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Setup output directory.

mkdir samtools_bowtie

If you do not have an alignment file in the SAM format you may want to start with Introduction to mapping.

cp bowtie/REL606.5.sam samtools_bowtie/ 
cp bowtie/REL606.5.fasta samtools_bowtie/

Index the reference file.

samtools faidx samtools_bowtie/REL606.5.fasta

Convert from SAM to BAM format.

samtools view -bS -o samtools_bowtie/REL606.5.bam bowtie/REL606.5.sam |borderStyle=solid}

Sort the BAM file.

samtools sort samtools_bowtie/REL606.5.bam samtools_bowtie/sorted_REL606.5

Output VCF file.

samtools mpileup -uf samtools_bowtie/REL606.5.fasta samtools_bowtie/sorted_REL606.5.bam \|bcftools view -vcg - \> samtools_bowtie/output.vcf 

Samtools Output

Exercise 1

VCF format has Allele Frequency tags denoted by AF1. Try the following command to see what values we have in our files.

cat input.vcf | grep AF1

For the data we are dealing with, predictions with an allele frequency not equal to 1 are not really applicable. How can we remove these lines from the file and continue on?

What does the -v flag do in grep?

grep -v *something*


Filtering Allele Frequencies Output

Determining Differences Between Mappers.

Setup output directory and then change into it.

mkdir output
cp samtools_bowtie/output.vcf output/bowtie.vcf
cp samtools_bwa/output.vcf output/bwa.vcf
cd output

Bedtools is a suite of utility programs that work on a variety of file formats, one of which is conveniently VCF format. Using intersectBed and subtractBed we can find equal and different predictions between mappers.

Load Bedtools.

module load bedtools

Finding alike mutations.

intersectBed -a bowtie.vcf -b bwa.vcf > intersect.vcf

Finding unique mutations for each mapper.

subtractBed -a bowtie.vcf -b intersect.vcf > unique_bowtie.vcf
subtractBed -a bwa.vcf -b intersect.vcf > unique_bwa.vcf

Samtools Output

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