London Calling 2016

A revolution is occurring in genomic epidemiology. Recently, real-time portable genome sequencing using the Oxford Nanopore MinION device was successfully used to characterize the genetic diversity of the Ebola virus outbreak in Guinea. We are extending this ground-breaking achievement to Zika virus by establishing up the ZiBRA project - a mobile portable genome sequencing laboratory in Brazil. Through collaboration with Instituto Evandro Chagas and Fundação Oswaldo Cruz public health laboratories in Brazil we will perform real-time genomic surveillance of Zika, Chikungunya and dengue viruses, covering a broad geographical region including historical samples, and from patients with a range of clinical presentations. Data will be subject to open release as it is generated with project progress and discussed via the applicants Virological.org forum. This effort will serve as a beacon for open science during a public health emergency.

The eXtreme Microbiome Project (XMP) is a global scientific collaboration to characterize, discover, and develop new pipelines and protocols for studying novel microorganisms in extreme environments, including “The Door to Hell” gas crater in Turkmenistan, permafrost soils beside frozen mammoths, blood-red waterfalls from Antarctica, and even samples from the International Space Station. Included in this study is Lake Hillier, a bright-pink hypersaline lake located on a remote island off the Western Australian coast. While the pink colour is thought to be caused by salt-loving algae, the actual microbial composition of the lake is unknown. The XMP collected and analysed lake water and sediment samples using a variety of metagenomics techniques, including the Oxford Nanopore MinION. The analysis revealed a surprising range of microbial diversity in the lake, and indicate that many algal, bacterial, and archaeal halophiles contribute to the persistent pigmentation of this pink paradise.

Why does the military need sequencing, and how will they use it? Recent advances in DNA sequencing technology, such as the Oxford Nanopore MiNION, could transform a previously cumbersome and fixed-location task into a truly mobile prospect. The Defence Science and Technology Laboratory works both to develop its own scientific solutions and to integrate the innovations of the wider community into fieldable technologies for defence and security. My presentation will explore some of the work Dstl is doing to overcome some of the current limitations and how we could apply this work to some example scenarios. I will explore sample collection such as our man-worn aerosol samplers and larger scale environmental collectors. I will discuss our work to develop a simple DNA extraction device, which can be operated whilst wearing bulky personal protective equipment. I will discuss what we need to do to complete a system and how you can help us achieve this.

The MinION replaces the conventional model of "sequence followed by analysis to final result" with instant access to data before the completion of a sequencing run. This instant access extends to the analysis of sequence "squiggle" data even before a read has finished traversing the nanopore. We have developed a suite of tools, minoTour, to analyse MinION data in real time and extended them to exploit both ‘Run Until’ and, soon, ‘Read Until’. "Run until" allows the sequencer to switch off after achieving a specific goal, such as depth of coverage. "Read Until" allows individual reads to be rejected from the pore and free that pore to sequence an alternative preferred read. We have developed methods for "Run Until" and "Read Until" in a number of different scenarios including selective small genome sequencing, barcode normalisation and amplicon sequencing. Although read until implementations are limited by compute resource and the ever increasing speed of sequencing, potential applications are increased by enabling the sequencing of regions of DNA from a background pool.

Acute myeloid leukemia is a devastating cancer of the myeloid blood cells where the cells cannot differentiate normally. This disease accounts for ~1% of cancer deaths annually, however the mortality rate exceeds 50% and left untreated can lead to death within a few weeks. Copy number variations (CNV) contribute to cancer pathogenesis, patient prognosis and therapeutic response. Clinical sequencing often takes weeks before actionable results are generated, time which patients may not have. The MinION device could provide clinicians with a tool to generate rapid copy number profiles in a point-of-care setting. Our pipeline generates CNV profiles from cancer samples in three days. Using the widely rearranged SKBR3 cancer cell line as a model we have generated >300k short reads on during a single MinION run followed by CNV analysis by Ginkgo. These profiles identify nearly all CNVs called by Ginkgo from MiSeq libraries. This approach can be used by clinicians to accurately and rapidly characterize CNV profiles from clinical samples and help in informing patient treatment.

Nanopore strand sequencing is uniquely suited to analysis of long DNA fragments and base modifications. In this presentation, we will discuss recent experiments that demonstrate 99% consensus accuracy for 150kb+ DNA fragments in single MinION runs. We will also discuss single nanopore experiments that detect base modifications on individual native tRNA strands.

Unbiased diagnosis of all pathogens in a single test by metagenomic next-generation sequencing is now feasible, but has been limited to date by concerns regarding sensitivity and sample-to-answer turnaround times. Here we will describe the development, validation, and implementation of a rapid, field-ready assay for differential diagnosis of acute febrile illness on an Oxford Nanopore MinION sequencer that can be performed in under 6 hours. Nanopore sequencing data will be analyzed in real-time on a laptop computer using SURPIrt, a portable version of the SURPI computational pipeline that is currently being implemented for precision medicine diagnosis in hospitals. The eventual goal of these studies is clinical performance validation and deployment at field sites for use in clinical diagnosis and public health surveillance in patients with any unknown febrile illness — including but not limited to infections by Zika virus, Ebola virus, Lassa virus, chikungunya virus, dengue virus, and the malarial parasite Plasmodium falciparum.

Diagnostic and public health microbiology depends on identifying the species, determining the antimicrobial resistance and categorising involvement in an outbreak of infecting pathogens. This is now accessible in one step using whole genome sequencing. There is substantial proof-of-principle work in viruses, bacteria and parasites indicating the advantages of whole genome sequencing of pathogens. The key features required for large scale adoption are achieving greater speed, lower cost and high accuracy of processing clinical samples, if possible, direct from the patient. What is needed is a small, speedier cheaper sequencer requiring nucleic acids prepared by simple quick extraction and fast computationally ‘light-weight’ processing software. The prospects will be highlighted by work on multi-resistant Enterobacteriaceae and TB.

Though culture methods are the gold standard for detecting clinical pathogenic microorganisms, followed by testing with PCR and microarrays of known pathogens, they are still very limiting. Culture is time consuming, confined by culturability of microorganisms, and gives very narrow information about the sample. We are applying the MinION for shotgun metagenomic sequencing, shortening the detection time and yielding information about substrains and therapeutic resistance from clinical infections. We used nanopore sequencing to detect infectious microorganisms in 3 separate cases: 1) detecting vancomycin resistance in remnant rectal swab cultures, 2) analyzing a clinical timeseries of multidrug resistant hypermucovirulent K. pneumonia to understand antibiotic resistance and containment, and 3) measuring substrain variations of cultured and clinical isolates of influenza strains for real-time surveillance and monitoring of transmission and evolution.

KeyGene operates at the forefront with respect to new technologies and innovations and participates as such in the MinION™ and PromethION™ Early Access Programs. Genome sequencing initiatives of large complex genomes typically yield considerably fragmented genome assemblies. Using the Oxford Nanopore sequencing technology that offers ultra- long reads KeyGene aims to produce contiguous, high-quality genome assemblies of plant pathogens and complex plant genomes. In order to obtain high quality libraries for sequencing the DNA integrity and quality are crucial. KeyGene has developed specific knowledge in this area. In 2016 KeyGene started to sequence and de novo assemble the plant pathogen Rhizoctonia solani with a genome size of ~85 Mb. Using the PromethION the next step will be to sequence and assemble the genome of a melon variety (~450 Mb). Progress and challenges in these projects will be presented and discussed during the breakout session.