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WO2025085784A1 - Biomarqueurs génomiques et de méthylation permettant de déterminer le risque de maladie cardiaque d'un patient et de nouvelles cibles de médicament génomique et épigénomique pour diminuer le risque de maladie cardiaque et/ou améliorer le résultat pour un patient après un infarctus du myocarde ou une lésion cardiaque - Google Patents

Biomarqueurs génomiques et de méthylation permettant de déterminer le risque de maladie cardiaque d'un patient et de nouvelles cibles de médicament génomique et épigénomique pour diminuer le risque de maladie cardiaque et/ou améliorer le résultat pour un patient après un infarctus du myocarde ou une lésion cardiaque Download PDF

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WO2025085784A1
WO2025085784A1 PCT/US2024/052026 US2024052026W WO2025085784A1 WO 2025085784 A1 WO2025085784 A1 WO 2025085784A1 US 2024052026 W US2024052026 W US 2024052026W WO 2025085784 A1 WO2025085784 A1 WO 2025085784A1
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data
individuals
methylation
information
data repository
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Sean Gordon
Catalin Barbacioru
Daniel GAILE
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Guardant Health Inc
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Guardant Health Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • LVEF left ventricular ejection fraction
  • Genomic and epigenetic data was compared between case and controls groups to identify biomarkers that may be valuable for use in diagnosing whether patients are/will develop heart disease including risk of AF and HF as well as increased risk of poor cardiac remodeling and myocardial interstitial fibrosis following myocardial infarction. These biomarkers may also be novel targets for novel drugs that manipulate the levels of the biomarkers reported here in order to decrease patient risk of heart disease and improve patient outcome after myocardial infarction or injury. [0005] Genomic and epigenetic biomarkers identified here could be used for more sensitive or more convenient diagnosis of heart disease risk than currently. Patients being genotyped or monitored for cancer disease and receiving cancer treatments known to increase risk of heart disease could be simultaneously be monitored for the development of heart disease before it happens.
  • Described herein is a method including: detecting methylation in at least one of a plurality of sites in nucleic acids in a cell-free DNA (cfDNA) sample from a subject; generating a plurality of methylation calls for each of the plurality of sites; determining one or more metrics from the methylation calls; and processing the one or more metrics to calculate a risk probability for a cardiac event for the subject.
  • cfDNA cell-free DNA
  • the at least one of the plurality of sites includes one or more genes, and/or association with one or more genes, selected from the group consisting of SIX2, KBTBD8, TAFA4, EVC, NCA, SNCA-AS, ARAP4, NT5E, XPO1, TMEM181, NRG1, DYDC2, CACUL1, 0ORF888, FAM, FAM222A, MIR9-3HG, BCKDK, MAPK7, A1G, CACNA1, MXRA7, ADCYAP1, EPOR, MAST1, DKKL1, PCSK2, CGB7, EMB, TGFBI, COX6B1, ZNF347, and DKKL1.
  • the at least one of the plurality of sites includes one or more genes, and/or association with one or more genes, selected from the group consisting of SIX2, KBTBD8, TAFA4, EVC, NCA, SNCA-AS, CGB7, EMB, TGFBI, COX6B1, ZNF347, and DKKL1.
  • the cardiac event includes one or more of stroke, transient ischemic attack (TIA), myocardial infarction (MI), angina, transient ischemic attack (TIA), stroke, and acute coronary syndrome.
  • the cardiac event is associated with cardiomyopathy selected from the group consisting of dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and arrhythmogenic right ventricular dysplasia.
  • cardiomyopathy includes inherited cardiomyopathy.
  • the cardiomyopathy includes acquired cardiomyopathy.
  • the cardiomyopathy is associated with an agent..
  • the agent is a drug.
  • the drug is Letrozole, Palbociclib, Capecitabine, Tamoxifen, Trastuzumab, T-DM1, Pertuzumab, Ado-trastuzumab emtansine, Neratinib, Lapatinib, Tucatinib, Margetuximab, and Trastuzumab deruxtecan.
  • determining one or more metrics from the methylation calls includes determining counts of methylated molecules for the at least one of the plurality of sites. In various embodiments, the counts are peak counts. In various embodiments, processing the one or more metrics includes selection on the basis of tumor fraction and/or methylated molecule counts.
  • processing the one or more metrics includes application of a model.
  • the model is a probabilistic model.
  • processing the one or more metrics includes determining probabilities based on the one or more metrics.
  • the probabilities based on the one or more metrics generates calculation of the risk probability for the cardiac event for the subject.
  • the risk probability for the subject is an increased risk for a cardiac event.
  • the cardiomyopathy is a comorbidity with another disease or condition in the subject.
  • the disease or condition in the subject is selected from one or more diseases or conditions from the group consisting of: cancer, coronary heart disease, heart attack, high blood pressure, diabetes, thyroid disease, viral hepatitis, HIV1, and viral infections.
  • Described herein is a method, including: detecting methylation in at least one of a plurality of sites in nucleic acids in a cell-free DNA (cfDNA) sample from a subject afflicted with cancer receiving treatment; generating a plurality of methylation calls for each of the plurality of sites; determining one or more metrics from the methylation calls; and processing the one or more metrics to calculate a risk probability for a cardiac event for the subject; and determining whether to maintain or alter treatment based on the calculated risk probability.
  • treatment includes administration of an agent.
  • the agent includes one or more of: Letrozole, Palbociclib, Capecitabine, Tamoxifen, Trastuzumab, T-DM1, Pertuzumab, Ado-trastuzumab emtansine, Neratinib, Lapatinib, Tucatinib, Margetuximab, and Trastuzumab deruxtecan.
  • the method includes obtaining the sample or having obtained the sample from the subject.
  • the at least one of the plurality of sites includes one or more genes, and/or association with one or more genes, selected from the group consisting of: SIX2, KBTBD8, TAFA4, EVC, NCA, SNCA-AS, ARAP4, NT5E, XPO1, TMEM181, NRG1, DYDC2, CACUL1, OORF888, FAM, FAM222A, MIR9-3HG, BCKDK, MAPK7, A1G, CACNA1, MXRA7, ADCYAP1, EPOR, MAST1, DKKL1, PCSK2, CGB7, EMB, TGFBI, COX6B1, ZNF347, and DKKL1.
  • the at least one of the plurality of sites includes one or more genes, and/or association with one or more genes, selected from the group consisting of: SIX2, KBTBD8, TAFA4, EVC, NCA, SNCA-AS, CGB7, EMB, TGFBI, C0X6B1, ZNF347, and DKKL1.
  • the subject afflicted with cancer is one of: HR+/HER2-, HR+/HER2+, HR-/HER2+, and HR-/HER2-.
  • Described herein is a method, including: detecting methylation in at least one of a plurality of sites in nucleic acids in a cell-free DNA (cfDNA) sample from a subject; generating a plurality of methylation calls for each of the plurality of sites; determining one or more metrics from the methylation calls; and processing the one or more metrics to calculate a risk probability for a cardiac event for the subject; and diagnosing a patient as susceptible to a cardiomyopathy and/or cardiac event and/or prognosing patient likelihoods suffering cardiomyopathy and/or a cardiac event.
  • cfDNA cell-free DNA
  • the cardiac event includes one or more of: stroke, transient ischemic attack (TIA), myocardial infarction (MI), angina, transient ischemic attack (TIA), stroke, and acute coronary syndrome.
  • the cardiomyopathy is selected from the group consisting of: dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and arrhythmogenic right ventricular dysplasia.
  • the cardiomyopathy includes inherited cardiomyopathy.
  • the cardiomyopathy includes acquired cardiomyopathy.
  • a method including: detecting one or more biomarkers in a sample including cell-free DNA (cfDNA); and characterizing the sample based on detection of the one or more biomarkers in the sample.
  • detecting the one or more biomarkers includes measuring the methylation level in one or more of a plurality of sites.
  • the sites comprise a custom panel.
  • the custom panel is configured in an in silico panel.
  • the custom panel is configured in a physical panel.
  • the panel includes a set of oncogenes, promoter regions for a set of oncogenes, HRR genes, immuno-oncology (IO) genes, a cancer pathway, methylation peaks found in cancer or methylation peaks found in clinical samples.
  • oncogenes include a set of oncogenes, promoter regions for a set of oncogenes, HRR genes, immuno-oncology (IO) genes, a cancer pathway, methylation peaks found in cancer or methylation peaks found in clinical samples.
  • the biomarkers comprise, the presence, absence, or increased expression of one or more genes, and/or association with one or more genes, selected from the group consisting of: SIX2, KBTBD8, TAFA4, EVC, NCA, SNCA-AS, ARAP4, NT5E, XPO1, TMEM181, NRG1, DYDC2, CACUL1, 0ORF888, FAM, FAM222A, MIR9-3HG, BCKDK, MAPK7, A1G, CACNA1, MXRA7, ADCYAP1, EPOR, MAST1, DKKL1, PCSK2, CGB7, EMB, TGFBI, COX6B1, ZNF347, and DKKL1.
  • the at least one of the plurality of sites includes one or more genes, and/or association with one or more genes, selected from the group consisting of: SIX2, KBTBD8, TAFA4, EVC, NCA, SNCA-AS, CGB7, EMB, TGFBI, C0X6B1, ZNF347, and DKKL1.
  • Described herein is a method, including: detecting methylation in a sample obtained from a subject; normalizing the detected methylation; applying a probabilistic model; calculating a test statistic based on one or more expected and/or detection values; and generating a quantitative score for cardiomyopathy and/or cardiac event risk for the subject.
  • the method includes permuting a plurality a sample and applying the test statistic.
  • the is a pseudo test statistic.
  • the probabilistic model includes detection of methylation from a plurality of samples, each obtained from a plurality of subjects;
  • the expected value includes negative observations.
  • the detection values comprise a sum of probabilities.
  • the probabilistic model includes a plurality of probabilities for the plurality of samples.
  • the method includes applying logistic regression. [0012] Described herein is a method, including: determining a state of biological molecules obtained from a sample derived from a human subject.
  • the method includes combining a plurality of nucleic acid molecules derived from a subject with a solution including an amount of methyl binding domain (MBD) proteins to produce a nucleic acid-MBD protein solution; and performing a plurality of washes of the nucleic acid-MBD protein solution with a salt solution to produce a number of nucleic acid fractions, individual nucleic acid fractions having a threshold number of methylated cytosines in regions of the plurality of nucleic acids having at least the threshold cytosine-guanine content.
  • MBD methyl binding domain
  • the wash of the plurality of washes is performed with a solution having a concentration of sodium chloride (NaCl) and produces a nucleic acid fraction of the number of nucleic acid fractions having a range of binding strengths to MBD proteins.
  • NaCl sodium chloride
  • the method includes combining at least a portion of the number of nucleic acid fractions with an amount of restriction enzyme that cleaves molecules with one or more unmethylated cytosines to produce at least a portion of the plurality of samples used to produce the sequencing reads, wherein the threshold amount of methylated cytosines corresponds to a minimum frequency of methylated cytosines within a region having at least the threshold cytosine-guanine content.
  • the method includes combining at least a portion of the number of nucleic acid fractions with an amount of a restriction enzyme that cleaves molecules with one or more methylated cytosines to produce at least a portion of the plurality of samples used to produce the sequencing reads, wherein the threshold amount of unmethylated cytosines corresponds to a maximum frequency of methylated cytosines that are not cleaved within a region having at least the threshold cytosine-guanine content.
  • the method includes sequencing nucleic acid molecules derived from a sample obtained from a subject, analyzing sequence reads derived from the sequencing to identify one or more driver mutations in the nucleic acid molecules, and using information about the presence, absence, or amount of the one or more driver mutations in the nucleic acid molecules to identify a tumor in the subject.
  • the nucleic acid molecules are cell-free DNA.
  • the sample is at least one of blood, serum, plasma or tissue.
  • the method includes determination of treatment for the subject.
  • a limit of detection for the model to determine tumor fraction of samples is no greater than 0.05%.
  • the one or more driver mutations includes a somatic variant detected at a mutant allele frequency (MAF) of no more than 0.05%. In other embodiments, the one or more driver mutations includes a fusion detected at a mutant allele frequency (MAF) of no more than 0.1%. In other embodiments, the method includes detecting mutation distributions for each of one or more driver mutations, wherein the mutation distribution for each of the one or more driver mutations is detected with a correlation of at least 0.99 to a mutation distribution of the driver mutation detected in a cohort of the subject by tissue genotyping. In other embodiments, the method detects the tumor in the subject with a sensitivity of at least 85%, a specificity of at least 99%, and a diagnostic accuracy of at least 99%.
  • the method includes identifying cell free (cfDNA) and one or more driver mutations in the cfDNA.
  • the method includes obtaining, by a computing system having one or more hardware processors and memory, testing sequence data from a subject, the testing sequence data including testing sequencing reads derived from a sample of the subject, analyzing, by the computing system, the testing sequencing reads to determine a first quantitative measure derived from the testing sequencing reads to genomic regions of a reference genome, analyzing, by the computing system, the testing sequencing reads to determine a second quantitative measure derived from the testing sequencing reads to genomic regions of a reference genome, determining, by the computing system, a metric based on the first quantitative and the second quantitative measure, and generating, by the computing system, an input vector that includes the metrics determining, by the computing system, an indication of cancer status in the subject by providing the input vector to a model that implements one or more machine learning techniques to generate indications of cancer status in subjects, the model including weights for individual classification regions of a pluralit
  • the individual testing sequencing reads include a nucleotide sequence corresponding to a fragment of a nucleic acid included in the sample and the individual testing sequencing reads correspond to molecules having a threshold amount of methylated cytosines included in regions of the nucleotide sequence having at least the threshold cytosine-guanine content, the first quantitative measure derived from the testing sequencing reads that correspond to individual classification regions of a plurality of classification regions at least a portion of the individual classification regions of the plurality of classification regions corresponding to genomic regions of a reference genome that have the threshold amount of methylated cytosines in subjects in which cancer is detected and that have at least the threshold cytosine-guanine content, the second quantitative measure derived from the testing sequencing reads that correspond to individual control regions a plurality of control regions, individual control regions of the plurality of control regions corresponding to additional genomic regions of the reference genome that have at least the threshold cytosine-guanine content and that have at least the threshold amount of methylated cytosines in subjects
  • the method includes obtaining, by the computing system having one or more hardware processors and memory, training sequence data including training sequencing reads derived from a plurality of samples of a plurality of training subjects, individual training sequencing reads including a nucleotide sequence corresponding to a fragment of a nucleic acid included in a sample of the plurality of samples and individual training sequencing reads corresponding to molecules having a threshold amount of methylated cytosines included in regions of the nucleotide sequence having at least a threshold cytosine- guanine content, analyzing, by the computing system, the training sequencing reads to determine an additional first quantitative measure derived from the training sequencing reads that corresponds to individual classification regions of the plurality of classification regions, analyzing, by the computing system, the training sequencing reads to determine an additional second quantitative measure derived from the training sequencing reads that correspond to a plurality of control regions, determining, by the computing system, an additional metric for the individual classification regions of the plurality of classification regions based on the additional first quantitative measure for
  • the one or more machine learning algorithms include one or more classification algorithms. In other embodiments, the one or more machine learning algorithms include one or more regression algorithms, and the indication corresponds to an estimate of tumor fraction of the sample.
