Error: invalid feature coordinates (end<start!) at line:

[5Tony]

hello ,
I want to mapping gencode.v43.transcripts.fa and GRCh38.primary_assembly.genome.fa, I have some problems during using uLTRA, I would like to ask why there is such an error?

code:

 uLTRA index  --disable_infer  /home/data/t050326/reference/GRCh38.primary_assembly.genome.fa /home/data/t050326/reference/new/gencode.v43.annotation.gtf  /home/data/t050326/index/uLTRA
uLTRA align --t 10 --index ~/index/uLTRA --prefix ultra --max_intron 1000000  ~/reference/GRCh38.primary_assembly.genome.fa ~/reference/gencode.v43.transcripts.fa  ~/result/uLTRA/t1
samtools view -bS ultra.sam > ultra.bam
bedtools bamtobed -bed12 -i ~/result/uLTRA/t1/ultra.bam > ~/result/uLTRA/t1/ultra.bed12
bedToGenePred ~/result/uLTRA/t1/ultra.bed12 ~/result/uLTRA/t1/ultra.GenePred
genePredToGtf -utr -honorCdsStat file ~/result/uLTRA/t1/ultra.GenePred ~/result/uLTRA/t1/ultra.gtf
gffcompare -T -r ~/reference/gencode.v43.annotation.gtf -o ~/result/uLTRA/gffcompare/t1/ultra ~/result/uLTRA/t1/ultra.gtf

warning:

Error: invalid feature coordinates (end<start!) at line:
chr2    /home/data/t050326/result/uLTRA/t1/ultra.GenePred       exon    111196368       111196367       .       -       .       gene_id "ENST00000642451.1|ENSG00000222041.13|OTTHUMG00000130273.15|OTTHUMT00000330387.4|CYTOR-216|CYTOR|7512|lncRNA|_2"; transcript_id "ENST00000642451.1|ENSG00000222041.13|OTTHUMG00000130273.15|OTTHUMT00000330387.4|CYTOR-216|CYTOR|7512|lncRNA|_2"; exon_number "1"; exon_id "ENST00000642451.1|ENSG00000222041.13|OTTHUMG00000130273.15|OTTHUMT00000330387.4|CYTOR-216|CYTOR|7512|lncRNA|_2.1";
Error: invalid feature coordinates (end<start!) at line:
chr2    /home/data/t050326/result/uLTRA/t1/ultra.GenePred       exon    95834978        95834977        .       -       .       gene_id "ENST00000612307.4|ENSG00000277701.5|OTTHUMG00000188202.4|OTTHUMT00000476587.2|ENST00000612307|ENSG00000277701|530|lncRNA|_3"; transcript_id "ENST00000612307.4|ENSG00000277701.5|OTTHUMG00000188202.4|OTTHUMT00000476587.2|ENST00000612307|ENSG00000277701|530|lncRNA|_3"; exon_number "4"; exon_id "ENST00000612307.4|ENSG00000277701.5|OTTHUMG00000188202.4|OTTHUMT00000476587.2|ENST00000612307|ENSG00000277701|530|lncRNA|_3.4";
Error: invalid feature coordinates (end<start!) at line:
chr5    /home/data/t050326/result/uLTRA/t1/ultra.GenePred       exon    178039387       178039386       .       +       .       gene_id "ENST00000697483.1|ENSG00000289731.1|-|-|FAM153B-206|FAM153B|721|lncRNA|_3"; transcript_id "ENST00000697483.1|ENSG00000289731.1|-|-|FAM153B-206|FAM153B|721|lncRNA|_3"; exon_number "4"; exon_id "ENST00000697483.1|ENSG00000289731.1|-|-|FAM153B-206|FAM153B|721|lncRNA|_3.4";
Error: invalid feature coordinates (end<start!) at line:
chr5    /home/data/t050326/result/uLTRA/t1/ultra.GenePred       exon    176119655       176119654       .       +       .       gene_id "ENST00000650646.1|ENSG00000204677.13|OTTHUMG00000163459.11|OTTHUMT00000502047.1|FAM153CP-213|FAM153CP|1179|lncRNA|_2"; transcript_id "ENST00000650646.1|ENSG00000204677.13|OTTHUMG00000163459.11|OTTHUMT00000502047.1|FAM153CP-213|FAM153CP|1179|lncRNA|_2"; exon_number "15"; exon_id "ENST00000650646.1|ENSG00000204677.13|OTTHUMG00000163459.11|OTTHUMT00000502047.1|FAM153CP-213|FAM153CP|1179|lncRNA|_2.15";
Error: invalid feature coordinates (end<start!) at line:
chr10   /home/data/t050326/result/uLTRA/t1/ultra.GenePred       exon    87342323        87342322        .       -       .       gene_id "ENST00000653620.1|ENSG00000225484.7|OTTHUMG00000018585.17|OTTHUMT00000522829.1|NUTM2B-AS1-214|NUTM2B-AS1|4086|lncRNA|_2"; transcript_id "ENST00000653620.1|ENSG00000225484.7|OTTHUMG00000018585.17|OTTHUMT00000522829.1|NUTM2B-AS1-214|NUTM2B-AS1|4086|lncRNA|_2"; exon_number "3"; exon_id "ENST00000653620.1|ENSG00000225484.7|OTTHUMG00000018585.17|OTTHUMT00000522829.1|NUTM2B-AS1-214|NUTM2B-AS1|4086|lncRNA|_2.3";
Error: invalid feature coordinates (end<start!) at line:
chr10   /home/data/t050326/result/uLTRA/t1/ultra.GenePred       exon    79826485        79826484        .       -       .       gene_id "ENST00000663683.1|ENSG00000223482.10|OTTHUMG00000018671.22|OTTHUMT00000523167.1|NUTM2A-AS1-225|NUTM2A-AS1|1398|lncRNA|_2"; transcript_id "ENST00000663683.1|ENSG00000223482.10|OTTHUMG00000018671.22|OTTHUMT00000523167.1|NUTM2A-AS1-225|NUTM2A-AS1|1398|lncRNA|_2"; exon_number "5"; exon_id "ENST00000663683.1|ENSG00000223482.10|OTTHUMG00000018671.22|OTTHUMT00000523167.1|NUTM2A-AS1-225|NUTM2A-AS1|1398|lncRNA|_2.5";
Error: invalid feature coordinates (end<start!) at line:
chr16   /home/data/t050326/result/uLTRA/t1/ultra.GenePred       exon    22374887        22374886        .       +       .       gene_id "ENST00000522480.5|ENSG00000180747.16|OTTHUMG00000164329.2|OTTHUMT00000378303.1|SMG1P3-201|SMG1P3|4262|transcribed_unprocessed_pseudogene|_2"; transcript_id "ENST00000522480.5|ENSG00000180747.16|OTTHUMG00000164329.2|OTTHUMT00000378303.1|SMG1P3-201|SMG1P3|4262|transcribed_unprocessed_pseudogene|_2"; exon_number "1"; exon_id "ENST00000522480.5|ENSG00000180747.16|OTTHUMG00000164329.2|OTTHUMT00000378303.1|SMG1P3-201|SMG1P3|4262|transcribed_unprocessed_pseudogene|_2.1";
  263660 query transfrags loaded.

