Oxford Nanopore MinION Sequencing and Genome Assembly

The revolution of genome sequencing is continuing after the successful second-generation sequencing (SGS) technology. The third-generation sequencing (TGS) technology, led by Pacific Biosciences (PacBio), is progressing rapidly, moving from a technology once only capable of providing data for small genome analysis, or for performing targeted screening, to one that pro-mises high quality de novo assembly and structural variation detection for human-sized genomes. In 2014, the MinION, the first commercial sequencer using nanopore technology, was released by Oxford Nanopore Technologies (ONT). MinION identifies DNA bases by measuring the changes in electrical conductivity generated as DNA strands pass through a biological pore. Its portability, affordability, and speed in data production makes it suitable for real-time applications, the release of the long read sequencer MinION has thus generated much excitement and interest in the geno-mics community. While de novo genome assemblies can be cheaply produced from SGS data, assem-bly continuity is often relatively poor, due to the limited ability of short reads to handle long repeats. Assembly quality can be greatly improved by using TGS long reads, since repetitive regions can be easily expanded into using longer sequencing lengths, despite having higher error rates at the base level. The potential of nanopore sequencing has been demonstrated by various studies in gen-ome surveillance at locations where rapid and reliable sequencing is needed, but where resources are limited.     

纳米孔测序信号处理及其在DNA数据存储的应用

随着高通量测序技术的不断更新,可以在单个分子水平读取核苷酸序列的第三代测序技术迅速发展,纳米孔测序技术是其具有代表性的单分子测序技术,该技术通过检测DNA单链分子穿过纳米孔时引起的跨膜电流信号的变化,实现碱基识别.纳米孔测序仪在便携性、碱基读取速度、测序读段长度等方面较传统的第一代与第二代测序技术都有明显优势.随着纳米...     

纳米孔测序技术应用于食品微生物检测的可行性分析

近些年来DNA测序技术发展迅速,已经从第一代生化测序发展到第三代单分子测序。作为第三代测序技术中的一种不同于当前流行的其他测序技术,纳米孔测序技术是基于电信号的一种物理方法测序。许多研究者通常将高通量测序技术应用于食品微生物的研究,但是将纳米孔测序技术应用于食品中微生物的检测却鲜有报道。Oxford Nanopore Technologies(牛津纳米孔科技公司)研发的DNA测序仪MinION,是世界首例用于商业测序的纳米孔测序仪,经过不断完善,近年来MinION在DNA测序中被广泛应用。MinION 测序一次需要的DNA量约1μg,其标准识别速度为一秒钟识别250个碱基,平均读长可至13kb~20kb,测序准确率可以达到98%。纳米孔测序的高识别速度和高准确率,完全满足快速检测的要求,将其应用于食品中微生物检测是完全可行的。     

Nanopore sequencing technology: research trends and applications

Nanopore sequencing is one of the most promising technologies being developed as a cheap and fast alternative to the conventional Sanger sequencing method. Protein or synthetic nanopores have been used to detect DNA or RNA molecules. Although none of the technologies to date has shown single-base resolution for de novo DNA sequencing, there have been several reports of alpha-hemolysin protein nanopores being used for basic DNA analyses, and various synthetic nanopores have been fabricated. This review will examine current nanopore sequencing technologies, including recent developments of new applications.     

Recent patents of nanopore DNA sequencing technology: progress and challenges

DNA sequencing techniques witnessed fast development in the last decades, primarily driven by the Human Genome Project. Among the proposed new techniques, Nanopore was considered as a suitable candidate for the single DNA sequencing with ultrahigh speed and very low cost. Several fabrication and modification techniques have been developed to produce robust and well-defined nanopore devices. Many efforts have also been done to apply nanopore to analyze the properties of DNA molecules. By comparing with traditional sequencing techniques, nanopore has demonstrated its distinctive superiorities in main practical issues, such as sample preparation, sequencing speed, cost-effective and read-length. Although challenges still remain, recent researches in improving the capabilities of nanopore have shed a light to achieve its ultimate goal: Sequence individual DNA strand at single nucleotide level. This patent review briefly highlights recent developments and technological achievements for DNA analysis and sequencing at single molecule level, focusing on nanopore based methods.     

