Issues with custom genome and GeneScoreMatrix creation in ArchR

[hayatkhanndsu]

I am trying to analyze single-cell ATAC-seq data using ArchR with a custom genome, but I am repeatedly encountering errors at multiple steps. My data were processed using Cell Ranger ATAC, and I am using a fragments.tsv.gz file along with a custom FASTA and GTF annotation.

The main issues I am facing include:

Errors related to chromosome/contig mismatches between the fragments file and the custom FASTA, including warnings about extra chromosomes in the reference.

Repeated failures during Arrow file creation, with different errors appearing across runs.

Problems with gene annotation, where the GeneScoreMatrix or gene-related matrices are not created successfully.

Errors related to seqnames and matrix generation, which prevent downstream analysis.

At this point, I would like to properly validate my data and reference setup. I would greatly appreciate guidance on:

How to correctly filter and prepare a custom FASTA reference for ArchR.

How to ensure compatibility between the FASTA, GTF, and fragments files.

A recommended workflow or example code for creating a custom genome and generating Arrow files, including successful GeneScoreMatrix creation.

Thank you very much for your time and support.