  • the training sequencing reads comprise a first portion of the training sequence data and additional training sequencing reads comprise a second portion of the training sequence data, wherein the additional training sequencing reads are different from the training sequencing reads; and the method including analyzing, by the computing system, at least one of the first portion of the training sequence data or the second portion of the training sequence data to determine an individual frequency of a plurality of variants present in an individual sample of the plurality of samples, determining, by the computing system and for the individual sample, a variant of the plurality of variants having a maximum frequency that corresponds to the individual frequency having a greatest value among individual frequencies derived from an individual sample, and determining, by the computing system, individual measures of tumor fraction for an individual sample based on the greatest value of the individual frequencies derived from the individual sample.
  • the training data includes the individual measures of tumor fraction for the individual samples of the plurality of samples, and the model is generated based on the individual measures of tumor fraction for the individual samples of the plurality of samples.
  • the method includes generating, by a computing system including processing circuitry and memory, a data file including first tokens generated using a first hash function, individual first tokens corresponding to a respective individual of a group of individuals having data stored by a molecular data repository, sending, by the computing system, the data file to a health insurance claims data management system, obtaining, by the computing system and from the health insurance claims data management system, in response to the data file, health data corresponding to the group of individuals, generating, by the computing system, a number of identifiers using a second hash function that is different from the first hash function, each identifier corresponds to one or more tokens related to each individual of the group of individuals, obtaining, by the computing system and using the number of identifiers, second data from the molecular data repository
  • the method includes determining, by the computing system, a first set of data processing instructions that are executable in relation to first data stored by the integrated data repository, causing, by the computing system, the first set of data processing instructions to be executed to analyze first health insurance claims codes included in the first data to determine a first subset of the group of individuals in which a biological condition is present and generating, by the computing system, a first dataset indicating the subset of the group of individuals in which the biological condition is present.
  • the method includes determining, by the computing system, a second set of data processing instructions that are executable in relation to second data stored by the integrated data repository, causing, by the computing system, the second set of data processing instructions to be executed to analyze the second health insurance claims codes included in the second data to determine one or more treatments provided to a second subset of the group of individuals, and generating, by the computing system, a second dataset indicating the one or more treatments provided to the second subset of the group of individuals.
  • the method includes determining, by the computing system, a third subset of the group of individuals that includes a portion of the first subset of the group of individuals that overlaps with a portion of the second subset of the group of individuals, receiving, by the computing system, a request to perform an analysis of the first dataset and the second dataset in relation to the third subset of the group of individuals, and analyzing, by the computing system and in response to the request, the first dataset and the second dataset with respect to the third subset of the group of individuals to determine a measure of significance of a characteristic of the third subset of the group of individuals with respect to the biological condition.
  • the method includes determining, by the computing system, one or more genomic mutations present in the third subset of the group of individuals, determining, by the computing system, a plurality of treatments provided to the third subset of the group of individuals, and determining, by the computing system, respective survival rates for the third subset of the group of individuals.
  • the measure of significance corresponds to survival rate with respect to a treatment of the plurality of treatments and a genomic mutation of the one or more genomic mutations.
  • the method includes determining, by the computing system and based on measure of significance, an effectiveness of the treatment for the third subset of the group of individuals.
  • the method includes determining, by the computing system, individuals in third subset of the group of individuals that have not received the treatment. In other embodiments, the method includes administering one or more therapeutically effective amounts of the treatment to the individuals in the third subset that have not received the treatment.
  • the integrated data repository is arranged according to a data repository schema that includes a plurality of data tables and a plurality of logical links between the plurality of data tables, individual logical links of the plurality of logical links indicating one or more rows of a data table of the plurality of data tables that correspond to one or more additional rows of an additional data table of the plurality of data tables.
  • the plurality of data tables include a first data table that stores genomics data of the group of individuals, a second data that stores data related to one or more patient visits by individuals to one or more healthcare providers, a third data table that stores information corresponding to respective services provided to individuals with respect to one or more patient visits to one or more healthcare providers indicated by the second data table, a fourth data table that stores personal information of the group of individuals, a fifth data table that stores information related to a health insurance company or governmental entity that made payment for services provided to the group of individuals, a sixth data table storing information corresponding to health insurance coverage information for the group of individuals, and a seventh data table that stores information related to pharmaceutical treatments obtained by the group of individuals.
  • the method includes storing, by the computing system, the one or more data structures in an intermediate data repository, performing, by the computing system, one or more de-identification operations with respect to the identifiers of the portion of the subset of the additional group of individuals before modifying the integrated data repository to store at least a portion of the additional information of the medical records of the portion of the subset of the additional group of individuals in relation to the number of identifiers.
  • the molecular data repository stores at least one or more of genomic information, genetic information, metabolomic information, transcriptomic information, fragmentomic information, immune receptor information, methylation information, epigenomic information, or proteomic information.
  • the method includes a cfDNA test, real world evidence (RWE), or both.
  • the methods include administering a therapy to the subject to treat the cardiomyopathy and/or cardiac event (including reducing the risk of developing the cardiomyopathy and/or the cardiac event from occurring).
  • the therapy includes a treatment including one or more of apixaban, rivaroxaban, dabigatran, edoxaban, solalol hydrochloride, ibutilide fumarate injection, flecainide acetate, digoxin, tafamidis, tafamidis meglumine, mavacamten, spironolactone, eplerenone, isosorbide dinitrate, dapagliflozin, tenecteplase, clopidogrel, abciximab, tirofiban, eptifibatide, colchicine, flurpiridaz Fl 8, ticagrelor, valsartan, hydrochlorothiazide, can
  • the treatment includes antiplatelet agents, anticoagulant agents, beta-blockers, nitrates, fibrinolytics, or any combination thereof.
  • the therapy includes administering one or more treatments to the subject to treat both the cancer and the cardiac event.
  • Methylation regions can be identified by testing normal vs. cancer samples from large cohorts, including plasma from 3,000 cancer-free donors established regions where DNA does not have hypermethylation signal, Plasma from 1,700+ CRC, breast, lung and bladder cancer patients was used to detect tumor methylation mutation molecules. A subset of all regions can be used for tumor detection and quantification, >15K regions, even if not used for tumor detection and quantification, can be used for applications such as adverse event detection. Adverse event detection distinguishes tumor signal from non-tumor signal released due to adverse event.
  • TGFBI hypermethylation calls more frequent in case (positives) than controls.
  • Plots show raw counts of methylated molecules (x-axis) for select genes in case (patient develops heart failure, orange) versus control (patient does not develop heart failure, blue) versus y-axis which displays the tumor content of the sample.
  • Many implicated genes such as those shown below have signal that differentiate case versus control and do not have significant association with the presence of tumor (potential confounding factor when evaluating likelihood of developing heart failure in patients with cancer. Only a subset of genes are shown here.
  • Figure 7. CGB7 hypermethylation calls more frequent in case (positives) than controls.
  • Figure 15 Example logistic regression (LR) model (all samples). Logistic regressionbased association of individual gene methylation to heart failure status. Gene methylated is displayed on the x-axis and corresponding p-value for association with heart failure is shown on the y-axis.
  • FIG. 1 CGB7, EMB, TGFBI, COX6B1, ZNF347, DKKL1 are among the genes with most significant p-value associations among methylated genes associated with patients that later go onto have heart failure as compared to matched patients negative for heart failure.
  • Figure 19 Validation cohort selection. Breast cancer patients unselected except for requirement on prior treatment history; both HER2 targeted treatment and non-HER2 targeted sub-cohorts.
  • Figure 20 Patients with documented heart failure in the validation cohort.
  • Figure 21 Promoter methylation of NT5E and XPO1, genes in which cardiac health function has been documented, were significantly higher in the group positive for heart failure.
  • Predictive model included NT5E, XPO1, left panel, and XPO1, right panel.
  • FIG. 23 Heatmap show the amount of methylation of 30 genes (promoter regions) used in the model where blue is higher methylation. Row are normalized methylation signal from selected DMRs. Columns are patients.
  • the term “at least” and its grammatical equivalents in relation to a reference numerical value can include the reference numerical value and greater than that value.
  • the amount “at least 10” can include the value 10 and any numerical value above 10, such as 11, 100, and 1,000.
  • the term “at most” and its grammatical equivalents in relation to a reference numerical value can include the reference numerical value and less than that value.
  • the amount “at most 10” can include the value 10 and any numerical value under 10, such as 9, 8, 5, 1, 0.5, and 0.1.
  • DNA methylation profiling can be used to detect regions with different extents of methylation (“differentially methylated regions” or “DMRs”) of the genome that are altered during development or that are perturbed by disease, for example, cancer or any cancer-associated disease.
  • the genome of cancer cells harbor imbalance in the above DNA methylation patterns, and therefore in functional packaging of the DNA.
  • the abnormalities of chromatin organization are therefore coupled with methylation changes and may contribute to enhanced cancer profiling when analyzed jointly.
  • Combining MBD-partitioning with fragmentomic data, such as fragment mapped starts and stops positions (correlated with nucleosome positions) , fragment length and associated nucleosome occupancy, can be used for chromatin structure analysis in hypermethylation studies with the aim to improve biomarker detection rate.
  • Methylation profiling can involve determining methylation patterns across different regions of the genome. For example, after partitioning molecules based on extent of methylation (e.g., relative number of methylated sites per molecule) and sequencing, the sequences of molecules in the different partitions can be mapped to a reference genome. This can show regions of the genome that, compared with other regions, are more highly methylated or are less highly methylated. In this way, genomic regions, in contrast to individual molecules, may differ in their extent of methylation.
  • extent of methylation e.g., relative number of methylated sites per molecule
  • a characteristic of nucleic acid molecules may be a modification, which may include various chemical or protein modifications (i.e. epigenetic modifications).
  • chemical modification may include, but are not limited to, covalent DNA modifications, including DNA methylation.
  • DNA methylation includes addition of a methyl group to a cytosine at a CpG site (a cytosine followed by a guanine in a nucleic acid sequence).
  • DNA methylation includes addition of a methyl group to adenine, such as in N6-methyladenine.
  • DNA methylation is 5- methylation (modification of the 5th carbon of the 6 carbon ring of cytosine).
  • 5-methylation includes addition of a methyl group to the 5C position of the cytosine to create 5-methylcytosine (m5c).
  • methylation includes a derivative of m5c.
  • Derivatives of m5c include, but are not limited to, 5 -hydroxymethylcytosine (5-hmC), 5 -formylcytosine (5-fC), and 5-caryboxylcytosine (5-caC).
  • DNA methylation is 3C methylation (modification of the 3rd carbon of the 6 carbon ring of cytosine).
  • 3C methylation includes addition of a methyl group to the 3C position of the cytosine to generate 3 -methylcytosine (3mC).
  • Other examples include N6- methyladenine or glycosylation.
  • DNA methylation includes addition of methyl groups to DNA (e.g. CpG) and can change the expression of methylated DNA region. Methylation can also occur at non CpG sites, for example, methylation can occur at a CpA, CpT, or CpC site.
  • DNA methylation can change the activity of methylated DNA region. For example, when DNA in a promoter region is methylated, transcription of the gene may be repressed. DNA methylation is critical for normal development and abnormality in methylation may disrupt epigenetic regulation. The disruption, e.g., repression, in epigenetic regulation may cause diseases, such as cancer. Promoter methylation in DNA may be indicative of cancer.
  • a CpG dyad is the dinucleotide CpG (cytosine-phosphate-guanine, i.e. a cytosine followed by a guanine in a 5’ - 3’ direction of the nucleic acid sequence) on the sense strand and its complementary CpG on the antisense strand of a double-stranded DNA molecule.
  • CpG dyads can be either fully methylated or hemi-methylated (methylated on one strand only).
  • the CpG dinucleotide is underrepresented in the normal human genome, with the majority of CpG dinucleotide sequences being transcriptionally inert (e.g. DNA heterochromatic regions in pericentromeric parts of the chromosome and in repeat elements) and methylated. However, many CpG islands are protected from such methylation especially around transcription start sites (TSS).
  • TSS transcription start sites
  • Protein modifications include binding to components of chromatin, particularly histones including modified forms thereof, and binding to other proteins, such as proteins involved in replication or transcription.
  • the disclosure provides methods of processing and analyzing nucleic acids with different extents of modification, such that the nature of their original modification is correlated with a nucleic acid tag and can be decoded by sequencing the tag when nucleic acids are analyzed. Genetic variation of sample nucleic acid modifications can then be associated with the extent of modification (epigenetic variation) of that nucleic acid in the original sample, include single stranded (e.g., ssDNA or RNA) or double stranded molecules (e.g., dsDNA).
  • single stranded e.g., ssDNA or RNA
  • double stranded molecules e.g., dsDNA
  • the loss of DNA can reduce the presence of one or more types of DNA such that the presence of the one or more types of DNA such as cfDNA, is difficult to detect.
  • existing methods to measure DNA methylation such as enrichment or depletion methods, can have a relatively high level of resolution, such as about 100 base pairs (bp) to about 200 bp that can make accurately determining an amount of methylation of DNA difficult.
  • the accuracy with which DNA methylation is determined can impact the accuracy of estimates of tumor fraction for samples. Since tumor fraction can be used to determine whether a sample is derived from a subject in which a tumor is present or not, the accuracy of determinations of tumor fraction estimates can impact diagnosis and/or treatment decisions for individuals.
  • Genomic and epigenetic biomarkers identified here could be used to predict whether a patient is at high risk of cardiovascular disease before they have a heart attack or severe myocarditis. They could then be given existing drugs to reduce risk of heart attack or severe myocarditis.
  • Cardiac adverse events are a recognized toxicity for patients with breast cancer receiving anti-human epidermal growth factor receptor-2 (HER2) therapy.
  • HER2 anti-human epidermal growth factor receptor-2
  • Large phase III clinical trials have shown an incidence of left ventricular ejection fraction (LVEF) decrease >10% of 7.1-18.6% and overt heart failure of 1.7-4.1%.2
  • 3 Retrospective analyses from large registries such as the Surveillance, Epidemiology and End Results (SEER) and Cancer Research Network have reported an even higher incidence of heart failure at 41.9% and 20.1%, respectively.
  • Drugs targeting the genes and epigenetic loci identified here could be used to reduce risk of heart disease and/or improve patient outcome after myocardial infarction via influencing cardiac remodeling and myocardial interstitial fibrosis following myocardial infarction.
  • valsartan significantly attenuated the increase in periostin expression and may be part of its mechanism of action.
  • the inhibition of periostin by valsartan might contribute to its beneficial effects on cardiac remodeling after myocardial infarction.
  • TGFBI hypermethylation in patients that ultimately had heart failure and/or severe myocarditis (case group) relative to controls.