Nanopore-based Fourth-generation DNA Sequencing Technology

Nanopore-based sequencers, as the fourth-generation DNA sequencing technology, have the potential to quickly and reliably sequence the entire human genome for less than $1000, and possibly for even less than $100. The single-molecule techniques used by this technology allow us to further study the interaction between DNA and protein, as well as between protein and protein.Nanopore analysis opens a new door to molecular biology investigation at the single-molecule scale.In this article, we have reviewed academic achievements in nanopore technology from the past as well as the latest advances, including both biological and solid-state nanopores, and discussed their recent and potential applications.     

第三代测序基本原理

文章阐述了以单分子实时测序和纳米孔技术为标志第三代测序的基本原理,介绍了Helicos的Heliscope单分子测序仪、Pacific Bioscience的SMRT技术和Oxford Nanopore Technologies公司正在研究的纳米孔单分子测序技术。与其他测序技术进行了简单的对比以并提出一些单分子测序仍需面对的问题以及对未来单分子测序的展望。     

单分子测序技术及应用研究进展

从DNA双螺旋结构的发现开始,生命科学研究进入分子水平,在20世纪70年代出现的测序技术为破译遗传密码作出了巨大贡献.近几年出现的单分子测序技术,可以在单个分子水平读取核苷酸序列,也被称为第三代测序技术,主要代表有HeliScope、Nanopore和PacBio等.与传统的第一代和第二代测序技术相比,第三代测序能够产生更长的碱基读长,能直接对RNA进行测序,无需逆转录,测序速度极快,同时其中某些技术所涉及的设备可以小型化,可便携至野外现场测序.第三代测序技术在生命科学基础理论研究及生物医学临床实践中,具有广泛的应用.本文重点介绍了各种单分子测序技术的原理、优缺点,及其应用研究进展.     

Genome assembly using Nanopore-guided long and error-free DNA reads

Background

Long-read sequencing technologies were launched a few years ago, and in contrast with short-read sequencing technologies, they offered a promise of solving assembly problems for large and complex genomes. Moreover by providing long-range information, it could also solve haplotype phasing. However, existing long-read technologies still have several limitations that complicate their use for most research laboratories, as well as in large and/or complex genome projects. In 2014, Oxford Nanopore released the MinION® device, a small and low-cost single-molecule nanopore sequencer, which offers the possibility of sequencing long DNA fragments.

Results

The assembly of long reads generated using the Oxford Nanopore MinION® instrument is challenging as existing assemblers were not implemented to deal with long reads exhibiting close to 30% of errors. Here, we presented a hybrid approach developed to take advantage of data generated using MinION® device. We sequenced a well-known bacterium, Acinetobacter baylyi ADP1 and applied our method to obtain a highly contiguous (one single contig) and accurate genome assembly even in repetitive regions, in contrast to an Illumina-only assembly. Our hybrid strategy was able to generate NaS (Nanopore Synthetic-long) reads up to 60 kb that aligned entirely and with no error to the reference genome and that spanned highly conserved repetitive regions. The average accuracy of NaS reads reached 99.99% without losing the initial size of the input MinION® reads.

Conclusions

We described NaS tool, a hybrid approach allowing the sequencing of microbial genomes using the MinION® device. Our method, based ideally on 20x and 50x of NaS and Illumina reads respectively, provides an efficient and cost-effective way of sequencing microbial or small eukaryotic genomes in a very short time even in small facilities. Moreover, we demonstrated that although the Oxford Nanopore technology is a relatively new sequencing technology, currently with a high error rate, it is already useful in the generation of high-quality genome assemblies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1519-z) contains supplementary material, which is available to authorized users.     

