The first genome assembly of fungal pathogen Pyrenophora tritici-repentis race 1 isolate using Oxford Nanopore MinION sequencing

Objectives: The assembly of fungal genomes using short-reads is challenged by long repetitive and low GC regions. However, long-read sequencing technologies, such as PacBio and Oxford Nanopore, are able to overcome many problematic regions, thereby providing an opportunity to improve fragmented genome assemblies derived from short reads only. Here, a necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr) isolate 134 (Ptr134), which causes tan spot disease on wheat, was sequenced on a MinION using Oxford Nanopore Technologies (ONT), to improve on a previous Illumina short-read genome assembly and provide a more complete genome resource for pan-genomic analyses of Ptr. Results: The genome of Ptr134 sequenced on a MinION using ONT was assembled into 28 contiguous sequences with a total length of 40.79 Mb and GC content of 50.81%. The long-read assembly provided 6.79 Mb of new sequence and 2846 extra annotated protein coding genes as compared to the previous short-read assembly. This improved genome sequence represents near complete chromosomes, an important resource for large scale and pan genomic comparative analyses

Oxford Nanopore sequencing, hybrid error correction, and de novo assembly of a eukaryotic genome

Monitoring the progress of DNA molecules through a membrane pore has been postulated as a method for sequencing DNA for several decades. Recently, a nanopore-based sequencing instrument, the Oxford Nanopore MinION, has become available, and we used this for sequencing the Saccharomyces cerevisiae genome. To make use of these data, we developed a novel open-source hybrid error correction algorithm Nanocorr specifically for Oxford Nanopore reads, because existing packages were incapable of assembling the long read lengths (5-50 kbp) at such high error rates (between approximately 5% and 40% error). With this new method, we were able to perform a hybrid error correction of the nanopore reads using complementary MiSeq data and produce a de novo assembly that is highly contiguous and accurate: The contig N50 length is more than ten times greater than an Illumina-only assembly (678 kb versus 59.9 kbp) and has >99.88% consensus identity when compared to the reference. Furthermore, the assembly with the long nanopore reads presents a much more complete representation of the features of the genome and correctly assembles gene cassettes, rRNAs, transposable elements, and other genomic features that were almost entirely absent in the Illumina-only assembly

Shotgun metagenome data of a defined mock community using Oxford Nanopore, PacBio and Illumina technologies.

Metagenomic sequence data from defined mock communities is crucial for the assessment of sequencing platform performance and downstream analyses, including assembly, binning and taxonomic assignment. We report a comparison of shotgun metagenome sequencing and assembly metrics of a defined microbial mock community using the Oxford Nanopore Technologies (ONT) MinION, PacBio and Illumina sequencing platforms. Our synthetic microbial community BMock12 consists of 12 bacterial strains with genome sizes spanning 3.2-7.2 Mbp, 40-73% GC content, and 1.5-7.3% repeats. Size selection of both PacBio and ONT sequencing libraries prior to sequencing was essential to yield comparable relative abundances of organisms among all sequencing technologies. While the Illumina-based metagenome assembly yielded good coverage with few misassemblies, contiguity was greatly improved by both, Illumina + ONT and Illumina + PacBio hybrid assemblies but increased misassemblies, most notably in genomes with high sequence similarity to each other. Our resulting datasets allow evaluation and benchmarking of bioinformatics software on Illumina, PacBio and ONT platforms in parallel

MinION Analysis and Reference Consortium: Phase 1 data release and analysis

The advent of a miniaturized DNA sequencing device with a high-throughput contextual sequencing capability embodies the next generation of large scale sequencing tools. The MinION™ Access Programme (MAP) was initiated by Oxford Nanopore Technologies™ in April 2014, giving public access to their USB-attached miniature sequencing device. The MinION Analysis and Reference Consortium (MARC) was formed by a subset of MAP participants, with the aim of evaluating and providing standard protocols and reference data to the community. Envisaged as a multi-phased project, this study provides the global community with the Phase 1 data from MARC, where the reproducibility of the performance of the MinION was evaluated at multiple sites. Five laboratories on two continents generated data using a control strain of Escherichia coli K-12, preparing and sequencing samples according to a revised ONT protocol. Here, we provide the details of the protocol used, along with a preliminary analysis of the characteristics of typical runs including the consistency, rate, volume and quality of data produced. Further analysis of the Phase 1 data presented here, and additional experiments in Phase 2 of E. coli from MARC are already underway to identify ways to improve and enhance MinION performance

Applications and Challenges of Real-time Mobile DNA Analysis

The DNA sequencing is the process of identifying the exact order of nucleotides within a given DNA molecule. The new portable and relatively inexpensive DNA sequencers, such as Oxford Nanopore MinION, have the potential to move DNA sequencing outside of laboratory, leading to faster and more accessible DNA-based diagnostics. However, portable DNA sequencing and analysis are challenging for mobile systems, owing to high data throughputs and computationally intensive processing performed in environments with unreliable connectivity and power. In this paper, we provide an analysis of the challenges that mobile systems and mobile computing must address to maximize the potential of portable DNA sequencing, and in situ DNA analysis. We explain the DNA sequencing process and highlight the main differences between traditional and portable DNA sequencing in the context of the actual and envisioned applications. We look at the identified challenges from the perspective of both algorithms and systems design, showing the need for careful co-design

