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Launch_PASA_pipeline.pl
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Launch_PASA_pipeline.pl
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#!/usr/bin/env perl
use strict;
use warnings;
use Time::localtime;
use FindBin;
use lib ("$FindBin::Bin/PerlLib");
use Pasa_init;
use ConfigFileReader;
use Getopt::Long qw(:config no_ignore_case bundling pass_through);
use Cwd;
use File::Basename qw(fileparse);
use DB_connect;
use Data::Dumper;
use Pipeliner;
use File::Basename;
#my $VERSION = "__BLEEDING_EDGE__";
my $VERSION = "2.5.2";
$ENV{PATH} = "$FindBin::Bin/bin:$ENV{PATH}";
my ($opt_c, $opt_C, $opt_r, $opt_R, $opt_A, $opt_g, $opt_t, $opt_f, $opt_T, $opt_u, $opt_d, $opt_h, $opt_x,
$ALT_SPLICE, $INVALIDATE_SINGLE_EXON_ESTS, $IMPORT_CUSTOM_ALIGNMENTS_GFF3,
$splice_graph_assembler_flag,
$ALIGNED_IS_TRANSCRIBED_ORIENT,
$ANNOTS_FILE, $opt_L, $STRINGENT_ALIGNMENT_OVERLAP, $GENE_OVERLAP,
$SIM4_CHASER, $genetic_code, $TRANSDECODER, @PRIMARY_ALIGNERS,
$PASACONF,
$SHOW_VERSION_INFO
);
my $MAX_INTRON_LENGTH = 500000;
my $CPU = 2;
my $NUM_TOP_ALIGNMENTS = 1;
my $TDN_file; # file containing list of Trinity (full) de novo transcriptome assemblies. (used to find 'missing' genes in genome, among other types)
my %SUPPORTED_PRIMARY_ALIGNERS = map { + $_ => 1 } qw (gmap blat minimap2);
my $PASA_PIPELINE_CMD = join(" ", $0, @ARGV);
my $CUFFLINKS_GTF;
&GetOptions ( 'config|c=s' => \$opt_c,
'create|C' => \$opt_C,
'replace|r' => \$opt_r,
'run|R' => \$opt_R,
'annot_compare|A' => \$opt_A,
'genome|g=s' => \$opt_g,
'transcripts|t=s' => \$opt_t,
'f=s' => \$opt_f,
'T' => \$opt_T,
'u=s' => \$opt_u,
'd' => \$opt_d,
'h' => \$opt_h,
'x' => \$opt_x,
'INVALIDATE_SINGLE_EXON_ESTS' => \$INVALIDATE_SINGLE_EXON_ESTS,
'IMPORT_CUSTOM_ALIGNMENTS_GFF3=s' => \$IMPORT_CUSTOM_ALIGNMENTS_GFF3,
'USE_SPLICE_GRAPH_ASSEMBLER' => \$splice_graph_assembler_flag,
'MAX_INTRON_LENGTH|I=i' => \$MAX_INTRON_LENGTH,
'APPLY_SIM4_CHASER' => \$SIM4_CHASER,
'TRANSDECODER' => \$TRANSDECODER,
'CPU=i' => \$CPU,
'TDN=s' => \$TDN_file,
'N=i' => \$NUM_TOP_ALIGNMENTS,
'ALIGNERS=s' => \@PRIMARY_ALIGNERS,
'trans_gtf=s' => \$CUFFLINKS_GTF,
'PASACONF=s' => \$PASACONF,
## RNA-Seq options
'transcribed_is_aligned_orient' => \$ALIGNED_IS_TRANSCRIBED_ORIENT,
'ALT_SPLICE' => \$ALT_SPLICE,
## Annotation compare opts
'L' => \$opt_L,
'annots=s' => \$ANNOTS_FILE,
'GENETIC_CODE=s' => \$genetic_code,
## clustering options:
'stringent_alignment_overlap=f' => \$STRINGENT_ALIGNMENT_OVERLAP,
'gene_overlap=f' => \$GENE_OVERLAP,
'version' => \$SHOW_VERSION_INFO,
);
if (@ARGV) {
die "Error, do not recognize opts: @ARGV\n";
}
our $SEE = 0;
my $usage = <<_EOH_;
############################# Options ###############################
#
# * indicates required
#
#
# --config|-c * <filename> alignment assembly configuration file
#
# // spliced alignment settings
# --ALIGNERS <string> aligners (available options include: gmap, blat, minimap2... can run using several, ie. 'gmap,blat,minimap2')