High resolution copy number inference in cancer using short-molecule nanopore sequencing

Genome copy number is an important source of genetic variation in health and disease. In cancer, Copy Number Alterations (CNAs) can be inferred from short-read sequencing data, enabling genomics-based precision oncology. Emerging Nanopore sequencing technologies offer the potential for broader clinical utility, for example in smaller hospitals, due to lower instrument cost, higher portability, and ease of use. Nonetheless, Nanopore sequencing devices are limited in the number of retrievable sequencing reads/molecules compared to short-read sequencing platforms, limiting CNA inference accuracy. To address this limitation, we targeted the sequencing of short-length DNA molecules loaded at optimized concentration in an effort to increase sequence read/molecule yield from a single nanopore run. We show that short-molecule nanopore sequencing reproducibly returns high read counts and allows high quality CNA inference. We demonstrate the clinical relevance of this approach by accurately inferring CNAs in acute myeloid leukemia samples. The data shows that, compared to traditional approaches such as chromosome analysis/cytogenetics, short molecule nanopore sequencing returns more sensitive, accurate copy number information in a cost effective and expeditious manner, including for multiplex samples. Our results provide a framework for short-molecule nanopore sequencing with applications in research and medicine, which includes but is not limited to, CNAs.     

不同DNA聚合酶对运用ARTIC工作流的新型冠状病毒纳米孔测序的影响

摘要 目的：为了验证不同高保真DNA聚合酶是否会对运用ARTIC工作流进行新型冠状病毒纳米孔测序产生影响。方法：使用英国Nanopore公司MinION测序仪对2份已获得全基因组序列的新冠肺炎确诊病例核酸样本分别采用KAPA HiFi HotStart ReadyMix,PrimeSTAR?誖GXL DNA Polymerase和NEBNext High-Fidelity 2X PCR Master Mix进行ARTIC工作流的多重PCR扩增,对扩增产物进行测序,并对测序质量进行分析。结果：不同高保真DNA聚合酶在相同扩增条件下,扩增产物的质检结果和测序质量均不相同,NEBNext High-Fidelity 2X PCR Master Mix在覆盖度和测序深度上明显好于另外两种酶。结论：NEBNext High-Fidelity 2X PCR Master Mix在纳米孔新型冠状病毒ARTIC快速测序工作流中的应用效果较好。     

固态纳米孔:下一代DNA测序技术——原理、工艺与挑战

固态纳米孔测序技术作为新兴的第四代DNA测序技术,具有低成本、高读长、易集成等优势.如今,随着半导体工艺技术的飞速发展,小型化、高速度、大通量的纳米孔测序芯片的实现成为可能.相比传统的测序技术,固态纳米孔测序技术在成本、速度等方面有着十分巨大的优势.然而,作为一种新兴的测序技术,固态纳米孔在制造、测序、集成等方面也存在着诸多挑战.本文主要介绍了纳米孔测序技术的原理、制备工艺和面临的挑战,并展望了未来纳米孔测序技术的发展前景.     

DNA sequencing: an overview of solid-state and biological nanopore-based methods

The field of sequencing is a topic of significant interest since its emergence and has become increasingly important over time. Impressive achievements have been obtained in this field, especially in relations to DNA and RNA sequencing. Since the first achievements by Sanger and colleagues in the 1950s, many sequencing techniques have been developed, while others have disappeared. DNA sequencing has undergone three generations of major evolution. Each generation has its own specifications that are mentioned briefly. Among these generations, nanopore sequencing has its own exciting characteristics that have been given more attention here. Among pioneer technologies being used by the third-generation techniques, nanopores, either biological or solid-state, have been experimentally or theoretically extensively studied. All sequencing technologies have their own advantages and disadvantages, so nanopores are not free from this general rule. It is also generally pointed out what research has been done to overcome the obstacles. In this review, biological and solid-state nanopores are elaborated on, and applications of them are also discussed briefly.     

Protein sequence analysis using Hewlett-Packard biphasic sequencing cartridges in an applied biosystems 473A protein sequencer

Protein sequence analysis using an adsorptive biphasic sequencing cartridge, a set of two coupled columns introduced by Hewlett-Packard for protein sequencing by Edman degradation, in an Applied Biosystems 473A protein sequencer has been demonstrated. Samples containing salts, detergents, excipients, etc. (e.g., formulated protein drugs) can be easily analyzed using the ABI sequencer. Simple modifications to the ABI sequencer to accommodate the cartridge extend its utility in the analysis of difficult samples. The ABI sequencer solvents and reagents were compatible with the HP cartridge for sequencing. Sequence information up to ten residues can be easily generated by this nonoptimized procedure, and it is sufficient for identifying proteins by database search and for preparing a DNA probe for cloning novel proteins.     

