MAESTRO-Pool Enables Highly Parallel and Specific Mutation-Enrichment Sequencing for Minimal Residual Disease Detection in Cohort Studies.

BACKGROUND: Tracing patient-specific tumor mutations in cell-free DNA (cfDNA) for minimal residual disease (MRD) detection is promising but challenging. Assaying more mutations and cfDNA stands to improve MRD detection but requires highly accurate, efficient sequencing methods and proper calibration to prevent false detection with bespoke tests. METHODS: MAESTRO (Minor Allele Enriched Sequencing Through Recognition Oligonucleotides) uses mutation-specific oligonucleotide probes to enrich cfDNA libraries for tumor mutations and enable their accurate detection with minimal sequencing. A new approach, MAESTRO-Pool, which entails pooling MAESTRO probes for all patients and applying these to all samples from all patients, was used to screen for 22 333 tumor mutations from 9 melanoma patients in 98 plasma samples. This enabled quantification of MRD detection in patient-matched samples and false detection in unmatched samples from other patients. To detect MRD, a new dynamic MRD caller was used that computes a probability for MRD detection based on the number of mutations and cfDNA molecules sequenced, thereby calibrating for variations in each bespoke test. RESULTS: MAESTRO-Pool enabled sensitive detection of MRD down to 0.78 parts per million (ppm), reflecting a 10- to 100-fold improvement over existing tests. Of the 8 MRD positive samples with ultra-low tumor fractions <10 ppm, 7 were either in upward-trend preceding recurrence or downward-trend aligning with response. Of 784 patient-unmatched tests, only one was found as MRD positive (tumor fraction = 2.7 ppm), suggesting high specificity. CONCLUSIONS: MAESTRO-Pool enables massively parallel, tumor-informed MRD testing with concurrent benchmarking of bespoke MRD tests. Meanwhile, our new MRD caller enables more mutations and cfDNA molecules to be tested without compromising specificity. These improve the ability for detecting traces of MRD from blood.

A nuclease-polymerase chain reaction enables amplification of probes used for capture-based DNA target enrichment.

DNA target enrichment via hybridization capture is a commonly adopted approach which remains expensive due in-part to using biotinylated-probe panels. Here we provide a novel isothermal amplification reaction to amplify rapidly existing probe panels without knowledge of the sequences involved, thereby decreasing a major portion of the overall sample preparation cost. The reaction employs two thermostable enzymes, BST-polymerase and duplex-specific nuclease DSN. DSN initiates random 'nicks' on double-stranded-DNA which enable BST to polymerize DNA by displacing the nicked-strand. Displaced strands re-hybridize and the process leads to an exponential chain-reaction generating biotinylated DNA fragments within minutes. When starting from single-stranded-DNA, DNA is first converted to double-stranded-DNA via terminal-deoxynucleotidyl-transferase (TdT) prior to initiation of BST-DSN reaction. Biotinylated probes generated by TdT-BST-DSN (TBD) reactions using panels of 33, 190 or 7186 DNA targets are used for hybrid-capture-based target enrichment from amplified circulating-DNA, followed by targeted re-sequencing. Polymerase-nuclease isothermal-chain-reactions generate random amplified probes with no apparent sequence dependence. One round of target-capture using TBD probes generates a modest on-target sequencing ratio, while two successive rounds of capture generate >80% on-target reads with good sequencing uniformity. TBD-reactions generate enough capture-probes to increase by approximately two to three orders-of-magnitude the target-enrichment experiments possible from an initial set of probes.