  • TGFBI over-expression acts similar to periostin over-expression, in that it appears to support a greater cardiac growth response (1)
  • drugs targeting TGFBI expression may be useful to reduce risk of heart failure or improve cardiac remodeling after myocardial infarction.
  • a sample can be any biological sample isolated from a subject.
  • a sample can be a bodily sample.
  • Samples can include body tissues, such as known or suspected solid tumors, whole blood, platelets, serum, plasma, stool, red blood cells, white blood cells or leucocytes, endothelial cells, tissue biopsies, cerebrospinal fluid synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid, the fluid in spaces between cells, including gingival crevicular fluid, bone marrow, pleural effusions, cerebrospinal fluid, saliva, mucous, sputum, semen, sweat, urine. Samples are preferably body fluids, particularly blood and fractions thereof, and urine.
  • a sample can be in the form originally isolated from a subject or can have been subjected to further processing to remove or add components, such as cells, or enrich for one component relative to another.
  • a preferred body fluid for analysis is plasma or serum containing cell-free nucleic acids.
  • a sample can be isolated or obtained from a subject and transported to a site of sample analysis. The sample may be preserved and shipped at a desirable temperature, e.g., room temperature, 4°C, -20°C, and/or -80°C.
  • a sample can be isolated or obtained from a subject at the site of the sample analysis.
  • the subject can be a human, a mammal, an animal, a companion animal, a service animal, or a pet.
  • the subject may have a cancer.
  • the subject may not have cancer or a detectable cancer symptom.
  • the subject may have been treated with one or more cancer therapy, e.g., any one or more of chemotherapies, antibodies, vaccines or biologies.
  • the subject may be in remission.
  • the subject may or may not be diagnosed as being susceptible to cardiovascular disease such as heart failure, cancer or any cancer-associated genetic mutations/disorders.
  • the volume of plasma can depend on the desired read depth for sequenced regions. Exemplary volumes are 0.4-40 ml, 5-20 ml, 10-20 ml. For example, the volume can be 0.5 mL, 1 mL, 5 mL 10 mL, 20 mL, 30 mL, or 40 mL. The volume of sampled plasma may be 5 to 20 mL.
  • a sample can comprise various amounts of nucleic acid that contains genome equivalents.
  • a sample of about 30 ng DNA can contain about 10,000 (104) haploid human genome equivalents and, in the case of cfDNA, about 200 billion (2x1011) individual polynucleotide molecules.
  • a sample of about 100 ng of DNA can contain about 30,000 haploid human genome equivalents and, in the case of cfDNA, about 600 billion individual molecules.
  • a sample can comprise nucleic acids from different sources, e.g., from cells and cell-free of the same subject, from cells and cell-free of different subjects.
  • a sample can comprise nucleic acids carrying mutations.
  • a sample can comprise DNA carrying germline mutations and/or somatic mutations.
  • Germline mutations refer to mutations existing in germline DNA of a subject.
  • Somatic mutations refer to mutations originating in somatic cells of a subject, e.g., cancer cells.
  • a sample can comprise DNA carrying cancer-associated mutations (e.g., cancer-associated somatic mutations).
  • a sample can comprise an epigenetic variant (i.e.
  • the epigenetic variant is associated with the presence of a genetic variant such as a cancer-associated mutation.
  • the sample includes an epigenetic variant associated with the presence of a genetic variant, wherein the sample does not comprise the genetic variant.
  • Exemplary amounts of cell-free nucleic acids in a sample before amplification range from about 1 fg to about 1 pg, e.g., 1 pg to 200 ng, 1 ng to 100 ng, 10 ng to 1000 ng.
  • the amount can be up to about 600 ng, up to about 500 ng, up to about 400 ng, up to about 300 ng, up to about 200 ng, up to about 100 ng, up to about 50 ng, or up to about 20 ng of cell-free nucleic acid molecules.
  • the amount can be at least 1 fg, at least 10 fg, at least 100 fg, at least 1 pg, at least 10 pg, at least 100 pg, at least 1 ng, at least 10 ng, at least 100 ng, at least 150 ng, or at least 200 ng of cell-free nucleic acid molecules.
  • the amount can be up to 1 femtogram (fg), 10 fg, 100 fg, 1 picogram (pg), 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 150 ng, or 200 ng of cell-free nucleic acid molecules.
  • the method can comprise obtaining 1 femtogram (fg) to 200 ng-
  • Cell-free nucleic acids are nucleic acids not contained within or otherwise bound to a cell or in other words nucleic acids remaining in a sample after removing intact cells.
  • Cell-free nucleic acids include DNA, RNA, and hybrids thereof, including genomic DNA, mitochondrial DNA, siRNA, miRNA, circulating RNA (cRNA), tRNA, rRNA, small nucleolar RNA (snoRNA), Pi wi -interacting RNA (piRNA), long non-coding RNA (long ncRNA), or fragments of any of these.
  • Cell-free nucleic acids can be double-stranded, single-stranded, or a hybrid thereof.
  • a cell-free nucleic acid can be released into bodily fluid through secretion or cell death processes, e.g., cellular necrosis and apoptosis.
  • Some cell-free nucleic acids are released into bodily fluid from cancer cells e.g., circulating tumor DNA, (ctDNA). Others are released from healthy cells.
  • cfDNA is cell-free fetal DNA (cffDNA)
  • cell free nucleic acids are produced by tumor cells.
  • cell free nucleic acids are produced by a mixture of tumor cells and non-tumor cells.
  • Cell-free nucleic acids have an exemplary size distribution of about 100-500 nucleotides, with molecules of 110 to about 230 nucleotides representing about 90% of molecules, with a mode of about 168 nucleotides and a second minor peak in a range between 240 to 440 nucleotides.
  • Cell-free nucleic acids can be isolated from bodily fluids through a fractionation or partitioning step in which cell-free nucleic acids, as found in solution, are separated from intact cells and other non-soluble components of the bodily fluid. Partitioning may include techniques such as centrifugation or filtration. Alternatively, cells in bodily fluids can be lysed and cell-free and cellular nucleic acids processed together.
  • nucleic acids can be precipitated with alcohol. Further clean up steps may be used such as silica based columns to remove contaminants or salts.
  • Non-specific bulk carrier nucleic acids such as Cot-1 DNA, DNA or protein for bisulfite sequencing, hybridization, and/or ligation, may be added throughout the reaction to optimize certain aspects of the procedure such as yield.
  • samples can include various forms of nucleic acid including double stranded DNA, single stranded DNA and single stranded RNA.
  • single stranded DNA and RNA can be converted to double stranded forms so they are included in subsequent processing and analysis steps.
  • Analytes can include nucleic acid analytes, and non-nucleic acid analytes.
  • the disclosure provides for detecting genetic variations in biological samples from a subject.
  • Biological samples may include polynucleotides from cancer cells. Polynucleotides may be DNA (e.g., genomic DNA, cDNA), RNA (e.g., mRNA, small RNAs), or any combination thereof.
  • Biological samples may include tumor tissue, e.g., from a biopsy. In some cases, biological samples may include blood or saliva. In particular cases, biological samples may comprise cell free DNA (“cfDNA”) or circulating tumor DNA (“ctDNA”). Cell free DNA can be present in, e.g., blood.
  • cfDNA cell free DNA
  • ctDNA circulating tumor DNA
  • non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquity lati on variants of proteins, sulfation variants of proteins, viral proteins (e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.), extracellular and intracellular proteins, antibodies, and antigen binding fragments.
  • viral proteins e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.
  • a posttranslational modification e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation or lipidation
  • the systems, apparatus, methods, and compositions can be used to analyze any number of analytes, further including both nucleic acid analytes and non-nucleic acid analytes.
  • the number of analytes that are analyzed can be at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 100, at least about 1,000, at least about 10,000, at least about 100,000 or more different analytes present in a region of the sample or within an individual feature of the substrate. Methods for performing multiplexed assays to analyze two or more different analytes will be discussed in a subsequent section of this disclosure.
  • nucleic acid analytes and/or non-nucleic acid analytes constitute a set of molecular interactions in a biological system under study (e.g., cells), which may be regarded as “interactome” - the molecular interactions that occur between molecules belonging to different biochemical families (proteins, nucleic acids, lipids, carbohydrates, etc.) and also within a given family.
  • an interactome is a protein-DNA interactome (network formed by transcription factors (and DNA or chromatin regulatory proteins) and their target genes.
  • interactome refers to protein-protein interaction network (PPI), or protein interaction network (PIN).
  • PPI protein-protein interaction network
  • PIN protein interaction network
  • the present methods can be used to diagnose presence of conditions, particularly cancer, in a subject, to characterize conditions (e.g., staging cancer or determining heterogeneity of a cancer), monitor response to treatment of a condition, effect prognosis risk of developing a condition or subsequent course of a condition.
  • the present disclosure can also be useful in determining the efficacy of a particular treatment option.
  • Successful treatment options may increase the amount of copy number variation or rare mutations detected in subject's blood if the treatment is successful as more cancers may die and shed DNA. In other examples, this may not occur.
  • certain treatment options may be correlated with genetic profiles of cancers over time. This correlation may be useful in selecting a therapy.
  • the present methods can be used to monitor residual disease or recurrence of disease.
  • the types and number of cancers that may be detected may include blood cancers, brain cancers, lung cancers, skin cancers, nose cancers, throat cancers, liver cancers, bone cancers, lymphomas, pancreatic cancers, skin cancers, bowel cancers, rectal cancers, thyroid cancers, bladder cancers, kidney cancers, mouth cancers, stomach cancers, solid state tumors, heterogeneous tumors, homogenous tumors and the like.
  • Type and/or stage of cancer can be detected from genetic variations including mutations, rare mutations, indels, copy number variations, transversions, translocations, inversion, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structure alterations, gene fusions, chromosome fusions, gene truncations, gene amplification, gene duplications, chromosomal lesions, DNA lesions, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, and abnormal changes in nucleic acid 5-methylcytosine.
  • Genetic and other analyte data can also be used for characterizing a specific form of cancer. Cancers are often heterogeneous in both composition and staging.
  • Genetic profile data may allow characterization of specific sub-types of cancer that may be important in the diagnosis or treatment of that specific sub-type. This information may also provide a subject or practitioner clues regarding the prognosis of a specific type of cancer and allow either a subject or practitioner to adapt treatment options in accord with the progress of the disease. Some cancers can progress to become more aggressive and genetically unstable. Other cancers may remain benign, inactive or dormant. The system and methods of this disclosure may be useful in determining disease progression.
  • the present analyses are also useful in determining the efficacy of a particular treatment option.
  • Successful treatment options may increase the amount of copy number variation or rare mutations detected in subject's blood if the treatment is successful as more cancers may die and shed DNA. In other examples, this may not occur.
  • certain treatment options may be correlated with genetic profiles of cancers over time. This correlation may be useful in selecting a therapy.
  • the present methods can be used to monitor residual disease or recurrence of disease.
  • the present methods can also be used for detecting genetic variations in conditions other than cancer.
  • Immune cells such as B cells
  • Clonal expansions may be monitored using copy number variation detection and certain immune states may be monitored.
  • copy number variation analysis may be performed over time to produce a profile of how a particular disease may be progressing.
  • Copy number variation or even rare mutation detection may be used to determine how a population of pathogens changes during the course of infection. This may be particularly important during chronic infections, such as HIV/AIDS or Hepatitis infections, whereby viruses may change life cycle state and/or mutate into more virulent forms during the course of infection.
  • the present methods may be used to determine or profile rejection activities of the host body, as immune cells attempt to destroy transplanted tissue to monitor the status of transplanted tissue as well as altering the course of treatment or prevention of rejection.
  • numerous types of malfunctions and abnormalities that commonly occur in the cardiovascular system wherein failure to diagnose or treat, will progressively decrease the body's ability to supply sufficient oxygen to satisfy the coronary oxygen demand when the individual encounters stress.
  • the progressive decline in the cardiovascular system's ability to supply oxygen under stress conditions will ultimately culminate in a heart attack, i.e., myocardial infarction event that is caused by the interruption of blood flow through the heart resulting in oxygen starvation of the heart muscle tissue (i.e., myocardium).
  • myocardial infarction event that is caused by the interruption of blood flow through the heart resulting in oxygen starvation of the heart muscle tissue (i.e., myocardium).
  • permanent damage will occur to the cells including the myocardium that will subsequently predispose the individual's suscept
  • Methods of the disclosure can characterize malfunctions and abnormalities associated with the heart muscle and valve tissues (e.g., hypertrophy), the decreased supply of blood flow and oxygen supply to the heart are often secondary symptoms of debilitation and/or deterioration of the blood now and supply system caused by physical and biochemical stresses.
  • cardiovascular diseases that are directly affected by these types of stresses include atherosclerosis, coronary artery disease, peripheral vascular disease and peripheral artery disease, along with various cardias and arrythmias which may represent other forms of disease and dysfunction.
  • the present methods can be used to generate or profile, fingerprint or set of data that is a summation of genetic information derived from different cells in a heterogeneous disease. This set of data may comprise copy number variation and mutation analyses alone or in combination.
  • the present methods can be used to diagnose, prognose, monitor or observe cancers, or other diseases. In some embodiments, the methods herein do not involve the diagnosing, prognosing or monitoring a fetus and as such are not directed to non-invasive prenatal testing. In other embodiments, these methodologies may be employed in a pregnant subject to diagnose, prognose, monitor or observe cancers or other diseases in an unborn subject whose DNA and other polynucleotides may co-circulate with maternal molecules.
  • bisulfite sequencing examples include, but are not limited to oxidative bisulfite sequencing (OX-BS-seq), Tet-assisted bisulfite sequencing (TAB-seq), and reduced bisulfite sequencing (redBS-seq).
  • OX-BS-seq oxidative bisulfite sequencing
  • TAB-seq Tet-assisted bisulfite sequencing
  • redBS-seq reduced bisulfite sequencing
  • Oxidative bisulfite sequencing (OX-BS-seq) is used to distinguish between 5mC and 5hmC, by first converting the 5hmC to 5fC, and then proceeding with bisulfite sequencing as previously described.
  • Tet-assisted bisulfite sequencing (TAB-seq) can also be used to distinguish 5mc and 5hmC.
  • TAB-seq 5hmC is protected by glucosylation.
  • a Tet enzyme is then used to convert 5mC to 5caC before proceeding with bisulfite sequencing, as previously described.
  • Reduced bisulfite sequencing is used to distinguish 5fC from modified cytosines.
  • cytosine sequencing a nucleic acid sample is divided into two aliquots and one aliquot is treated with bisulfite.
  • the bisulfite converts native cytosine and certain modified cytosine nucleotides (e.g. 5-formylcytosine or 5-carboxylcytosine) to uracil whereas other modified cytosines (e.g., 5- methylcytosine, 5-hydroxylmethylcystosine) are not converted.
  • modified cytosines e.g., 5- methylcytosine, 5-hydroxylmethylcystosine
  • Comparison of nucleic acid sequences of molecules from the two aliquots indicates which cytosines were and were not converted to uracils. Consequently, cytosines which were and were not modified can be determined.
