Shotgun sequencing

Gene

Exome

Deep sequencing

Genome

Ion semiconductor sequencing

Exome sequencing

Single cell sequencing

Massive parallel sequencing

Mutation

DNA sequencing

Genetics

Computational biology

Sanger sequencing

Locus (genetics)

Biology

Nature Biomedical Engineering

Assaying for large numbers of low-frequency mutations requires sequencing at extremely high depth and accuracy. Increasing sequencing depth aids the detection of low-frequency mutations yet limits the number of loci that can be simultaneously probed. Here we report a method for the accurate tracking of thousands of distinct mutations that requires substantially fewer reads per locus than conventional hybrid-capture duplex sequencing. The method, which we named MAESTRO (for minor-allele-enriched sequencing through recognition oligonucleotides), combines massively parallel mutation enrichment with duplex sequencing to track up to 10,000 low-frequency mutations, with up to 100-fold fewer reads per locus. We show that MAESTRO can be used to test for chimaerism by tracking donor-exclusive single-nucleotide polymorphisms in sheared genomic DNA from human cell lines, to validate whole-exome sequencing and whole-genome sequencing for the detection of mutations in breast-tumour samples from 16 patients, and to monitor the patients for minimal residual disease via the analysis of cell-free DNA from liquid biopsies. MAESTRO improves the breadth, depth, accuracy and efficiency of mutation testing by sequencing. The combination of massively parallel mutation enrichment and duplex sequencing allows for the tracking of up to 10,000 low-frequency mutations with up to 100-fold fewer reads per locus than conventional duplex sequencing.

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