A Streamlined and High-Throughput Error-Corrected Next-Generation Sequencing Method for Low Variant Allele Frequency Quantitation

Quantifying mutant or variable allele frequencies (VAFs) of ≤10−3 using next-generation sequencing (NGS) has utility in both clinical and nonclinical settings. Two common approaches for quantifying VAFs using NGS are tagged single-strand sequencing and duplex sequencing. While duplex sequencing is reported to have sensitivity up to 10−8 VAF, it is not a quick, easy, or inexpensive method. We report a method for quantifying VAFs that are ≥10−4 that is as easy and quick for processing samples as standard sequencing kits, yet less expensive than the kits. The method was developed using PCR fragment-based VAFs of Kras codon 12 in log10 increments from 10−5 to 10−1, then applied and tested on native genomic DNA. For both sources of DNA, there is a proportional increase in the observed VAF to input VAF from 10−4 to 100% mutant samples. Variability of quantitation was evaluated within experimental replicates and shown to be consistent across sample preparations. The error at each successive ba...