  • the initial splitting of the sample into two aliquots is disadvantageous for samples containing only small amounts of nucleic acids, and/or composed of heterogeneous cell/tissue origins such as bodily
  • the present disclosure provides methods allowing bisulfite sequencing and variants thereof. These methods work by linking nucleic acids in a population to a capture moiety, i.e., a label that can be captured or immobilized.
  • Capture moieties include, without limitation, biotin, avidin, streptavidin, a nucleic acid including a particular nucleotide sequence, a hapten recognized by an antibody, and magnetically attractable particles.
  • the extraction moiety can be a member of a binding pair, such as biotin/ streptavidin or hapten/antibody.
  • a capture moiety that is attached to an analyte is captured by its binding pair which is attached to an isolatable moiety, such as a magnetically attractable particle or a large particle that can be sedimented through centrifugation.
  • the capture moiety can be any type of molecule that allows affinity separation of nucleic acids bearing the capture moiety from nucleic acids lacking the capture moiety.
  • Exemplary capture moieties are biotin which allows affinity separation by binding to streptavidin linked or linkable to a solid phase or an oligonucleotide, which allows affinity separation through binding to a complementary oligonucleotide linked or linkable to a solid phase.
  • the capture moiety can be linked to sample nucleic acids as a component of an adapter, which may also provide amplification and/or sequencing primer binding sites.
  • sample nucleic acids are linked to adapters at both ends, with both adapters bearing a capture moiety.
  • any cytosine residues in the adapters are modified, such as by 5methylcytosine, to protect against the action of bisulfite.
  • the capture moieties are linked to the original templates by a cleavable linkage (e.g., photocleavable desthiobiotin- TEG or uracil residues cleavable with USERTM enzyme, Chem. Commun. (Camb). 2015 Feb 21; 51(15): 3266-3269), in which case the capture moieties can, if desired, be removed.
  • the amplicons are denatured and contacted with an affinity reagent for the capture tag.
  • Original templates bind to the affinity reagent whereas nucleic acid molecules resulting from amplification do not. Thus, the original templates can be separated from nucleic acid molecules resulting from amplification.
  • the respective populations of nucleic acids can be subjected to bisulfite treatment with the original template population receiving bisulfite treatment and the amplification products not.
  • the amplification products can be subjected to bisulfite treatment and the original template population is not.
  • the respective populations can be amplified (which in the case of the original template population converts uracils to thymines).
  • the populations can also be subjected to biotin probe hybridization for enrichment. The respective populations are then analyzed and sequences compared to determine which cytosines were 5-methylated (or 5-hydroxylmethylated) in the original.
  • Detection of a T nucleotide in the template population indicates an unmodified C.
  • the presence of C's at corresponding positions of the original template and amplified populations indicates a modified C in the original sample.
  • a method uses sequential DNA-seq and bisulfite-seq (BlS-seq) NGS library preparation of molecular tagged DNA libraries. This process is performed by labeling of adapters (e.g., biotin), DNA-seq amplification of whole library, parent molecule recovery (e.g. streptavidin bead pull down), bisulfite conversion and BlS-seq.
  • the method identifies 5-methylcytosine with single-base resolution, through sequential NGS-preparative amplification of parent library molecules with and without bisulfite treatment.
  • sample DNA molecules are adapter ligated, and amplified (e.g., by PCR). As only the parent molecules will have a labeled adapter end, they can be selectively recovered from their amplified progeny by label-specific capture methods (e.g., streptavidin-magnetic beads).
  • label-specific capture methods e.g., streptavidin-magnetic beads.
  • the bisulfite treated library can be combined with a non-treated library prior to enrichment/NGS by addition of a sample tag DNA sequence in standard multiplexed NGS workflow.
  • bioinformatics analysis can be carried out for genomic alignment and 5- methylated base identification. In sum, this method provides the ability to selectively recover the parent, ligated molecules, carrying 5-methylcytosine marks, after library amplification, thereby allowing for parallel processing for bisulfite converted DNA.
  • the disclosure provides alternative methods for analyzing modified nucleic acids (e.g., methylated, linked to histones and other modifications discussed above).
  • modified nucleic acids e.g., methylated, linked to histones and other modifications discussed above.
  • a population of nucleic acids bearing the modification to different extents e.g., 0, 1, 2, 3, 4, 5 or more methyl groups per nucleic acid molecule
  • adapters attach to either one end or both ends of nucleic acid molecules in the population.
  • the adapters include different tags of sufficient numbers that the number of combinations of tags results in a low probability e.g., 95, 99 or 99.9% of two nucleic acids with the same start and stop points receiving the same combination of tags.
  • the nucleic acids are amplified from primers binding to the primer binding sites within the adapters.
  • Adapters, whether bearing the same or different tags, can include the same or different primer binding sites, but preferably adapters include the same primer binding site.
  • the nucleic acids are contacted with an agent that preferably binds to nucleic acids bearing the modification (such as the previously described such agents).
  • the nucleic acids are separated into at least two partitions differing in the extent to which the nucleic acids bear the modification from binding to the agents. For example, if the agent has affinity for nucleic acids bearing the modification, nucleic acids overrepresented in the modification (compared with median representation in the population) preferentially bind to the agent, whereas nucleic acids underrepresented for the modification do not bind or are more easily eluted from the agent.
  • the different partitions can then be subject to further processing steps, which typically include further amplification, and sequence analysis, in parallel but separately. Sequence data from the different partitions can then be compared.
  • Nucleic acids can be linked at both ends to Y-shaped adapters including primer binding sites and tags.
  • the molecules are amplified.
  • the amplified molecules are then fractionated by contact with an antibody preferentially binding to 5-methylcytosine to produce two partitions.
  • One partition includes original molecules lacking methylation and amplification copies having lost methylation.
  • the other partition includes original DNA molecules with methylation.
  • the two partitions are then processed and sequenced separately with further amplification of the methylated partition.
  • the sequence data of the two partitions can then be compared.
  • tags are not used to distinguish between methylated and unmethylated DNA but rather to distinguish between different molecules within these partitions so that one can determine whether reads with the same start and stop points are based on the same or different molecules.
  • the disclosure provides further methods for analyzing a population of nucleic acid in which at least some of the nucleic acids include one or more modified cytosine residues, such as 5-methylcytosine and any of the other modifications described previously.
  • the population of nucleic acids is contacted with adapters including one or more cytosine residues modified at the 5C position, such as 5-methylcytosine.
  • cytosine residues in such adapters are also modified, or all such cytosines in a primer binding region of the adapters are modified.
  • Adapters attach to both ends of nucleic acid molecules in the population.
  • the adapters include different tags of sufficient numbers that the number of combinations of tags results in a low probability e.g., 95, 99 or 99.9% of two nucleic acids with the same start and stop points receiving the same combination of tags.
  • the primer binding sites in such adapters can be the same or different, but are preferably the same.
  • the nucleic acids are amplified from primers binding to the primer binding sites of the adapters.
  • the amplified nucleic acids are split into first and second aliquots.
  • the first aliquot is assayed for sequence data with or without further processing.
  • the sequence data on molecules in the first aliquot is thus determined irrespective of the initial methylation state of the nucleic acid molecules.
  • the nucleic acid molecules in the second aliquot are treated with bisulfite. This treatment converts unmodified cytosines to uracils.
  • the bisulfite treated nucleic acids are then subjected to amplification primed by primers to the original primer binding sites of the adapters linked to nucleic acid. Only the nucleic acid molecules originally linked to adapters (as distinct from amplification products thereof) are now amplifiable because these nucleic acids retain cytosines in the primer binding sites of the adapters, whereas amplification products have lost the methylation of these cytosine residues, which have undergone conversion to uracils in the bisulfite treatment. Thus, only original molecules in the populations, at least some of which are methylated, undergo amplification. After amplification, these nucleic acids are subject to sequence analysis. Comparison of sequences determined from the first and second aliquots can indicate among other things, which cytosines in the nucleic acid population were subject to methylation.
  • a population of different forms of nucleic acids can be physically partitioned based on one or more characteristics of the nucleic acids prior to further analysis, e.g., differentially modifying or isolating a nucleobase, tagging, and/or sequencing. This approach can be used to determine, for example, whether certain sequences are hypermethylated or hypomethylated.
  • hypermethylation variable epigenetic target regions are analyzed to determine whether they show hypermethylation characteristic of tumor cells and/or hypomethylation variable epigenetic target regions are analyzed to determine whether they show hypomethylation characteristic of tumor cells.
  • partitioning a heterogeneous nucleic acid population one may increase rare signals, e.g., by enriching rare nucleic acid molecules that are more prevalent in one fraction (or partition) of the population. For example, a genetic variation present in hyper-methylated DNA but less (or not) in hypomethylated DNA can be more easily detected by partitioning a sample into hyper-methylated and hypo-methylated nucleic acid molecules.
  • a multi-dimensional analysis of a single locus of a genome or species of nucleic acid can be performed and hence, greater sensitivity can be achieved.
  • a heterogeneous nucleic acid sample is partitioned into two or more partitions (e.g., at least 3, 4, 5, 6 or 7 partitions).
  • each partition is differentially tagged.
  • Tagged partitions can then be pooled together for collective sample prep and/or sequencing. The partitioning-tagging-pooling steps can occur more than once, with each round of partitioning occurring based on a different characteristics (examples provided herein) and tagged using differential tags that are distinguished from other partitions and partitioning means.
  • partitioning examples include sequence length, methylation level, nucleosome binding, sequence mismatch, immunoprecipitation, and/or proteins that bind to DNA.
  • Resulting partitions can include one or more of the following nucleic acid forms: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), shorter DNA fragments and longer DNA fragments.
  • partitioning based on a cytosine modification (e.g., cytosine methylation) or methylation generally is performed and is optionally combined with at least one additional partitioning step, which may be based on any of the foregoing characteristics or forms of DNA.
  • a heterogeneous population of nucleic acids is partitioned into nucleic acids with one or more epigenetic modifications and without the one or more epigenetic modifications.
  • epigenetic modifications include presence or absence of methylation; level of methylation; type of methylation (e.g., 5- methylcytosine versus other types of methylation, such as adenine methylation and/or cytosine hydroxymethylation); and association and level of association with one or more proteins, such as histones.
  • a heterogeneous population of nucleic acids can be partitioned into nucleic acid molecules associated with nucleosomes and nucleic acid molecules devoid of nucleosomes.
  • a heterogeneous population of nucleic acids may be partitioned into single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA).
  • a heterogeneous population of nucleic acids may be partitioned based on nucleic acid length (e.g., molecules of up to 160 bp and molecules having a length of greater than 160 bp).
  • each partition representsative of a different nucleic acid form
  • the partitions are differentially labelled, and the partitions are pooled together prior to sequencing.
  • the different forms are separately sequenced.
  • a population of different nucleic acids is partitioned into two or more different partitions.
  • Each partition is representative of a different nucleic acid form, and a first partition (also referred to as a subsample) includes DNA with a cytosine modification in a greater proportion than a second subsample.
  • Each partition is distinctly tagged.
  • the first subsample is subjected to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first subsample, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base pairing specificity.
  • the tagged nucleic acids are pooled together prior to sequencing. Sequence reads are obtained and analyzed, including to distinguish the first nucleobase from the second nucleobase in the DNA of the first subsample, in silico. Tags are used to sort reads from different partitions.
  • Analysis to detect genetic variants can be performed on a partition-by-partition level, as well as whole nucleic acid population level.
  • analysis can include in silico analysis to determine genetic variants, such as CNV, SNV, indel, fusion in nucleic acids in each partition.
  • in silico analysis can include determining chromatin structure.
  • coverage of sequence reads can be used to determine nucleosome positioning in chromatin. Higher coverage can correlate with higher nucleosome occupancy in genomic region while lower coverage can correlate with lower nucleosome occupancy or nucleosome depleted region (NDR).
  • Samples can include nucleic acids varying in modifications including post-replication modifications to nucleotides and binding, usually noncovalently, to one or more proteins.
  • the population of nucleic acids is one obtained from a serum, plasma or blood sample from a subject suspected of having neoplasia, a tumor, or cancer or previously diagnosed with neoplasia, a tumor, or cancer.
  • the population of nucleic acids includes nucleic acids having varying levels of methylation. Methylation can occur from any one or more postreplication or transcriptional modifications. Post-replication modifications include modifications of the nucleotide cytosine, particularly at the 5-position of the nucleobase, e.g., 5- methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine.
  • the affinity agents can be antibodies with the desired specificity, natural binding partners or variants thereof (Bock et al., Nat Biotech 28: 1106-1114 (2010); Song et al., Nat Biotech 29: 68-72 (2011)), or artificial peptides selected e.g., by phage display to have specificity to a given target.
  • capture moieties contemplated herein include methyl binding domain (MBDs) and methyl binding proteins (MBPs) as described herein, including proteins such as MeCP2 and antibodies preferentially binding to 5-methylcytosine.
  • partitioning of different forms of nucleic acids can be performed using histone binding proteins which can separate nucleic acids bound to histones from free or unbound nucleic acids.
  • histone binding proteins examples include RBBP4, RbAp48 and SANT domain peptides.
  • binding to the agent may occur in an essentially all or none manner depending on whether a nucleic acid bears a modification, the separation may be one of degree.
  • nucleic acids overrepresented in a modification bind to the agent at a greater extent that nucleic acids underrepresented in the modification.
  • nucleic acids having modifications may bind in an all or nothing manner. But then, various levels of modifications may be sequentially eluted from the binding agent.
  • partitioning can be binary or based on degree/level of modifications.
  • all methylated fragments can be partitioned from unmethylated fragments using methyl-binding domain proteins (e.g., MethylMiner Methylated DNA Enrichment Kit (ThermoFisher Scientific)).
  • methyl-binding domain proteins e.g., MethylMiner Methylated DNA Enrichment Kit (ThermoFisher Scientific)
  • additional partitioning may involve eluting fragments having different levels of methylation by adjusting the salt concentration in a solution with the methyl-binding domain and bound fragments. As salt concentration increases, fragments having greater methylation levels are eluted.
  • the final partitions are representative of nucleic acids having different extents of modifications (overrepresentative or underrepresentative of modifications).
  • Overrepresentation and underrepresentation can be defined by the number of modifications born by a nucleic acid relative to the median number of modifications per strand in a population. For example, if the median number of 5-methylcytosine residues in nucleic acid in a sample is 2, a nucleic acid including more than two 5- methylcytosine residues is overrepresented in this modification and a nucleic acid with 1 or zero 5-methylcytosine residues is underrepresented.
  • the effect of the affinity separation is to enrich for nucleic acids overrepresented in a modification in a bound phase and for nucleic acids underrepresented in a modification in an unbound phase (i.e. in solution). The nucleic acids in the bound phase can be eluted before subsequent processing.
