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

Area of Science:

• Genomics and Molecular Biology
• Bioinformatics and Computational Biology
• Clinical Diagnostics and Prognostics

Background:

• Mutations, or changes in DNA, occur at varying frequencies in tissues, posing challenges for clinical applications due to their rarity.
• Standard Next-Generation Sequencing (NGS) has limitations in detecting ultralow-frequency mutations (VAFs < 0.5%), hindering accurate diagnosis and research.
• Inconsistent terminology and methodologies complicate the reliable characterization of mutation frequencies across different research groups.

Purpose of the Study:

• To define terms and characterize the challenges in measuring ultralow-frequency mutations.
• To review and describe recent innovations in sequencing methodologies designed to enhance the detection of rare DNA variants.
• To highlight the practical applications and implications of these advanced sequencing techniques.

Main Methods:

• Review and categorization of advanced sequencing techniques, including single-strand, tandem-strand, and ultrasensitive parent-strand consensus sequence methods.
• Detailed description of methods like Safe-SeqS, SiMSen-Seq, o2n-Seq, SMM-Seq, DuplexSeq, PacBio HiFi, and others.
• Discussion of approaches that quantify variant allele frequency (VAF) down to 10^-5 and mutation frequency (MF) down to 10^-7 per nucleotide, or even lower by analyzing large genomic regions.

Main Results:

• Ultrasensitive sequencing methods enable quantification of VAF down to 10^-5 and MF down to 10^-7 per nucleotide.
• Some methods can quantify MF below 10^-9 per nucleotide or <15 errors per haploid genome by analyzing extensive non-repeated sites.
• Ultrasensitive techniques demonstrate that many mutations previously observed at 0.5-1% VAF are spurious without these advanced methods.

Conclusions:

• Recent innovations in sequencing technologies significantly improve the ability to detect and quantify ultralow-frequency mutations.
• These ultrasensitive methods are essential for accurate clinical diagnosis, prognosis, toxicology, and identifying new disease causes.
• Distinguishing between clonal expansion and independent mutations requires clear reporting of mutation frequency metrics (MF_minI vs. MF_maxI).