Samtools output errors (ref is all 'N' in vcf)

[rajj]

I am working on using bowtie and samtools to take a fastq file, map to HG19 using bowtie, and use samtools to find SNPs and output a VCF file.

I am able to get decent .vcf output, but the vcf file created has all 'N's for ref. Is there an easy fix?

I am using the commands:

bowtie -S humanref " + patfile + " > bw_out
samtools view -S -u bw_out > bw_bam
samtools sort bw_bam bw_bamsort
samtools mpileup -u -D bw_bamsort.bam > bcf_out
bcftools view bcf_out > vcf_out

Thanks!

[swbarnes2]

You didn't tell mpileup what the reference was.

use -f to tell it what your reference is. It will make the reference index file if it doesn't exist.

[rajj]

Thank you for the quick response.

I have multiple reference files. How do I call all 22 of them?

[vivek_]

If you are mentioning 22 different chromosomes, you should concatenate them together and present as a single fasta file for samtools.

[rajj]

OK that worked great!

One last question, when I am using mpileup, my vcf file only has information for chromosome 1 to 19. The last chromosomes are not included. Is this due to a maximum depth error, or maybe memory? And how do I fix this?