Parameters used to assemble Symmetrospora coprosmae UCD350

Skewer v0.2.2
skewer -m pe -q 15 -n

SPADES v3.11.1
spades.py 


Pipeline for variant analysis using SAMtools v1.1.19, BWA mem v0.7.12-r1039, PicardTools and Genome Analysis Toolkit v4.0.1. 


#Generate index files for reference assemblies
bwa index ASSEMBLY.fasta

samtools faidx ASSEMBLY.fasta

picard-tools CreateSequenceDictionary REFERENCE=ASSEMBLY.fasta OUTPUT=ASSEMBLY.dict

#Align reads
bwa mem -M -Y -R "@RG\tID:RGID\tSM:SAMPLE_NAME\tPL:illumina\tLB:lib1\tPU:unit1" ASSEMBLY.fasta READS1.fastq READS2.fastq > SAMPLE.sam

samtools view -S -b SAMPLE.sam > SAMPLE.bam

samtools sort SAMPLE.bam -o SAMPLE.sorted

samtools index SAMPLE.sorted

#Call variants
picard-tools MarkDuplicates INPUT=SAMPLE.sorted OUTPUT=SAMPLE_dedup.bam METRICS_FILE=SAMPLE_duplicates_metrics.txt VALIDATION_STRINGENCY=LENIENT

picard-tools BuildBamIndex INPUT=SAMPLE_dedup.bam VALIDATION_STRINGENCY=LENIENT


gatk HaplotypeCaller --reference ASSEMBLY.fasta --input SAMPLE_dedup.bam --output SAMPLE.vcf

#Index VCFs
gatk IndexFeatureFile -F SAMPLE.vcf

#Run cluster filter
gatk VariantFiltration -R ASSEMBLY.fasta -V SAMPLE.vcf -O SAMPLE_ClusterFilter.vcf --cluster-size 5 --cluster-window-size 20

#Run GATK recommended filters
gatk VariantFiltration -R ASSEMBLY.fasta -V SAMPLE_ClusterFilter.vcf -O SAMPLE_ClusterFilter.filterGATK.vcf --filter-expression "QD < 2.0" --filter-name "GATKRecomFilterQD" --filter-expression "MQ < 40.0" --filter-name "GATKRecomFilterMQ" --filter-expression "FS > 60.0" --filter-name "GATKRecomFilterFS" --filter-expression "SOR > 3.0" --filter-name "GATKRecomFilterSOR" --filter-expression "MQRankSum < -12.5" --filter-name "GATKRecomFilterMQRankSum" --filter-expression "ReadPosRankSum < -8.0" --filter-name "GATKRecomFilterReadPosRankSum"

#Select SNPs

gatk SelectVariants -V SAMPLE_ClusterFilter.filterGATK.vcf -select-type SNP -O ASSEMBLY_ClusterFilter.filterGATK.snps.vcf

#Select het SNPs
gatk SelectVariants -V SAMPLE_ClusterFilter.filterGATK.snps.vcf -select 'vc.getGenotype("SAMPLE").isHet()' -O SAMPLE_ClusterFilter.filterGATK.snps.het.vcf

#Select INDELs
gatk SelectVariants -V SAMPLE_ClusterFilter.filterGATK.vcf -select-type INDEL -O SAMPLE_ClusterFilter.filterGATK.indels.vcf

#Select het indels
gatk SelectVariants -V SAMPLE_ClusterFilter.filterGATK.indels.vcf -select 'vc.getGenotype("SAMPLE").isHet()' -O SAMPLE_ClusterFilter.filterGATK.indels.het.vcf



General description of GATK filtering
gatk VariantFiltration \
-R ASSEMBLY.fasta \
-V SAMPLE.vcf \
-O SAMPLE_ClusterFilter.vcf \
--cluster-size 5 \
--cluster-window-size 20

gatk VariantFiltration \
-R ASSEMBLY.fasta \
-V SAMPLE_ClusterFilter.vcf \
-O SAMPLE_filterGATK.vcf \
--filter-expression "QD < 2.0" \
--filter-name "GATKRecomFilterQD" \
--filter-expression "MQ < 40.0" \
--filter-name "GATKRecomFilterMQ" \
--filter-expression "FS > 60.0" \
--filter-name "GATKRecomFilterFS" \
--filter-expression "SOR > 3.0" \
--filter-name "GATKRecomFilterSOR" \
--filter-expression "MQRankSum < -12.5" \
--filter-name "GATKRecomFilterMQRankSum" \
--filter-expression "ReadPosRankSum < -8.0" \
--filter-name "GATKRecomFilterReadPosRankSum"