变异，即生物的DNA序列无法逆转的改变，存在于组织中的变异等位基因频率（VAF）范围内，从～10^-8碱基对（bp）的创始变异到包含数个独立克隆的组织标本的组织学正常组织样品的VAF为～10^-3，而杂合肿瘤变异或多态性的VAF为1%-50%。这些事件的稀有性给准确的临床诊断和预后、毒理学以及新疾病病因的发现带来了挑战。标准的下一代测序（NGS）技术可以报告VAF低至0.5%每个碱基，但要可靠地观察到更罕见的前体事件，需要额外的先进技术来测量超低频变异。我们详细描述了这一挑战；定义了用于表征结果的术语，这些术语在实验室之间有所变化，有时在生物学家和生物信息学家之间存在冲突；并描述了最近改进了标准NGS方法的创新，包括：单链一致序列方法，如Safe-SeqS和SiMSen-Seq；串联链一致序列方法，如o2n-Seq和SMM-Seq；以及超灵敏母链一致序列方法， 如DuplexSeq、PacBio HiFi、SinoDuplex、OPUSeq、EcoSeq、BotSeqS、Hawk-Seq、NanoSeq、SaferSeq和CODEC。还介绍了实际应用。一些方法可以量化每个碱基的VAF低至10^-5，以及目标区域的突变频率（MF）低至每个碱基10^-7以下。通过扩展到从未观察到两次的超过1Mb位点，因此放弃VAF，其他方法可以量化每个碱基的MF <10^-9或<每个单倍体基因组15个错误。通过测序无法直接区分克隆扩张和独立变异，因此一篇论文报告其MF是否仅计算了不同的变异–最小独立变异频率（MFminI）–或包括重复出现的所有变异。超灵敏方法揭示了即使VAF为0.5% - 1%的变异通常也是虚假的，如果不使用超灵敏方法。 版权 ©2023，由Elsevier B.V.出版。

Mutations, the irreversible changes in an organism's DNA sequence, are present in tissues at a variant allele frequency (VAF) ranging from ~10-8 per bp for a founder mutation to ~10-3 for a histologically normal tissue sample containing several independent clones - compared to 1%-50% for a heterozygous tumor mutation or a polymorphism. The rarity of these events poses a challenge for accurate clinical diagnosis and prognosis, toxicology, and discovering new disease etiologies. Standard Next-Generation Sequencing (NGS) technologies report VAFs as low as 0.5% per nt, but reliably observing rarer precursor events requires additional sophistication to measure ultralow-frequency mutations. We detail the challenge; define terms used to characterize the results, which vary between laboratories and sometimes conflict between biologists and bioinformaticists; and describe recent innovations to improve standard NGS methodologies including: single-strand consensus sequence methods such as Safe-SeqS and SiMSen-Seq; tandem-strand consensus sequence methods such as o2n-Seq and SMM-Seq; and ultrasensitive parent-strand consensus sequence methods such as DuplexSeq, PacBio HiFi, SinoDuplex, OPUSeq, EcoSeq, BotSeqS, Hawk-Seq, NanoSeq, SaferSeq, and CODEC. Practical applications are also noted. Several methods quantify VAF down to 10-5 at a nt and mutation frequency (MF) in a target region down to 10-7 per nt. By expanding to >1Mb of sites never observed twice, thus forgoing VAF, other methods quantify MF <10-9 per nt or <15 errors per haploid genome. Clonal expansion cannot be directly distinguished from independent mutations by sequencing, so it is essential for a paper to report whether its MF counted only different mutations - the minimum independent-mutation frequency MFminI - or all mutations observed including recurrences - the larger maximum independent-mutation frequency MFmaxI which may reflect clonal expansion. Ultrasensitive methods reveal that, without their use, even mutations with VAF 0.5% - 1% are usually spurious.Copyright © 2023. Published by Elsevier B.V.