SQANTI3 for non-model organisms

[Maxim-Karpov]

Hello, I would like to use SQUANTI3 for processing my long read RNAseq data, however there is no reference annotation for the organism I am working on - the earthworm. What options do I have in this situation? Is it feasible/sensible to produce my own annotation for use in SQUANTI3?

[carolinamonzo]

Hi @Maxim-Karpov, SQANTI3 (not SQUANTI3) needs a reference annotation to be able to give information on the quality of your long-reads transcriptome. You should generate your own annotation.

[Maxim-Karpov]

Hi @carolinamonzo, Thanks for your response. I have a gene prediction GTF file from BRAKER3 which isn't recognised by SQANTI3 as a GTF file, as stated by the following error:

**** Parsing Reference Transcriptome....
/media/Maxim/SSD/BRAKER3/braker_lr_lrRNAseq/braker_chrs.gtf doesn't appear to be a GTF file (GFF not supported by this program)

The running process crashes as the refAnnotation_SQANTI.genePred is not produced:

  File "/media/Maxim/SSD/SQANTI3/SQANTI3-5.2.1/sqanti3_qc.py", line 2525, in <module>
    main()
  File "/media/Maxim/SSD/SQANTI3/SQANTI3-5.2.1/sqanti3_qc.py", line 2508, in main
    run(args)
  File "/media/Maxim/SSD/SQANTI3/SQANTI3-5.2.1/sqanti3_qc.py", line 1856, in run
    refs_1exon_by_chr, refs_exons_by_chr, junctions_by_chr, junctions_by_gene, start_ends_by_gene = reference_parser(args, list(genome_dict.keys()))
  File "/media/Maxim/SSD/SQANTI3/SQANTI3-5.2.1/sqanti3_qc.py", line 657, in reference_parser
    for r in genePredReader(referenceFiles):
  File "/media/Maxim/SSD/SQANTI3/SQANTI3-5.2.1/sqanti3_qc.py", line 121, in __init__
    self.f = open(filename)
FileNotFoundError: [Errno 2] No such file or directory: '/media/Maxim/SSD/SQANTI3/lrRNAseq1/refAnnotation_SQANTI.genePred'

Do you have any suggestions regarding this error? Is there a way I can parse/edit my BRAKER3 GTF file (current formatting demonstrated below) such that it is accepted by SQANTI3 i.e. what is missing and what is not needed here?

BRAKER3 GTF format:

1	AUGUSTUS	gene	1	9439	.	-	.	g1
1	AUGUSTUS	transcript	1	9439	0.33	-	.	g1.t1
1	AUGUSTUS	intron	1	483	0.57	-	.	transcript_id "g1.t1"; gene_id "g1";
1	AUGUSTUS	CDS	484	622	0.49	-	0	transcript_id "g1.t1"; gene_id "g1";
1	AUGUSTUS	exon	484	622	.	-	.	transcript_id "g1.t1"; gene_id "g1";
1	AUGUSTUS	intron	623	1618	0.49	-	.	transcript_id "g1.t1"; gene_id "g1";
1	AUGUSTUS	CDS	1619	1758	0.94	-	2	transcript_id "g1.t1"; gene_id "g1";
1	AUGUSTUS	exon	1619	1758	.	-	.	transcript_id "g1.t1"; gene_id "g1";
1	AUGUSTUS	intron	1759	6505	0.93	-	.	transcript_id "g1.t1"; gene_id "g1";
1	AUGUSTUS	CDS	6506	6635	0.99	-	0	transcript_id "g1.t1"; gene_id "g1";