Nanopore Sequencing Technology and Tools for Genome Assembly: Computational Analysis of the Current State, Bottlenecks and Future Directions

Nanopore sequencing technology has the potential to render other sequencing technologies obsolete with its ability to generate long reads and provide portability. However, high error rates of the technology pose a challenge while generating accurate genome assemblies. The tools used for nanopore sequence analysis are of critical importance as they should overcome the high error rates of the technology. Our goal in this work is to comprehensively analyze current publicly available tools for nanopore sequence analysis to understand their advantages, disadvantages, and performance bottlenecks. It is important to understand where the current tools do not perform well to develop better tools. To this end, we 1) analyze the multiple steps and the associated tools in the genome assembly pipeline using nanopore sequence data, and 2) provide guidelines for determining the appropriate tools for each step. We analyze various combinations of different tools and expose the tradeoffs between accuracy, performance, memory usage and scalability. We conclude that our observations can guide researchers and practitioners in making conscious and effective choices for each step of the genome assembly pipeline using nanopore sequence data. Also, with the help of bottlenecks we have found, developers can improve the current tools or build new ones that are both accurate and fast, in order to overcome the high error rates of the nanopore sequencing technology.Comment: To appear in Briefings in Bioinformatics (BIB), 201

De novo Assembly of the Brugia malayi Genome Using Long Reads from a Single MinION Flowcell

Filarial nematode infections cause a substantial global disease burden. Genomic studies of filarial worms can improve our understanding of their biology and epidemiology. However, genomic information from field isolates is limited and available reference genomes are often discontinuous. Single molecule sequencing technologies can reduce the cost of genome sequencing and long reads produced from these devices can improve the contiguity and completeness of genome assemblies. In addition, these new technologies can make generation and analysis of large numbers of field isolates feasible. In this study, we assessed the performance of the Oxford Nanopore Technologies MinION for sequencing and assembling the genome of Brugia malayi, a human parasite widely used in filariasis research. Using data from a single MinION flowcell, a 90.3 Mb nuclear genome was assembled into 202 contigs with an N50 of 2.4 Mb. This assembly covered 96.9% of the well-defined B. malayi reference genome with 99.2% identity. The complete mitochondrial genome was obtained with individual reads and the nearly complete genome of the endosymbiotic bacteria Wolbachia was assembled alongside the nuclear genome. Long-read data from the MinION produced an assembly that approached the quality of a well-established reference genome using comparably fewer resources

DeepNano: Deep Recurrent Neural Networks for Base Calling in MinION Nanopore Reads

Motivation: The MinION device by Oxford Nanopore is the first portable sequencing device. MinION is able to produce very long reads (reads over 100~kBp were reported), however it suffers from high sequencing error rate. In this paper, we show that the error rate can be reduced by improving the base calling process. Results: We present the first open-source DNA base caller for the MinION sequencing platform by Oxford Nanopore. By employing carefully crafted recurrent neural networks, our tool improves the base calling accuracy compared to the default base caller supplied by the manufacturer. This advance may further enhance applicability of MinION for genome sequencing and various clinical applications. Availability: DeepNano can be downloaded at http://compbio.fmph.uniba.sk/deepnano/. Contact: [email protected]

Oxford nanopore MinION sequencing enables rapid whole genome assembly of rickettsia typhi in a resource-limited setting

The infrastructure challenges and costs of next-generation sequencing have been largely overcome, for many sequencing applications, by Oxford Nanopore Technologies’ portable MinION sequencer. However, the question remains open whether MinION-based bacterial whole genome sequencing is by itself sufficient for the accurate assessment of phylogenetic and epidemiological relationships between isolates and whether such tasks can be undertaken in resource-limited settings. To investigate this question, we sequenced the genome of an isolate of Rickettsia typhi, an important and neglected cause of fever across much of the tropics and subtropics, for which only three genomic sequences previously existed. We prepared and sequenced libraries on a MinION in Vientiane, Lao PDR, using v9.5 chemistry, and in parallel, we sequenced the same isolate on the Illumina platform in a genomics laboratory in the United Kingdom. The MinION sequence reads yielded a single contiguous assembly, in which the addition of Illumina data revealed 226 base-substitution and 5,856 indel errors. The combined assembly represents the first complete genome sequence of a human R. typhi isolate collected in the last 50 years and differed from the genomes of existing strains collected over a 90-year time period at very few sites, with no rearrangements. Filtering based on the known error profile of MinION data improved the accuracy of the nanopore-only assembly. However, the frequency of false-positive errors remained greater than true sequence divergence from recorded sequences. Although nanopore-only sequencing cannot yet recover phylogenetic signals in R. typhi, such an approach may be applicable for more diverse organism