Alyssa Barry: MinION whole genome sequencing of malaria field isolates
Associate Professor Alyssa Barry, of the Walter+Eliza Hall Institute of Medical Research, Melbourne, described how she and her team developed a protocol for whole genome sequencing of Plasmodium field isolates on the MinION device, to help further understanding of evolution of the parasite and the biology, transmission and prevention of malaria.
Alyssa described the scale and worldwide burden of malaria which, in the space of a year, caused 429,000 deaths, with 212 million clinical cases; she noted that “more than half the world’s population live in at-risk areas.” There is currently no broadly effective vaccine. Since 2000, global efforts to control malaria have reduced the number of cases and deaths from malaria by 50%, with 17 countries where the disease was previously endemic now declared malaria-free since 2010. However, this progress has since stalled; malaria remains endemic in 91 countries, and many, including Papua New Guinea, are now experiencing a resurgence in the disease. The 2016-30 Global Technical Strategy for Malaria aims to reduce deaths and cases by 90%, eliminate malaria from a further 35 countries and prevent its resurgence in those now malaria free. A combination of lack of funding and poor healthcare infrastructure means that only half of these countries are on track; Alyssa stressed that hitting this target will take new tools, better surveillance and “investment in more efficient and novel strategies.” These strategies must also be able to identify the smaller, more clustered infections as transmission decreases, and be sensitive enough to detect asymptomatic infections.
Alyssa introduced the benefits of using the MinION device for malaria genomic epidemiology, describing how the portable, real-time technology allows for multiplexed outbreak surveillance and drug resistance typing, potentially in less than three hours. The long read output enables whole genome assemblies, spanning regions of high complexity and resolving large structural variants. She especially highlighted the potential of the technology for capacity building, enabling sequencing by those who are most affected and most passionate about malaria eradication.
Alyssa and her team prepared two P. falciparum lab isolate DNA libraries, Xha_k and BB12, using the 1D ligation sequencing kit (SQK_LSK108) and sequenced them on the MinION device. Reads were basecalled via Albacore, quality-checked with NanoPLOT, trimmed using Porechop and aligned via Minimap. 30x coverage was generated for 96% and 95% of Xha_k and BB12 respectively, with 100% of the aligned DNA representing parasite, rather than host, DNA. De novo assembly was achieved via Canu, then polished using Nanopolish, Contigs were then aligned against the reference isolate 3D7 via nucmer. Xha was assembled in 15 contigs, with a mean coverage of 75x, and BB12 in 17, with mean coverage at 106x. The protocol was then optimised for use on field isolates using mock infections, comprised of ~1% 3D7 DNA spiked into a human DNA background. After WGA, treatment of libraries with T7 endonuclease to remove hyperbranching was shown to dramatically increase enrichment of P. falciparum vs human background, for improved breadth and depth of coverage of the parasite genome. The team then used the optimised protocol to sequence and assemble multiple field isolates from several parts of the world using the MinION device. They were able to call mutations in genes leading to reduced sensitivity to a number of drugs, especially lumefantrine, but none for artemisinin.
Alyssa then described the case of a patient admitted to the Royal Melbourne Hospital, having recently returned from the Ivory Coast. After diagnosis of clinical malaria, the patient was treated with Artemether Lumefantrine and returned home. A month later, he returned; the patient was again treated and returned home. To investigate whether this second infection was caused by the same clone or represented the emergence of a minor drug-resistant clone following the first treatment, and to identify drug resistance markers, the team used their rapid sequencing workflow. DNA from patient blood samples taken at each hospital visit were sequenced on a MinION, and in 45 minutes generated sufficient data for ~5x coverage of the P. falciparum genome: this was sufficient for resistance variant calling. Analysis of the aligned data showed a high proportion of heterozygous sites, despite P. falciparum being haploid, indicating infection with more than one clone. Analysis of highly polymorphic regions subsequently suggested that at least two distinct haplotypes were present in each infection. Drug resistance markers were then identified for lumefantrine, but not for artemisinin, a short-acting drug; this suggested that the infection re-emerged once artemisinin treatment wore off, and due to lumefantrine resistance. Alyssa highlighted how this rapid sequencing test produced results significant in patient follow-up and in personalised treatment.
Alyssa concluded that the use of the MinION device in generating a rapid malaria parasite genome sequencing protocol enabled whole genome assembly, with long reads allowing resolution of complex regions, generation of reference genome scaffolds and determination of both structural variants and drug resistance markers. She noted that field isolates could be sequenced in multiplex at a cost of ~$300/sample: “this technology really has potential for real-time clinical genomics.”