if ($java_version =~ /(java|openjdk) version \"1\.(\d)\./) {
my $version_id = $2;
if ($version_id != 7) {
die "Error, Trinity requires access to Java version 1.7. Currently installed version is: $java_version";
}
}
else {
print STDERR "\n\n\n********************************************************************\n"
. "** Warning, Trinity cannot determine which version of Java is being used. Version 1.7 is required. \n\nAttempting to continue in 30 seconds\n"
. "********************************************************************\n\n\n";
sleep(30);
}
}
unless ($NO_VERSION_CHECK || $TRINITY_COMPLETE_FLAG) {
&version_check();
}
# Give the variable with memory size and a user-oriented name
sub bfly_check {
my ($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 ($bflyHeapSpaceMax).\n";
}
if ($inchworm_cpu > $CPU) {
$inchworm_cpu = $CPU;
}
if ($bflyCalculateCPU && $jellyfish_ram) {
$bflyCPU = int ($jellyfish_ram * 0.80 / $bflyHeapSpaceMaxBytes);
}
$bflyCPU = $CPU if !defined $bflyCPU;
if (defined($bflyGCThreads) && $bflyGCThreads > 32) {
die "Error, you probably want fewer than $bflyGCThreads java garbage collection threads. Try a number less than 32.";
}
if ($genome_guided_bam) {
## genome-guided mode.
unless ($genome_guided_max_intron) {
die "Error, must specifiy --genome_guided_max_intron <int> for genome-guided mode.\n";
}
}
$ENV{OMP_NUM_THREADS} = $CPU; ## for Inchworm and Chrysalis
my $PAIRED_MODE = ( (@left_files && @right_files) || $run_as_paired_flag) ? 1:0;
if ($PAIRED_MODE && (!$NO_RUN_CHRYSALIS_FLAG) && (!$NO_BOWTIE)) {
## be sure we can find 'bowtie', since we use it as part of the iworm pair scaffolding step
my $bowtie_path = `which bowtie`;
my $bowtie_build_path = `which bowtie-build`;
if ($bowtie_path =~ /\w/ && $bowtie_build_path =~ /\w/) {
print "Paired mode requires bowtie. Found bowtie at: $bowtie_path\n and bowtie-build at $bowtie_build_path\n\n" if $SEE;
}
else {
die "Error, cannot find path to bowtie ($bowtie_path) or bowtie-build ($bowtie_build_path), which is now needed as part of Chrysalis' read scaffolding step. If you should choose to not run bowtie, include the --no_bowtie in your Trinity command.\n\n";
}
my $samtools_path = `which samtools`;
if ($samtools_path =~ /\w/) {
print "Found samtools at: $samtools_path\n" if $SEE;
}
else {
die "Error, cannot find samtools. Please be sure samtools is installed and included in your PATH setting.\n";
}
unless ($path_reinforcement_distance) {
$path_reinforcement_distance = $PE_path_reinforcement_distance;
}
}
else {
unless ($path_reinforcement_distance) {
$path_reinforcement_distance = $SE_path_reinforcement_distance;
}
}
my $MKDIR_OUTDIR_FLAG = 0; ## only purging output_directory if we create it in this run.
## Regular run. Name the output based on the butterfly reconstruction mode.
my $butterfly_output_filename = "Trinity.fasta";
if ($PASAFLY_MODE) {
$butterfly_output_filename = "Trinity.Pasafly.fasta";
}
elsif ($CUFFFLY_MODE) {
$butterfly_output_filename = "Trinity.Cufffly.fasta";
}
my $PARALLEL_SAMTOOLS_SORT_TOKEN = "-\@ $CPU";
unless (basename($output_directory) =~ /trinity/i) {
die "Error, output directory must contain the word 'trinity' as a safety precaution, given that auto-deletion can take place.\n";
}
if ($grid_conf_file) {
# Prep for HPC parafly use.
$ENV{PATH} = dirname($PARAFLY) . ":$ENV{PATH}";
&HPC::GridRunner::use_parafly();
}
main: {
$ENV{OMP_NUM_THREADS} = $CPU;
unless ($NO_RUN_INCHWORM_FLAG || $NO_RUN_CHRYSALIS_FLAG) {
print STDERR "-since butterfly will eventually be run, lets test for proper execution of java\n" if $SEE;
&test_java_failure_capture();
}
unless ($genome_guided_bam) {
if (basename($chrysalis_output_dir) !~ /chrysalis/i) {
die "Error, chrysalis output directory name must include 'chrysalis' in the name."; # lets try to prevent bad things from happening... (security issue)
}
if ($FULL_CLEANUP && basename($output_directory) !~ /\w/) {
die "Error, working in full-cleanup mode. Specify a named directory for the output. The directory and contents are purged at end of a successful run.";
}
if ($chrysalis_output_dir !~ /^\//) {
$chrysalis_output_dir = "$output_directory/$chrysalis_output_dir";
}
$chrysalis_output_dir = &create_full_path($chrysalis_output_dir, 0);
if ($component_directory){
# does a component directory exist from a previous run?
if (-e $chrysalis_output_dir.'/Component_bins'){
if (-l $chrysalis_output_dir.'/Component_bins'){
$component_directory = readlink($chrysalis_output_dir.'/Component_bins');
}else{
$component_directory = $chrysalis_output_dir.'/Component_bins';
}
warn "Reusing existing component directory $component_directory\n";
}else{
$component_directory .= "/Trinity.$$";
mkdir($component_directory) || die ("component directory cannot be created or already exists!\n");
die "Cannot create component directory $component_directory" unless -d $component_directory;
$component_directory .= "/Component_bins";
mkdir($component_directory) || die ("component directory cannot be created or already exists!\n");
die "Cannot create component directory $component_directory" unless -d $component_directory;
}
# so that users know where it is/remember to remove it if manually done?
symlink($component_directory,$chrysalis_output_dir.'/Component_bins') unless -e $chrysalis_output_dir.'/Component_bins';
}else{
$component_directory = $chrysalis_output_dir . '/Component_bins';
$component_directory = &create_full_path($component_directory,0);