A sequencer coming of age: De novo genome assembly using MinION reads

Lannoy, Carlos de; Ridder, Dick de; Risse, Judith

doi:10.12688/f1000research.12012.1

Cited by 29 publications

(22 citation statements)

References 41 publications

(18 reference statements)

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“…AAAAAAA) is challenging. Both of these problems make deletions the dominant error of nanopore sequencing [25,26]. Thus, basecalling is the most important step of the pipeline that plays a critical role in decreasing the error rate.…”

Section: Basecallingmentioning

confidence: 99%

“…Previous genome assembly methods designed for accurate and short reads (i.e. de Bruijn graph approach [40,41]) are not suitable for nanopore reads because of the high error rates of the current nanopore sequencing devices [9,26,42,43]. Instead, OLC algorithms [44] are used for nanopore sequencing reads, as they perform better with longer, error-prone reads.…”

Section: Read-to-read Overlap Findingmentioning

confidence: 99%

“…Polishing, i.e. postassembly error-correction, improves the accuracy of the draft assembly by mapping the reads to the assembly and changing the assembly to increase local similarity with the reads [26,50,51]. The first step of polishing is mapping the basecalled reads to the generated draft assembly from the previous step.…”

Section: Read Mapping and Polishingmentioning

confidence: 99%

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Nanopore sequencing technology and tools for genome assembly: computational analysis of the current state, bottlenecks and future directions

Cali

Kim

Ghose

et al. 2018

Briefings in Bioinformatics

165

128

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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. Based on our analyses, we make four key observations: (1) the choice of the tool for basecalling plays a critical role in overcoming the high error rates of nanopore sequencing technology. (2) Read-to-read overlap finding tools, GraphMap and Minimap, perform similarly in terms of accuracy. However, Minimap has a lower memory usage, and it is faster than GraphMap. (3) There is a trade-off between accuracy and performance when deciding on the appropriate tool for the assembly step. The fast but less accurate assembler Miniasm can be used for quick initial assembly, and further polishing can be applied on top of it to increase the accuracy, which leads to faster overall assembly. (4) The state-of-the-art polishing tool, Racon, generates high-quality consensus sequences while providing a significant speedup over another polishing tool, Nanopolish. We analyze various combinations of different tools and expose the trade-offs 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, to overcome the high error rates of the nanopore sequencing technology.

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Section: Basecallingmentioning

confidence: 99%

Section: Read-to-read Overlap Findingmentioning

confidence: 99%

See 1 more Smart Citation

Nanopore sequencing technology and tools for genome assembly: computational analysis of the current state, bottlenecks and future directions

Cali

Kim

Ghose

et al. 2018

Briefings in Bioinformatics

165

128

View full text Add to dashboard Cite

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“…But as a major drawback genome sequence-based analyses have to be conducted in an advanced laboratory setting, requiring highly specialized bioinformatics expertise or expensive commercial software ( Franz et al, 2014 ; Eppinger and Cebula, 2015 ; Parsons et al, 2016 ; Newell and La Ragione, 2018 ). Furthermore as long as portable sequencing devices like Oxford nanopore are not commonly used and error-prone ( Laver et al, 2015 ; Lu et al, 2016 ; de Lannoy et al, 2017 ), other ubiquitously usable and cheap DNA-based methods need to be developed for an on-site hazard characterization.…”

Section: Introductionmentioning

confidence: 99%

Identification of Novel Biomarkers for Priority Serotypes of Shiga Toxin-Producing Escherichia coli and the Development of Multiplex PCR for Their Detection

