Real time, field-deployable whole genome sequencing of malaria parasites using nanopore technology

Abstract

Malaria parasite genomes have been generated predominantly using short read sequencing technology which can be slow, requires advanced laboratory training and does not adequately interrogate complex genomic regions that harbour important malaria virulence determinants. The portable Oxford Nanopore Technologies MinION platform generates long reads in real time and may overcome these limitations. We present compelling evidence that Nanopore sequencing delivers valuable additional information for malaria parasites with comparable data fidelity for single nucleotide variant (SNV) calls, compared to standard Illumina whole genome sequencing. We demonstrate this through sequencing of pure Plasmodium falciparum DNA, mock infections and natural isolates. Nanopore has low error rates for haploid SNV genotyping and identifies structural variants (SVs) not detected with short reads. Nanopore genomes are directly comparable to publically available genomes and produce high quality end to end chromosome assemblies. Nanopore sequencing will expedite genomic surveillance of malaria and provide new insights into parasite genome biology.

Selective whole-genome sequencing ofPlasmodiumparasites directly from blood samples by Nanopore adaptive sampling

“…Ideally, WGS is carried out on fresh samples and in endemic settings where patients would benefit directly from clinical genomics. Sequencing devices of Oxford Nanopore Technologies (ONT) have enabled mobile and real-time sequencing 8,9 . During Nanopore sequencing, DNA is translocated via a protein pore through which an ionic current flows.…”

The sulfonylpiperazine MMV020291 prevents red blood cell invasion by the malaria parasitePlasmodium falciparumthrough interference with actin-1/profilin dynamics

“…1B). Genomic DNA was extracted from these resistant clones, along with a parental 3D7 reference strain, and whole genome sequencing was performed using the Oxford Nanopore MinION platform (Razook et al, 2020). Here, a minimum of 10x depth coverage with 70% of the reads to support the called allele was required for verification (Razook et al, 2020).…”

“…Candidate SNPs in blacklisted genomic regions (Miles et al, 2016) (that is, subtelomeric, centromeric or internal hypervariable regions) were removed using bedtools subtract (V2.29.2) to retain SNPs in the core genome only. Quality filtration of SNP calls, enforcing a minimum depth of coverage of 10X and requiring at least 70% of reads to support the called allele (Razook et al, 2020), was performed using custom awk script.…”

Nanopore sequencing for real-time genomic surveillance ofPlasmodium falciparum

“…Nanopore has previously been applied to P. falciparum amplicon sequencing for drug resistance genes [71,72] and to whole genome sequencing [73]. Here, we demonstrate that Nanopore can be used prospectively for real-time genomic analysis from clinical malaria samples in an endemic setting using the latest ONT chemistry, which reports single read accuracy of >99% [74].…”