Hello,

I have this bam file that originally contained paired sequences, after converting it into fastq sequences I got 2 different files. My idea is to make an alignment with a chromosome sequence, and from there obtain a VCF. My question is if the sequence that I will use will matter in my future results?, there is a way to know which sequence should I use?

I got the sequences using samtools:

    bam2fastq -o blah_aligned_R#.fastq --no-unaligned Bamfilename.bam

EDIT:

Thank you very much for the comments, I will explain myself better (and sorry for any misunderstanding, but this is the first time that I am working in bioinformatics).

I am wanting to see the occurrence of KRAS-variant mutation (as described by Ratner et al, in http://www.nature.com/onc/journal/v31/n42/pdf/onc2011539a.pdf) in chromosome 12 in ovarian cancer cell lines. For this, I have downloaded some bam files from the nci60-project (http://watson.nci.nih.gov/projects/nci60/wes/BAMS/). These bam files are presented as paired sequences, so after extracting chromosome 12 information from them (using the bai files that are in the same webpage as index) by the code:

    $ samtools view  IGR-OV1_reord_mdups_ralgn_fmate_recal.bam chr12>chr12.bam

Then, I sorted the bam file, and instead of using the original code that I posted at the beginning, I converted them to fastq using:

```
$ bedtools bamtofastq -i chr12.qsort.bam \
                      -fq chr12.end1.fq \
                      -fq2 chr12.end2.fq
```

So now, I have two sequences in fastq format per each original bam file.

My following steps, that I have in mind, is to align these fastq files with the human assembly 37, convert then again to bam files, and make the variant calling with mpileup so from there start with the annotation using annovar or something alike. So my questions in fact are:

1. Is my sort of pipeline acceptable?
2. Should I use both sequences for the variant calling?

Thank you in advance for your help and comments
<p>The steps sound fine, and as others have suggested, using both read1 and read2 for alignment is more efficient. The paired mapping allows for better read placement, especially over repetitive or homologous regions.</p>

<p>Am not sure why you dont directly use the aligned BAM file to call variants on chr12 and then annotate them or intersect for any falling in KRAS?</p>

<p>If it is just a single position in KRAS, seems the paper you ref its a 3&#39; UTR mutation, you can simply genotype that coordinate (chromosome-position) on all the BAM files and get a quick answer if the mutation is present/absent.</p>

<p>Hope this helps.</p>
