Rapid Methods for Precise Validation of Germline and Mosaic Genetics Mutations
This invention provides a rapid, precise, and sensitive approach for identifying and validating genetic mutations
Background
Previous methods and studies have clearly shown the important role of mosaic mutations in disease pathogenesis but quantifying the exact level of these mutations in a tissue have traditionally relied on slow, low-throughput, and costly methods such as ddPCR (requiring expensive allele-specific probes) and topo-cloning (requiring 100s of sanger sequencing reactions for an estimate for mosaicism). While others have begun using ultradeep amplicon sequencing for validation of mosaic alleles, these studies remain limited due to the well-known issues of PCR-induced allelic dropout and other amplification biases. These issues result in the potential to miss or inaccurately quantitate a mutation. Therefore, the gold-standard has been the single allele testing by ddPCR, which can be expensive, limiting its application in the general clinical and scientific community.
Technology Overview
This invention combats the above weaknesses. It allows for flexible scalability to rapidly test nearly unlimited numbers of alleles in a single assay by combining the power of multiple different techniques, along with built-in replicates for statistical power. The novel approach for mosaic and germline testing described in this invention, based on generating deep coverage of overlapping amplicons of a target nucleic acid sequence, are cost-effective and time-effective approaches that are not available in the present market.
Further Details:
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Doan RN, Miller MB, Kim SN, Rodin RE, Ganz J, Bizzotto S, Morillo KS, Huang AY, Digumarthy R, Zemmel Z, Walsh CA. MIPP-Seq: ultra-sensitive rapid detection and validation of low-frequency mosaic mutations. BMC Med Genomics. 2021 Feb 12;14(1):47. doi: 10.1186/s12920-021-00893-3. PMID: 33579278; PMCID: PMC7881461.
Benefits
This invention is cost-effective and time-effective when compared to the current methods on the market, which are very slow, low-throughput, inefficient, and costly. This invention allows for the flexible scalability to rapidly test nearly unlimited numbers of alleles in a single assay by combining the power of different techniques, along with built in replicates for statistical power. The invention’s method is cost-effective, quicker, and efficient, and its unlimited allele testing ability surpasses the current standard method’s single allele testing ability.
Applications
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The detection of low frequency genetic variants, such as somatic mosaic variants. Genetic variation is an important variable in disease etiology and progression, evolution, and population genetics.
- The detection and monitoring of diseases, such as cancer. The detection and characterization of disease-associated variants, including somatic mosaic variants, can provide information relevant for diagnosing a disease, determining the progression or regression of disease, and treating disease. Changes in the allele frequency of a disease marker before, during, and after a disease has been detected can signal disease presence, growth, or regression.