# -N <int> max number of top scoring alignments (default: 1)
# --MAX_INTRON_LENGTH|-I <int> (max intron length parameter passed to GMAP or BLAT) (default: 100000)
# --IMPORT_CUSTOM_ALIGNMENTS_GFF3 <filename> :only using the alignments supplied in the corresponding GFF3 file.
# --trans_gtf <filename> :incorporate cufflinks or stringtie--generated transcripts
#
#
# // actions
# --create|-C flag, create database
# --replace|-r flag, drop database if -C is also given. This will DELETE all your data and it is irreversible.
# --run|-R flag, run alignment/assembly pipeline.
# --annot_compare|-A (see section below; can use with opts -L and --annots) compare to annotated genes.
# --ALT_SPLICE flag, run alternative splicing analysis
# // input files
# --genome|-g * <filename> genome sequence FASTA file (should contain annot db asmbl_id as header accession.)
# --transcripts|-t * <filename> transcript db
# -f <filename> file containing a list of fl-cdna accessions.
# --TDN <filename> file containing a list of accessions corresponding to Trinity (full) de novo assemblies (not genome-guided)
#
# // polyAdenylation site identification ** highly recommended **
# -T flag,transcript db were trimmed using the TGI seqclean tool.
# -u <filename> value, transcript db containing untrimmed sequences (input to seqclean)
# <a filename with a .cln extension should also exist, generated by seqclean.>
#
#
#
# Misc:
# --TRANSDECODER flag, run transdecoder to identify candidate full-length coding transcripts
# --CPU <int> multithreading (default: $CPU)
# --PASACONF <string> path to a user-defined pasa.conf file containing mysql connection info
# (used in place of the \$PASAHOME/pasa_conf/conf.txt file)
# (and allows for users to have their own unique mysql connection info)
# (instead of the pasa role account)
#
# -d flag, Debug
# -h flag, print this option menu and quit
#
#########
#
# // Transcript alignment clustering options (clusters are fed into the PASA assembler):
#
# By default, clusters together transcripts based on any overlap (even 1 base!).
#
# Alternatives:
#
# --stringent_alignment_overlap <float> (suggested: 30.0) overlapping transcripts must have this min % overlap to be clustered.
#
# --gene_overlap <float> (suggested: 50.0) transcripts overlapping existing gene annotations are clustered. Intergenic alignments are clustered by default mechanism.
# * if --gene_overlap, must also specify --annots with annotations in recognizable format (gtf, gff3, or data adapted) (just examines 'gene' rows, though).
#
#
#
# --INVALIDATE_SINGLE_EXON_ESTS :invalidates single exon ests so that none can be built into pasa assemblies.
#
#
# --transcribed_is_aligned_orient flag for strand-specific RNA-Seq assemblies, the aligned orientation should correspond to the transcribed orientation.
#
#
################
#
# // Annotation comparison options (used in conjunction with -A at top).
#
# -L load annotations (use in conjunction with --annots)
# --annots <filename> existing gene annotations in recognized format (gtf, gff3, or custom adapted).
# --GENETIC_CODE (default: universal, options: Euplotes, Tetrahymena, Candida, Acetabularia)
#
###################### Process Args and Options #####################
_EOH_
;
# --USE_SPLICE_GRAPH_ASSEMBLER (use at own risk! alpha-state) // likely will cause the alt-splice analysis to break...
if ($opt_h) {die $usage;}
if ($SHOW_VERSION_INFO) {
print STDERR "PASA version: $VERSION\n";
exit(0);
}
if ($PASACONF) {
$ENV{PASACONF} = $PASACONF;
}
require "Pasa_conf.pm";
my $DEBUG = $opt_d;
my $full_length_cdna_listing = $opt_f || "NULL"; ## NULL filename results in graceful exit, used when nothing exists to load.
