Hi dears all,
I am going to do SNP calling using bcftools mpileup for WGS data on fish. Before SNP calling I did this preprocessing as follow:

   samtools sort -n F0-F06-10.bam F0-F06-10ns.bam

# Add ms and MC tags for markdup to use later

    samtools fixmate -m F0-F06-10ns.bam F0-F06-10-fixmate.bam

# Markdup needs position order

    samtools sort -o F0-F06-10PS.bam F0-F06-10-fixmate.bam

# Finally mark duplicates

    samtools markdup F0-F06-10PS.bam F0-F06-10-markdup.bam

Then using samtools faidx the fasta reference file got indexed and bcftools mpileup was used for SNP calling. For the last step (bcftools mpileup) I got involved with an error.

    samtools faidx /home2/omid/alignment/ref/GCF_001858045.2_O_niloticus_UMD_NMBU_genomic.fa

    bcftools mpileup -Ou -f GCF_001858045.2_O_niloticus_UMD_NMBU_genomic.fa.fai F0-F06-10-markdup.bam

> error:    [E::fai_build_core] Format error, unexpected "N" at line 1

I will be so grateful if any one can help me to solve this problem.

Cheers
I'm quite sure that the reference file you are using in your `bcftools` command is the index file of your reference and not the reference file itself.

The index file must be located in the same location like the reference file. The file you specify with the `-f` parameter must be the reference genome file.

fin swimmer