Next-generation sequencing methodologies to detect low-frequency mutations: "Catch me if you can"

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.