Difference of transcripts between my GFF file and the IGV results
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Entering edit mode
3.5 years ago
pablo ▴ 300

Hi,

I aligned my transcripts reads generated by the Isoseq tool, against my reference. Then, I generated a GFF file.

When I look at the number of transcripts in the GFF file and with IGV, there is a difference. For example, I focus on a specific scaffold (named Super-Scaffold_100047) of the reference.

GFF file :

cat out.gff | grep "Super-Scaffold_100047" | awk '{print $3}' | grep "transcript" | wc -l

21 transcripts

IGV :

I count 25 transcripts as you can see on the image.

IGV

I share the GFF file corresponding to the scaffold in question (only the beginning because of the size of the file ) :

Super-Scaffold_100047   PacBio  transcript      281156  287937  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.1";
Super-Scaffold_100047   PacBio  exon    281156  281855  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.1";
Super-Scaffold_100047   PacBio  exon    282017  282094  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.1";
Super-Scaffold_100047   PacBio  exon    282323  282446  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.1";
Super-Scaffold_100047   PacBio  exon    283108  283380  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.1";
Super-Scaffold_100047   PacBio  exon    284686  284805  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.1";
Super-Scaffold_100047   PacBio  exon    287372  287937  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.1";
Super-Scaffold_100047   PacBio  transcript      281168  287895  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.2";
Super-Scaffold_100047   PacBio  exon    281168  281855  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.2";
Super-Scaffold_100047   PacBio  exon    282017  282094  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.2";
Super-Scaffold_100047   PacBio  exon    282323  282446  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.2";
Super-Scaffold_100047   PacBio  exon    283108  283380  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.2";
Super-Scaffold_100047   PacBio  exon    284686  284805  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.2";
Super-Scaffold_100047   PacBio  exon    287372  287895  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.2";
Super-Scaffold_100047   PacBio  transcript      281217  288458  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.3";
Super-Scaffold_100047   PacBio  exon    281217  281855  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.3";
Super-Scaffold_100047   PacBio  exon    282017  282094  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.3";
Super-Scaffold_100047   PacBio  exon    282323  282446  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.3";
Super-Scaffold_100047   PacBio  exon    283108  283380  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.3";
Super-Scaffold_100047   PacBio  exon    284686  284805  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.3";
Super-Scaffold_100047   PacBio  exon    287372  288458  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.3";
Super-Scaffold_100047   PacBio  transcript      281287  288086  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.4";
Super-Scaffold_100047   PacBio  exon    281287  281855  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.4";
Super-Scaffold_100047   PacBio  exon    282017  282094  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.4";
Super-Scaffold_100047   PacBio  exon    282323  282446  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.4";
Super-Scaffold_100047   PacBio  exon    283108  283380  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.4";
Super-Scaffold_100047   PacBio  exon    284686  284805  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.4";
Super-Scaffold_100047   PacBio  exon    287372  288086  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.4";
Super-Scaffold_100047   PacBio  transcript      281544  288081  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.5";
Super-Scaffold_100047   PacBio  exon    281544  281855  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.5";
Super-Scaffold_100047   PacBio  exon    282017  282094  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.5";
Super-Scaffold_100047   PacBio  exon    282323  282446  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.5";
Super-Scaffold_100047   PacBio  exon    283108  283380  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.5";
Super-Scaffold_100047   PacBio  exon    284686  284805  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.5";
Super-Scaffold_100047   PacBio  exon    287372  288081  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.5";
Super-Scaffold_100047   PacBio  transcript      281590  286734  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.6";
Super-Scaffold_100047   PacBio  exon    281590  281855  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.6";
Super-Scaffold_100047   PacBio  exon    282017  282094  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.6";
Super-Scaffold_100047   PacBio  exon    282323  282446  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.6";
Super-Scaffold_100047   PacBio  exon    283108  283380  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.6";
Super-Scaffold_100047   PacBio  exon    284686  286734  .       +       .       gene_id "PB.8691"; transcript_id "PB.8691.6";
Super-Scaffold_100047   PacBio  transcript      220944  223787  .       -       .       gene_id "PB.8692"; transcript_id "PB.8692.1";
Super-Scaffold_100047   PacBio  exon    220944  223787  .       -       .       gene_id "PB.8692"; transcript_id "PB.8692.1";
Super-Scaffold_100047   PacBio  transcript      311770  318491  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.1";
Super-Scaffold_100047   PacBio  exon    311770  312629  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.1";
Super-Scaffold_100047   PacBio  exon    312770  312942  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.1";
Super-Scaffold_100047   PacBio  exon    314316  314634  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.1";
Super-Scaffold_100047   PacBio  exon    314763  315028  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.1";
Super-Scaffold_100047   PacBio  exon    315299  315530  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.1";
Super-Scaffold_100047   PacBio  exon    315681  315995  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.1";
Super-Scaffold_100047   PacBio  exon    316110  316180  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.1";
Super-Scaffold_100047   PacBio  exon    316280  316494  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.1";
Super-Scaffold_100047   PacBio  exon    316582  316945  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.1";
Super-Scaffold_100047   PacBio  exon    317141  317314  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.1";
Super-Scaffold_100047   PacBio  exon    317520  317738  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.1";
Super-Scaffold_100047   PacBio  exon    317816  318154  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.1";
Super-Scaffold_100047   PacBio  exon    318223  318491  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.1";
Super-Scaffold_100047   PacBio  transcript      311770  318501  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.2";
Super-Scaffold_100047   PacBio  exon    311770  312050  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.2";
Super-Scaffold_100047   PacBio  exon    312246  312629  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.2";
Super-Scaffold_100047   PacBio  exon    312770  312942  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.2";
Super-Scaffold_100047   PacBio  exon    314316  314634  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.2";
Super-Scaffold_100047   PacBio  exon    314763  315028  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.2";
Super-Scaffold_100047   PacBio  exon    315299  315530  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.2";
Super-Scaffold_100047   PacBio  exon    315681  315995  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.2";
Super-Scaffold_100047   PacBio  exon    316110  316180  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.2";
Super-Scaffold_100047   PacBio  exon    316280  316494  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.2";
Super-Scaffold_100047   PacBio  exon    316582  316945  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.2";
Super-Scaffold_100047   PacBio  exon    317141  317314  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.2";
Super-Scaffold_100047   PacBio  exon    317520  317738  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.2";
Super-Scaffold_100047   PacBio  exon    317816  318154  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.2";
Super-Scaffold_100047   PacBio  exon    318223  318501  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.2";
Super-Scaffold_100047   PacBio  transcript      311770  316826  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.3";
Super-Scaffold_100047   PacBio  exon    311770  312629  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.3";
Super-Scaffold_100047   PacBio  exon    312770  312942  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.3";
Super-Scaffold_100047   PacBio  exon    314316  314634  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.3";
Super-Scaffold_100047   PacBio  exon    314763  315028  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.3";
Super-Scaffold_100047   PacBio  exon    315299  315530  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.3";
Super-Scaffold_100047   PacBio  exon    315681  315995  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.3";
Super-Scaffold_100047   PacBio  exon    316110  316180  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.3";
Super-Scaffold_100047   PacBio  exon    316280  316494  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.3";
Super-Scaffold_100047   PacBio  exon    316582  316826  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.3";
Super-Scaffold_100047   PacBio  transcript      311771  317638  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.4";
Super-Scaffold_100047   PacBio  exon    311771  312629  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.4";
Super-Scaffold_100047   PacBio  exon    312770  312942  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.4";
Super-Scaffold_100047   PacBio  exon    314316  314634  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.4";
Super-Scaffold_100047   PacBio  exon    314763  315028  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.4";
Super-Scaffold_100047   PacBio  exon    315299  315530  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.4";
Super-Scaffold_100047   PacBio  exon    315681  315995  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.4";
Super-Scaffold_100047   PacBio  exon    316110  316180  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.4";
Super-Scaffold_100047   PacBio  exon    316280  316494  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.4";
Super-Scaffold_100047   PacBio  exon    316582  316945  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.4";
Super-Scaffold_100047   PacBio  exon    317141  317314  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.4";
Super-Scaffold_100047   PacBio  exon    317520  317638  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.4";
Super-Scaffold_100047   PacBio  transcript      311771  316452  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.5";
Super-Scaffold_100047   PacBio  exon    311771  312629  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.5";
Super-Scaffold_100047   PacBio  exon    312770  312942  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.5";
Super-Scaffold_100047   PacBio  exon    314316  314634  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.5";
Super-Scaffold_100047   PacBio  exon    314763  315028  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.5";
Super-Scaffold_100047   PacBio  exon    315299  315530  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.5";
Super-Scaffold_100047   PacBio  exon    315681  315995  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.5";
Super-Scaffold_100047   PacBio  exon    316110  316180  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.5";
Super-Scaffold_100047   PacBio  exon    316280  316452  .       -       .       gene_id "PB.8693"; transcript_id "PB.8693.5";

