Subject: Handling Multiple Reference FASTA Files in nf-core RNA-seq I’m using the nf-core/rnaseq pipeline with the Macaca fasicularis Macaca_fascicularis_6.0 genome, which is split across multiple .fa.gz files (one per chromosome). Questions: 1. Should I concatenate all the chromosome FASTA files into a single .fa.gz before using --fasta, or can the pipeline handle multiple FASTA files (e.g., with a wildcard)? 2. If concatenation is needed, will the pipeline handle indexing without issues?

Yes, you'll need to combine the chromosomes- though normally Ensembl etc do provide a whole-genome file.