my $RUN_PIPELINE = $opt_R;
#$ALT_SPLICE = $RUN_PIPELINE; ## always run alt-splice analysis in alignment-assembly pipeline.
my $COMPARE_TO_ANNOT = $opt_A;
my $CREATE_DB = $opt_C;
my $PRINT_CMDS_ONLY = $opt_x;
my $configfile = $opt_c or die $usage;
if ($splice_graph_assembler_flag) {
$splice_graph_assembler_flag = "-X";
}
else {
$splice_graph_assembler_flag = "";
}
## Read configuration file.
my %config = &readConfig($configfile);
my $database = $config{DATABASE} or die "Error, couldn't extract DATABASE from config file " . cwd() . "/$configfile\n";
&DB_connect::configure_db_driver($database);
my $mysql_server = &Pasa_conf::getParam("MYSQLSERVER");
my $user = &Pasa_conf::getParam("MYSQL_RW_USER");
my $password = &Pasa_conf::getParam("MYSQL_RW_PASSWORD");
my $DBname = fileparse($database); # just the filename if SQLite
my %advanced_prog_opts = &parse_advanced_prog_opts();
## Add a few env variables:
my $UTILDIR = "$ENV{PASAHOME}/scripts";
my $PLUGINS_DIR = "$ENV{PASAHOME}/pasa-plugins";
unless ($RUN_PIPELINE || $COMPARE_TO_ANNOT || $ALT_SPLICE || $CREATE_DB) {
print STDERR "Sorry, nothing to do here.\n";
exit(1);
}
my $checkpts_dir = "__pasa_" . basename($database) . "_$ENV{DBI_DRIVER}_chkpts";
unless (-d $checkpts_dir) {
mkdir $checkpts_dir or die "Error, cannot create checkpoints dir: $checkpts_dir";
}
my $pipeliner = new Pipeliner(-verbose=>2,
-checkpoint_dir=>$checkpts_dir,
-cmds_log=> "$checkpts_dir.cmds_log",
);
my @cmds; # stores all commands to be executed.
if ($CREATE_DB) {
if (&Pasa_conf::getParam("USE_PASA_DB_SETUP_HOOK") =~ /true/i) {
&execute_custom_PASA_DB_setup_hook();
}
else {
## going the old fashioned way
my ($schema_file, $prog);
if (exists($ENV{DBI_DRIVER}) and $ENV{DBI_DRIVER} eq 'mysql') {
$schema_file = "$ENV{PASAHOME}/schema/cdna_alignment_mysqlschema";
$prog = "$UTILDIR/create_mysql_cdnaassembly_db.dbi";
} else { # default to SQLite
$schema_file = "$ENV{PASAHOME}/schema/cdna_alignment_sqliteschema";
$prog = "$UTILDIR/create_sqlite_cdnaassembly_db.dbi";
}
my $params = "-c $opt_c -S '$schema_file'";
$params .= ' -r' if $opt_r;
push (@cmds, {
prog => $prog,
params => $params,
input => undef,
output => undef,
chkpt => "create_db.ok",
}
);
}
}
## directory to store voluminous logging info from pasa processes
my $PASA_LOG_DIR = "pasa_run.log.dir";
if (! -d $PASA_LOG_DIR) {
mkdir ($PASA_LOG_DIR) or die "Error, cannot mkdir $PASA_LOG_DIR";
