Thread 8 terminated abnormally: Error, spliced orient in db (-) differs from calculated spliced orient (+)

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Xuping Zhou

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Dec 16, 2023, 6:48:05 AM12/16/23
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Hello, everyone
I used the PASA (v2.5.3) pipeline to update the annotations from EVM with the following command: /vol2/YL_group/YL/software/PASApipeline-master/Launch_PASA_pipeline.pl -c alignAssembly.config -A -g /vol3/YL_group/YL/zhou/01.ont/1226/00.repaet/1226.fa -t /vol3/YL_group/YL/zhou/01.ont/1226/03.re_gene.anno/02.PASA/trinity_filter.fa .clean --CPU 96
The following error was reported during the run:
  • Thread 8 terminated abnormally: Error, spliced orient in db (-) differs from calculated spliced orient (+) at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/Ath1_cdnas.pm line 152 thread 8, <$fh> line 215747.
    Ath1_cdnas::create_alignment_obj(DB_connect=HASH(0x55dc7ed1a650), 1093979, SCALAR(0x55dc7f17c138)) called at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/Ath1_cdnas.pm line 175 thread 8
    Ath1_cdnas::get_alignment_obj_via_align_acc(DB_connect=HASH(0x55dc7ed1a650), "asmbl_86579", SCALAR(0x55dc7f17c138)) called at /vol2/YL_group/YL/software/PASApipeline-master/scripts/cDNA_annotation_comparer.dbi line 1592 thread 8
    main::get_alignment_assemblies_via_subcluster_id(DB_connect=HASH(0x55dc7ed1a650), 29859, SCALAR(0x55dc7f17c138), HASH(0x55dc7f175740)) called at /vol2/YL_group/YL/software/PASApipeline-master/scripts/cDNA_annotation_comparer.dbi line 400 thread 8
    main::process_contig("U") called at /vol2/YL_group/YL/software/PASApipeline-master/scripts/cDNA_annotation_comparer.dbi line 311 thread 8
    eval {...} called at /vol2/YL_group/YL/software/PASApipeline-master/scripts/cDNA_annotation_comparer.dbi line 311 thread 8
    substr outside of string at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/Gene_obj.pm line 1315.
    Use of uninitialized value in concatenation (.) or string at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/Gene_obj.pm line 1315.
    substr outside of string at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/Gene_obj.pm line 1315.
    Use of uninitialized value in concatenation (.) or string at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/Gene_obj.pm line 1315.
    substr outside of string at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/Gene_obj.pm line 1315.
    Use of uninitialized value in concatenation (.) or string at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/Gene_obj.pm line 1315.
    substr outside of string at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/Gene_obj.pm line 1315.
    Use of uninitialized value in concatenation (.) or string at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/Gene_obj.pm line 1315.
    No incoming CDS sequence to set to
    TU_feat_name: PC314770
    classification: annotated_genes
    source: .
    com_name: PC314770.1
    is_5prime_partial: 0
    gene_type: protein-coding
    strand: -
    asmbl_id: V
    gene_length: 2422
    is_pseudogene: 0
    Model_feat_name: PC314770.1
    num_exons: 4
    mid_pt: 15736116
    is_3prime_partial: 0
    gene_synonyms:
    mRNA_coords
    CDS_coords
    RNA-exon: 15737327, 15737239 CDS-exon: 15737327, 15737239
    RNA-exon: 15736923, 15736818 CDS-exon: 15736923, 15736818
    RNA-exon: 15736176, 15735647 CDS-exon: 15736176, 15735647
    RNA-exon: 15735143, 15734906 CDS-exon: 15735143, 15734906
    gene_span: 15737327-15734906
    model_span: 15737327-15734906

No incoming protein sequence to set to.
TU_feat_name: PC314770
classification: annotated_genes
source: .
com_name: PC314770.1
is_5prime_partial: 0
gene_type: protein-coding
strand: -
asmbl_id: V
gene_length: 2422
is_pseudogene: 0
Model_feat_name: PC314770.1
num_exons: 4
mid_pt: 15736116
is_3prime_partial: 0
gene_synonyms:
mRNA_coords
CDS_coords
RNA-exon: 15737327, 15737239 CDS-exon: 15737327, 15737239
RNA-exon: 15736923, 15736818 CDS-exon: 15736923, 15736818
RNA-exon: 15736176, 15735647 CDS-exon: 15736176, 15735647
RNA-exon: 15735143, 15734906 CDS-exon: 15735143, 15734906
gene_span: 15737327-15734906
model_span: 15737327-15734906

