PacBio long-read amplicon sequencing enables scalable high-resolution population allele typing of the complex CYP2D6 locus.

The CYP2D6 enzyme is estimated to metabolize 25% of commonly used pharmaceuticals and is of intense pharmacogenetic interest due to the polymorphic nature of the CYP2D6 gene. Accurate allele typing of CYP2D6 has proved challenging due to frequent copy number variants (CNVs) and paralogous pseudogenes. SNP-arrays, qPCR and short-read sequencing have been employed to interrogate CYP2D6, however these technologies are unable to capture longer range information. Long-read sequencing using the PacBio Single Molecule Real Time (SMRT) sequencing platform has yielded promising results for CYP2D6 allele typing. However, previous studies have been limited in scale and have employed nascent data processing pipelines. We present a robust data processing pipeline "PLASTER" for accurate allele typing of SMRT sequenced amplicons. We demonstrate the pipeline by typing CYP2D6 alleles in a large cohort of 377 Solomon Islanders. This pharmacogenetic method will improve drug safety and efficacy through screening prior to drug administration.

Surveillance of molecular markers of Plasmodium falciparum artemisinin resistance (kelch13 mutations) in Papua New Guinea between 2016 and 2018.

Plasmodium falciparum resistance to artemisinin-based combination therapy (ACT) is a global threat to malaria control and elimination efforts. Mutations in the P. falciparum kelch13 gene (Pfk13) that are associated with delayed parasite clearance have emerged on the Thai-Cambodian border since 2008. There is growing evidence of widespread Pfk13 mutations throughout South-East Asia and they have independently emerged in other endemic regions. In Papua New Guinea (PNG), Pfk13 "C580Y" mutant parasites with reduced in vitro sensitivity to artemisinin have been isolated in Wewak, a port town in East Sepik Province. However, the extent of any local spread of these mutant parasites in other parts of PNG is unknown. We investigated the prevalence of Pfk13 mutations in multiple malaria-endemic regions of PNG. P. falciparum isolates (n = 1152) collected between 2016 and 2018 and assessed for Pfk13 variation by sequencing. Of 663 high quality Pfk13 sequences a total of five variants were identified. They included C580Y, a mutation at a previously documented polymorphic locus: N499K, and three previously undescribed mutations: R471C, K586E and Y635C. All variants were found in single isolates, indicating that these Pfk13 mutations were rare in the areas surveyed. Notably, C580Y was absent from Maprik district, which neighbours Wewak where C580Y mutant parasites were previously identified. The single C580Y isolate was found in the port town of Lae, Morobe Province, a potential entry site for the importation of drug resistant parasites into PNG. Although sample size in this location was small (n = 5), our identification of a C580Y mutant in this second location is concerning, highlighting the urgent need for further surveillance in Lae. Other Pfk13 mutants were rare in PNG between 2016 and 2018. Continued surveillance for molecular markers of drug resistance is critically important to inform malaria control in PNG.

SNP barcodes provide higher resolution than microsatellite markers to measure Plasmodium vivax population genetics.

BACKGROUND: Genomic surveillance of malaria parasite populations has the potential to inform control strategies and to monitor the impact of interventions. Barcodes comprising large numbers of single nucleotide polymorphism (SNP) markers are accurate and efficient genotyping tools, however may need to be tailored to specific malaria transmission settings, since 'universal' barcodes can lack resolution at the local scale. A SNP barcode was developed that captures the diversity and structure of Plasmodium vivax populations of Papua New Guinea (PNG) for research and surveillance. METHODS: Using 20 high-quality P. vivax genome sequences from PNG, a total of 178 evenly spaced neutral SNPs were selected for development of an amplicon sequencing assay combining a series of multiplex PCRs and sequencing on the Illumina MiSeq platform. For initial testing, 20 SNPs were amplified in a small number of mono- and polyclonal P. vivax infections. The full barcode was then validated by genotyping and population genetic analyses of 94 P. vivax isolates collected between 2012 and 2014 from four distinct catchment areas on the highly endemic north coast of PNG. Diversity and population structure determined from the SNP barcode data was then benchmarked against that of ten microsatellite markers used in previous population genetics studies. RESULTS: From a total of 28,934,460 reads generated from the MiSeq Illumina run, 87% mapped to the PvSalI reference genome with deep coverage (median = 563, range 56-7586) per locus across genotyped samples. Of 178 SNPs assayed, 146 produced high-quality genotypes (minimum coverage = 56X) in more than 85% of P. vivax isolates. No amplification bias was introduced due to either polyclonal infection or whole genome amplification (WGA) of samples before genotyping. Compared to the microsatellite panels, the SNP barcode revealed greater variability in genetic diversity between populations and geographical population structure. The SNP barcode also enabled assignment of genotypes according to their geographic origins with a significant association between genetic distance and geographic distance at the sub-provincial level. CONCLUSIONS: High-throughput SNP barcoding can be used to map variation of malaria transmission dynamics at sub-national resolution. The low cost per sample and genotyping strategy makes the transfer of this technology to field settings highly feasible.

Monitoring Plasmodium falciparum and Plasmodium vivax using microsatellite markers indicates limited changes in population structure after substantial transmission decline in Papua New Guinea.

Monitoring the genetic structure of pathogen populations may be an economical and sensitive approach to quantify the impact of control on transmission dynamics, highlighting the need for a better understanding of changes in population genetic parameters as transmission declines. Here we describe the first population genetic analysis of two major human malaria parasites, Plasmodium falciparum (Pf) and Plasmodium vivax (Pv), following nationwide distribution of long-lasting insecticide-treated nets (LLINs) in Papua New Guinea (PNG). Parasite isolates from pre- (2005-2006) and post-LLIN (2010-2014) were genotyped using microsatellite markers. Despite parasite prevalence declining substantially (East Sepik Province: Pf = 54.9%-8.5%, Pv = 35.7%-5.6%, Madang Province: Pf = 38.0%-9.0%, Pv: 31.8%-19.7%), genetically diverse and intermixing parasite populations remained. Pf diversity declined modestly post-LLIN relative to pre-LLIN (East Sepik: Rs  = 7.1-6.4, HE  = 0.77-0.71; Madang: Rs  = 8.2-6.1, HE  = 0.79-0.71). Unexpectedly, population structure present in pre-LLIN populations was lost post-LLIN, suggesting that more frequent human movement between provinces may have contributed to higher gene flow. Pv prevalence initially declined but increased again in one province, yet diversity remained high throughout the study period (East Sepik: Rs  = 11.4-9.3, HE  = 0.83-0.80; Madang: Rs  = 12.2-14.5, HE  = 0.85-0.88). Although genetic differentiation values increased between provinces over time, no significant population structure was observed at any time point. For both species, a decline in multiple infections and increasing clonal transmission and significant multilocus linkage disequilibrium post-LLIN were positive indicators of impact on the parasite population using microsatellite markers. These parameters may be useful adjuncts to traditional epidemiological tools in the early stages of transmission reduction.