  • methylation When using MethylMiner Methylated DNA Enrichment Kit (ThermoFisher Scientific) various levels of methylation can be partitioned using sequential elutions. For example, a hypom ethylated partition (e.g., no methylation) can be separated from a methylated partition by contacting the nucleic acid population with the MBD from the kit, which is attached to magnetic beads. The beads are used to separate out the methylated nucleic acids from the non- methylated nucleic acids. Subsequently, one or more elution steps are performed sequentially to elute nucleic acids having different levels of methylation.
  • a hypom ethylated partition e.g., no methylation
  • a first set of methylated nucleic acids can be eluted at a salt concentration of 160 mM or higher, e.g., at least 150 mM, at least 200 mM, at least 300 mM, at least 400 mM, at least 500 mM, at least 600 mM, at least 700 mM, at least 800 mM, at least 900 mM, at least 1000 mM, or at least 2000 mM.
  • magnetic separation is once again used to separate higher levels of methylated nucleic acids from those with lower level of methylation.
  • the elution and magnetic separation steps can repeat themselves to create various partitions such as a hypomethylated partition (representative of no methylation), a methylated partition (representative of low level of methylation), and a hyper methylated partition (representative of high level of methylation).
  • nucleic acids bound to an agent used for affinity separation are subjected to a wash step.
  • the wash step washes off nucleic acids weakly bound to the affinity agent.
  • nucleic acids can be enriched in nucleic acids having the modification to an extent close to the mean or median (i.e., intermediate between nucleic acids remaining bound to the solid phase and nucleic acids not binding to the solid phase on initial contacting of the sample with the agent).
  • the affinity separation results in at least two, and sometimes three or more partitions of nucleic acids with different extents of a modification.
  • the nucleic acids of at least one partition, and usually two or three (or more) partitions are linked to nucleic acid tags, usually provided as components of adapters, with the nucleic acids in different partitions receiving different tags that distinguish members of one partition from another.
  • the tags linked to nucleic acid molecules of the same partition can be the same or different from one another. But if different from one another, the tags may have part of their code in common so as to identify the molecules to which they are attached as being of a particular partition.
  • portioning nucleic acid samples based on characteristics such as methylation see WO2018/119452, which is incorporated herein by reference.
  • the nucleic acid molecules can be fractionated into different partitions based on the nucleic acid molecules that are bound to a specific protein or a fragment thereof and those that are not bound to that specific protein or fragment thereof.
  • Nucleic acid molecules can be fractionated based on DNA-protein binding.
  • Protein-DNA complexes can be fractionated based on a specific property of a protein. Examples of such properties include various epitopes, modifications (e.g., histone methylation or acetylation) or enzymatic activity. Examples of proteins which may bind to DNA and serve as a basis for fractionation may include, but are not limited to, protein A and protein G. Any suitable method can be used to fractionate the nucleic acid molecules based on protein bound regions.
  • Examples of methods used to fractionate nucleic acid molecules based on protein bound regions include, but are not limited to, SDS-PAGE, chromatin-immuno-precipitation (ChIP), heparin chromatography, and asymmetrical field flow fractionation (AF4).
  • ChIP chromatin-immuno-precipitation
  • AF4 asymmetrical field flow fractionation
  • partitioning of the nucleic acids is performed by contacting the nucleic acids with a methylation binding domain (“MBD”) of a methylation into fractions with different extents of methylation can be performed by eluting fractions by increasing the NaCl concentration, binding protein (“MBP”).
  • MBD binds to 5-methylcytosine (5mC).
  • MBD is coupled to paramagnetic beads, such as Dynabeads® M-280 Streptavidin via a biotin linker.
  • An exemplary method for molecular tag identification of MBD-bead partitioned libraries through NGS is as follows:
  • genomic regions of interest e.g., cancer-specific genetic variants and differentially methylated regions.
  • Re-amplification of the enriched total DNA library appending a sample tag. Different samples are pooled and assayed in multiplex on an NGS instrument.
  • MBPs contemplated herein include, but are not limited to:
  • MeCP2 is a protein preferentially binding to 5-methyl-cytosine over unmodified cytosine.
  • FOXK1, FOXK2, FOXP1, FOXP4 and FOXI3 preferably bind to 5-formyl-cytosine over unmodified cytosine (lurlaro et al., Genome Biol. 14: R119 (2013)).
  • elution is a function of number of methylated sites per molecule, with molecules having more methylation eluting under increased salt concentrations.
  • salt concentration can range from about 100 nM to about 2500 mM NaCl.
  • the process results in three (3) partitions. Molecules are contacted with a solution at a first salt concentration and including a molecule including a methyl binding domain, which molecule can be attached to a capture moiety, such as streptavidin.
  • a population of molecules will bind to the MBD and a population will remain unbound.
  • the unbound population can be separated as a “hypomethylated” population.
  • a first partition representative of the hypomethylated form of DNA is that which remains unbound at a low salt concentration, e.g., 100 mM or 160 mM.
  • a second partition representative of intermediate methylated DNA is eluted using an intermediate salt concentration, e.g., between 100 mM and 2000 mM concentration. This is also separated from the sample.
  • a third partition representative of hypermethylated form of DNA is eluted using a high salt concentration, e.g., at least about 2000 mM.
  • the disclosure provides further methods for analyzing a population of nucleic acids in which at least some of the nucleic acids include one or more modified cytosine residues, such as 5-methylcytosine and any of the other modifications described previously.
  • the subsamples of nucleic acids are contacted with adapters including one or more cytosine residues modified at the 5C position, such as 5-methylcytosine.
  • cytosine residues in such adapters are also modified, or all such cytosines in a primer binding region of the adapters are modified.
  • Adapters attach to both ends of nucleic acid molecules in the population.
  • the adapters include different tags of sufficient numbers that the number of combinations of tags results in a low probability e.g., 95, 99 or 99.9% of two nucleic acids with the same start and stop points receiving the same combination of tags.
  • the primer binding sites in such adapters can be the same or different, but are preferably the same.
  • the nucleic acids are amplified from primers binding to the primer binding sites of the adapters.
  • the amplified nucleic acids are split into first and second aliquots.
  • the first aliquot is assayed for sequence data with or without further processing.
  • the sequence data on molecules in the first aliquot is thus determined irrespective of the initial methylation state of the nucleic acid molecules.
  • the nucleic acid molecules in the second aliquot are subjected to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA, wherein the first nucleobase includes a cytosine modified at the 5 position, and the second nucleobase includes unmodified cytosine.
  • This procedure may be bisulfite treatment or another procedure that converts unmodified cytosines to uracils.
  • the nucleic acids subjected to the procedure are then amplified with primers to the original primer binding sites of the adapters linked to nucleic acid.
  • nucleic acid molecules originally linked to adapters are now amplifiable because these nucleic acids retain cytosines in the primer binding sites of the adapters, whereas amplification products have lost the methylation of these cytosine residues, which have undergone conversion to uracils in the bisulfite treatment.
  • amplification products have lost the methylation of these cytosine residues, which have undergone conversion to uracils in the bisulfite treatment.
  • amplification products have lost the methylation of these cytosine residues, which have undergone conversion to uracils in the bisulfite treatment.
  • amplification products have lost the methylation of these cytosine residues, which have undergone conversion to uracils in the bisulfite treatment.
  • only original molecules in the populations, at least some of which are methylated undergo amplification.
  • these nucleic acids are subject to sequence analysis. Comparison of sequences determined from the first and second aliquots can indicate among other things, which cytos
  • methylated DNA is linked to Y-shaped adapters at both ends including primer binding sites and tags.
  • the cytosines in the adapters are modified at the 5 position (e.g., 5- methylated).
  • the modification of the adapters serves to protect the primer binding sites in a subsequent conversion step (e.g., bisulfite treatment, TAP conversion, or any other conversion that does not affect the modified cytosine but affects unmodified cytosine).
  • the DNA molecules are amplified.
  • the amplification product is split into two aliquots for sequencing with and without conversion. The aliquot not subjected to conversion can be subjected to sequence analysis with or without further processing.
  • the other aliquot is subjected to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA, wherein the first nucleobase includes a cytosine modified at the 5 position, and the second nucleobase includes unmodified cytosine.
  • This procedure may be bisulfite treatment or another procedure that converts unmodified cytosines to uracils. Only primer binding sites protected by modification of cytosines can support amplification when contacted with primers specific for original primer binding sites. Thus, only original molecules and not copies from the first amplification are subjected to further amplification. The further amplified molecules are then subjected to sequence analysis. Sequences can then be compared from the two aliquots. As in the separation scheme discussed above, nucleic acid tags in adapters are not used to distinguish between methylated and unmethylated DNA but to distinguish nucleic acid molecules within the same partition.
  • Methods disclosed herein comprise a step of subjecting the first subsample to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first subsample, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base pairing specificity.
  • the second nucleobase is a modified or unmodified adenine; if the first nucleobase is a modified or unmodified cytosine, then the second nucleobase is a modified or unmodified cytosine; if the first nucleobase is a modified or unmodified guanine, then the second nucleobase is a modified or unmodified guanine; and if the first nucleobase is a modified or unmodified thymine, then the second nucleobase is a modified or unmodified thymine (where modified and unmodified uracil are encompassed within modified thymine for the purpose of this step).
  • the first nucleobase is a modified or unmodified cytosine
  • the second nucleobase is a modified or unmodified cytosine.
  • first nucleobase may comprise unmodified cytosine (C) and the second nucleobase may comprise one or more of 5- methylcytosine (mC) and 5-hydroxymethylcytosine (hmC).
  • the second nucleobase may comprise C and the first nucleobase may comprise one or more of mC and hmC.
  • Other combinations are also possible, as indicated, e.g., in the Summary above and the following discussion, such as where one of the first and second nucleobases includes mC and the other includes hmC.
  • the procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first subsample includes bisulfite conversion.
  • Treatment with bisulfite converts unmodified cytosine and certain modified cytosine nucleotides (e.g. 5-formyl cytosine (fC) or 5-carboxylcytosine (caC)) to uracil whereas other modified cytosines (e.g., 5-methylcytosine, 5-hydroxylmethylcystosine) are not converted.
  • modified cytosine nucleotides e.g. 5-formyl cytosine (fC) or 5-carboxylcytosine (caC)
  • fC 5-formyl cytosine
  • caC 5-carboxylcytosine
  • the first nucleobase includes one or more of unmodified cytosine, 5-formyl cytosine, 5-carboxylcytosine, or other cytosine forms affected by bisulfite
  • the second nucleobase may comprise one or more of mC and hmC, such as mC and optionally hmC.
  • Sequencing of bisulfite-treated DNA identifies positions that are read as cytosine as being mC or hmC positions. Meanwhile, positions that are read as T are identified as being T or a bisulfite-susceptible form of C, such as unmodified cytosine, 5-formyl cytosine, or 5-carboxylcytosine.
  • the procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first subsample includes oxidative bisulfite (Ox-BS) conversion. In some embodiments, the procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first subsample includes Tet-assisted bisulfite (TAB) conversion.
  • Ox-BS oxidative bisulfite
  • TAB Tet-assisted bisulfite
  • the procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first subsample includes Tet-assisted conversion with a substituted borane reducing agent, optionally wherein the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane.
  • a substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane.
  • the procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first subsample includes chemical-assisted conversion with a substituted borane reducing agent, optionally wherein the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane.
  • the procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first subsample includes APOBEC-coupled epigenetic (ACE) conversion.
  • ACE APOBEC-coupled epigenetic
  • procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first subsample includes enzymatic conversion of the first nucleobase, e.g., as in EM-Seq. See, e.g., Vaisvila R, et al. (2019) EM-seq: Detection of DNA methylation at single base resolution from picograms of DNA. bioRxiv; DOI: 10.1101/2019.12.20.884692, available at www.biorxiv.org/content/10.1101/2019.12.20.884692vl.
  • TET2 and T4-PGT can be used to convert 5mC and 5hmC into substrates that cannot be deaminated by a deaminase (e.g., APOBEC3A), and then a deaminase (e.g., APOBEC3A) can be used to deaminate unmodified cytosines converting them to uracils.
  • a deaminase e.g., APOBEC3A
  • APOBEC3A a deaminase
  • the procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first subsample includes separating DNA originally including the first nucleobase from DNA not originally including the first nucleobase.
  • the first nucleobase is a modified or unmodified adenine
  • the second nucleobase is a modified or unmodified adenine.
  • the modified adenine is N6-methyladenine (mA).
  • the modified adenine is one or more of N6-methyladenine (mA), N6-hydroxymethyladenine (hmA), or N6-formyladenine (fA).
  • methylated DNA immunoprecipitation can be used to separate DNA containing modified bases such as mA from other DNA. See, e.g., Kumar et al., Frontiers Genet. 2018; 9: 640; Greer et al., Cell 2015; 161 : 868-878. An antibody specific for mA is described in Sun et al., Bioessays 2015; 37:1155-62. Antibodies for various modified nucleobases, such as forms of thymine/uracil including halogenated forms such as 5- bromouracil, are commercially available. Various modified bases can also be detected based on alterations in their base-pairing specificity.
  • hypoxanthine is a modified form of adenine that can result from deamination and is read in sequencing as a G. See, e.g., US Patent 8,486,630; Brown, Genomes, 2nd Ed., John Wiley & Sons, Inc., New York, N.Y., 2002, chapter 14, “Mutation, Repair, and Recombination.”
  • methods disclosed herein comprise a step of capturing one or more sets of target regions of DNA, such as cfDNA. Capture may be performed using any suitable approach known in the art. In some embodiments, capturing includes contacting the DNA to be captured with a set of target-specific probes.
  • the set of target-specific probes may have any of the features described herein for sets of target-specific probes, including but not limited to in the embodiments set forth above and the sections relating to probes below. Capturing may be performed on one or more subsamples prepared during methods disclosed herein.
  • DNA is captured from at least the first subsample or the second subsample, e.g., at least the first subsample and the second subsample.
  • a separation step e.g., separating DNA originally including the first nucleobase (e.g., hmC) from DNA not originally including the first nucleobase, such as hmC-seal
  • capturing may be performed on any, any two, or all of the DNA originally including the first nucleobase (e.g., hmC), the DNA not originally including the first nucleobase, and the second subsample.
  • the subsamples are differentially tagged (e.g., as described herein) and then pooled before undergoing capture.
  • the capturing step may be performed using conditions suitable for specific nucleic acid hybridization, which generally depend to some extent on features of the probes such as length, base composition, etc. Those skilled in the art will be familiar with appropriate conditions given general knowledge in the art regarding nucleic acid hybridization. In some embodiments, complexes of target-specific probes and DNA are formed.
  • a method described herein includes capturing cfDNA obtained from a test subject for a plurality of sets of target regions.
  • the target regions comprise epigenetic target regions, which may show differences in methylation levels and/or fragmentation patterns depending on whether they originated from a tumor or from healthy cells.
  • the target regions also comprise sequence-variable target regions, which may show differences in sequence depending on whether they originated from a tumor or from healthy cells.
  • the capturing step produces a captured set of cfDNA molecules, and the cfDNA molecules corresponding to the sequencevariable target region set are captured at a greater capture yield in the captured set of cfDNA molecules than cfDNA molecules corresponding to the epigenetic target region set.
