How to correctly use ragtag to improve mapping comparing a reference

[rahulnutron]

Dear RagTag team,

Thanks for developing this useful tool!

This post is a question and not an issue. I try to get an answer from other places like Biostar and didn't get an answer. So I though I can post it here as the question involve RagaTag and may be it will be useful for others.

I have a scRNA-seq data which is generated using a cultivar for which gene annotation (gff or gtf files) are not available. I have my_cultivar_ragtag.scaffold.fasta, my_cultivar_ragtag.scaffold.fasta.fai, my_cultivar_p_ctg.fa, my_cultivar_p_ctg.fa.fai files for the cultivar that was used for experiment, but not gtf or gff files. On the other hand, I have reference genome and annotation for another cultivar (reference).

I used

ragtag.py scaffold my_cultivar_ragtag.scaffold.fasta reference.fa -o ragtag_scaffold ragtag.py updategff reference.gff3 ragtag_scaffold/ragtag.scaffold.agp > updated_ragtag_scf.gff3

Then used I used ragtag_scaffold/ragtag.scaffold.fasta and updated_ragtag_scf.gff3 for indexing and mapping with cell ranger, however the reads mapped to the genome remains below 40% (usually >90%).

I also tried ragtag correct in the same way, without improvement of mapping percentage.

I will be grateful if you could let me know how to correctly use ragtag fasta and other files that I already have for the cultivar used in experiment and correct them (with reference) or reference (with cultivar) for improved mapping.

Thanks in advance, RS