Genomics-based identification and characterization of rare cell types
Inventors
Armour, Christopher • Lum, Pek Yee
Assignees
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Publication Number
US-12286622-B2
Publication Date
2025-04-29
Expiration Date
Abstract
This disclosure provides genomics-based methods that can be used to identify, quantify, and characterize rare cell types, including circulating tumor cells.
Core Innovation
The invention relates to a method of identifying the presence of a rare cell type in a biological sample by generating, for each of a plurality of nucleic acid subsets, a subset genomic library comprising barcoded double-stranded genomic DNA constructs. Each gDNA construct includes first and second gDNA strands carrying universal next generation sequencing primer sequences and a nucleic acid subset-specific molecular barcode configured to identify the subset origin.
Sequencing is performed on a pooled combined sequencing library obtained from the subset genomic libraries, and the presence of the rare cell type is determined by detecting a nucleic acid subset containing a rare-cell gDNA sequence using the nucleic acid subset-specific molecular barcode. The method addresses enrichment and detection of rare cells by using less stringent selection followed by physical partitioning of enriched cells into microplate wells, thereby improving per-well signal-to-noise.
Identification is genomically supported by incorporating well-specific molecular barcodes into gDNA/cDNA libraries, pooling, sequencing, and calling wells positive when cancer-driver mutations or RNA signatures are detected. The disclosed examples include detection of circulating tumor cells in blood, with CD45 depletion together with microplate arraying increasing mutation signal above background across wells.
Claims Coverage
The provided claims define one independent barcoded, subset-based genomic identification workflow, with dependent claims narrowing rare cell type categories, subset generation constraints, and nucleic-acid alteration features. The core inventive coverage centers on barcoded double-stranded gDNA subset genomic libraries, pooling, sequencing, and barcode-based identification of a nucleic acid subset containing the rare-cell gDNA sequence.
Subset genomic library generation with barcoded double-stranded gDNA constructs
Generating, for each of a plurality of nucleic acid subsets, a subset genomic library comprising barcoded double-stranded gDNA constructs, where the constructs include first and second gDNA strands, universal NGS primer sequences, and a nucleic acid subset-specific molecular barcode.
Pooling, sequencing, and barcode-based subset detection
Pooling the subset genomic libraries to form a combined sequencing library, obtaining DNA sequencing reads, and identifying by means of the nucleic acid subset-specific molecular barcode a nucleic acid subset comprising the gDNA sequence of the rare cell type to thereby identify the presence of the rare cell type in the biological sample.
Rare cell type selection among defined categories
Selecting the rare cell type from circulating tumor cells, circulating epithelial cells, stem cells, progenitor cells, or rare immune cells.
Rare cell type as a circulating tumor cell cluster with defined composition
Characterizing the rare cell type as a circulating tumor cell cluster made up of two or more tumor cells and one or more cancer stromal cells.
Cell-count constrained nucleic acid subset generation
Generating the nucleic acid subsets from subsets of the biological sample containing 10–10,000 cells per subset.
Nucleic-acid alteration definition for the rare cell type
Altering a nucleic acid specific to a rare cell type using a single-nucleotide variant or other specified DNA changes including insertion, deletion, methylation or demethylation, or changes in DNA copy number.
The inventive coverage centers on creating barcoded double-stranded gDNA subset genomic libraries from multiple nucleic acid subsets, pooling and sequencing the combined library, and using a nucleic-acid-subset-specific molecular barcode to identify the subset containing the rare-cell gDNA sequence. Dependent claims further narrow the rare cell type categories, constrain nucleic acid subset generation by cell-count range, and define nucleic-acid alterations associated with the rare cell type.
Stated Advantages
Improves per-well signal-to-noise by using less stringent selection followed by physical partitioning of enriched cells into microplate wells.
Increases mutation signal above background across wells in examples using CD45 depletion with microplate arraying.
Maintains signal integrity across a titration series, with RNA tumor markers and RNA tumor signatures detectable at approximately 1% tumor content.
Documented Applications
Detecting the presence of circulating tumor cells in blood samples, including detection of MCF7 cells spiked into blood.
Calling wells positive for cancer-driver mutations or RNA signatures based on barcoded subset analysis.
RNA marker detection in low-input rare-cell contexts, including EPCAM and CDH1 tumor markers and RNA tumor signatures.
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