  • a method described herein includes contacting cfDNA obtained from a test subject with a set of target-specific probes, wherein the set of target-specific probes is configured to capture cfDNA corresponding to the sequence-variable target region set at a greater capture yield than cfDNA corresponding to the epigenetic target region set.
  • the volume of data needed to determine fragmentation patterns (e.g., to test fsor perturbation of transcription start sites or CTCF binding sites) or fragment abundance (e.g., in hypermethylated and hypomethylated partitions) is generally less than the volume of data needed to determine the presence or absence of cancer-related sequence mutations.
  • Capturing the target region sets at different yields can facilitate sequencing the target regions to different depths of sequencing in the same sequencing run (e.g., using a pooled mixture and/or in the same sequencing cell).
  • the methods further comprise sequencing the captured cfDNA, e.g., to different degrees of sequencing depth for the epigenetic and sequence-variable target region sets, consistent with the discussion herein.
  • complexes of targetspecific probes and DNA are separated from DNA not bound to target-specific probes.
  • a washing or aspiration step can be used to separate unbound material.
  • the complexes have chromatographic properties distinct from unbound material (e.g., where the probes comprise a ligand that binds a chromatographic resin), chromatography can be used.
  • the set of target-specific probes may comprise a plurality of sets such as probes for a sequence-variable target region set and probes for an epigenetic target region set.
  • the capturing step is performed with the probes for the sequence- variable target region set and the probes for the epigenetic target region set in the same vessel at the same time, e.g., the probes for the sequence-variable and epigenetic target region sets are in the same composition.
  • the concentration of the probes for the sequence-variable target region set is greater that the concentration of the probes for the epigenetic target region set.
  • the capturing step is performed with the sequence-variable target region probe set in a first vessel and with the epigenetic target region probe set in a second vessel, or the contacting step is performed with the sequence- variable target region probe set at a first time and a first vessel and the epigenetic target region probe set at a second time before or after the first time.
  • This approach allows for preparation of separate first and second compositions including captured DNA corresponding to the sequence-variable target region set and captured DNA corresponding to the epigenetic target region set.
  • the compositions can be processed separately as desired (e.g., to fractionate based on methylation as described elsewhere herein) and recombined in appropriate proportions to provide material for further processing and analysis such as sequencing.
  • the DNA is amplified. In some embodiments, amplification is performed before the capturing step. In some embodiments, amplification is performed after the capturing step.
  • adapters are included in the DNA. This may be done concurrently with an amplification procedure, e.g., by providing the adapters in a 5’ portion of a primer, e.g., as described above. Alternatively, adapters can be added by other approaches, such as ligation.
  • tags which may be or include barcodes, are included in the DNA. Tags can facilitate identification of the origin of a nucleic acid. For example, barcodes can be used to allow the origin (e.g., subject) whence the DNA came to be identified following pooling of a plurality of samples for parallel sequencing.
  • amplification procedure e.g., by providing the barcodes in a 5’ portion of a primer, e.g., as described above.
  • adapters and tags/barcodes are provided by the same primer or primer set.
  • the barcode may be located 3’ of the adapter and 5’ of the target-hybridizing portion of the primer.
  • barcodes can be added by other approaches, such as ligation, optionally together with adapters in the same ligation substrate.
  • a captured set of DNA (e.g., cfDNA) is provided.
  • the captured set of DNA may be provided, e.g., by performing a capturing step after a partitioning step as described herein.
  • the captured set may comprise DNA corresponding to a sequence-variable target region set, an epigenetic target region set, or a combination thereof.
  • the quantity of captured sequence-variable target region DNA is greater than the quantity of the captured epigenetic target region DNA, when normalized for the difference in the size of the targeted regions (footprint size).
  • first and second captured sets may be provided, including, respectively, DNA corresponding to a sequence-variable target region set and DNA corresponding to an epigenetic target region set.
  • the first and second captured sets may be combined to provide a combined captured set.
  • the DNA corresponding to the sequence-variable target region set may be present at a greater concentration than the DNA corresponding to the epigenetic target region set, e.g., a 1.1 to 1.2-fold greater concentration, a 1.2- to 1.4-fold greater concentration, a 1.4- to 1.6-fold greater concentration, a 1.6- to 1.8-fold greater concentration, a 1.8- to 2.0-fold greater concentration, a 2.0- to 2.2-fold greater concentration, a 2.2- to 2.4-fold greater concentration a 2.4- to 2.6-fold greater concentration, a 2.6- to 2.8-fold greater concentration, a 2.8- to 3.0-fold greater concentration, a 3.0- to 3.5-fold greater concentration, a 3.5- to 4.0, a 4.0- to 4.5-fold greater concentration, a 4.5- to 5.0-fold
  • the epigenetic target region set may comprise one or more types of target regions likely to differentiate DNA from neoplastic (e.g., tumor or cancer) cells and from healthy cells, e.g., non-neoplastic circulating cells. Exemplary types of such regions are discussed in detail herein.
  • the epigenetic target region set may also comprise one or more control regions, e.g., as described herein. In some embodiments, the epigenetic target region set has a footprint of at least 100 kb, e.g., at least 200 kb, at least 300 kb, or at least 400 kb.
  • the epigenetic target region set has a footprint in the range of 100-1000 kb, e.g., 100-200 kb, 200- 300 kb, 300-400 kb, 400-500 kb, 500-600 kb, 600-700 kb, 700-800 kb, 800-900 kb, and 900- 1,000 kb.
  • the epigenetic target region set includes one or more hypermethylation variable target regions.
  • hypermethylation variable target regions refer to regions where an increase in the level of observed methylation, e.g., in a cfDNA sample, indicates an increased likelihood that a sample (e.g., of cfDNA) contains DNA produced by neoplastic cells, such as tumor or cancer cells.
  • a sample e.g., of cfDNA
  • hypermethylation of promoters of tumor suppressor genes has been observed repeatedly. See, e.g., Kang et al., Genome Biol. 18:53 (2017) and references cited therein.
  • hypermethylation variable target regions can include regions that do not necessarily differ in methylation in cancerous tissue relative to DNA from healthy tissue of the same type, but do differ in methylation (e.g., have more methylation) relative to cfDNA that is typical in healthy subjects.
  • methylation e.g., have more methylation
  • the presence of a cancer results in increased cell death such as apoptosis of cells of the tissue type corresponding to the cancer
  • such a cancer can be detected at least in part using such hypermethylation variable target regions.
  • hypermethylation variable target regions include one or more genomic regions, where the cfDNA molecules in those regions do not differ in methylation state in cancer subjects relative to cfDNA from healthy subjects, but the presence/increased quantity of hypermethylated cfDNA in those regions is indicative of a particular tissue type (e.g., cancer origin) and is presented as cfDNA with increased apoptosis (e.g. tumor shedding) into circulation.
  • tissue type e.g., cancer origin
  • apoptosis e.g. tumor shedding
  • Hypermethylation target regions may be obtained, e.g., from the Cancer Genome Atlas. Kang et al., Genome Biology 18:53 (2017), describe construction of a probabilistic method called CancerLocator using hypermethylation target regions from breast, colon, kidney, liver, and lung.
  • the hypermethylation target regions can be specific to one or more types of cancer.
  • the hypermethylation target regions include one, two, three, four, or five subsets of hypermethylation target regions that collectively show hypermethylation in one, two, three, four, or five of breast, colon, kidney, liver, and lung cancers.
  • the probes for the epigenetic target region set comprise probes specific for one or more hypermethylation variable target regions.
  • the hypermethylation variable target regions may be any of those set forth above.
  • the probes specific for hypermethylation variable target regions comprise probes specific for a plurality of loci listed in Table 1, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the loci listed in Table 1.
  • the probes specific for hypermethylation variable target regions comprise probes specific for a plurality of loci listed in Table 2, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the loci listed in Table 2.
  • the probes specific for hypermethylation variable target regions comprise probes specific for a plurality of loci listed in Table 1 or Table 2, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the loci listed in Table 1 or Table 2.
  • the one or more probes bind within 300 bp of the listed position, e.g., within 200 or 100 bp.
  • a probe has a hybridization site overlapping the position listed above.
  • the probes specific for the hypermethylation target regions include probes specific for one, two, three, four, or five subsets of hypermethylation target regions that collectively show hypermethylation in one, two, three, four, or five of breast, colon, kidney, liver, and lung cancers. Hypomethylation Variable Target Regions
  • the epigenetic target region set includes hypomethylation variable target regions, where a decrease in the level of observed methylation indicates an increased likelihood that a sample (e.g., of cfDNA) contains DNA produced by neoplastic cells, such as tumor or cancer cells.
  • hypomethylation variable target regions can include regions that do not necessarily differ in methylation state in cancerous tissue relative to DNA from healthy tissue of the same type, but do differ in methylation (e.g., are less methylated) relative to cfDNA that is typical in healthy subjects.
  • hypomethylation variable target regions include one or more genomic regions, where the cfDNA molecules in those regions do not differ in methylation state in cancer subjects relative to cfDNA from healthy subjects, but the presence/increased quantity of hypom ethylated cfDNA in those regions is indicative of a particular tissue type (e.g., cancer origin) and is presented as cfDNA with increased apoptosis (e.g. tumor shedding) into circulation.
  • tissue type e.g., cancer origin
  • hypomethylation variable target regions include repeated elements and/or intergenic regions.
  • repeated elements include one, two, three, four, or five of LINE 1 elements, Alu elements, centromeric tandem repeats, pericentromeric tandem repeats, and/or satellite DNA.
  • Exemplary specific genomic regions that show cancer-associated hypomethylation include nucleotides 8403565-8953708 and 151104701-151106035 of human chromosome 1.
  • the hypomethylation variable target regions overlap or comprise one or both of these regions.
  • the probes for the epigenetic target region set comprise probes specific for one or more hypomethylation variable target regions.
  • the hypomethylation variable target regions may be any of those set forth above.
  • the probes specific for one or more hypomethylation variable target regions may include probes for regions such as repeated elements, e.g., LINE1 elements, Alu elements, centromeric tandem repeats, pericentromeric tandem repeats, and satellite DNA, and intergenic regions that are ordinarily methylated in healthy cells may show reduced methylation in tumor cells.
  • probes specific for hypomethylation variable target regions include probes specific for repeated elements and/or intergenic regions.
  • probes specific for repeated elements include probes specific for one, two, three, four, or five of LINE1 elements, Alu elements, centromeric tandem repeats, pericentromeric tandem repeats, and/or satellite DNA.
  • Exemplary probes specific for genomic regions that show cancer-associated hypomethylation include probes specific for nucleotides 8403565-8953708 and/or 151104701- 151106035 of human chromosome 1.
  • the probes specific for hypomethylation variable target regions include probes specific for regions overlapping or including nucleotides 8403565-8953708 and/or 151104701-151106035 of human chromosome
  • Probes for detecting the panel of regions can include those for detecting genomic regions of interest (hotspot regions) as well as nucleosome-aware probes (e.g., KRAS codons 12 and 13) and may be designed to optimize capture based on analysis of cfDNA coverage and fragment size variation impacted by nucleosome binding patterns and GC sequence composition. Regions used herein can also include non-hotspot regions optimized based on nucleosome positions and GC models. Subjects
  • the DNA is obtained from a subject having a cancer. In some embodiments, the DNA (e.g., cfDNA) is obtained from a subject suspected of having a cancer. In some embodiments, the DNA (e.g., cfDNA) is obtained from a subject having a tumor. In some embodiments, the DNA (e.g., cfDNA) is obtained from a subject suspected of having a tumor. In some embodiments, the DNA (e.g., cfDNA) is obtained from a subject having neoplasia. In some embodiments, the DNA (e.g., cfDNA) is obtained from a subject suspected of having neoplasia.
  • the DNA (e.g., cfDNA) is obtained from a subject in remission from a tumor, cancer, or neoplasia (e.g., following chemotherapy, surgical resection, radiation, or a combination thereof).
  • the cancer, tumor, or neoplasia or suspected cancer, tumor, or neoplasia may be of the lung, colon, rectum, kidney, breast, prostate, or liver.
  • the cancer, tumor, or neoplasia or suspected cancer, tumor, or neoplasia is of the lung.
  • the cancer, tumor, or neoplasia or suspected cancer, tumor, or neoplasia is of the colon or rectum. In some embodiments, the cancer, tumor, or neoplasia or suspected cancer, tumor, or neoplasia is of the breast. In some embodiments, the cancer, tumor, or neoplasia or suspected cancer, tumor, or neoplasia is of the prostate. In any of the foregoing embodiments, the subject may be a human subject.
  • the sequence-variable target region probe set has a footprint of at least 0.5 kb, e.g., at least 1 kb, at least 2 kb, at least 5 kb, at least 10 kb, at least 20 kb, at least 30 kb, or at least 40 kb.
  • the epigenetic target region probe set has a footprint in the range of 0.5-100 kb, e.g., 0.5-2 kb, 2-10 kb, 10-20 kb, 20-30 kb, 30-40 kb, 40-50 kb, 50-60 kb, 60-70 kb, 70-80 kb, 80-90 kb, and 90-100 kb.
  • the probes specific for the sequence-variable target region set comprise probes specific for target regions from at least 10, 20, 30, or 35 cancer-related genes, such as AKT1, ALK, BRAF, CCND1, CDK2A, CTNNB1, EGFR, ERBB2, ESRI, FGFR1, FGFR2, FGFR3, FOXL2, GATA3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MED12, MET, MYC, NFE2L2, NRAS, PDGFRA, PIK3CA, PPP2R1 A, PTEN, RET, STK11, TP53, and U2AF1.
  • cancer-related genes such as AKT1, ALK, BRAF, CCND1, CDK2A, CTNNB1, EGFR, ERBB2, ESRI, FGFR1, FGFR2, FGFR3, FOXL2, GATA3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, K
  • the first population may comprise or be derived from DNA with a cytosine modification in a greater proportion than the second population.
  • the first population may comprise a form of a first nucleobase originally present in the DNA with altered base pairing specificity and a second nucleobase without altered base pairing specificity, wherein the form of the first nucleobase originally present in the DNA prior to alteration of base pairing specificity is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase, and the form of the first nucleobase originally present in the DNA prior to alteration of base pairing specificity and the second nucleobase have the same base pairing specificity.
  • the second population does not comprise the form of the first nucleobase originally present in the DNA with altered base pairing specificity.
  • the cytosine modification is cytosine methylation.
  • the first nucleobase is a modified or unmodified cytosine and the second nucleobase is a modified or unmodified cytosine.
  • the first and second nucleobase may be any of those discussed herein in the Summary or with respect to subjecting the first subsample to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first subsample.
  • the first population includes a sequence tag selected from a first set of one or more sequence tags and the second population includes a sequence tag selected from a second set of one or more sequence tags, and the second set of sequence tags is different from the first set of sequence tags.
  • the sequence tags may comprise barcodes.
  • the first population includes protected hmC, such as glucosylated hmC.
