samtools problems with reference in pileup file

[skingan]

No. All of the positions in the pileup file are N's and my reference sequences do not consist of all N's.
Sarah

[nilshomer]

my command:

samtools pileup -f myref.fas my_sorted_assembly.bam -c > mydata.pileup

my output (e.g.):

NT_033777 27895205 N A 24 0 6 17 aaaaaaaaaaaaaaaaa $))))))()')))")))

Is there an N at that position in the reference?

[skingan]

my command:

samtools pileup -f myref.fas my_sorted_assembly.bam -c > mydata.pileup

my output (e.g.):

NT_033777 27895205 N A 24 0 6 17 aaaaaaaaaaaaaaaaa $))))))()')))")))

[reneeg36]

Post the command you are using so we can help

Did you specify the FASTA file with the "-f" option?

Yes, here is what I have been trying:

command:
samtools pileup -f reference.fa sorted.bam > raw.txt

output (e.g.):
chr7:150849-150991 22 N 2 GG CC


Renee

[nilshomer]

Hello,

I am using samtools to generate a pileup file from a Mosaik genome assembly. My problem is that the pileup file contains all "N's" in the reference sequence field (column 3). I have double checked that I am using the exact same multi-sequence fasta file as the reference for both Mosaik and samtools.

Has anyone ever run into this before? I am using MosaikSort and MosaikText to generate a sorted bam file for input into samtools. Without the reference, I cannot get SNP quality scores, which is the whole point of my analysis.

Thanks for your help,
Sarah
[email protected]

Post the command you are using so we can help

Hi,

I am having the same problem. I am mapping reads to a list of contigs from a de novo assembly using bwa. I have converted the bwa sam alignment file to a sorted bam file using samtools. When I try running pileup to identify variants, I get only N's in the reference base position. I have checked the reference fasta file and don't find anything wrong.

Thanks!

Renee
[email protected]

Did you specify the FASTA file with the "-f" option?

[reneeg36]

Hi,

I am having the same problem. I am mapping reads to a list of contigs from a de novo assembly using bwa. I have converted the bwa sam alignment file to a sorted bam file using samtools. When I try running pileup to identify variants, I get only N's in the reference base position. I have checked the reference fasta file and don't find anything wrong.

Thanks!

Renee
[email protected]

[skingan]

Hello,

I am using samtools to generate a pileup file from a Mosaik genome assembly. My problem is that the pileup file contains all "N's" in the reference sequence field (column 3). I have double checked that I am using the exact same multi-sequence fasta file as the reference for both Mosaik and samtools.

Has anyone ever run into this before? I am using MosaikSort and MosaikText to generate a sorted bam file for input into samtools. Without the reference, I cannot get SNP quality scores, which is the whole point of my analysis.

Thanks for your help,
Sarah
[email protected]