Hello,

I am wondering if someone would be able to help me. I am currently trying to do RNA-seq analysis on a couple of different bacterial species using DESeq2.

In brief I am using bwa to map my fastq files to my reference, samtools to convert to sorted bam files then using coverageBed to count the reads mapping to each filter, generating .txt files for DESeq2. I have had no issue when using reference genbank and fasta files downloaded from NCBI for one of the species. However for the other species, I want to use a reference I have created (using spades, mauve and prokka). When I try using coveragebed for this reference it gives the following error:

It looks as though you have less than 3 columns at line: 9734. Are you sure your files are tab-delimited?

I have tried using the .gff file created by prokka and converting the .gbk file to a .gff file. I assume the issue is something to do with prokka? the settings I used for this were as follows:

prokka1.11 --cpus 12 --compliant --addgenes --locustag bacter -o bacter contigs.fasta

any help would be greatly appreciated.