  • the first population was subjected to any of the conversion procedures discussed herein, such as bisulfite conversion, Ox-BS conversion, TAB conversion, ACE conversion, TAP conversion, TAPSP conversion, or CAP conversion.
  • the first population was subjected to protection of hmC followed by deamination of mC and/or C.
  • the first population includes or was derived from DNA with a cytosine modification in a greater proportion than the second population and the first population includes first and second subpopulations
  • the first nucleobase is a modified or unmodified nucleobase
  • the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase
  • the first nucleobase and the second nucleobase have the same base pairing specificity.
  • the second population does not comprise the first nucleobase.
  • the first nucleobase is a modified or unmodified cytosine
  • the second nucleobase is a modified or unmodified cytosine, optionally wherein the modified cytosine is mC or hmC.
  • the first nucleobase is a modified or unmodified adenine
  • the second nucleobase is a modified or unmodified adenine, optionally wherein the modified adenine is mA.
  • the first nucleobase (e.g., a modified cytosine) is biotinylated.
  • the first nucleobase e.g., a modified cytosine
  • the captured DNA may comprise cfDNA.
  • the captured DNA may have any of the features described herein concerning captured sets, including, e.g., a greater concentration of the DNA corresponding to the sequence-variable target region set (normalized for footprint size as discussed above) than of the DNA corresponding to the epigenetic target region set.
  • the DNA of the captured set includes sequence tags, which may be added to the DNA as described herein. In general, the inclusion of sequence tags results in the DNA molecules differing from their naturally occurring, untagged form.
  • the combination may further comprise a probe set described herein or sequencing primers, each of which may differ from naturally occurring nucleic acid molecules.
  • a probe set described herein may comprise a capture moiety
  • sequencing primers may comprise a non-naturally occurring label.
  • Methods of the present disclosure can be implemented using, or with the aid of, computer systems.
  • such methods may comprise: partitioning the sample into a plurality of subsamples, including a first subsample and a second subsample, wherein the first subsample includes DNA with a cytosine modification in a greater proportion than the second subsample; subjecting the first subsample to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first subsample, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base pairing specificity; and sequencing DNA in the first subsample and DNA in the second subsample in a manner that distinguishes the first nucleobase from the second nucleobase in the DNA of
  • the present disclosure provides a non-transitory computer-readable medium including computer-executable instructions which, when executed by at least one electronic processor, perform at least a portion of a method including: collecting cfDNA from a test subject; capturing a plurality of sets of target regions from the cfDNA, wherein the plurality of target region sets includes a sequence-variable target region set and an epigenetic target region set, whereby a captured set of cfDNA molecules is produced; sequencing the captured cfDNA molecules, wherein the captured cfDNA molecules of the sequence-variable target region set are sequenced to a greater depth of sequencing than the captured cfDNA molecules of the epigenetic target region set; obtaining a plurality of sequence reads generated by a nucleic acid sequencer from sequencing the captured cfDNA molecules; mapping the plurality of sequence reads to one or more reference sequences to generate mapped sequence reads; and processing the mapped sequence reads corresponding to the sequence-variable target region set and to the epi
  • the code can be pre-compiled and configured for use with a machine with a processer adapted to execute the code or can be compiled during runtime.
  • the code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.
  • the architecture may include a data integration and analysis system.
  • the data integration and analysis system may obtain data from a number of data sources and integrate the data from the data sources into an integrated data repository.
  • the data integration and analysis system may obtain data from a health insurance claims data repository.
  • the data integration and analysis system and the health insurance claims data repository may be created and maintained by different entities.
  • the data integration and analysis system and the health insurance claims data repository may be created and maintained by the same entity.
  • the data integration and analysis system may be implemented by one or more computing devices.
  • the one or more computing devices may include one or more server computing devices, one or more desktop computing devices, one or more laptop computing devices, one or more tablet computing devices, one or more mobile computing devices, or combinations thereof.
  • at least a portion of the one or more computing devices may be implemented in a distributed computing environment.
  • at least a portion of the one or more computing devices may be implemented in a cloud computing architecture.
  • processing operations may be performed concurrently by multiple virtual machines.
  • the data integration and analysis system may implement multithreading techniques. The implementation of a distributed computing architecture and multithreading techniques cause the data integration and analysis system to utilize fewer computing resources in relation to computing architectures that do not implement these techniques.
  • the health insurance claims data repository may store information obtained from one or more health insurance companies that corresponds to insurance claims made by subscribers of the one or more health insurance companies.
  • the health insurance claims data repository may be arranged (e.g., sorted) by patient identifier.
  • the patient identifier may be based on the patient’s first name, last name, date of birth, social security number, address, employer, and the like.
  • the data stored by the health insurance claims data repository may include structured data that is arranged in one or more data tables.
  • the one or more data tables storing the structured data may include a number of rows and a number of columns that indicate information about health insurance claims made by subscribers of one or more health insurance companies in relation to procedures and/or treatments received by the subscribers from healthcare providers.
  • At least a portion of the rows and columns of the data tables stored by the health insurance claims data repository may include health insurance codes that may indicate diagnoses of biological conditions, and treatments and/or procedures obtained by subscribers of the one or more health insurance companies.
  • the health insurance codes may also indicate diagnostic procedures obtained by individuals that are related to one or more biological conditions that may be present in the individuals.
  • a diagnostic procedure may provide information used in the detection of the presence of a biological condition.
  • a diagnostic procedure may also provide information used to determine a progression of a biological condition.
  • a diagnostic procedure may include one or more imaging procedures, one or more assays, one or more laboratory procedures, one or more combinations thereof, and the like.
  • the data integration and analysis system may also obtain information from a molecular data repository.
  • the molecular data repository may store data of a number of individuals related to genomic information, genetic information, metabolomic information, transcriptomic information, fragmentomic information, immune receptor information, methylation information, epigenomic information, and/or proteomic information.
  • the data integration and analysis system and the molecular data repository may be created and maintained by different entities.
  • the data integration and analysis system and the molecular data repository may be created and maintained by a same entity.
  • the genomic information may indicate one or more mutations corresponding to genes of the individuals.
  • a mutation to a gene of individuals may correspond to differences between a sequence of nucleic acids of the individuals and one or more reference genomes.
  • the reference genome may include a known reference genome, such as hgl9.
  • a mutation of a gene of an individual may correspond to a difference in a germline gene of an individual in relation to the reference genome.
  • the reference genome may include a germline genome of an individual.
  • a mutation to a gene of an individual may include a somatic mutation.
  • genomic information stored by the molecular data repository may include genomic profiles of tumor cells present within individuals.
  • the genomic information may be derived from an analysis of genetic material, such as deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) from a sample, including, but not limited to, a tissue sample or tumor biopsy, circulating tumor cells (CTCs), exosomes or efferosomes, or from circulating nucleic acids (e.g., cell-free DNA) found in blood samples of individuals that is present due to the degradation of tumor cells present in the individuals. .
  • the genomic information of tumor cells of individuals may correspond to one or more target regions.
  • One or more mutations present with respect to the one or more target regions may indicate the presence of tumor cells in individuals.
  • the genomic information stored by the molecular data repository may be generated in relation to an assay or other diagnostic test that may determine one or more mutations with respect to one or more target regions of the reference genome.
  • the data integration and analysis system may obtain information from one or more additional data repositories.
  • the one or more additional data repositories may store data related to electronic medical records of individuals for which data is present in at least one of the health insurance claims data repository or the molecular data repository .
  • the one or more additional data repositories may store data related to pathology reports of individuals for which data is present in at least one of the health insurance claims data repository or the molecular data repository .
  • the one or more additional data repositories may store data related to biological conditions and/or treatments for biological conditions.
  • the data integration and analysis system and at least a portion of the one or more additional data repositories may be created and maintained by different entities.
  • the data integration and analysis system and at least a portion of the one or more additional data repositories may be created and maintained by a same entity.
  • the data integration and analysis system may obtain information from one or more reference information data repositories.
  • the one or more reference information data repositories may store information that includes definitions, standards, protocols, vocabularies, one or more combinations thereof, and the like.
  • the information stored by the one or more reference information data repositories may correspond to biological conditions and/or treatments for biological conditions.
  • the one or more reference information data repositories may include RxNorm.
  • the data integration and analysis system and at least a portion of the one or more reference information data repositories may be created and maintained by different entities. In one or more further examples, the data integration and analysis system and at least a portion of the one or more reference information data repositories may be created and maintained by a same entity.
  • the data integration and analysis system may obtain data from at least one of the health insurance claims data repository , the molecular data repository , the one or more additional data repositories , or the reference information data repositories via one or more communication networks accessible to the data integration and analysis system and accessible to at least one of the health insurance claims data repository , the molecular data repository , the one or more additional data repositories , or the reference information data repositories .
  • the data integration and analysis system may also obtain data from at least one of the health insurance claims data repository, the molecular data repository, the one or more additional data repositories , or the reference information data repositories via one or more secure communication channels.
  • the data integration and analysis system may obtain data from at least one of the health insurance claims data repository, the molecular data repository , the one or more additional data repositories , or the reference information data repositories via one or more calls of an application programming interface (API).
  • API application programming interface
  • the data integration and analysis system may include a data integration system.
  • the data integration system may obtain data from the health insurance claims data repository and the molecular data repository to generate the integrated data repository .
  • the data integration system may also obtain data from the one or more additional data repositories to generate the integrated data repository .
  • the data integration system may implement one or more natural language processing techniques to integrate data from the one or more additional data repositories into the integrated data repository.
  • the data integration system may generate one or more tokens to identify individuals that have data stored in the health insurance claims data repository and that have data stored in the molecular data repository.
  • the data integration system may generate one or more tokens by implementing one or more hash functions.
  • the data integration system may implement the one or more hash functions to generate the one or more tokens based on information stored by at least one of the health insurance claims data repository or the molecular data repository .
  • the information used by the data integration system to generate individual tokens by implementing a hash function may include at least one of an identifiers of respective individuals, date of birth of the respective individuals, a postal code of the respective individuals, date of birth of the respective individuals, or a gender of the respective individuals.
  • the identifiers of the respective individuals may include a combination of at least a portion of a first name of the respective individuals and at least a portion of the last name of the respective individuals.
  • Tokens generated using data from different data repositories may correspond to the same or similar information or the same or similar type stored by the different data repositories.
  • tokens may be generated using a portion of names of individuals, date of birth, at least a portion of a postal code, and gender obtained from the health insurance claims data repository and the molecular data repository.
  • the data integration system may integrate data from a number of different data sources by analyzing tokens generated by implementing one or more hash functions using data obtained from the number of different data sources. For example, the data integration system may obtain one or more first tokens generated from data stored by the health insurance claims data repository and one or more second tokens generated from data stored by the molecular data repository. The data integration system may analyze the one or more first tokens with respect to the one or more second tokens to determine individual first tokens that correspond to individual second tokens. In one or more illustrative examples, the data integration system may identify individual first tokens that match individual second tokens. A first token may match a second token when the data of the first token has at least a threshold amount of similarity with respect to the data of the second token.
  • a first token may match a second token when the data of the first token is the same as the data of the second token.
  • a first token may match a second token when an alphanumeric string of the first token is the same as an alphanumeric string of the second token.
  • the data integration system may identify an individual having data that is stored in both the health insurance claims data repository and in the molecular data repository . In this way, the data integration system may obtain data from the health insurance claims data repository from a number of individuals and data from the molecular data repository from the same number of individuals and store the health insurance claims data and the molecular data for the number of individuals in the integrated data repository .
  • the data integration system may also integrate data stored by the one or more additional data repositories with data from the health insurance claims data repository and the molecular data repository to generate the integrated data repository.
  • the data integration system may obtain one or more thirds of tokens generated from data stored by an additional data repository, such as a data repository storing data corresponding to pathology reports.
  • the data integration system may analyze the one or more third tokens with respect to the first tokens generated using information stored by the health insurance claims data repository and the second tokens generated using information stored by the molecular data repository to determine respective third tokens that correspond to individuals first tokens and individual second tokens.
  • the data integration system may identify third tokens generated using one or more hash functions and a common set of information obtained from the health insurance claims data repository, the molecular data repository, and the additional data repository .
  • the data integration system may identify an individual having data that is stored in the health insurance claims data repository , the molecular data repository , and in an additional data repository . In this way, the data integration system may obtain data from the health insurance claims data repository from a number of individuals and data from the molecular data repository and an additional data repository from the same number of individuals and store the health insurance claims data, the molecular data, and the additional data for the number of individuals in the integrated data repository .
  • the data stored by the integrated data repository for the number of individuals may be accessible using respective identifiers of individuals.
  • the data integration system may implement a number of techniques as part of a de-identification process with respect to storing and retrieving information of individuals in the integrated data repository.
  • the identifiers of individuals may correspond to keys that are generated using at least one hash function.
  • the identifiers of the individuals may also be generated by implementing one or more salting processes with respect to the keys generated using the at least one hash function, the tokens generated using one or more hash functions and a common set of information obtained from the health insurance claims data repository, the molecular data repository, and/or the additional data repository.
  • the identifiers generated by the data integration system to access information for respective individuals that is stored by the integrated data repository may be unique for each individual.
  • the identifiers of the individuals may be generated using at least a portion of the information used to generate the tokens related to the individuals.
  • the identifiers of the individuals may be generated using different information from the information used to generate the tokens related to the individuals.
  • the data integration system may also generate the integrated data repository from a number of different combinations of data repositories in a similar manner. For example, the data integration system may obtain tokens generated from information stored by the health insurance claims data repository and additional tokens generated from information stored by one or more additional data stores. The data integration system may determine individual tokens generated from information stored by the health insurance claims data repository that correspond to individual additional tokens generated from information stored by the one or more additional data repositories. By determining tokens generated using data stored by the health insurance claims data repository that correspond to additional tokens generated using data stored by an additional data repository, the data integration system may identify individuals having data that is stored in both the health insurance claims data repository and in the additional data repository .
  • the data integration system may obtain data from the health insurance claims data repository from a number of individuals and data from the additional data repository from the same number of individuals and store the health insurance claims data and the additional data for the number of individuals in the integrated data repository.
  • the health insurance claims data and the additional data stored by the integrated data repository for the number of individuals may be accessible using respective identifiers of individuals.
  • the data integration system may obtain tokens generated from information stored by the molecular data repository and tokens generated from information stored by one or more additional data stores.
  • the data integration system may determine individual tokens generated from information stored by the molecular data repository that correspond to individual additional tokens generated from information stored by the one or more additional data repositories. By determining tokens generated using data stored by the molecular data repository that correspond to additional tokens generated using data stored by an additional data repository, the data integration system may identify individuals having data that is stored in both the molecular data repository and in the additional data repository .
  • the data integration system may obtain data from the molecular data repository from a number of individuals and data from the additional data repository from the same number of individuals and store the molecular data and the additional data for the number of individuals in the integrated data repository .
  • the molecular data and the additional data stored by the integrated data repository for the number of individuals may be accessible using respective identifiers of individuals.