Thread 9 terminated abnormally: Error, coords are out of bounds of sequence length: 15452089:
$VAR1 = {
'15737522' => 15737239,
'15736176' => 15735647,
'15735143' => 15734527,
'15736923' => 15736818
};
at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/Exons_to_geneobj.pm line 115 thread 9.
Exons_to_geneobj::get_cdna_seq(HASH(0x55dc7f6664d0), SCALAR(0x55dc7f5746a8)) called at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/Exons_to_geneobj.pm line 29 thread 9
Exons_to_geneobj::create_gene_obj(HASH(0x55dc7f6664d0), SCALAR(0x55dc7f5746a8), HASH(0x2ac1245319f0)) called at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/CDNA/CDNA_alignment.pm line 1304 thread 9
CDNA::CDNA_alignment::_get_gene_obj_via_alignment_by_orient(CDNA::CDNA_alignment=HASH(0x2ac124419690), "-", HASH(0x2ac1245319f0)) called at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/CDNA/CDNA_alignment.pm line 1261 thread 9
CDNA::CDNA_alignment::get_gene_obj_via_alignment(CDNA::CDNA_alignment=HASH(0x2ac124419690), HASH(0x2ac1245319f0)) called at /vol2/YL_group/YL/software/PASApipeline-master/scripts/cDNA_annotation_comparer.dbi line 2309 thread 9
main::incorporate_compatible_nonFL_alignments_into_gene_annotations(DB_connect=HASH(0x55dc7f105cb0), ARRAY(0x2ac12453bb10), ARRAY(0x55dc7f331a70), HASH(0x55dc7f331a28), ARRAY(0x55dc7f331a10), HASH(0x55dc7f3319f8), HASH(0x55dc7f3319e0), HASH(0x55dc7f3319c8), ...) called at /vol2/YL_group/YL/software/PASApipeline-master/scripts/cDNA_annotation_comparer.dbi line 936 thread 9
main::analyze_non_fl_assemblies(DB_connect=HASH(0x55dc7f105cb0), ARRAY(0x2ac12453bb10), ARRAY(0x55dc7f56e328), SCALAR(0x55dc7f5746a8)) called at /vol2/YL_group/YL/software/PASApipeline-master/scripts/cDNA_annotation_comparer.dbi line 454 thread 9
main::process_contig("V") called at /vol2/YL_group/YL/software/PASApipeline-master/scripts/cDNA_annotation_comparer.dbi line 311 thread 9
eval {...} called at /vol2/YL_group/YL/software/PASApipeline-master/scripts/cDNA_annotation_comparer.dbi line 311 thread 9
ERROR, thread 8 exited with error Error, spliced orient in db (-) differs from calculated spliced orient (+) at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/Ath1_cdnas.pm line 152 thread 8, <$fh> line 215747.
Ath1_cdnas::create_alignment_obj(DB_connect=HASH(0x55dc7ed1a650), 1093979, SCALAR(0x55dc7f17c138)) called at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/Ath1_cdnas.pm line 175 thread 8
Ath1_cdnas::get_alignment_obj_via_align_acc(DB_connect=HASH(0x55dc7ed1a650), "asmbl_86579", SCALAR(0x55dc7f17c138)) called at /vol2/YL_group/YL/software/PASApipeline-master/scripts/cDNA_annotation_comparer.dbi line 1592 thread 8
main::get_alignment_assemblies_via_subcluster_id(DB_connect=HASH(0x55dc7ed1a650), 29859, SCALAR(0x55dc7f17c138), HASH(0x55dc7f175740)) called at /vol2/YL_group/YL/software/PASApipeline-master/scripts/cDNA_annotation_comparer.dbi line 400 thread 8
main::process_contig("U") called at /vol2/YL_group/YL/software/PASApipeline-master/scripts/cDNA_annotation_comparer.dbi line 311 thread 8
eval {...} called at /vol2/YL_group/YL/software/PASApipeline-master/scripts/cDNA_annotation_comparer.dbi line 311 thread 8

