VCF file REF Column outputs all 'N'

Question

[mhasa006]

I am doing SNP calling using samtools. The way I'm doing is following.

• I had a .bam file that contains information for all chromosome together.
• I extracted information for each chromosome and make separate bam file for each chromosome.
• I made a snpcall using mpileup. Separately each chromosome with the whole genome. Following is the result for Chromosome 1. Below is a section of my output.

GeneDB|Pf3D7_01_v3    107    .    N    G    68.5    .    DP=4;VDB=9.421102e-03;AF1=1;AC1=2;DP4=0,0,4,0;MQ=58;FQ=-39    GT:PL:GQ    1/1:101,12,0:21
GeneDB|Pf3D7_01_v3    108    .    N    A    148    .    DP=12;VDB=3.982228e-05;AF1=1;AC1=2;DP4=0,0,12,0;MQ=54;FQ=-63    GT:PL:GQ    1/1:181,36,0:69
GeneDB|Pf3D7_01_v3    109    .    N    A    152    .    DP=13;VDB=3.441712e-05;AF1=1;AC1=2;DP4=0,0,13,0;MQ=54;FQ=-66    GT:PL:GQ    1/1:185,39,0:75

All the REF values are 'N'. what could be the problem? I used the following command:

samtools mpileup -uf genome.fa in.bam | bcftools view -bvcg - > out.raw.bcf

[Pierre Lindenbaum]

'genome.fa' is a wrong REFerence.

[mhasa006]

Thanks for the reply. here genome.fa is used for general purpose. I have the fasta file of the original genome. Is that what you meant? I also checked the positions in the original genome. There was no 'N' there. What could be the problem?

[mkulecka]

Is the genome.fa the same file which was used for mapping in bam files? Maybe chromosome names are different?