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 sequencing technology: nanopore preparations

For the past decade, nanometer-scale pores have been developed as a powerful technique for sensing biological macromolecules. Various potential applications using these nanopores have been reported at the proof-of-principle stage, with the eventual aim of using them as an alternative to de novo DNA sequencing. Currently, there have been two general approaches to prepare nanopores for nucleic acid analysis: organic nanopores, such as alpha-hemolysin pores, are commonly used for DNA analysis, whereas synthetic solid-state nanopores have also been developed using various conventional and non-conventional fabrication techniques. In particular, synthetic nanopores with pore sizes smaller than the alpha-hemolysin pores have been prepared, primarily by electron-beam-assisted techniques: these are more robust and have better dimensional adjustability. This review will examine current methods of nanopore preparation, ranging from organic pore preparations to recent developments in synthetic nanopore fabrications.     

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。     

Membrane platforms for biological nanopore sensing and sequencing

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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...     

利用纳米孔测序鉴定蓖麻蚕病害

蓖麻蚕Samia ricini为大蚕蛾科樗蚕属的绢丝与食用的非滞育型经济昆虫,且是少数可以室内大量圈养的绢丝昆虫,全年可累代饲养.在蓖麻蚕室内大批量圈养的生产过程可能遭受毁灭性病害的侵害,包括蓖麻蚕微粒子病、脓病、软化病等,且随着累代对病原物的不停积累蚕病会呈现愈发严重的趋势,病原愈发难以清除.因此,对蚕病的监测是防控蚕病爆发的重要手段之一.掌上纳米孔测序仪MinION是一款便携式高通量测序平台,便于实地进行DNA测序并对病原物进行检测.本文通过MinION测序仪对蓖麻蚕病蚕DNA进行测序,并对未知病原物DNA序列进行分析,探讨了利用MinION测序仪鉴定蓖麻蚕病原的可行性,对蚕病的监测提供了保障.     

Long-read whole-genome methylation patterning using enzymatic base conversion and nanopore sequencing

Long-read whole-genome sequencing analysis of DNA methylation would provide useful information on the chromosomal context of gene expression regulation. Here we describe the development of a method that improves the read length generated by using the bisulfite-sequencing-based approach. In this method, we combined recently developed enzymatic base conversion, where an unmethylated cytosine (C) should be converted to thymine (T), with nanopore sequencing. After methylation-sensitive base conversion, the sequencing library was constructed using long-range polymerase chain reaction. This type of analysis is possible using a minimum of 1 ng genomic DNA, and an N50 read length of 3.4–7.6 kb is achieved. To analyze the produced data, which contained a substantial number of base mismatches due to sequence conversion and an inaccurate base read of the nanopore sequencing, a new analytical pipeline was constructed. To demonstrate the performance of long-read methylation sequencing, breast cancer cell lines and clinical specimens were subjected to analysis, which revealed the chromosomal methylation context of key cancer-related genes, allele-specific methylated genes, and repetitive or deletion regions. This method should convert the intractable specimens for which the amount of available genomic DNA is limited to the tractable targets.     

基于纳米孔测序技术的柑橘黄龙病检测方法建立

柑橘黄龙病(Huanglongbing)是柑橘生产上最具毁灭性的病害,及时快速地进行早期检测和诊断是防控黄龙病的关键措施之一.本文利用掌上纳米孔测序仪MinION对携带黄龙病菌Candidatus Liberibacter asiaticus的DNA样品进行测序,并利用Blast、GraphMap、minimap2以及两种bwa的比对方法将测序结果比对到黄龙病菌基因组上,比对结果均匀的比对到黄龙病菌基因组上,并未发现严重的偏倚现象,验证了其测序结果的可靠性.本技术可弥补因柑橘木虱Diaphorina citri(Kuwayama)虫体过小或损坏难以进行光学识别的不足,并可同时检测虫体是否携带有黄龙病菌,对有黄龙病发生风险但尚未有黄龙病实际发生的柑橘种植区提供实时实地的监控与预警.     

