How to create REFCLASSIF file, when run SQANTi3 Rescue

[MR-D-CJ]

When sqanti.py rescue is run, the -k (--refClassif) parameter exists, requiring input of the reference SQANTi3 QC result file. For the production of this file, the following three ways are correct, the code is as follows:

according to the transcript sequence
python /PATH/SQANTI3-5.2.2/sqanti3_qc.py \ /PATH/GRCh38/genecode/gencode.v47.transcripts.fa \ /PATH/GRCh38/genecode/gencode.v46.annotation.gtf \ /PATH/GRCh38/GCA_000001405.15_GRCh38_no_alt/GCA_000001405.15_GRCh38_no_alt_analysis_set.fa \ --CAGE_peak /PATH/polyA_info/human.refTSS_v3.1.hg38.sorted.bed \ --polyA_motif_list /PATH/polyA_info/mouse_and_human.polyA_motif.txt \ -o GCA_000001405.15_GRCh38_no_alt \ -d /PATH/SQANTi3_ref/GRCh38 \ --fasta \ --force_id_ignore \ --cpus 20 --report html

according to the transcript annotation file
python /PATH/SQANTI3-5.2.2/sqanti3_qc.py \ /PATH/GRCh38/genecode/gencode.v47.transcripts.gtf \ /PATH/GRCh38/genecode/gencode.v46.annotation.gtf \ /PATH/GRCh38/GCA_000001405.15_GRCh38_no_alt/GCA_000001405.15_GRCh38_no_alt_analysis_set.fa \ --CAGE_peak /PATH/polyA_info/human.refTSS_v3.1.hg38.sorted.bed \ --polyA_motif_list /PATH/polyA_info/mouse_and_human.polyA_motif.txt \ -o GCA_000001405.15_GRCh38_no_alt \ -d /PATH/SQANTi3_ref/GRCh38 \ --fasta \ --force_id_ignore \ --cpus 20 --report html

according to the genome annotation file
python /PATH/SQANTI3-5.2.2/sqanti3_qc.py \ /PATH/GRCh38/genecode/gencode.v46.annotation.gtf \ /PATH/GRCh38/genecode/gencode.v46.annotation.gtf \ /PATH/GRCh38/GCA_000001405.15_GRCh38_no_alt/GCA_000001405.15_GRCh38_no_alt_analysis_set.fa \ --CAGE_peak /PATH/polyA_info/human.refTSS_v3.1.hg38.sorted.bed \ --polyA_motif_list /PATH/polyA_info/mouse_and_human.polyA_motif.txt \ -o GCA_000001405.15_GRCh38_no_alt \ -d /PATH/SQANTi3_ref/GRCh38 \ --fasta \ --force_id_ignore \ --cpus 20 --report html

which one is true?

[lkondratova]

Hi @MR-D-CJ,
For this step, you should compare the annotation file against itself. Assuming you used gencode.v46.annotation.gtf for SQANTI3 on your samples, the first two arguments should be gencode.v46.annotation.gtf gencode.v46.annotation.gtf (as stated in your third option).
Please let us know if you have any further questions.