et al. 2018

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It would be desirable to have an unambiguous scheme for the typing of Shiga toxin-producing Escherichia coli (STEC) isolates to subpopulations. Such a scheme should take the high genomic plasticity of E. coli into account and utilize the stratification of STEC into subgroups, based on serotype or phylogeny. Therefore, our goal was to identify specific marker combinations for improved classification of STEC subtypes. We developed and evaluated two bioinformatic pipelines for genomic marker identification from larger sets of bacterial genome sequences. Pipeline A performed all-against-all BLASTp analyses of gene products predicted in STEC genome test sets against a set of control genomes. Pipeline B identified STEC marker genes by comparing the STEC core proteome and the “pan proteome” of a non-STEC control group. Both pipelines defined an overlapping, but not identical set of discriminative markers for different STEC subgroups. Differential marker prediction resulted from differences in genome assembly, ORF finding and inclusion cut-offs in both workflows. Based on the output of the pipelines, we defined new specific markers for STEC serogroups and phylogenetic groups frequently associated with outbreaks and cases of foodborne illnesses. These included STEC serogroups O157, O26, O45, O103, O111, O121, and O145, Shiga toxin-positive enteroaggregative E. coli O104:H4, and HUS-associated sequence type (ST)306. We evaluated these STEC marker genes for their presence in whole genome sequence data sets. Based on the identified discriminative markers, we developed a multiplex PCR (mPCR) approach for detection and typing of the targeted STEC. The specificity of the mPCR primer pairs was verified using well-defined clinical STEC isolates as well as isolates from the ECOR, DEC, and HUSEC collections. The application of the STEC mPCR for food analysis was tested with inoculated milk. In summary, we evaluated two different strategies to screen large genome sequence data sets for discriminative markers and implemented novel marker genes found in this genome-wide approach into a DNA-based typing tool for STEC that can be used for the characterization of STEC from clinical and food samples.

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“…Two main strategies have been used to assemble bacterial genomes using MinION sequencing [27, 28]. In the first, MinION reads are used to enhance genome assemblies that are generated from short-read Illumina data.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing

Goldstein

Beka

Graf

et al. 2018

Preprint

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BackgroundShort-read sequencing technologies have made microbial genome sequencing cheap and accessible. However, closing genomes is often costly and assembling short reads from genomes that are repetitive and/or have extreme %GC content remains challenging. Long-read, single-molecule sequencing technologies such as the Oxford Nanopore MinION have the potential to overcome these difficulties, although the best approach for harnessing their potential remains poorly evaluated.ResultsWe sequenced nine bacterial genomes spanning a wide range of GC contents using Illumina MiSeq and Oxford Nanopore MinION sequencing technologies to determine the advantages of each approach, both individually and combined. Assemblies using only MiSeq reads were highly accurate but lacked contiguity, a deficiency that was partially overcome by adding MinION reads to these assemblies. Even more contiguous genome assemblies were generated by using MinION reads for initial assembly, but these were more error-prone and required further polishing. This was especially pronounced when Illumina libraries were biased, as was the case for our strains with both high and low GC content. Increased genome contiguity dramatically improved the annotation of insertion sequences and secondary metabolite biosynthetic gene clusters, likely because long-reads can disambiguate these highly repetitive but biologically important genomic regions.ConclusionsGenome assembly using short-reads is challenged by repetitive sequences and extreme GC contents. Our results indicate that these difficulties can be largely overcome by using single-molecule, long-read sequencing technologies such as the Oxford Nanopore MinION. Using MinION reads for assembly followed by polishing with Illumina reads generated the most contiguous genomes and enabled the accurate annotation of important but difficult to sequence genomic features such as insertion sequences and secondary metabolite biosynthetic gene clusters. The combination of Oxford Nanopore and Illumina sequencing is cost effective and dramatically advances studies of microbial evolution and genome-driven drug discovery.

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A sequencer coming of age: De novo genome assembly using MinION reads

Cited by 29 publications

References 41 publications

Nanopore sequencing technology and tools for genome assembly: computational analysis of the current state, bottlenecks and future directions

Nanopore sequencing technology and tools for genome assembly: computational analysis of the current state, bottlenecks and future directions

Identification of Novel Biomarkers for Priority Serotypes of Shiga Toxin-Producing Escherichia coli and the Development of Multiplex PCR for Their Detection

Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing

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