}
## Analyze pipeline run options.
my $genome_db = $opt_g or die "Must specify genome_db.\n\n$usage\n";
unless (-s $genome_db) {
die "Can't find $genome_db\n\n";
}
my $transcript_db = $opt_t or die "Must specify transcript_db.\n\n$usage\n";
unless (-s $transcript_db) {
die "Can't find $transcript_db\n";
}
my $POLYA_IDENTIFICATION = $opt_T;
my $untrimmed_transcript_db = $opt_u;
if ($POLYA_IDENTIFICATION) {
unless (-s $untrimmed_transcript_db) {
die "ERROR, cannot find untrimmed transcript database ($untrimmed_transcript_db)\n";
}
if ($transcript_db eq $untrimmed_transcript_db) {
die "ERROR, your transcript db and untrimmed-transcript db are named identically.\n";
}
unless ($transcript_db =~ /$untrimmed_transcript_db/) {
print STDERR "WARNING: The transcript database ($transcript_db) name appears unrelated to the untrimmed transcript database ($untrimmed_transcript_db)\n";
print STDERR "press cntrl-c to stop the job and rerun using different parameters.\n";
sleep(10);
}
unless ($transcript_db =~ /\.clean/) {
print STDERR "WARNING: The transcript database ($transcript_db) lacks the .clean extension generated by seqclean. Are you certain you are using a set of trimmed transcript sequences?\n";
print STDERR "press cntrl-c to stop the job and rerun using different parameters.\n";
sleep(10);
}
unless (-s "$untrimmed_transcript_db.cln") {
die "ERROR: I cannot locate the .cln file generated by seqclean, expecting $untrimmed_transcript_db.cln\n\n";
}
}
unless (-s "$genome_db.fai") {
push (@cmds, { prog => "samtools",
params => "faidx $genome_db",
input => undef,
output => undef,
chkpt => "samtools_faidx_genome.ok",
});
}
unless (-s "$transcript_db.fai") {
push (@cmds, { prog => "samtools",
params => "faidx $transcript_db",
input => undef,
output => undef,
chkpt => "samtools_faidx_transcriptome.ok",
});
}
if ($RUN_PIPELINE) {
## Build Pipeline Command List
@PRIMARY_ALIGNERS = split(/,/,join(',',@PRIMARY_ALIGNERS)); # unpack list of aligners in case commas are used.
unless (@PRIMARY_ALIGNERS || $IMPORT_CUSTOM_ALIGNMENTS_GFF3) {
die "Error, must specify at least one primary aligner via --ALIGNERS or imported via --IMPORT...";
}
foreach my $aligner (@PRIMARY_ALIGNERS) {
unless ($SUPPORTED_PRIMARY_ALIGNERS{$aligner}){
die "Error, do not recognize aligner: [$aligner] ";
}
}
my $TDN_param = "";
if ($TDN_file) {
$TDN_param = "-T $TDN_file";
}
push (@cmds, { prog => "$UTILDIR/upload_transcript_data.dbi",
params => "-M '$database' -t $transcript_db $TDN_param -f $full_length_cdna_listing ",
input => undef,
output => undef,
chkpt => "upload_transcripts.ok",
}
);
if (@PRIMARY_ALIGNERS) {
# split the threads among the different aligners.
push (@cmds, { prog => "$UTILDIR/run_spliced_aligners.pl",
params => "--aligners " . join(",", @PRIMARY_ALIGNERS) . " --genome $genome_db"
. " --transcripts $transcript_db -I $MAX_INTRON_LENGTH -N $NUM_TOP_ALIGNMENTS --CPU $CPU",
input => undef,
output => undef,
chkpt => "align_transcripts.ok",
} );
foreach my $aligner (@PRIMARY_ALIGNERS) {
push (@cmds, { prog => "$UTILDIR/import_spliced_alignments.dbi",
params => "-M '$database' -A $aligner -g $aligner.spliced_alignments.gff3",
input => undef,
output => undef,
chkpt => "import_alignments.$aligner.ok",
},
);
}
}
if ($IMPORT_CUSTOM_ALIGNMENTS_GFF3) {
push (@cmds, { prog => "$UTILDIR/import_spliced_alignments.dbi",
params => "-M '$database' -A custom -g $IMPORT_CUSTOM_ALIGNMENTS_GFF3",
input => undef,
output => undef,
chkpt => "import_custom_alignments.ok",
},
);
push (@PRIMARY_ALIGNERS, "custom");
}
if ($CUFFLINKS_GTF) {
push (@cmds,
# first convert it to gff3-alignment format
{ prog => "$UTILDIR/../misc_utilities/cufflinks_gtf_to_alignment_gff3.pl",
params => "$CUFFLINKS_GTF",
input => undef,
output => "$CUFFLINKS_GTF.gff3",
chkpt => "cuff_gtf_to_gff3.ok",
},
# convert to fasta format:
{
prog => "$UTILDIR/../misc_utilities/cufflinks_gtf_genome_to_cdna_fasta.pl",
params => "$CUFFLINKS_GTF $genome_db",
input => undef,
output => "$CUFFLINKS_GTF.fasta",
chkpt => "cuff_gtf_to_fasta.ok",
},
# upload fasta entries into DB
{
prog => "$UTILDIR/upload_transcript_data.dbi",
params => "-M '$database' -t $CUFFLINKS_GTF.fasta",
input => undef,
output => undef,
chkpt => "upload_transcript_fasta.ok",
},
# upload the cufflinks transcript structures
{
prog => "$UTILDIR/import_spliced_alignments.dbi",
params => "-M '$database' -A cufflinks -g $CUFFLINKS_GTF.gff3",
input => undef,
output => undef,
chkpt => "import_cuff_gtf.ok",
},
# combine the cufflinks transcripts with the other transcripts for use in validation and other downstream studies
{
prog => "cat",
params => "$transcript_db $CUFFLINKS_GTF.fasta",
input => undef,
output => "__all_transcripts.fasta",
chkpt => "merge_in_cuff_transcripts.ok",
}
);
## reset the transcript db to the combined database
$transcript_db = "__all_transcripts.fasta";