Nucleotide discrimination with DNA immobilized in the MspA nanopore

Nanopore sequencing has the potential to become a fast and low-cost DNA sequencing platform. An ionic current passing through a small pore would directly map the sequence of single stranded DNA (ssDNA) driven through the constriction. The pore protein, MspA, derived from Mycobacterium smegmatis, has a short and narrow channel constriction ideally suited for nanopore sequencing. To study MspA's ability to resolve nucleotides, we held ssDNA within the pore using a biotin-NeutrAvidin complex. We show that homopolymers of adenine, cytosine, thymine, and guanine in MspA exhibit much larger current differences than in α-hemolysin. Additionally, methylated cytosine is distinguishable from unmethylated cytosine. We establish that single nucleotide substitutions within homopolymer ssDNA can be detected when held in MspA's constriction. Using genomic single nucleotide polymorphisms, we demonstrate that single nucleotides within random DNA can be identified. Our results indicate that MspA has high signal-to-noise ratio and the single nucleotide sensitivity desired for nanopore sequencing devices.     

单分子纳米孔测序技术及其应用研究进展

测序技术在通量和成本方面有了较大的改进,以单分子纳米孔测序技术为代表的第三代测序技术更是以其超长读长、实时检测和可以直接检测碱基甲基化修饰等优势在医学及生命科学等领域作出了较大贡献。文中就单分子纳米孔测序技术的原理进行了简要描述,并对其在临床、动物、植物、细菌及病毒等领域的应用和其未来的发展方向进行了讨论。     

Breakpoint mapping of a novel de novo translocation t(X;20)(q11.1;p13) by positional cloning and long read sequencing

Disease associated chromosomal rearrangements often have break points located within disease causing genes or in their vicinity. The purpose of this study is to characterize a balanced reciprocal translocation in a girl with intellectual disability and seizures by positional cloning and whole genome sequencing. The translocation was identification by G- banding and confirmed by WCP FISH. Fine mapping using BAC clones and whole genome sequencing using Oxford nanopore long read sequencing technology for a 1.46 X coverage of the genome was done. The positional cloning showed split signals with BAC RP11-943 J20. Long read sequencing analysis of chimeric reads carrying parts of chromosomes X and 20 helped to identify the breakpoints to be in intron 2 of ARHGEF9 gene on Xp11.1 and on 20p13 between RASSF2 and SLC23A2 genes. This is the first report of translocation which successfully delineated to single base resolution using Nanopore sequencing. The genotype-phenotype correlation is discussed.     

纳米孔测序技术在环境微生物研究中的应用

高通量测序技术是研究环境微生物的有效手段,而以纳米孔测序为代表的第三代测序技术以其测序读长长、测序速度快、测序数据实时监控、仪器方便携带、无GC偏好性、无需经过PCR扩增等显著优势有力推动了环境微生物研究的发展.本文对纳米孔测序技术的技术原理和特点进行了简要概述,重点介绍了纳米孔测序技术在环境微生物扩增子测序、宏基因组...     

纳米孔测序技术在病毒性传染病检测及研究中的应用

快速、准确鉴定出病原体是临床感染性疾病诊断和传染病预防控制的基础。高通量测序基因检测技术突破了传统检测手段的时效性、灵敏度等的局限，为病原体检测和研究提供了便捷、高效的途径。本综述以高通量测序技术发展过程为基础，回顾纳米孔三代测序技术，及其在病毒性传染病检测鉴定及研究中的应用，并对该技术的应用前景及可能存在的问题进行阐述，期望它能在病毒性传染病的防控方面发挥更大的作用。     

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site, but also into unintended off-target sites. DAJIN, a novel nanopore sequencing data analysis tool, identifies and quantifies allele numbers and their mutation patterns, reporting consensus sequences and visualizing mutations in alleles at single-nucleotide resolution.