Last Updated: 17/02/2023
MinION sequencing for rapid detection of imported P. vivax cases
Objectives
To establish laboratory-based markers for the 33-SNP barcode using MinION, a novel cutting-edge portable sequencer, that will enable National Malaria Elimination Programs to investigate the origin of vivax infections cost-efficiently and quickly and also develop standalone software to support endemic country partners to process data generated on the MinION and integrate this with the vivaxGEN platform for maximum ease of data analysis and interpretation.
There are an estimated 14 million cases of vivax malaria each year, with P. vivax becoming the predominant cause of malaria outside of sub-Saharan Africa. Imported cases present a considerable challenge to the elimination of malaria. Traditionally, patient travel history has been used to distinguish between imported and locally transmitted cases, but the dormant liver stages of Plasmodium vivax confound this approach. Molecular tools offer an alternative approach to identify and map imported cases. In previous work, our team identified a parsimonious 33-SNP molecular barcode with demonstrated accuracy to determine the country of origin of P. vivax infections. We also established an open-access online software (vivaxGEN-GEO) to enable endemic country partners to identify and map imported vivax cases using locally generated data at the 33-SNP barcode. These rapid tools will transform surveillance of imported malaria in countries such as China, where surveillance requires confirmation of imported cases within a week of case reporting.
1. We will use the PCR protocols established by Dr Kattenberg in A/Prof Rosanas-Urgell’s team to generate amplicons (each of ~200bp) comprising the target SNPs . The product will be ligated, purified, quantified, and sequenced using an adaptation of the protocols described by Dr Razook and colleagues in A/Prof Barry’s team for multiplex MinION sequencing of short-read amplicons for malaria .
2. We will apply the workflow generated in step 1 to a selection of 150 test samples from 12 vivax-endemic countries in the Asia-Pacific, Horn of Africa, and Americas.
3. A snakemake bioinformatics pipeline will be established for base calling, identifying and separating the subreads and aligning against the PvP01 P. vivax reference genome. The data will be polished and variant calling will be performed. We will test different software for each step to determine the most suitable components for this type of data. Dr Kleinecke and Dr McCann have previous experience with MinION sequence processing. Dr Kleinecke is currently establishing a similar pipeline for an approved ACREME seed grant (PI Mueller) on G6PD MinION sequencing.
4. The selected test samples have corresponding whole genome sequencing (WGS) and Illumina amplicon sequencing data to facilitate the validation of the genotyping calls. We will use the data from these independent platforms for concordance assessment against the genotype calls generated in step 3 to determine the accuracy and precision of the MinION-based variant calling to validify the assay.
5. A standalone software will be established to integrate the MinION genotyping pipeline with the vivaxGEN-GEO software. Dr Auburn’s team have established similar software for the vivaxGEN suite of tools and will support this process.
Nov 2021 — Oct 2023
$15,000