When I added --isoseq to the mapping process, I still got the error.

code:

uLTRA align --t 10 --index ~/index/uLTRA --prefix ultra --max_intron 1000000 --isoseq ~/reference/GRCh38.primary_assembly.genome.fa ~/reference/gencode.v43.transcripts.fa  ~/result/uLTRA/t2
samtools view -bS ultra.sam > ultra.bam
bedtools bamtobed -bed12 -i ~/result/uLTRA/t2/ultra.bam > ~/result/uLTRA/t2/ultra.bed12
bedToGenePred ~/result/uLTRA/t2/ultra.bed12 ~/result/uLTRA/t2/ultra.GenePred
genePredToGtf -utr -honorCdsStat file ~/result/uLTRA/t2/ultra.GenePred ~/result/uLTRA/t2/ultra.gtf
gffcompare -T -r ~/reference/gencode.v43.annotation.gtf -o ~/result/uLTRA/gffcompare/t2/ultra ~/result/uLTRA/t2/ultra.gtf

warning:

  252913 reference transcripts loaded.
  1443 duplicate reference transcripts discarded.
Error: invalid feature coordinates (end<start!) at line:
chr5    /home/data/t050326/result/uLTRA/t2/ultra.GenePred       exon    178039387       178039386       .       +       .       gene_id "ENST00000697483.1|ENSG00000289731.1|-|-|FAM153B-206|FAM153B|721|lncRNA|_3"; transcript_id "ENST00000697483.1|ENSG00000289731.1|-|-|FAM153B-206|FAM153B|721|lncRNA|_3"; exon_number "4"; exon_id "ENST00000697483.1|ENSG00000289731.1|-|-|FAM153B-206|FAM153B|721|lncRNA|_3.4";
  262577 query transfrags loaded.

Looking forward to your reply!

[ksahlin]

Interesting, thanks for reporting! If you provide me the link to the transcript and genome that you use I can try to exactly replicate what you did which will make it easier for me to figure out what is going on.

Are you using uLTRA v0.0.4.2? This version contains a bugfix, but I am not sure if what you are observing is related to that fix.