# US 2021/0172022 A1 · Methods and Systems for Analyzing Nucleic Acid Molecules > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2021/0172022 A1 - Document ID: US20210172022A1 - Status: Published application - Kind: A1 - Application number: 17/107,668 - Filed: 2020-11-30 - Published: 2021-06-10 - Earliest claimed: 2019-11-06 - Assignees: The Board of Trustees of the Leland Stanford Junior University - Applicants: The Board of Trustees of the Leland Stanford Junior University - Inventors: David M. Kurtz; Maximilian Diehn; Arash Ash Alizadeh - Web page: https://patentgrep.com/patent/US20210172022A1 ## CPC - C12Q 1/6883: for diseases caused by alterations of genetic material - C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575) - C12N 15/1089: {Design, preparation, screening or analysis of libraries using computer algorithms} - C12Q 1/6827: for detection of mutation or polymorphism - C12Q 1/6869: Methods for sequencing - C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation - G16B 20/00: ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations - G16B 20/10: Ploidy or copy number detection - G16B 20/20: Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection - G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids - G16B 30/10: Sequence alignment; Homology search - G16B 35/20: Screening of libraries - G16B 40/00: ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding - G16H 10/40: for data related to laboratory analysis, e.g. patient specimen analysis - G16H 20/10: relating to drugs or medications, e.g. for ensuring correct administration to patients - G16H 50/20: for computer-aided diagnosis, e.g. based on medical expert systems - G16H 50/30: for calculating health indices; for individual health risk assessment - G16H 50/70: for mining of medical data, e.g. analysing previous cases of other patients - G16H 70/60: relating to pathologies - C12Q 2600/112: Disease subtyping, staging or classification - C12Q 2600/156: Polymorphic or mutational markers - C12Q 2600/158: Expression markers - G01N 2800/7028: Cancer - G16H 10/60: for patient-specific data, e.g. for electronic patient records - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY - G16H: HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract Processes and materials to detect cancer from a biopsy are described. In some cases, cell-free nucleic acids can be sequenced, and the sequencing result can be utilized to detect sequences derived from a neoplasm. Detection of somatic variants occurring in phase can indicate the presence of cancer in a diagnostic scan and a clinical intervention can be performed. ## Claims ### Claim 1 A method comprising: (a) obtaining, by a computer system, sequencing data derived from a plurality of cell-free nucleic acid molecules that is obtained or derived from a subject; (b) processing, by the computer system, the sequencing data to identify one or more cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules, wherein each of the one or more cell-free nucleic acid molecules comprises a plurality of phased variants relative to a reference genomic sequence, wherein at least about 10% of the one or more cell-free nucleic acid molecules comprises a first phased variant of the plurality of phased variants and a second phased variant of the plurality of phased variants that are separated by at least one nucleotide; and (c) analyzing, by the computer system, the identified one or more cell-free nucleic acid molecules to determine a condition of the subject. ### Claim at position 2 -115. (canceled) ### Claim 116 (depends on 1) The method of claim 1, further comprising separating, in silico, (i) at least a portion of the identified one or more cell-free nucleic acid molecules from (ii) one or more other cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules that are not identified to comprise the plurality of phased variants. ### Claim 117 (depends on 116) The method of claim 116, further comprising analyzing, by the computer system, (i) and (ii) as different variables. ### Claim 118 (depends on 1) The method of claim 1, wherein the analyzing of the identified one or more cell-free nucleic acid molecules is not based on other cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules that are not identified to comprise the plurality of phased variants. ### Claim 119 (depends on 1) The method of claim 1, wherein the sequencing data is obtained without in silico removal or suppression of (i) background error or (ii) sequencing error. ### Claim 120 (depends on 1) The method of claim 1, wherein a number of the plurality of phased variants from the identified one or more cell-free nucleic acid molecules is indicative of the condition of the subject. ### Claim 121 (depends on 120) The method of claim 120, wherein a ratio of (i) the number of the plurality of phased variants from the identified one or more cell-free nucleic acid molecules and (ii) a number of single nucleotide variants (SNVs) from the identified one or more cell-free nucleic acid molecules is indicative of the condition of the subject. ### Claim 122 (depends on 1) The method of claim 1, wherein a frequency of the plurality of phased variants in the identified one or more cell-free nucleic acid molecules is indicative of the condition of the subject. ### Claim 123 (depends on 1) The method of claim 1, wherein the at least about 10% of the one or more cell-free nucleic acid molecules comprises at least about 50% of the one or more cell-free nucleic acid molecules. ### Claim 