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US-12624393-B2 - Deep sequencing profiling of tumors

US12624393B2US 12624393 B2US12624393 B2US 12624393B2US-12624393-B2

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.

Inventors

  • Nelson R Alexander
  • Daniel Burgess
  • Heidi J Rosenbaum
  • Stacey Stanislaw

Assignees

  • Roche Sequencing Solutions, Inc.
  • VENTANA MEDICAL SYSTEMS, INC.

Dates

Publication Date
20260512
Application Date
20230406

Claims (8)

  1. 1 . A method of measuring allele frequency and/or copy number variation without introducing bias during sequencing comprising: (i) isolating at least about 10 micrograms of genomic material from a homogenized sample, and wherein no amplification cycles are performed prior to enrichment, wherein the homogenized sample is prepared by mechanically shearing an obtained tumor sample with a blender or an ultra sonicator, wherein the obtained tumor sample is derived from a tumor of a human subject, and wherein the obtained tumor sample comprises at least 40% of the tumor derived from the human subject, wherein any heterogeneity of cells within the obtained tumor sample is substantially distributed within the homogenized sample, and wherein any portion of the homogenized sample expresses the heterogeneity of the obtained tumor sample; (ii) capturing one or more target nucleic acid molecules from the isolated genomic material, wherein an amount of captured one or more target nucleic acid molecules is at least about 90 ng; (iii) sequencing the captured one or more target nucleic acid molecules; and (iv) measuring allele frequency and/or copy number variation.
  2. 2 . The method of claim 1 , wherein the captured one or more target nucleic acid molecules are sequenced without performing post-capture amplification.
  3. 3 . The method of claim 1 , wherein the capturing of the one or more target nucleic acid molecules comprises: (i) introducing one or more probes to the isolated genomic material; (ii) removing non-target nucleic acids; and (iii) releasing the target nucleic acid molecules from the capture probes.
  4. 4 . The method of claim 1 , wherein the sequencing comprises next generation sequencing.
  5. 5 . The method of claim 1 , wherein the human subject was previously diagnosed with cancer.
  6. 6 . The method of claim 1 , wherein the whole obtained tumor sample is homogenized.
  7. 7 . The method of claim 1 , wherein the whole obtained tumor is homogenized except for one or more biopsy samples used for conducting diagnostic tests.
  8. 8 . The method of claim 1 , wherein the sample is homogenized through mechanical shearing.

Description

CROSS REFERENCE TO RELATED APPLICATIONS The present application is a continuation of U.S. patent application Ser. No. 16/034,392 filed on Jul. 13, 2018, which application is a continuation of International Application No. PCT/US2016/060835, which application claims the benefit of the filing date of U.S. Provisional Patent Application No. 62/415,952, filed Nov. 1, 2016 and the benefit of the filing date of U.S. Provisional Patent Application No. 62/279,126, filed Jan. 15, 2016, the disclosures of which are hereby incorporated by reference herein in their entireties. FIELD OF THE SUBJECT DISCLOSURE The present disclosure provides a targeted representational sequencing workflow. BACKGROUND Current diagnostic oncology utilizes information taken from a fraction of a tumor and is predicated on the assumption that tumors are composed of cells that are uniform in their composition. Rather than being uniform in composition, many tumors are heterogeneous. Indeed, it has been reported that some solid tumors, rather than being homogeneous, are composed of multiple genetically distinct, spatially segregated populations of cancer cells. See Gerlinger et al., NEJM (2012) 366:883-92; and Yachida et al. Nature (2010) 467(7319):1114-1117. Conventional histological methodologies address this heterogeneity with the selection of multiple biopsy samples for analysis, e.g., based on morphology and other characteristics. For example, biopsy samples are taken from multiple regions of the tumor, wherein each sample taken comprises about 0.1 cubic centimeter of tissue. These methods survey more of the tumor tissue and different spatial areas of the tumor; however, the vast majority of the tumor assayed using such methods remains un-sampled. Similarly, conventional methods sample only a small portion of the lymph nodes from cancer patients and do not sample the vast majority of the tissue. The small size of these samples can also be limiting on the further diagnostic steps that are utilized, such as sequencing. Solid tumors contain hundreds to thousands of mutant alleles that are spatially segregated throughout the three-dimensional tumor mass. Traditional methods for sequence capture utilize extremely small amounts of input DNA (about 5 to about 200 nanograms) isolated from formalin fixed, paraffin embedded tissue sections (e.g. from biopsy specimens), such as depicted in FIG. 2. Typical sequence capture methods have evolved to fit the input DNA requirements in today's clinical pathology labs. Due to the small amounts of input DNA, and loss of DNA at several steps in the sequence capture workflow, the DNA fragments must be amplified or too little will remain at the end of the capture workflow for sequencing to be performed. This amplification generally is performed twice, a first time prior to the specific probe capture, and a second time following the specific probe capture of the selected targets (see FIGS. 1 and 2). While this amplification is useful for increasing the absolute mass of the DNA available for subsequent protocol steps, it does not increase the amount of information present. Rather, and without wishing to be bound by any particular theory, when a population of different DNA fragments is amplified in the same reaction (i.e. multiplex PCR), the process of amplification can alter the information that was contained within the original sample. For example, if two different DNA fragments, A and B, are initially present in a sample at one copy each (a 1:1 numerical ratio), PCR may result in an amplified sample that contains 1,000 copies of DNA fragment A and 2,000 copies of DNA fragment B (a 1:2 numerical ratio). It is believed that the risk of introducing bias to the original information is increased when smaller numbers of individual molecules are used as input into the amplification process and when the amount of amplification is increased (i.e. a greater number of PCR cycles are applied). BRIEF SUMMARY OF THE DISCLOSURE 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 that minimal or no amplification processes are required prior to sequencing. In some embodiments, the input sample is derived from an intact tumor or from lymph nodes. In some embodiments, the input sample is obtained through homogenization of an intact tumor sample (whole or partial) and/or one or more lymph nodes obtained from a patient or mammalian subject, as discussed further herein. In some embodiments, the input sample is derived from a sufficient quantity of blood, including whole blood or any fraction thereof. In some embodiments, the input sample is derived from cancerous tissue. In some embodiments, the input sample is derived from pre-cancerous tissue. In some embodiments, the targeted sequencing workflow comprises one or more amplification steps (e.g. a pre-capture amplification step, an amplification step post-capture) prior to