Oral Presentation 8th International Conference on Plasmodium vivax Research 2022

Development of microhaplotype-based amplicon sequencing assays to determine P. vivax relatedness   (80290)

Mariana Kleinecke 1 , Sasha V. Siegel 2 , Jutta Marfurt 1 , Hidayat Trimarsanto 3 , Richard Pearson 2 , Angela Rumaseb 1 , Ric N. Price 1 4 5 , Sarah Auburn 1 4 5
  1. Global and Tropical Health Division, Menzies School of Health Research and Charles Darwin University, Darwin, Northern Territory, Australia
  2. Wellcome Sanger Institute, Hinxton, Cambridge CB10 1SA, UK
  3. Eijkman Institute for Molecular Biology, Jakarta, Indonesia
  4. Mahidol‐Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok 10400, Thailand
  5. Centre for Tropical Medicine and Global Health,Nuffield Department of Medicine, University of Oxford, OX3 7LJ, UK

Characterising P. vivax relatedness can inform spatiotemporal evaluation and differentiation between relapse, recrudescence and reinfection. We previously identified panels of P. vivax microhaplotypes that are globally diverse, with high accuracy in inferring relatedness between infections. Here, we describe new, amplicon-based sequencing assays for a genome-wide panel of microhaplotypes. 

A single Illumina rhAmpSeq multiplex was designed to encompass 146 candidate microhaplotypes, 4 drug resistance targets and 2 mitochondrial amplicons providing information on Plasmodium spp. Multiplex and indexing PCRs were conducted on 192 P. vivax samples from 10 countries, including 168 (87.5%) with corresponding genomic data, and 16 control samples. The resultant libraries were sequenced on a MiSeq run generating 150bp paired-end reads as a pilot to test the sensitivity and specificity of the assays. Read mapping and genotype calling were undertaken using bwa, samtools, picard and GATK. Standard population genetic measures were conducted using the scikit-allel package. 

Six (3.1%) P. vivax samples exhibited low yields (<1,000 reads) and were excluded from the analysis. A mean of 76,227 reads were generated from the remaining 186 samples. Overall, 144 (94.7%) microhaplotypes demonstrated high specificity to P. vivax, with <1% reads mapping to human DNA or other Plasmodium species. The mitochondrial markers discriminated Plasmodium species accurately. Amongst the 144 high-specificity microhaplotypes, the average read-depth at the target loci was 1000, with 96.5% concordance of major allele calls against the genomic data. The assays performed well in all countries. Population genetic analyses confirmed high diversity in all countries and clustering by geography. 

Further evaluations and optimisations are underway, including removal of redundant markers to achieve a final panel of 100 microhaplotypes. The final product will be used to inform on recurrence patterns in clinical trials, and spatiotemporal transmission analysis in P. vivax.