Do you have an explanation?

Best
gff igv transcripts • 1.2k views
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0
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These these are giant sequences perhaps there is a secondary alignment with one or two of reads=transcripts?

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0
Entering edit mode

Actually, these are full length transcripts obtained from PacBio sequencing. That's why they are giant.

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0
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Is there a secondary alignment with one or two of them? Leading to two extra alignments you see.

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0
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There's not. I check each transcript : they are all unique and align only once on the genome.

Also, when I count the number of transcripts specific to that scaffold in my alignment.bam file, I got 25 sequences.

Do you know if there is a way to count the number of alignments with IGV? I could verify with other scaffols but there are much more alignments , so boring to count..

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0
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IGV is only a viewer. You can count number of alignments easily with samtools idxstats.

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0
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Hi,

Could you provide the mentioned GFF file?

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1
Entering edit mode

I updated my post as you can see with the mentioned GFF file.

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0
Entering edit mode

Maybe identical isoforms that are collapsed by IGV. Could you run your gff file through AGAT to check if there is any identical isoform?:

agat_convert_sp_gxf2gxf.pl --gff input.gff -o output.gff
ADD REPLY •
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Entering edit mode

I will install that tools. Actually, when I run samtools inxstats my_alignment.bam , I find the right number of reads/isoforms for the scaffold in question Super-Scaffold_100047 379068 25 0 . Then, the problem comes from the GFF file which is bad created.

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