Deep sequencing profiling of tumors
Inventors
Alexander, Nelson R • Burgess, Daniel • Rosenbaum, Heidi J • Stanislaw, Stacey
Assignees
Ventana Medical Systems Inc • Roche Sequencing Solutions Inc
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Publication Number
US-12624393-B2
Publication Date
2026-05-12
Expiration Date
Abstract
In one aspect of the present disclosure is a targeted sequencing workflow where an input sample comprising a sufficient quantity of genomic material is provided such minimal or no amplification cycles are utilized prior to sequencing.
Core Innovation
The invention relates to a method of measuring allele frequency and/or copy number variation without introducing bias during sequencing. It applies to genomic material isolated from a homogenized sample derived from a tumor of a human subject, where the homogenized sample is prepared by mechanically shearing an obtained tumor sample so that heterogeneity of cells within the obtained tumor sample is substantially distributed within the homogenized sample and any portion of the homogenized sample expresses the heterogeneity of the obtained tumor sample.
In the method, no amplification cycles are performed prior to enrichment. One or more target nucleic acid molecules are captured from the isolated genomic material, and the captured target nucleic acid molecules are sequenced. Allele frequency and/or copy number variation are then measured from the resulting sequence data.
The approach further includes probe-based target capture, including introducing probes to isolated genomic material, removing non-target nucleic acids, and releasing target nucleic acid molecules from capture probes. Sequencing is performed without post-capture amplification, and dependent refinements also describe preserving one or more biopsy samples for diagnostic testing.
Claims Coverage
The partial set provided identifies one independent claim for the method of measuring allele frequency and/or copy number variation without introducing bias. The dependent claims refine capture, sequencing constraints, and homogenization scope and mechanism, adding additional inventive features to the baseline method.
Measuring allele frequency and/or copy number variation without introducing sequencing bias via capture-first sequencing of homogenized human tumor material
Isolating genomic material from a homogenized human tumor sample in which tumor cell heterogeneity is substantially distributed across the homogenized sample; performing no amplification cycles prior to enrichment; capturing one or more target nucleic acid molecules; sequencing the captured target nucleic acid molecules; and measuring allele frequency and/or copy number variation.
Probe-based target capture with removal of non-target nucleic acids and release of target nucleic acids
Capturing the one or more target nucleic acid molecules by introducing probes to isolated genomic material, removing non-target nucleic acids, and releasing target nucleic acid molecules from the capture probes.
Sequencing without post-capture amplification
Sequencing the captured one or more target nucleic acid molecules without performing post-capture amplification.
Next generation sequencing
Sequencing the captured one or more target nucleic acid molecules using next generation sequencing.
Mechanical shearing homogenization with distributed heterogeneity
Preparing the homogenized sample by mechanically shearing an obtained tumor sample such that heterogeneity within the obtained tumor sample is substantially distributed within the homogenized sample and expressed in any portion of the homogenized sample.
Homogenizing the entire obtained tumor while preserving biopsy samples for diagnostic testing
Further specifying that the entire obtained tumor is homogenized except that one or more biopsy samples are kept for diagnostic testing.
Overall, the claim coverage centers on bias-minimized allele frequency and/or copy number variation measurement by combining homogenized human tumor-derived genomic material with substantially distributed cellular heterogeneity, no amplification cycles prior to enrichment, and sequencing without post-capture amplification, with dependent refinements describing probe-based capture workflows and next generation sequencing.
Stated Advantages
Measuring allele frequency and/or copy number variation without introducing bias during sequencing.
Maintaining representation of tumor cell heterogeneity by distributing it within the homogenized sample and expressing it in any portion of the homogenized sample.
Enabling allele frequency and/or copy number variation measurement after capturing target nucleic acid molecules and sequencing the captured molecules.
Documented Applications
Cancer subtype identification via sequencing with limited amplification.
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