}
##############################
## done aligning transcripts.
##############################
if ($TRANSDECODER) {
## Identify likely full-length transcripts via TransDecoder plug-in
my $td_params = "-t $transcript_db ";
if ($genetic_code) {
$td_params .= " -G $genetic_code";
}
my $td_longorf_params = "";
if ($ALIGNED_IS_TRANSCRIBED_ORIENT) {
$td_longorf_params = " -S ";
}
push (@cmds, { prog => "$PLUGINS_DIR/transdecoder/TransDecoder.LongOrfs",
params => "$td_params $td_longorf_params",
input => undef,
output => undef,
chkpt => "transdecoder_longorfs.ok",
},
);
push (@cmds, { prog => "$PLUGINS_DIR/transdecoder/TransDecoder.Predict",
params => $td_params,
input => undef,
output => undef,
chkpt => "transdecoder_predict.ok",
},
);
## get the full-length entries
my $transdecoder_gff3_file = basename("$transcript_db.transdecoder.gff3");
my $td_full_length_file = "$transdecoder_gff3_file.fl_accs";
push (@cmds, { prog => "$UTILDIR/extract_FL_transdecoder_entries.pl",
params => $transdecoder_gff3_file,
input => undef,
output => $td_full_length_file,
chkpt => "transdecoder.extract_FL.ok",
},
## update the full-length status
{ prog => "$UTILDIR/update_fli_status.dbi",
params => "-M '$database' -f $td_full_length_file",
input => undef,
output => undef,
chkpt => "transdecoder.update_FL_status.ok",
},
);
}
push (@cmds,
# validate the alignment data:
{
prog => "$UTILDIR/validate_alignments_in_db.dbi",
params => "-M '$database' -g $genome_db -t $transcript_db --MAX_INTRON_LENGTH $MAX_INTRON_LENGTH --CPU $CPU ", # creates output file: $mysql_db.${map_program}_validations that is read in below.
