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 > 1 Mb 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 MF_minI – or all mutations observed including recurrences – the larger maximum independent-mutation frequency MF_maxI which may reflect clonal expansion. Ultrasensitive methods reveal that, without their use, even mutations with VAF 0.5–1% are usually spurious.

突变是生物体 DNA 序列中不可逆的变化，以变异等位基因频率 (VAF) 存在于组织中，范围从每 bp 约 10 ^-8的创始突变到约 10 ^-3包含多个独立克隆的组织学正常组织样本– 与杂合肿瘤突变或多态性的 1%− 50% 相比。这些事件的罕见性对准确的临床诊断和预后、毒理学以及发现新的疾病病因提出了挑战。标准下一代测序 (NGS) 技术报告 VAF 低至每个 0.5%，但可靠地观察罕见的前体事件需要额外的复杂性来测量超低频突变。我们详细介绍了挑战；定义用于表征结果的术语，这些术语在实验室之间有所不同，有时生物学家和生物信息学家之间会发生冲突；并描述改进标准 NGS 方法的最新创新，包括：单链共有序列方法，例如 Safe-SeqS 和 SiMSen-Seq；串联链共有序列方法，例如o2n-Seq和SMM-Seq；以及超灵敏亲本链共有序列方法，例如 DuplexSeq、PacBio HiFi、SinoDuplex、OPUSeq、EcoSeq、BotSeqS、Hawk-Seq、NanoSeq、SaferSeq 和 CODEC。还指出了实际应用。多种方法将每个 nt 的 VAF 量化至低至 10 ^-5，并将目标区域中的突变频率 (MF) 量化至每个 nt 10 ^{-7 。}通过扩展到 > 1 Mb 的位点，从未观察到两次，从而放弃 VAF，其他方法可以量化每个 nt 的MF < 10 ^{-9或每个单倍体基因组的 < 15 个错误。}克隆扩增无法通过测序直接与独立突变区分开来，因此论文必须报告其 MF 是否仅计算不同的突变（最小独立突变频率 MF minI）或观察到的所有突变（包括复发）（较大的最大独立突变_频率）突变频率 MF _maxI可能反映克隆扩张。超灵敏方法表明，如果不使用它们，即使 VAF 为 0.5-1% 的突变通常也是虚假的。