Hello, In my project I am going to compare bioinformatic tools for tumor clonary evolution analysis (sciClone and others). I have results of paired-end Whole Exom Sequencing, three samples from one patient: Control, Primary and Relapse tumor. Therefore, first I have to detect somatic mutations and I am going to do that with VarScan. I don't quite understand though what processing pipeline should I choose from the step of creating .mpileup files with Samtools.

I've found some possibilieties, which includes:

-creating .mpileup files separately for normal and tumor samples, and then make both of them an input to VarScan following (in short):

samtools mpileup -f hg19.fa nomal.bam > normal.bam.mpileup
samtools mpileup -f hg19.fa tumor.bam > tumor.bam.mpileup
java -jar VarScan.jar somatic normal.bam.mpileup tumor.bam.mpileup --output-snp snp --output-indel indel

(source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3971343/)

-creating .mpileup files for paired normal-tumor samples, and then give the result as the input to VarScan:

samtools mpileup –f reference.fasta normal.bam tumor.bam > normal-tumor.mpileup
java –jar VarScan.jar somatic normal-tumor.mpileup output.basename

(source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4278659/)

My problem is, that I don't quite understand which way should I choose - could someone please explain me what is the difference in those schemes? I am completely freshman in NGS data analysis, therefore the simplest words, the better :) And just to be sure - can I treat my "Control" sample as normal sample?

I would appreciate any help

Kind regards, Agata