Samtools
Setup output directory.
mkdir bowtie_samtools
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 bowtie_samtools/ cp bowtie/REL606.5.fasta bowtie_samtools/
Index the reference file.
samtools faidx bowtie_samtools/REL606.5.fasta
Convert from SAM to BAM format.
samtools view -bS -o bowtie_samtools/REL606.5.bam bowtie/REL606.5.sam |borderStyle=solid}
Sort the BAM file.
samtools sort bowtie_samtools/REL606.5.bam bowtie_samtools/sorted_REL606.5
Output VCF file.
samtools mpileup -uf bowtie_samtools/REL606.5.fasta bowtie_samtools/sorted_REL606.5.bam \|bcftools view -vcg - \> bowtie_samtools/output.vcf
Produces output.vcf from Bowtie and output.vcf from BWA.
Move all 3
bam files and all 3 vcf files to lonestar
introduce bedtools.
Developing a pipeline.
Entering all these commands to first create an alignment and then using Samtools to create a VCF format quickly becomes tedious... Let's create a simple bashscript to streamline things.
Mapping with Bowtie - bowtie_samtools.sh
#!/usr/bin/env bash #Bowtie. mkdir bowtie bp_seqconvert --from genbank --to fasta <REL606.5.gbk > bowtie/REL606.5.fasta bowtie-build bowtie/REL606.5.fasta bowtie/REL606.5 bowtie -t --sam bowtie/REL606.5 -1 SRR030257_1.fastq -2 SRR030257_2.fastq bowtie/REL606.5.sam #Samtools mkdir bowtie_samtools cp bowtie/REL606.5.sam bowtie_samtools/ cp bowtie/REL606.5.fasta bowtie_samtools/ samtools faidx bowtie_samtools/REL606.5.fasta samtools view -bS -o bowtie_samtools/REL606.5.bam bowtie/REL606.5.sam samtools sort bowtie_samtools/REL606.5.bam bowtie_samtools/sorted_REL606.5 samtools mpileup -uf bowtie_samtools/REL606.5.fasta bowtie_samtools/sorted_REL606.5.bam \|bcftools view -vcg - \> bowtie_samtools/output.vcf
Mapping with BWA - bwa_samtools.sh
#!/usr/bin/env bash #BWA mkdir bwa bp_seqconvert --from genbank --to fasta <REL606.5.gbk > bwa/REL606.5.fasta bwa index bwa/REL606.5.fasta bwa aln -f bwa/SRR030257_1.sai bwa/REL606.5.fasta SRR030257_1.fastq bwa aln -f bwa/SRR030257_2.sai bwa/REL606.5.fasta SRR030257_2.fastq bwa sampe -f bwa/REL606.5.sam bwa/REL606.5.fasta bwa/SRR030257_1.sai bwa/SRR030257_2.sai SRR030257_1.fastq SRR030257_2.fastq #Samtools mkdir bwa_samtools cp bwa/REL606.5.sam bwa_samtools/ cp bwa/REL606.5.fasta bwa_samtools/ samtools faidx bwa_samtools/REL606.5.fasta samtools view -bS -o bwa_samtools/REL606.5.bam bwa/REL606.5.sam samtools sort bwa_samtools/REL606.5.bam bwa_samtools/sorted_REL606.5 samtools mpileup -uf bwa_samtools/REL606.5.fasta bwa_samtools/sorted_REL606.5.bam \|bcftools view -vcg - \> bwa_samtools/output.vcf
Both are available at
/corral-repl/utexas/BioITeam/ngs_course/local/data/mapping_example/scripts
