Quantification of Genetic Heterogeneity Using Long‐Read Targeted Individual DNA Molecule Sequencing

Abstract

AbstractUnderstanding genetic heterogeneity is of paramount importance in unraveling the intricate functioning of biological systems, as it contributes to the diversity of phenotypes of gene‐environment interactions. We have developed a method termed targeted Individual DNA Molecule Sequencing (IDMseq) to accurately quantify genetic heterogeneity within cell populations, even those with rare variants present at low frequencies. IDMseq ensures that each original DNA molecule is distinctively represented by one unique molecule identifier (UMI) group, preventing false UMI groups and enabling precise quantification of allele frequency within the original population. IDMseq is a versatile sequencing technique that combines error correction and long‐read sequencing, enabling sensitive detection of various genetic variants, including single nucleotide variants and large structural variants in both basic and clinical research settings. This protocol provides a comprehensive, step‐by‐step guide to preparing samples and performing IDMseq to determine genetic variations. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol: UMI labeling and amplification of DNASupport Protocol 1: AMPure XP beads cleanupSupport Protocol 2: Suggested data analysis pipeline