Variant_call_Day5

# Structural Variation (SV) analysis

# Very general workflow for variant analysis
# 1. fastq files from your sample
# 2. Run FastQC for quality control
#  2a) if qualtify of bases (in particular at the end of reads) are bad, perform trimming.
#  2b) if there are adapter sequences present in your fastq files, remove adapter sequence.
# => OK. fastq quality is good. Lets go to step 3
# 3. Perform Alignemnt using your favorite aligner (e.g., BWA, etc.) and generate SAM or BAM files.
# 4. Sort bam file and generate an index file for your bam file.
# 5. Variant calling using your favorite SV detection tool
# 6. Now we have variant calls (e.g., a list of SVs). We want to annotate them to see whether those variants are reported by someone eles or associated with known disease phenotype and/or see what would be functional impact.
#  6a) We will use snpEff tool (http://snpeff.sourceforge.net) and please read snpEff readme file (http://snpeff.sourceforge.net/SnpEff.html)
# 7. Interpretation. Now we have annotated variant calls and need to make sense out of it.
# 8. It is important to visualize your bam file to double check whether variants called by computational algorithms are real(?).
# Utilmately, you want to validate your variants using Sanger or etc.

qrsh
mkdir day5
cd day5
cp -R /data/bootcamp/day5/* .

# Environmental variables
export PATH=/data/bootcamp/tools/bwa-0.7.12:/data/bootcamp/tools/delly-0.7.3/src:/data/bootcamp/tools/delly-0.7.3/src/bcftools:/data/bootcamp/tools/samtools-1.2/bin:/data/bootcamp/tools/jdk1.8.0_101/bin:$PATH

# Picard
export PICARD_DIRECTORY=/data/bootcamp/tools/picard-tools-1.96

#SHEAR: Structural Variation call algorithm (http://vk.cs.umn.edu/SHEAR/)
(http://bmcgenomics.biomedcentral.com/articles/10.1186/1471-2164-15-84)
export PATH=/data/bootcamp/seqprg/jre1.8.0_101/bin:$PATH
java -jar /data/bootcamp/tools/shear-v1.1.2.1/SHEAR.jar sv -p output_sample_2126 -f reference/chr19.fa -b sample_2126_T1.bam --bam-normal sample_2126_N1.bam

#You should be able to see the following results
#-bash-4.1$ ls | grep report
#output_sample_2126.all.report
#output_sample_2126.snpindel.report
#output_sample_2126.sv.report
#-bash-4.1$ more output_sample_2126.sv.report
#Type  Details                       Microhomology  Reference Bases      Variant Bases        Heterogeneity
#DEL   19:1220416-1221610 (1195 bp)              -  AGGGCATT...GGCTCCCT  -                           53.85%

#Lets download bam and bai file and visualize Deletion present in your sample.