ERROR, thread 9 exited with error Error, coords are out of bounds of sequence length: 15452089:
$VAR1 = {
'15737522' => 15737239,
'15736176' => 15735647,
'15735143' => 15734527,
'15736923' => 15736818
};
at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/Exons_to_geneobj.pm line 115 thread 9.
Exons_to_geneobj::get_cdna_seq(HASH(0x55dc7f6664d0), SCALAR(0x55dc7f5746a8)) called at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/Exons_to_geneobj.pm line 29 thread 9
Exons_to_geneobj::create_gene_obj(HASH(0x55dc7f6664d0), SCALAR(0x55dc7f5746a8), HASH(0x2ac1245319f0)) called at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/CDNA/CDNA_alignment.pm line 1304 thread 9
CDNA::CDNA_alignment::_get_gene_obj_via_alignment_by_orient(CDNA::CDNA_alignment=HASH(0x2ac124419690), "-", HASH(0x2ac1245319f0)) called at /vol2/YL_group/YL/software/PASApipeline-master/PerlLib/CDNA/CDNA_alignment.pm line 1261 thread 9
CDNA::CDNA_alignment::get_gene_obj_via_alignment(CDNA::CDNA_alignment=HASH(0x2ac124419690), HASH(0x2ac1245319f0)) called at /vol2/YL_group/YL/software/PASApipeline-master/scripts/cDNA_annotation_comparer.dbi line 2309 thread 9
main::incorporate_compatible_nonFL_alignments_into_gene_annotations(DB_connect=HASH(0x55dc7f105cb0), ARRAY(0x2ac12453bb10), ARRAY(0x55dc7f331a70), HASH(0x55dc7f331a28), ARRAY(0x55dc7f331a10), HASH(0x55dc7f3319f8), HASH(0x55dc7f3319e0), HASH(0x55dc7f3319c8), ...) called at /vol2/YL_group/YL/software/PASApipeline-master/scripts/cDNA_annotation_comparer.dbi line 936 thread 9
main::analyze_non_fl_assemblies(DB_connect=HASH(0x55dc7f105cb0), ARRAY(0x2ac12453bb10), ARRAY(0x55dc7f56e328), SCALAR(0x55dc7f5746a8)) called at /vol2/YL_group/YL/software/PASApipeline-master/scripts/cDNA_annotation_comparer.dbi line 454 thread 9
main::process_contig("V") called at /vol2/YL_group/YL/software/PASApipeline-master/scripts/cDNA_annotation_comparer.dbi line 311 thread 9
eval {...} called at /vol2/YL_group/YL/software/PASApipeline-master/scripts/cDNA_annotation_comparer.dbi line 311 thread 9

Error, there were 2 threads (contig jobs) that failed... See error messages above in order to troubleshoot furtherFailed thread (8) info:
Thread(8) FAILED CMD: unknown Time to complete: 46075 seconds
Failed thread (9) info:
Thread(9) FAILED CMD: unknown Time to complete: 46075 seconds
Error, cmd: /cDNA_annotation_comparer.dbi -G 1226.fa --CPU 96 -M 'moss.sqlite' > pasa_run.log.dir/moss.sqlite.annotation_compare.120672.out died with ret 256 No such file or directory at /PerlLib/Pipeliner.pm line 187.
Pipeliner::run(Pipeliner=HASH(0x180e9b8)) called at /Launch_PASA_pipeline.pl line 1061


In addition, there are a lot of errors like the following in the .out file:
STATUS: asmbl_87506 18 errors for . non-consensus splice pair [GG-TA] non-consensus splice pair [TG-TG] non-consensus splice pair [GA-GG] non-consensus splice pair [GT-GG] non-consensus splice pair [GG-CA] non-consensus splice pair [AA-AG] non-consensus splice pair [AT-AT] non-consensus splice pair [TG-TC] non-consensus splice pair [GG-GG] non-consensus splice pair [AA-TC] non-consensus splice pair [AG-TC] non-consensus splice pair [GA-TC] non-consensus splice pair [CC-AG] non-consensus splice pair [TT-AA] non-consensus splice pair [GG-CA] non-consensus splice pair [CT-GG] non-consensus splice pair [GA-TT] non-consensus splice pair [CT-AA] Stitched EST lacks compatibility with preexisting protein annotations; invalid and no alt-splice template available. EST assembly stitched into gene model fails validation. PC309490 0

It's worth noting that when I use the same command to update the other species it works fine, the only one that reports an error is species 1226.
I find this very strange, is anyone else experiencing the same error as me?

Best regards,
Xuping

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