Methods for using mosaicism in nucleic acids sampled distal to their origin

Patent metadata

Field	Value
Publication number	US-12516385-B2
Application number	US-202519073665-A
Country	US
Kind code	B2
Filing date	Mar 7, 2025
Priority date	Oct 30, 2014
Publication date	Jan 6, 2026
Grant date	Jan 6, 2026

Abstract

Disclosed herein are methods for improving detection and monitoring of human diseases. The methods can be used to provide spatial and/or developmental localization of the source of each differential mutation within the body. The methods can also be used to generate a mutation map of a subject. And the mutation map can be used to monitoring state(s) of health of one or more tissues of a subject.

First claim

What is claimed is:

1 . A method for detecting a presence or recurrence of a solid tumor, comprising:
(a) generating a first set of sequence reads and a second set of sequence reads by independently performing whole genome sequencing on: (i) nucleic acid molecules extracted from a tissue sample of a subject, wherein the tissue sample comprises a benign tissue, a malignant tissue, or a mixed tissue sample, and (ii) nucleic acid molecules extracted from leukocytes from a first blood sample, wherein the leukocytes are a normal sample; (b) comparing with a programmed computer processor the first set of sequence reads and the second set of sequence reads to identify mosaic variants, wherein the mosaic variants are present in the first set of sequence reads and are not present in the second set of sequence reads; (c) obtaining one or more blood samples from the subject at a later time point than the first tissue sample and the first blood sample; (d) extracting non-amplified nucleic acid molecules from the one or more blood samples obtained in step (c); (e) enriching nucleic acid molecules extracted from the one or more blood samples obtained in step (c) using a plurality of biotin-labeled capture probes in solution, wherein individual instances of the plurality of biotin-labeled capture probes are configured to hybridize to individual instances of the mosaic variants identified in step (b); (f) performing a sequencing assay on the nucleic acid molecules enriched in step (e) to identify the presence or absence of individual instances of the mosaic variants identified in step (b) one or more times over the life of the subject; and (g) providing a report based on the presence or absence of the individual instances of mosaic variants in the one or more blood samples, thereby detecting the presence or recurrence of the solid tumor in the subject.

2 . The method of claim 1 , further comprising, subsequent to the extracting of step (d) but prior to the enriching of step (e), amplifying the non-amplified nucleic acid molecules from the one or more blood samples from the subject.

3 . The method of claim 1 , wherein the mosaic variants are selected from the group consisting of: (i) copy number variation, (ii) small insertions and deletions (inDels), (iii) single nucleotide polymorphisms (SNPs), (iv) multiple nucleotide polymorphisms (MNPs), and (v) any combination thereof.

4 . The method of claim 1 , wherein the mosaic variants identified comprise somatic mosaicism.

5 . The method of claim 1 , wherein the mosaic variants are causal or predisposing for a solid tumor.

6 . The method of claim 1 , wherein the mosaic variants are non-causal for a solid tumor.

7 . The method of claim 1 , wherein the sequencing assay of step (f) comprises next generation sequencing, duplex sequencing, or redundant sequencing.

8 . The method of claim 1 , wherein the one or more blood samples are separated into at least a first component that includes cell-free or surface-bound nucleic acid molecules and a second component that includes leukocytes.

9 . The method of claim 8 , wherein the sequencing assay of step (f) is performed on the first component that includes cell-free or surface-bound nucleic acid molecules.

10 . The method of claim 9 , wherein the cell-free nucleic acid molecules comprise cell-free DNA.

11 . The method of claim 1 , wherein the report further comprises predicting, diagnosing, and/or prognosing a status or outcome of the solid tumor in the subject.

12 . The method of claim 11 , wherein the predicting, diagnosing, and/or prognosing the status or outcome of the solid tumor comprises: (i) diagnosing the solid tumor, (ii) identifying the solid tumor, (iii) determining the stage of the solid tumor, (iv) assessing the risk of the solid tumor, (v) assessing the risk of recurrence of the solid tumor, (vi) assessing the efficacy of a drug, (vii) assessing risk of an adverse drug reaction, (viii) predicting optimal drug dosage, (ix) predicting drug resistance, or (x) a combination thereof.

13 . The method of claim 12 , wherein the solid tumor comprises a sarcoma, a carcinoma, or a lymphoma.

14 . The method of claim 1 , wherein the report further comprises a determination of state of health of the tissue sample.

15 . The method of claim 14 , wherein the state of health of the tissue sample is determined by comparing a quantity of nucleic acid molecules from the tissue sample against a reference to identify a relative abundance of the nucleic acid molecules from the tissue sample.

16 . The method of claim 14 , wherein the state of health of the tissue sample is determined by aligning one or more nucleic acid sequences or portions thereof from the tissue sample to a genome or exome of the subject to identify a location of the one or more nucleic acid sequences or portions thereof from the tissue sample, which location is indicative of the state of health.

17 . The method of claim 14 , wherein the state of health of the tissue sample is determined by comparing one or more nucleic acid sequences or portions thereof from the tissue sample to a reference to determine an apoptotic pattern, necrotic pattern, or predetermined mutations.

18 . The method of claim 14 , wherein the state of health of the tissue sample is classified as unhealthy or potentially unhealthy.

19 . The method of claim 1 , further comprising:
(h) obtaining an additional blood or plasma sample from the subject at a time point later than the one or more blood samples; (i) repeating steps (d)-(f) using nucleic acid molecules isolated from the additional blood or plasma sample; and (j) providing an additional report based on the presence or absence of the mosaic variants in the additional blood or plasma sample, thereby detecting the presence or recurrence of the solid tumor in the subject.

20 . The method of claim 1 , wherein individual instances of the plurality of biotin-labeled capture probes each comprise different nucleic acid binding sites.

21 . The method of claim 1 , wherein individual instances of the plurality of biotin-labeled capture probes each comprise between about 10 to about 500 nucleotides.

22 . The method of claim 1 , wherein the enriching of step (e) comprises:
(i) hybridizing the non-amplified nucleic acid molecules from the one or more blood samples to the plurality of biotin-labeled capture probes in solution; (ii) separating capture probe-hybridized non-amplified nucleic acid molecules from capture-probe free non-amplified nucleic acid molecules.

23 . The method of claim 22 , wherein the enriching of step (e) further comprises:
(iii) conducting one or more elution reactions on the capture probe-hybridized non- amplified nucleic acid molecules.

24 . The method of claim 22 , wherein, prior to the separating of step (e)(ii), the nucleic acid molecules extracted from the one or more blood samples are further hybridized to a plurality of beads, and the method further comprises fragmenting bead bound nucleic acid molecules, wherein the ratio of the volume of the beads to the volume of the nucleic acid sample is about 0.10 or more.

25 . The method of claim 22 , wherein, prior to the separating of step (e)(ii), the nucleic acid molecules extracted from the one or more blood samples are further hybridized to a plurality of beads, and the method further comprises fragmenting bead bound nucleic acid molecules, wherein

Assignees

• Personalis Inc

Inventors

• West John

Classifications

• C12Q1/6827

  for detection of mutation or polymorphism · CPC title

• C12Q1/6809

  Methods for determination or identification of nucleic acids involving differential detection · CPC title

• G16B45/00

  ICT specially adapted for bioinformatics-related data visualisation, e.g. displaying of maps or networks · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• A61K31/7068 Primary

  having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid · CPC title