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APPENDIX A: Benchmark commands

BWA scaling test command

bwa mem -M -t [number of cores] -v 1 [reference] [read fastq 1] [read fastq 1] > [sam output file]

BWA-GATK commands

Phase 1. Pre-processing

Step 1. Aligning and sorting

bwa mem -c 250 -M -t [number of threads] -R ‘@RG\tID:noID\tPL:illumine\tLB:noLB\tSM:bar’ [reference chromosome] [read fastq

1] [read fastq 2] | samtools view -bu - | sambamba sort -t [number of threads] -m 30G --tmpdir [path/to/temp] -o [sorted bam output]

/dev/stdin

Step 2. Mark and remove duplicates

sambamba markdup -t [number of threads] --remove-duplicates --tmpdir=[path/to/temp] [input: sorted bam output] [output: bam

without duplicates]

Step 3. Generate realigning targets

java -d64 -Xms4g -Xmx30g -jar GenomeAnalysisTK.jar -T RealignerTargetCreator -nt [number of threads] -R [reference

chromosome] -o [target list file] -I [bam without duplicates] -known [reference vcf file]

Step 4. Realigning around InDel

java -d64 -Xms4g -Xmx30g -jar GenomeAnalysisTK.jar -T IndelRealigner -R [reference chromosome] -I [bam without duplicates] -

targetIntervals [target list file] -known [reference vcf file] -o [realigned bam]

Step 5. Base recalibration

java -d64 -Xms4g -Xmx30g -jar GenomeAnalysisTK.jar -T BaseRecalibrator -nct [number of threads] -l INFO -R [reference

chromosome] -I [realigned bam] -known [reference vcf file] -o [recalibrated data table]

Step 6. Print recalibrated reads - Optional

java -d64 -Xms8g -Xmx30g -jar GenomeAnalysisTK.jar -T PrintReads -nct [number of threads] -R [reference chromosome] -I

[realigned bam] -BQSR [recalibrated data table] -o [recalibrated bam]

Step 7. After base recalibration - Optional

java -d64 -Xms4g -Xmx30g -jar GenomeAnalysisTK.jar -T BaseRecalibrator -nct [number of threads] -l INFO -R [reference

chromosome] -I [recalibrated bam] -known [reference vcf file] -o [post recalibrated data table]

Step 8. Analyze covariates - Optional

java -d64 -Xms8g -Xmx30g -jar GenomeAnalysisTK.jar -T AnalyzeCovariates -R [reference chromosome] -before [recalibrated

data table] -after [post recalibrated data table] -plots [recalibration report pdf] -csv [recalibration report csv]

Phase 2. Variant discovery – Calling germline variants

Step 1. Haplotype caller

java -d64 -Xms8g -Xmx30g -jar GenomeAnalysisTK.jar -T HaplotypeCaller -nct [number of threads] -R [reference chromosome] -

ERC GVCF -BQSR [recalibrated data table] -L [reference vcf file] -I [recalibrated bam] -o [gvcf output]