Massively parallel enrichment of low-frequency alleles enables duplex sequencing at low depth.

Abstract

The ability to assay large numbers of low-frequency mutations is useful in biomedicine, yet, the technical hurdles of sequencing multiple mutations at extremely high depth, with accuracy, limits their detection in clinical practice. Low-frequency mutations can typically be detected by increasing the sequencing depth, however this limits the number of loci that can be probed for simultaneously. Here, we report a technique to accurately track thousands of distinct mutations with minimal reads, termed MAESTRO (minor allele enriched sequencing through recognition oligonucleotides), which employs massively-parallel mutation enrichment to enable duplex sequencing to track up to 10,000 low-frequency mutations, yet requiring up to 100-fold less sequencing. We show that MAESTRO could inform the mutation validation of whole-exome sequencing and whole genome sequencing data from tumor samples, enable chimerism testing, and is suitable for the monitoring of minimal residual disease via liquid biopsies. MAESTRO may improve the breadth, depth, accuracy, and efficiency of sequencing-based mutational testing.