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长距离PCR和纳米孔测序用于定位和分相变异:一个端到端的临床应用工作流程

Javad Jamshidi^1,2,3, Conor Rowntree^4,5,6, Shannon Fadaee⁶

¹Neuroscience Research Australia, Sydney, NSW, Australia. j.jamshidi@neura.edu.au.

BMC medical genomics

|November 19, 2025

概括

这项研究引入了一种新的工作流程,使用远程PCR和纳米孔测序来准确的变体分相和定位. 这种方法克服了临床环境中复杂的遗传分析的短读测序限制.

科学领域:

基因组学就是基因组学.
分子生物学分子生物学
生物信息学是一种生物信息学.

背景情况:

短读测序在分期遥远变异和分析同源区域方面存在局限性.
精确的变异分相对于识别化合物异构性至关重要.
长读测序为复杂的基因组区域提供了改进的单元型分析.

研究的目的:

开发和验证一个强大的工作流程,用于变量分阶段和本地化.
为了使长读测序用于遗传诊断的临床实施.
在复杂的基因组分析中克服短读测序的局限性.

主要方法:

优化远程PCR (LR-PCR) 用于使用四个套件的高达20kB的目标.
针对性的纳米孔测序在Flongle流动细胞上,带有条码的amplicons.
开发了一个内部生物信息管道,包括Clair3,WhatsHap和HapCUT2用于变异调用和分阶段.

主要成果:

超跑长距离PCR套件在DNA放大到22kb的过程中取得了90%的成功.
所有测试的异质性SNV对和小的InDels (距离最多21.4kb) 均以100%的一致性分阶段测试.
调用低可映射性基因的SNV显示精度和灵敏度为1;中位数的仿真读取值为2.80%.

结论:

建立了一种可靠且负担得起的临床工作流程,用于分阶段变异,最高可达20kb.
允许在短读序列无法访问的区域进行变体本地化.
综合方法改善了复杂的遗传发现解决方案和诊断中的变异解释.

关键词:

远程PCR是一种远程PCR.纳米孔长读序列测序阶段化是分阶段进行的.

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