124 (depends on 123) The method of claim 123, wherein the at least about 10% of the one or more cell-free nucleic acid molecules comprises at least about 100% of the one or more cell-free nucleic acid molecules. ### Claim 125 (depends on 1) The method of claim 1, wherein the first and second phased variants are separated by at least 2 nucleotides. ### Claim 126 (depends on 1) The method of claim 1, wherein the first phased variant and the second phased variant are separated by at most about 160 nucleotides. ### Claim 127 (depends on 1) The method of claim 1, wherein the reference genome sequence comprises at least a portion of hg19 human genome, hg18 genome, hg17 genome, hg16 genome, or hg38 genome. ### Claim 128 (depends on 1) The method of claim 1, wherein the reference genomic sequence is derived from a sample of the subject. ### Claim 129 (depends on 1) The method of claim 1, wherein the reference genomic sequence is derived from a healthy cell of the subject. ### Claim 130 (depends on 1) The method of claim 1, wherein the plurality of cell-free nucleic acid molecules comprise a plurality of cell-free deoxyribonucleic acid (DNA) molecules. ### Claim 131 (depends on 1) The method of claim 1, wherein the condition comprises neoplasm, cancer, or tumor. ### Claim 132 (depends on 1) The method of claim 1, wherein the condition comprises a solid tumor. ### Claim 133 (depends on 1) The method of claim 1, wherein the condition comprises a lymphoma. ### Claim 134 (depends on 1) The method of claim 1, further comprising determining, by the computer, that the identified one or more cell-free nucleic acid molecules are derived from a sample associated with the condition, based on performing a statistical model analysis of the identified one or more cell-free nucleic acid molecules. ### Claim 135 A method comprising: (a) providing a mixture comprising (1) a set of nucleic acid probes and (2) a plurality of cell-free nucleic acid molecules that is obtained or derived from a subject, wherein an individual nucleic acid probe of the set of nucleic acid probes is designed to hybridize to at least a portion of a target cell-free nucleic acid molecule comprising a plurality of phased variants relative to a reference genomic sequence that are separated by at least one nucleotide, and wherein the individual nucleic acid probe comprises an activatable reporter agent, activation of the activatable reporter agent being selected from the group consisting of: (i) hybridization of the individual nucleic acid probe to the plurality of phased variants and (ii) dehybridization of at least a portion of the individual nucleic acid probe that has been hybridized to the plurality of phased variants; (b) detecting the activatable reporter agent that is activated, to identify one or more cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules, wherein each of the one or more cell-free nucleic acid molecules comprises the plurality of phased variants; and (c) analyzing the identified one or more cell-free nucleic acid molecules to determine a condition of the subject. ### Claim 136 (depends on 135) The method of claim 135, wherein the activatable reporter agent is activated upon hybridization of the individual nucleic acid probe to the plurality of phased variants. ### Claim 137 (depends on 135) The method of claim 135, wherein the activatable reporter agent is activated upon dehybridization of at least a portion of the individual nucleic acid probe that has been hybridized to the plurality of phased variants. ### Claim 138 (depends on 135) The method of claim 135, wherein the activatable reporter agent is a fluorophore. ### Claim 139 (depends on 135) The method of claim 135, a number of the plurality of phased variants from the identified one or more cell-free nucleic acid molecules is indicative of the condition of the subject. ### Claim 140 A method comprising: (a) obtaining sequencing data derived from a plurality of cell-free nucleic acid molecules that is obtained or derived from a subject; (b) processing the sequencing data to identify one or more cell-free nucleic acid molecules of the plurality of cell-free nucleic acid molecules with a limit of detection of less than about 1 out of 50,000 observations from the sequencing data; and (c) analyzing the identified one or more cell-free nucleic acid molecules to determine a condition of the subject, wherein each of the one or more cell-free nucleic acid molecules comprises a plurality of phased variants relative to a reference genomic sequence. ### Claim 141 (depends on 140) The method of claim 140, wherein the limit of detection of the identification step is less than about 1 out of 300,000 observations from the sequencing data. ### Claim 142 (depends on 141) The method of claim 141, wherein the limit of detection of the identification step is less than about 1 out of 1,000,000 observations from the sequencing data. ### Claim 143 (depends on 140) The method of claim 140, wherein a first phased variant of the plurality of phased variants and a second phased variant of the plurality of phased variants are separated by at least one nucleotide. ### Claim 144 A composition comprising a bait set comprising a set of nucleic acid probes designed to capture cell-free DNA molecules derived from at least about 5% of genomic regions set forth in (i) the genomic regions identified in Table 1, (ii) the genomic regions identified in Table 3, or (iii) the genomic regions identified to have a plurality of phased variants in Table 3.