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。     

纳米孔测序技术在实蝇类害虫检疫鉴定中的应用

【目的】纳米孔测序技术是单分子实时测序技术的一种,正在被广泛应用于临床快速诊断及微生物检测等领域。本研究以实蝇这一类重要的检疫性有害生物为例,探究该技术在昆虫检疫鉴定中应用的可行性,为昆虫检疫鉴定提供新方法。【方法】分别采用一代测序技术和纳米孔测序技术,对14种经形态学鉴定的实蝇成虫进行DNA条形码测序,通过BOLD和NCBI数据库对测序结果进行比对分析,并比较2种测序技术所得序列结果准确性的差异。【结果】通过纳米孔测序,14个实蝇样品在44 min内获得181 Mb bases,每个样品平均得到11280条reads,单个reads的准确度为92.10%~94.53%;经一致性序列校正,所有实蝇样品均可得到与一代测序结果完全一致的序列,序列分析结果与形态学鉴定结果完全一致。【结论】采用本研究的实验流程和数据分析方法,纳米孔测序技术可以应用于实蝇类害虫的检疫鉴定,测序结果准确、高效;本研究提供的实验方案同样适用于基于扩增子测序的物种鉴定,满足大规模样品的高通量精准鉴定需求。     

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.     

Bioinformatics challenges of new sequencing technology

New DNA sequencing technologies can sequence up to one billion bases in a single day at low cost, putting large-scale sequencing within the reach of many scientists. Many researchers are forging ahead with projects to sequence a range of species using the new technologies. However, these new technologies produce read lengths as short as 35-40 nucleotides, posing challenges for genome assembly and annotation. Here we review the challenges and describe some of the bioinformatics systems that are being proposed to solve them. We specifically address issues arising from using these technologies in assembly projects, both de novo and for resequencing purposes, as well as efforts to improve genome annotation in the fragmented assemblies produced by short read lengths.     

An informatics approach to distinguish RNA modifications in nanopore direct RNA sequencing

Modifications in RNA can influence their structure, function, and stability and play essential roles in gene expression and regulation. Methods to detect RNA modifications rely on biophysical techniques such as chromatography or mass spectrometry, which are low throughput, or on high throughput short-read sequencing techniques based on selectively reactive chemical probes. Recent studies have utilized nanopore-based fourth-generation sequencing methods to detect modifications by directly sequencing RNA in its native state. However, these approaches are based on modification-associated mismatch errors that are liable to be confounded by SNPs. Also, there is a need to generate matched knockout controls for reference, which is laborious. In this work, we introduce an internal comparison strategy termed “IndoC,” where features such as ‘trace’ and ‘current signal intensity’ of potentially modified sites are compared to similar sequence contexts on the same RNA molecule within the sample, alleviating the need for matched knockout controls. We first show that in an IVT model, ‘trace’ is able to distinguish between artificially generated SNPs and true pseudouridine (Ψ) modifications, both of which display highly similar mismatch profiles. We then apply IndoC on yeast and human ribosomal RNA to demonstrate that previously reported Ψ sites show marked changes in their trace and signal intensity profiles compared with their unmodified counterparts in the same dataset. Finally, we perform direct RNA sequencing of RNA containing Ψ intact with a chemical probe adduct (N-cyclohexyl-N′-β-(4-methylmorpholinium) ethylcarbodiimide [CMC]) and show that CMC reactivity also induces changes in trace and signal intensity distributions in a Ψ specific manner, allowing their separation from high mismatch sites that display SNP-like behavior.     

ENDO-Pore: high-throughput linked-end mapping of single DNA cleavage events using nanopore sequencing

Mapping the precise position of DNA cleavage events plays a key role in determining the mechanism and function of endonucleases. ENDO-Pore is a high-throughput nanopore-based method that allows the time resolved mapping single molecule DNA cleavage events in vitro. Following linearisation of a circular DNA substrate by the endonuclease, a resistance cassette is ligated recording the position of the cleavage event. A library of single cleavage events is constructed and subjected to rolling circle amplification to generate concatemers. These are sequenced and used to produce accurate consensus sequences. To identify the cleavage site(s), we developed CSI (Cleavage Site Investigator). CSI recognizes the ends of the cassette ligated into the cleaved substrate and triangulates the position of the dsDNA break. We firstly benchmarked ENDO-Pore using Type II restriction endonucleases. Secondly, we analysed the effect of crRNA length on the cleavage pattern of CRISPR Cas12a. Finally, we mapped the time-resolved DNA cleavage by the Type ISP restriction endonuclease LlaGI that introduces random double-strand breaks into its DNA substrates.