input => undef,
output => "alignment.validations.output",
chkpt => "validate_alignments.ok",
},
# update the alignment validation results.
{
prog => "$UTILDIR/update_alignment_status.dbi",
params => "-M '$database'",
input => "alignment.validations.output",
output => "$PASA_LOG_DIR/alignment.validation_loading.output",
chkpt => "update_align_valid_status.ok",
},
);
foreach my $map_program (@PRIMARY_ALIGNERS) {
push (@cmds,
# write the gff3 file describing the valid alignments:
{
prog => "$UTILDIR/PASA_transcripts_and_assemblies_to_GFF3.dbi",
params => "-M '$database' -v -A -P ${map_program}",
input => undef,
output => "$DBname.valid_${map_program}_alignments.gff3",
chkpt => "$DBname.valid_${map_program}_alignments.gff3.ok",
},
# do again, but write in BED format
{
prog => "$UTILDIR/PASA_transcripts_and_assemblies_to_GFF3.dbi",
params => "-M '$database' -v -A -P ${map_program} -B ",
input => undef,
output => "$DBname.valid_${map_program}_alignments.bed",
chkpt => "$DBname.valid_${map_program}_alignments.bed.ok",
},
# do again, but write in GTF format
{
prog => "$UTILDIR/PASA_transcripts_and_assemblies_to_GFF3.dbi",
params => "-M '$database' -v -A -P ${map_program} -T ",
input => undef,
output => "$DBname.valid_${map_program}_alignments.gtf",
chkpt => "$DBname.valid_${map_program}_alignments.gtf.ok",
},
# write the gff3 file describing the failures:
{
prog => "$UTILDIR/PASA_transcripts_and_assemblies_to_GFF3.dbi",
params => "-M '$database' -f -A -P ${map_program}",
input => undef,
output => "$DBname.failed_${map_program}_alignments.gff3",
chkpt => "$DBname.failed_${map_program}_alignments.gff3.ok",
},
# do again, but write in BED format
{
prog => "$UTILDIR/PASA_transcripts_and_assemblies_to_GFF3.dbi",
params => "-M '$database' -f -A -P ${map_program} -B ",
input => undef,
output => "$DBname.failed_${map_program}_alignments.bed",
chkpt => "$DBname.failed_${map_program}_alignments.gff3.ok",
},
# do again, but write in BED format
{
prog => "$UTILDIR/PASA_transcripts_and_assemblies_to_GFF3.dbi",
params => "-M '$database' -f -A -P ${map_program} -T ",
input => undef,
output => "$DBname.failed_${map_program}_alignments.gtf",
chkpt => "$DBname.failed_${map_program}_alignments.gtf.ok",
},
);
}
if ($INVALIDATE_SINGLE_EXON_ESTS) {
push (@cmds,
{
prog => "$UTILDIR/invalidate_single_exon_ESTs.dbi",
params => "-M '$database'",
input => undef,
output => "$PASA_LOG_DIR/invalidating_single_exon_alignments.output",
chkpt => "invalidate_single_exon_ESTs.ok",
},
);
}
## PolyA-site analysis, help assign transcribed orientations to intron-less alignments.
if ($POLYA_IDENTIFICATION) {
push (@cmds, (
## Analyze polyA site-inferring transcripts
{
prog => "$UTILDIR/polyA_site_transcript_mapper.dbi",
params => "-M '$database' -c $untrimmed_transcript_db.cln "
. "-g $genome_db -t $untrimmed_transcript_db",
input => undef,
output => "$PASA_LOG_DIR/polyAsite_analysis.out",
chkpt => "polyAsite_analysis.ok",
},
## Summarize PolyA site findings:
{
prog => "$UTILDIR/polyA_site_summarizer.dbi",
params => "-M '$database' -g $genome_db ",
input => undef,
output => "$DBname.polyAsites.fasta",
chkpt => "polAsites.fasta.ok",
},
)
);
}
if ($ALIGNED_IS_TRANSCRIBED_ORIENT) {
push (@cmds,
{
prog => "$UTILDIR/set_spliced_orient_transcribed_orient.dbi",
params => "-M '$database'",
input => undef,
output => "$PASA_LOG_DIR/setting_aligned_as_transcribed_orientation.output",
chkpt => "set_spliced_orient_transcribed_orient.ok",
},
);
}
#####################################
## Cluster the transcript alignments.
#####################################
if ($STRINGENT_ALIGNMENT_OVERLAP) {
## require substantial overlap between neighboring transcript alignments for clustering.
push (@cmds,
{
prog => "$UTILDIR/assign_clusters_by_stringent_alignment_overlap.dbi",
params => "-M $database -L $STRINGENT_ALIGNMENT_OVERLAP", # require all alignments are valid here.
input => undef,
output => "$PASA_LOG_DIR/cluster_reassignment_by_stringent_overlap.out",
chkpt => "cluster_reassign_stringent_overlap.ok",
},
);
}
elsif ($GENE_OVERLAP) {
# define transcript overlap clusters based on mapping to overlapping annotated gene models (annotation-informed).
## transcripts in intergenic regions are clustered using the default method.
unless ($ANNOTS_FILE) {
die "Error, need --annots specified for clustering genes based on gene overlaps. ";
}
push (@cmds,
{
prog => "$UTILDIR/assign_clusters_by_gene_intergene_overlap.dbi",
params => "-M '$database' -G $ANNOTS_FILE -L $GENE_OVERLAP", # require all alignments are valid here.
