Optimization of multigene analysis of tumor samples
Inventors
Lipson, Doron • Otto, Geoffrey Alan • Parker, Alexander Nevin • Stephens, Philip James • Downing, Sean R. • Jarosz, Mirna • Shapiro, Mikhail G. • Yelensky, Roman
Assignees
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Publication Number
US-12180540-B2
Publication Date
2024-12-31
Expiration Date
Abstract
A method of analyzing a tumor sample comprising: (a) acquiring a library comprising a plurality of tumor members from a tumor sample;(b) contacting the library with a bait set to provide selected members;(c) acquiring a read for a subgenomic interval from a tumor member from said library;(d) aligning said read; and(e) assigning a nucleotide value (e.g., calling a mutation) from said read for the preselected nucleotide position, thereby analyzing said tumor sample.
Core Innovation
The invention relates to separating targeted nucleic acid molecules from a sample by forming nucleic acid hybrids using at least two different bait sets in the same solution. A nucleic acid library is combined with bait molecules comprising a binding entity and a sequence configured to capture a target sequence, and the resulting nucleic acid hybrids comprise a target nucleic acid molecule and a bait molecule. The plurality of nucleic acid hybrids is separated from a remainder of the library.
One bait set is configured to capture a target sequence comprising a point mutation, and another bait set is configured to capture a target sequence comprising a structural breakpoint. The approach integrates multiple bait sets in a single hybridization mixture, enabling targeted enrichment of different classes of alterations in the same workflow. Separating the nucleic acid hybrids enriches the library for nucleic acid molecules corresponding to the point-mutation target and the structural-breakpoint target.
The content also indicates per-target customization in alignment and Bayesian mutation calling using prior expectations and thresholds, including mutation present/read data and individualized priors. It further describes bait categories with different coverage-depth goals for low-frequency point mutations, higher-frequency mutations, pharmacogenomic and fingerprinting SNPs, intron targets for structural breakpoints, and sparse intron targeting for copy-number detection.
Claims Coverage
The provided documents include independent claims directed to forming nucleic acid hybrids from a library and at least two different bait sets in the same solution, where one bait set targets point mutations and another bait set targets structural breakpoints, followed by separating the hybrids from the remainder of the library. In total, the coverage centers on 4 inventive features.
Multiple bait sets in the same solution for hybrid formation
Combining, in the same solution, at least two different bait sets and a library comprising a plurality of nucleic acid molecules to form a plurality of nucleic acid hybrids, wherein each bait set comprises one or more bait molecules comprising a binding entity and a sequence configured to capture a target sequence.
Bait sets differentiated by target class (point mutation and structural breakpoint)
The at least two different bait sets comprise a first bait set comprising a bait molecule configured to capture a target sequence comprising a point mutation and a second bait set comprising a bait molecule configured to capture a target sequence comprising a structural breakpoint.
Hybrid separation from the remainder of the library
Separating the plurality of nucleic acid hybrids from a remainder of the library.
Defined hybrid composition for point-mutation vs structural-breakpoint targets
The plurality of nucleic acid hybrids comprises a first nucleic acid hybrid comprising a bait molecule from the first bait set and a target nucleic acid molecule comprising a point mutation and a second nucleic acid hybrid comprising a bait molecule from the second bait set and a target nucleic acid molecule comprising a structural breakpoint.
Overall, the independent claims cover an integrated targeted nucleic-acid enrichment strategy that uses at least two different bait sets in the same solution to form nucleic acid hybrids with defined hybrid contents for point mutations versus structural breakpoints, followed by separating those hybrids from the remainder of the library.
Stated Advantages
Separates targeted nucleic acid molecules by enriching nucleic acid hybrids from the remainder of the library.
The approach integrates multiple bait sets in a single hybridization mixture, enabling targeted enrichment of different classes of alterations in the same workflow.
Bayesian mutation calling uses prior expectation values per-site conditions or thresholds to obtain sensitivity and specificity for low-frequency somatic alterations.
Documented Applications
Targeted cancer and pharmacogenomic nucleic-acid subgenomic intervals to be captured and sequenced.
Downstream targeted analysis for tumor-related applications.
Workflows compatible with FFPE.
Gene panels that comprise cancer genes and pharmacogenomics (PGx) loci.
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