Zascavage Roxanne R, Hall Courtney L, Thorson Kelcie, Mahmoud Medhat, Sedlazeck Fritz J, Planz John V
Department of Criminology and Criminal Justice, University of Texas at Arlington, Arlington, Texas.
Department of Microbiology, Immunology and Genetics, University of North Texas Health Science Center, Fort Worth, Texas.
Curr Protoc Hum Genet. 2019 Dec;104(1):e94. doi: 10.1002/cphg.94.
Traditional approaches for interrogating the mitochondrial genome often involve laborious extraction and enrichment protocols followed by Sanger sequencing. Although preparation techniques are still demanding, the advent of next-generation or massively parallel sequencing has made it possible to routinely obtain nucleotide-level data with relative ease. These short-read sequencing platforms offer deep coverage with unparalleled read accuracy in high-complexity genomic regions but encounter numerous difficulties in the low-complexity homopolymeric sequences characteristic of the mitochondrial genome. The inability to discern identical units within monomeric repeats and resolve copy-number variations for heteroplasmy detection results in suboptimal genome assemblies that ultimately complicate downstream data analysis and interpretation of biological significance. Oxford Nanopore Technologies offers the ability to generate long-read sequencing data on a pocket-sized device known as the MinION. Nanopore-based sequencing is scalable, portable, and theoretically capable of sequencing the entire mitochondrial genome in a single contig. Furthermore, the recent development of a nanopore protein with dual reader heads allows for clear identification of nucleotides within homopolymeric stretches, significantly increasing resolution throughout these regions. The unrestricted read lengths, superior homopolymeric resolution, and affordability of the MinION device make it an attractive alternative to the labor-intensive, time-consuming, and costly mainstay deep-sequencing platforms. This article describes three approaches to extract, prepare, and sequence mitochondrial DNA on the Oxford Nanopore MinION device. Two of the workflows include enrichment of mitochondrial DNA prior to sequencing, whereas the other relies on direct sequencing of native genomic DNA to allow for simultaneous assessment of the nuclear and mitochondrial genomes. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Enrichment-free mitochondrial DNA sequencing Alternate Protocol 1: Mitochondrial DNA sequencing following enrichment with polymerase chain reaction (PCR) Alternate Protocol 2: Mitochondrial DNA sequencing following enrichment with PCR-free hybridization capture Support Protocol 1: DNA quantification and quality assessment using the Agilent 4200 TapeStation System Support Protocol 2: AMPure XP bead clean-up Support Protocol 3: Suggested data analysis pipeline.
传统的线粒体基因组检测方法通常需要繁琐的提取和富集流程，随后进行桑格测序。尽管样本制备技术仍然要求较高，但新一代测序或大规模平行测序技术的出现，使得相对轻松地常规获取核苷酸水平的数据成为可能。这些短读长测序平台在高复杂性基因组区域能提供深度覆盖且具有无与伦比的读取准确性，但在线粒体基因组特有的低复杂性同聚物序列中会遇到诸多困难。无法辨别单体重复内的相同单元以及解决异质性检测中的拷贝数变异，会导致基因组组装效果欠佳，最终使下游数据分析和生物学意义解读变得复杂。牛津纳米孔技术公司提供了在一种名为MinION的袖珍设备上生成长读长测序数据的能力。基于纳米孔的测序具有可扩展性、便携性，理论上能够在单个重叠群中对整个线粒体基因组进行测序。此外，最近开发的具有双读取头的纳米孔蛋白能够清晰识别同聚物延伸内的核苷酸，显著提高了这些区域的分辨率。MinION设备不受限制的读长、卓越的同聚物分辨率以及可承受的价格，使其成为劳动强度大、耗时且昂贵的主流深度测序平台的一个有吸引力的替代方案。本文介绍了在牛津纳米孔MinION设备上提取、制备和测序线粒体DNA的三种方法。其中两种工作流程包括在测序前富集线粒体DNA，而另一种则依赖于对天然基因组DNA进行直接测序，以便同时评估核基因组和线粒体基因组。© 2019约翰威立国际出版公司。基本方案：无富集线粒体DNA测序替代方案1：聚合酶链反应（PCR）富集后线粒体DNA测序替代方案2：无PCR杂交捕获富集后线粒体DNA测序支持方案1：使用安捷伦4200 TapeStation系统进行DNA定量和质量评估支持方案2：AMPure XP磁珠纯化支持方案3：建议的数据分析流程