input => undef,
output => "$PASA_LOG_DIR/alignment_cluster_reassignment.out",
chkpt => "alignment_cluster_reassignment.ok",
},
);
}
else {
## Default transcript clustring based on overlap piles (any overlap with same transcribed orientation)
push (@cmds,
{
prog => "$UTILDIR/reassign_clusters_via_valid_align_coords.dbi",
params => "-M '$database' ",
input => undef,
output => "$PASA_LOG_DIR/cluster_reassignment_by_valid_alignment_coords.default.out",
chkpt => "reassign_clusters_via_valid_align_coords.ok",
},
);
}
if ($NUM_TOP_ALIGNMENTS > 1 || scalar(@PRIMARY_ALIGNERS) == 0) {
## ensure only one valid alignment per cdna per cluster
# (doesn't make sense to assemble a blat and gsnap alignment for the same cDNA.
# Instead, just keep the highest scoring alignment of that cdna at that locus )
push (@cmds,
{
prog => "$UTILDIR/ensure_single_valid_alignment_per_cdna_per_cluster.pl",
params => "-M '$database'",
input => undef,
output => "$PASA_LOG_DIR/ensuring_single_valid_alignment_per_cdna_per_cluster.log",
chkpt => "ensuring_single_valid_alignment_per_cdna_cluster.ok",
},
);
}
####################################################
## PASA assembly of clustered transcript alignments
####################################################
push (@cmds,
# build the assemblies:
{
prog => "$UTILDIR/assemble_clusters.dbi",
params => "-G $genome_db -M '$database' $splice_graph_assembler_flag -T $CPU ",
input => undef,
output => "$DBname.pasa_alignment_assembly_building.ascii_illustrations.out",
chkpt => "assemble_clusters.ok",
},
# load the assemblies:
{
prog => "$UTILDIR/assembly_db_loader.dbi",
params => "-M '$database'",
input => undef,
output => "$PASA_LOG_DIR/alignment_assembly_loading.out",
chkpt => "alignment_assembly_loading.ok",
},
# build the subclusters:
{
prog => "$UTILDIR/subcluster_builder.dbi",
params => "-G $genome_db -M '$database' ",
input => undef,
output => "$PASA_LOG_DIR/alignment_assembly_subclustering.out",
chkpt => "alignment_assembly_subclustering.ok",
},
# populate the alignment field for assemblies
# and write the PASA assembly fasta file.
{
prog => "$UTILDIR/populate_mysql_assembly_alignment_field.dbi",
params => "-M '$database' -G $genome_db",
input => undef,
output => undef,
chkpt => "populate_db_assembly_alignment_field.ok",
},
# populate pasa assembly sequences:
{
prog => "$UTILDIR/populate_mysql_assembly_sequence_field.dbi",
params => "-M '$database' -G $genome_db",
input => undef,
output => undef,
chkpt => "populate_db_assembly_sequence_field.ok",
},
# load the subclusters:
{
prog => "$UTILDIR/subcluster_loader.dbi",
params => "-M '$database' ",
input => "$PASA_LOG_DIR/alignment_assembly_subclustering.out",
output => undef,
chkpt => "alignment_assembly_subcluster_loading.ok",
},
# create gene models based on pasa assembies and long orfs (note, this is only used in web displays!, see documentation for more robust de novo annotation based on transcripts.)
