RSEM ref genome problems

[Hope Kirby]

Hi!

So I am trying to do basic RNAseq using a reference genome I created witth RSEM

Code for creating reference genome:

~/Documents/RNAseq/bowtie2-2.2.6/RSEM-1.3.3/rsem-prepare-reference --gff3  ref/17978mff.gff3 --gff3-genes-as-transcripts --bowtie2 --bowtie2-path ~/Documents/RNAseq/bowtie2-2.2.6 ref/17978mff.fasta ref/17978mff_ref 

(It is for a bacterial genome so only gff3 is available. The reference genome seems successfully created but when I try to performed paired analysis (code below), it gives me the following error:

"The SAM/BAM file declares less than one reference sequence!")

I've tried to ensure the correct folders and notation multiple times so any help would be much appreciated.

Code for calculating expression:

~/Documents/RNAseq/bowtie2-2.2.6/RSEM-1.3.3/rsem-calculate-expression -p 8 --paired-end --bowtie2 --bowtie2-path ~/Documents/RNAseq/bowtie2-2.2.6 --estimate-rspd --append-names --output-genome-bam ./Clean/CWTL1_24/17978L1_S1_L001_R1_001_val_1.fq ./Clean/CWTL1_24/17978L1_S1_L001_R2_001_val_2.fq ./ref/17978mff_ref ./exp/WTL

Thank you in advance!