An Optimized GATK4 Pipeline for Plasmodium falciparum Whole Genome Sequencing Variant Calling and Analysis.

Accurate variant calls from whole genome sequencing (WGS) of infections are crucial in malaria population genomics. Here we optimized a falciparum variant calling pipeline based on GATK version 4 (GATK4) and applied it to 6,626 public Illumina WGS samples. We optimized parameters that control the heterozygosity, local assembly region size, ploidy, mapping and base quality in both GATK HaplotypeCaller and GenotypeGVCFs leveraging control WGS and accurate PacBio assemblies of 10 laboratory strains. From these controls we generated a high-quality training dataset to recalibrate the raw variant data. On current high-quality samples (read length = 250bp, insert size = 405 - 524 bp ), we show improved sensitivity (86.6 ± 1.7% for SNPs and 82.2 ± 5.9% for indels) compared to the default GATK4 pipeline (77.7 ± 1.3% for SNPs; and 73.1 ± 5.1% for indels, adjusted P < 0.001) and previous variant calling with GATK version 3 (GATK3, 70.3 ± 3.0% for SNPs and 59.7 ± 5.8% for indels, adjusted P < 0.001). The sensitivity of our pipeline on simulated mixed infection samples (80.8 ± 6.1% for SNPs and 78.3 ± 5.1% for indels) was again improved relative to default GATK4 (68.8 ± 6.0% for SNPs and 38.9 ± 0.7% for indels, adjusted P < 0.001). Precision was high and comparable across all pipelines on each type of data tested. We further show that using the combination of high-quality SNPs and indels increases the resolution of local population population structure detection in sub-Saharan Africa. We finally demonstrate that increasing ploidy improves the detection of drug resistance mutations and estimation of complexity of infection. Overall, we provide an optimized GATK4 pipeline and resource for variant calling which should help improve genomic studies of malaria.