{
prog => "$UTILDIR/alignment_assembly_to_gene_models.dbi",
params => "-M '$database' -G $genome_db",
input => undef,
output => undef,
chkpt => "alignment_assembly_to_gene_models.ok",
},
############################################################
# write summary GFF3 files for PASA assemblies:
# and other summary reports
############################################################
{ # gff3 format
prog => "$UTILDIR/PASA_transcripts_and_assemblies_to_GFF3.dbi",
params => "-M '$database' -a ",
input => undef,
output => "$DBname.pasa_assemblies.gff3",
chkpt => "pasa_assemblies_to_gff3.ok",
},
{ # bed format
prog => "$UTILDIR/PASA_transcripts_and_assemblies_to_GFF3.dbi",
params => "-M '$database' -a -B ",
input => undef,
output => "$DBname.pasa_assemblies.bed",
chkpt => "pasa_assemblies_to_bed.ok",
},
{ # gtf format
prog => "$UTILDIR/PASA_transcripts_and_assemblies_to_GFF3.dbi",
params => "-M '$database' -a -T ",
input => undef,
output => "$DBname.pasa_assemblies.gtf",
chkpt => "pasa_assemblies_to_gtf.ok",
},
# describe assemblies in the pasa alignment format, which can be used with accessory scripts.
{
prog => "$UTILDIR/describe_alignment_assemblies_cgi_convert.dbi",
params => "-M '$database' ",
input => undef,
output => "$DBname.pasa_assemblies_described.txt",
chkpt => "pasa_assemblies_described.ok",
},
);
}
######################################
## Annotation Comparison
######################################
if ($COMPARE_TO_ANNOT) {
if ($opt_L) {
unless ($ANNOTS_FILE) {
die "Error, must set --annots for auto-loading of gene annotations";
}
my $cmd = {
prog => "$UTILDIR/Load_Current_Gene_Annotations.dbi",
params => "-c $configfile -g $genome_db -P $ANNOTS_FILE ",
input => undef,
output => "$PASA_LOG_DIR/output.annot_loading.$$.out",
chkpt => "annot_loading.". time(),
};
push (@cmds, $cmd);
}
## compare to annotation:
my $genetic_code_opt = ($genetic_code) ? "--GENETIC_CODE $genetic_code" : "";
my $cmd = {
prog => "$UTILDIR/cDNA_annotation_comparer.dbi",
params => "-G $genome_db --CPU $CPU -M '$database' $genetic_code_opt",
input => undef,
output => "$PASA_LOG_DIR/$DBname.annotation_compare.$$.out", ## TODO: use compare_id and annot_version values for file naming. Ditto for below and other relevant places.
chkpt => "cDNA_annot_comparer." . time(),
};
push (@cmds, $cmd);
$cmd = {
prog => "$UTILDIR/dump_valid_annot_updates.dbi",
params => "-M '$database' -V -R -g $genome_db",
input => undef,
output => "$DBname.gene_structures_post_PASA_updates.$$.gff3",
chkpt => "dump_valid_annot_updates." . time(),
};
push (@cmds, $cmd);
## write it in BED format:
$cmd = {
prog => "$UTILDIR/../misc_utilities/gff3_file_to_bed.pl",
params => "$DBname.gene_structures_post_PASA_updates.$$.gff3",
input => undef,
output => "$DBname.gene_structures_post_PASA_updates.$$.bed",
chkpt => "gene_structures_post_PASA.bed." . time(),
};
push (@cmds, $cmd);
}
######################################
## Alternative Splicing Analysis
######################################
if ($ALT_SPLICE && !$COMPARE_TO_ANNOT) { #this has bitten me before. do alt-splice analysis separately from annot comparison. plus, only do it once.
my $cmd = {
prog => "$UTILDIR/classify_alt_splice_isoforms.dbi",
params => "-M '$database' -G $genome_db -T $CPU ",
input => undef,
output => "$PASA_LOG_DIR/alt_splicing_analysis.out",
chkpt => "alt_splicing_analysis.ok",
};
push (@cmds, $cmd);
$cmd = {
prog => "$UTILDIR/find_alternate_internal_exons.dbi",
params => "-M '$database' -G $genome_db",
input => undef,
output => "$PASA_LOG_DIR/alt_internal_exon_finding.out",
chkpt => "find_alternate_internal_exons.ok",
};
push (@cmds, $cmd);
$cmd = {
prog => "$UTILDIR/classify_alt_splice_as_UTR_or_protein.dbi",
params => "-M '$database' -G $genome_db",
input => undef,
output => "$PASA_LOG_DIR/alt_splice_FL_FL_compare",
chkpt => "alt_splice_FL_FL_compare.ok",
};
push (@cmds, $cmd);
$cmd = {
prog => "$UTILDIR/report_alt_splicing_findings.dbi",
params => "-M '$database' ",