1. BWA 

bwa index ref.fa
bwa aln -I ref.fa input.fq > aln_sa.sai
bwa samse ref.fa aln_sa.sai input.fq >aln-se.sam)


2. Picard 

java -Xmx4g -jar MarkDuplicates.jar I = input. bam O = input.dedup.bam M = input.bam.pcr REMOVE_DUPLICATES = true VALIDATION_STRINGENCY = LENIENT


3. GATK pipeline

Realignment: 
java -Xmx4g -jar GenomeAnalysisTK.jar -T RealignerTargetCreator -I input.bam -R reference.fasta -o forIndelRealinger.intervals
java -Xmx4g -jar GenomeAnalysisTK.jar -T IndelRealigner  -I input.bam -R reference.fasta -targetIntervals forIndelRealinger.intervals  -o input.realigned.bam.

Remove duplicates: 
java -Xmx4g -jar MarkDuplicates.jar I = input.realigned.bam O = input.realigned.dedup.bam M = input.realigned.bam.pcr REMOVE_DUPLICATES = false VALIDATION_STRINGENCY = LENIENT.

Recalibration: 
java -Xmx4g -jar GenomeAnalysisTK.jar -T BaseRecalibrator -I input.realigned.dedup.bam -R reference.fasta -o recal_data.grp -knownSites dbSNP.vcf; 
java -Xmx4g -jar GenomeAnalysisTK.jar -T PrintReads  -I input.realigned.dedup.bam -R reference.fasta -BQSR recal_data.grp  -o input.realigned.dedup.recal.bam.

Variants calling: 
java -Xmx4g -jar GenomeAnalysisTK.jar -T UnifiedGenotyper -R reference.fasta --dbsnp dbSNP.vcf -I input.realigned.dedup.recal.bam -o snps.raw.vcf -mbq 20 -stand_call_conf 30.0 -stand_emit_conf 10.0 -dcov 200 -metrics snps.raw.vcf.metrics --output_mode EMIT_VARIANTS_ONLY --annotation QualByDepth --annotation HaplotypeScore --annotation MappingQualityRankSumTest --annotation ReadPosRankSumTest --annotation FisherStrand --annotation RMSMappingQuality -glm BOTH -L targets.interval.list.

Variant quality score recalibration: 
java -Xmx4g -jar GenomeAnalysisTK.jar -T VariantRecalibrator -input snps.raw.vcf -R reference.fasta --maxGaussians 4 -resource: dbsnp, known=true, training=true, truth=true, prior=10.0 dbSNP.vcf -an QD -an HaplotypeScore -an MQRankSum -an ReadPosRankSum -an FS -an MQ -mode SNP -recalFile snps.raw.vcf.recal -tranchesFile snps.raw.vcf.tranches -rscriptFile snps.raw.vcf.plots.R; 
java -Xmx4g -jar GenomeAnalysisTK.jar -T ApplyRecalibration -R reference.fasta -input snps.raw.vcf -tranchesFile snps.raw.vcf.tranches -recalFile snps.raw.vcf.recal -o snps..recalibrated.filtered.vcf --ts_filter_level 99.90 -mode SNP.