I want to use bedtools to count reads mapping back to genes, ultimately to use for differential expression analysis in edgeR. I used trinity to assemble my metatranscriptomes de novo, and bowtie2 to map raw reads back to the assembly. I seemingly sorted and indexed the file without a problem (returned no errors at least). When I go to run the bedtools multicov command, I get the error: could not find indexes. The indexes are in the same file as the bam file. Used prodigal for ORF calling to create .gff file.

Commands used:

First I sorted

samtools sort bowtie_output.bam -o sorted_output.bam -T temp_prefix

Then I indexed

samtools index -b sorted_output.bam indexed_sample.bam.bai

Then I try to get read counts

bedtools multicov -bams bowtie_output.bam -bed prodigal_output.gff

I thought maybe I was supposed to use the bam.bai file instead of bowtie output bam file, so I tried

bedtools multicov -bams indexed_file.bam.bai -bed prodigal_output.gff

But still get 'could not find indexes'

Any idea what I am doing wrong? Disclaimer: very much a beginner!