Doubts regarding samtools mpileup - Call somatic mutations from a pair of samples ?

[swapnil2188]

I am trying to compare two samples for somatic mutations using Samtools mpileup. (RNAseq data)


The two .bam files or samples I am comparing include multiple samples merged together into them (3 .bam files in one and 8 in other- merged using samtools merge)

The command given in the manual
( " samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair - > var.bcf" ) doesnt specify the number of samples (inputs)

I used this command

samtools mpileup -DSuf ref.fa aln1.bam aln2.bam | bcftools view -bvcgT pair - > var.bcf

It gives me back the error saying
"[mpileup] 1 samples in 2 input files
<mpileup> Set max per-file depth to 8000
[afs] 0:0.000"

I also tried to make a sample.txt file for the two samples (merged files) and used this command

"samtools mpileup -DSuf ref.fa aln1.bam aln2.bam | bcftools view -bvcgT pair -s samples.txt - > var.bcf "

I still get an error (different error)

Is there anything wrong here ?

What should be the ideal steps to find out somatic variants between two samples ? (samples include multiple bam files merged together)

Please suggest me some steps to be followed in order to find out somatic variants by comparing two samples using samtools mpileup

Thanks!

[swapnil2188]

I am trying to compare two samples for somatic mutations using Samtools mpileup. (RNAseq data)


The two .bam files or samples I am comparing include multiple samples merged together into them (3 .bam files in one and 8 in other- merged using samtools merge)

The command given in the manual
( " samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair - > var.bcf" ) doesnt specify the number of samples (inputs)

I used this command

samtools mpileup -DSuf ref.fa aln1.bam aln2.bam | bcftools view -bvcgT pair - > var.bcf

It gives me back the error saying
"[mpileup] 1 samples in 2 input files
<mpileup> Set max per-file depth to 8000
[afs] 0:0.000"

I also tried to make a sample.txt file for the two samples (merged files) and used this command

"samtools mpileup -DSuf ref.fa aln1.bam aln2.bam | bcftools view -bvcgT pair -s samples.txt - > var.bcf "

I still get an error (different error)

Is there anything wrong here ?

What should be the ideal steps to find out somatic variants between two samples ? (samples include multiple bam files merged together)

Please suggest me some steps to be followed in order to find out somatic variants by comparing two samples using samtools mpileup

Thanks!

Please can Anyone help me with this ?

I am waiting for response !

Thank You !

[gaffa]

Do you have read group information specified in the two files?