  • the data stored by the integrated data repository may be stored according to one or more regulatory frameworks that protect the privacy and ensure the security of medical records, health information, and insurance information of individuals.
  • data may be stored by the integrated data repository in accordance with one or more governmental regulatory frameworks directed to protecting personal information, such as the Health Insurance Portability and Accountability Act (HIPAA) and/or the General Data Protection Regulation (GDPR).
  • HIPAA Health Insurance Portability and Accountability Act
  • GDPR General Data Protection Regulation
  • the integrated data repository also stores data in an anonymized and de-identified manner to ensure protection of the privacy of individuals that have data stored by the integrated data repository.
  • the data integration system may re-generate the integrated data repository periodically. For example, the data integration system may create the integrated data repository once per quarter.
  • the data integration system may generate the integrated data repository on a monthly basis, on a weekly basis, or once every two weeks.
  • the integrated data repository enhances privacy protection with respect to data stored by the integrated data repository . That is, in situations where data repositories are refreshed simply with new data, it may be possible to more easily track individuals associated with data that has been newly added to a data repository because the number of new individuals added at a given time is typically smaller than an existing number of individuals that already have data stored by the data repository.
  • data stored by the integrated data repository may be accessed via a database management system.
  • the integrated data repository may store data according to one or more database models.
  • the integrated data repository may store data according to one or more relational database technologies.
  • the integrated data repository may store data according to a relational database model.
  • the integrated data repository may store data according to an object-oriented database model.
  • the integrated data repository may store data according to an extensible markup language (XML) database model.
  • XML extensible markup language
  • the integrated data repository may store data according to a structured query language (SQL) database model.
  • the integrated data repository may store data according to an image database model.
  • the data integration system may generate the integrated data repository by generating a number of data tables and creating links between the data tables.
  • the links may indicate logical couplings between the data tables.
  • the data integration system may generate the data tables by extracting specified sets of data from the information obtained from the data repositories and storing the data in rows and columns of respective data tables.
  • the logical couplings between data tables may include at least one of a one-to-one link where a row of information in one data table corresponds to a row of information in another data table, a one-to- many link where a row of information in one data table corresponds to multiple rows of information in another data table, or a many-to-many link where multiple rows of information of one data table correspond to multiple rows of information in another data table.
  • the number of data tables may be arranged according to a data repository schema.
  • the data repository schema includes a first data table, a second data table, a third data table , a fourth data table , and a fifth data table .
  • the data repository schema may include more data tables or fewer data tables.
  • the data repository schema may also include links between the data tables. The links between the data tables may indicate that information retrieved from one of the data tables results in additional information stored by one or more additional data tables to be retrieved. Additionally, not all the data tables may be linked to each of the other data tables.
  • first data table is logically coupled to the second data table by a first link and the first data table is logically coupled to the fourth data table by a second link .
  • second data table is logically coupled to the third data table via a third link and the fourth data table is logically coupled to the fifth data table via a fourth link .
  • third data table is logically coupled to the fifth data table via a fifth link.
  • the integrated data repository may store data tables according to the data repository schema for at least a portion of the individuals for which the data integration system obtained information from a combination of at least two of the health insurance claims data repository , the molecular data repository , the one or more additional data repositories , and the one or more reference information data repositories .
  • the integrated data repository may store respective instances of the data tables according to the data repository schema for thousands, tens of thousands, up to hundreds of thousands or more individuals.
  • the data integration and analysis system may also include a data pipeline system.
  • the data pipeline system may include a number of algorithms, software code, scripts, macros, or other bundles of computer-executable instructions that process information stored by the integrated data repository to generate additional datasets.
  • the additional datasets may include information obtained from one or more of the data tables.
  • the additional datasets may also include information that is derived from data obtained from one or more of the data tables.
  • the components of the data pipeline system implemented to generate a first additional dataset may be different from the components of the data pipeline system used to generate a second additional dataset.
  • the data pipeline system may generate a dataset that indicates pharmacy treatments received by a number of individuals.
  • the data pipeline system may analyze information stored in at least one of the data tables to determine health insurance codes corresponding to pharmaceutical treatments received by a number of individuals.
  • the data pipeline system may analyze the health insurance codes corresponding to pharmaceutical treatments with respect to a library of data that indicates specified pharmaceutical treatments that correspond to one or more health insurance codes to determine names of pharmaceutical treatments that have been received by the individuals.
  • the data pipeline system may analyze information stored by the integrated data repository to determine medical procedures received by a number of individuals. To illustrate, the data pipeline system may analyze information stored by one of the data tables to determine treatments received by individuals via at least one injection or intravenously.
  • the data pipeline system may analyze information stored by the integrated data repository to determine episodes of care for individuals, lines of therapy received by individuals, progression of a biological condition, or time to next treatment.
  • the datasets generated by the data pipeline system may be different for different biological conditions.
  • the data pipeline system may generate a first number of datasets with respect to a first type of cancer, such as lung cancer, and a second number of datasets with respect to a second type of cancer, such as colorectal cancer.
  • the data pipeline system may also determine one or more confidence levels to assign to information associated with individuals having data stored by the integrated data repository.
  • the respective confidence levels may correspond to different measures of accuracy for information associated with individuals having data stored by the integrated data repository.
  • the information associated with the respective confidence levels may correspond to one or more characteristics of individuals derived from data stored by the integrated data repository. Values of confidence levels for the one or more characteristics may be generated by the data pipeline system in conjunction with generating one or more datasets from the integrated data repository.
  • a first confidence level may correspond to a first range of measures of accuracy
  • a second confidence level may correspond to a second range of measures of accuracy
  • a third confidence level may correspond to a third range of measures of accuracy.
  • the second range of measures of accuracy may include values that are less values of the first range of measures of accuracy and the third range of measures of accuracy may include values that are less than values of the second range of measures of accuracy.
  • information corresponding to the first confidence level may be referred to as Gold standard information
  • information corresponding to the second confidence level may be referred to as Silver standard information
  • information corresponding to the third confidence level may be referred to as Bronze standard information.
  • the data pipeline system may determine values for the confidence levels of characteristics of individuals based on a number of factors. For example, a respective set of information may be used to determine characteristics of individuals. The data pipeline system may determine the confidence levels of characteristics of individuals based on an amount of completeness of the respective set of information used to determine a characteristic for an individual. In situations where one or more pieces of information are missing from the set of information associated with a first number of individuals, the confidence levels for a characteristic may be lower than for a second number of individuals where information is not missing from the set of information. In one or more examples, an amount of missing information may be used by the data pipeline system to determine confidence levels of characteristics of individuals.
  • a greater amount of missing information used to determine a characteristic of an individual may cause confidence levels for the characteristic to be lower than in situations where the amount of missing information used to determine the characteristic is lower.
  • different types of information may correspond to various confidence levels for a characteristic.
  • the presence of a first piece of information used to determine a characteristic of an individual may result in confidence levels for the characteristic being higher than the presence of a second piece of information used to determine the characteristic.
  • the data pipeline system may determine a number of individuals included in a cohort with a primary diagnosis of lung cancer (or other biological condition).
  • the data pipeline system may determine confidence levels for respective individuals with respect to being classified as having a primary diagnosis of lung cancer.
  • the data pipeline system may use information from a number of columns included in the data tables to determine a confidence level for the inclusion of individuals within a lung cancer cohort.
  • the number of columns may include health insurance codes related to diagnosis of biological conditions and/or treatments of biological conditions. Additionally, the number of columns may correspond to dates of diagnosis and/or treatment for biological conditions.
  • the data pipeline system may determine that a confidence level of an individual being characterized as being part of the lung cancer cohort is higher in scenarios where information is available for each of the number of columns or at least a threshold number of columns than in instances where information is available for less than a threshold number of columns. Further, the data pipeline system may determine confidence levels for individuals included in a lung cancer cohort based on the type of information and availability of information associated with one or more columns.
  • the data pipeline system may determine that the confidence level of including the group of individuals in the lung cancer cohort is greater than in situations where at least one of the diagnosis codes is absent and the treatment codes used to determine whether individuals are included in the lung cancer cohort are present.
  • the data integration and analysis system may include a data analysis system.
  • the data analysis system may receive integrated data repository requests from one or more computing devices, such as an example computing device.
  • the one or more integrated data repository requests may cause data to be retrieved from the integrated data repository.
  • the one or more integrated data repository requests may cause data to be retrieved from one or more datasets generated by the data pipeline system.
  • the integrated data repository requests may specify the data to be retrieved from the integrated data repository and/or the one or more datasets generated by the data pipeline system.
  • the integrated data repository requests may include one or more prebuilt queries that correspond to computer-executable instructions that retrieve a specified set of data from the integrated data repository and/or one or more datasets generated by the data pipeline system.
  • the data analysis system may analyze data retrieved from at least one of the integrated data repository or one or more datasets generated by the data pipeline system to generate data analysis results .
  • the data analysis results may be sent to one or more computing devices, such as example computing devices.
  • the data analysis results may be received by a same computing device that sent the one or more integrated data repository requests .
  • the data analysis results may be displayed by one or more user interfaces rendered by the computing device or the computing device.
  • the data analysis system may implement at least one of one or more machine learning techniques or one or more statistical techniques to analyze data retrieved in response to one or more integrated data repository requests.
  • the data analysis system may implement one or more artificial neural networks to analyze data retrieved in response to one or more integrated data repository requests.
  • the data analysis system may implement at least one of one or more convolutional neural networks or one or more residual neural networks to analyze data retrieved from the integrated data repository in response to one or more integrated data repository requests.
  • the data analysis system may implement one or more random forests techniques, one or more support vector machines, or one or more Hidden Markov models to analyze data retrieved in response to one or more integrated data repository requests.
  • One or more statistical models may also be implemented to analyze data retrieved in response to one or more integrated data repository requests to identify at least one of correlations or measures of significance between characteristics of individuals. For example, log rank tests may be applied to data retrieved in response to one or more integrated data repository requests. In addition, Cox proportional hazards models may be implemented with respect to date retrieved in response to one or more integrated data repository requests. Further, Wilcoxon signed rank tests may be applied to data retrieved in response to one or more integrated data repository requests. In still other examples, a z-score analysis may be performed with respect to data retrieved in response to one or more integrated data repository requests. In still additional examples, a Kaplan Meier analysis may be performed with respect to data retrieved in response to one or more integrated data repository requests. In at least some examples, one or more machine learning techniques may be implemented in combination with one or more statistical techniques to analyze data retrieved in response to one or more integrated data repository requests.
  • the data analysis system may determine a rate of survival of individuals in which lung cancer is present in response to one or more treatments. In one or more additional illustrative examples, the data analysis system may determine a rate of survival of individuals having one or more genomic region mutations in which lung cancer is present in response to one or more treatments. In various examples, the data analysis system may generate the data analysis results in situations where the data retrieved from at least one of the integrated data repository or the one or more datasets generated by the data pipeline system satisfies one or more criteria. For example, the data analysis system may determine whether at least a portion of the data retrieved in response to one or more integrated data repository requests satisfies a threshold confidence level.
  • the data analysis system may refrain from generating at least a portion of data analysis results. In scenarios where the confidence level for at least a portion of the data retrieved in response to one or more integrated data repository requests is at least a threshold confidence level, the data analysis system may generate at least a portion of the data analysis results.
  • the threshold confidence level may be related to the type of data analysis results being generated by the data analysis system.
  • the data analysis system may receive an integrated data repository request to generate data analysis results that indicate a rate of survival of one or more individuals. In these instances, the data analysis system may determine whether the data stored by the integrated data repository and/or by one or more datasets generated by the data pipeline system satisfies a threshold confidence level, such as a Gold standard confidence level. In one or more additional examples, the data analysis system may receive an integrated data repository request to generate data analysis results that indicate a treatment received by one or more individuals. In these implementations, the data analysis system may determine whether the data stored by the integrated data repository and/or by one or more datasets generated by the data pipeline system satisfies a lower threshold confidence level, such as a Bronze standard confidence level.
  • a threshold confidence level such as a Gold standard confidence level
  • the data analysis system may receive an integrated data repository request to determine individuals having one or more genomic mutations and that have received one or more treatments for a biological condition.
  • the data analysis system can determine a survival rate of individuals with the one or more genomic mutations in relation to the one or more treatments received by the individuals.
  • the data analysis system can then identify based on the survival rate of individuals and effectiveness of treatments for the individuals in relation to genomic mutations that may be present in the individuals. In this way, health outcomes of individuals may be improved by identifying prospective treatments that may be more effective for populations of individuals having one or more genomic mutations than current treatments being provided to the individuals.
  • Described herein is a framework corresponding to an arrangement of data tables in an integrated data repository, according to one or more implementations.
  • the framework includes a data repository schema that includes a first data table , a second data table , a third data table , a fourth data table a fifth data table , a sixth data table , and a seventh data table .
  • the data repository schema may include more data tables or fewer data tables.
  • the data repository schema may also include links between the data tables. The links between the data tables may indicate that information retrieved from one of the data tables results in additional information stored by one or more additional data tables to be retrieved.
  • not all the data tables may be linked to each of the other data tables.
  • the first data table is logically coupled to the second data table by a first link and the third data table is logically coupled to the second data table by a second link .
  • the second data table is also logically coupled to the fourth data table by a third link
  • the second data table is logically coupled to the fifth data table by a fourth link
  • the second data table is logically coupled to the sixth data table by a fifth link
  • the fifth data table is logically coupled to the sixth data table by a sixth link
  • the sixth data table is logically coupled to the seventh data table by a seventh link .
  • the seventh data table is logically coupled to the fourth data table by an eighth link .
  • additional links between data tables may be added to or removed from the data repository schema .
  • the integrated data repository may store data tables according to the data repository schema for at least a portion of the individuals for which the data integration system obtained information from a combination of at least two of the health insurance claims data repository , the molecular data repository , and the one or more additional data repositories .
  • the integrated data repository may store respective instances of the data tables according to the data repository schema for thousands, tens of thousands, up to hundreds of thousands or more individuals.
  • the first data table may store data corresponding to genomics and genomics testing for individuals.
  • the first data table may include columns that include information corresponding to a panel used to generate genomics data, mutations of genomic regions, types of mutations, copy numbers of genomic regions, coverage data indicating numbers of nucleic acid molecules identified in a sample having one or more mutations, testing dates, and patient information.
  • the first data table may also include one or more columns that include health insurance data codes that may correspond to one or more diagnosis codes.
  • the information in the first data table may include at least one identifier for an individual that is associated with an instance of the first data table .
  • the second data table may store data related to one or more patient visits by individuals to one or more healthcare providers.
  • the third data table may store information corresponding to respective services provided to individuals with respect to one or more patient visits to one or more healthcare providers indicated by the second data table .
  • an individual may visit a healthcare provider and multiple services may be performed with respect to the individual at the visit.
  • a second data table may include columns indicating information for each of the multiple services performed during the patient visit.
  • Multiple third data tables may be generated with respect to the patient visit that include columns indicating information on a more granular level for a respective service provided during the patient visit than the information stored by the second data table related to the patient visit.
  • the second data table may include multiple columns indicating a health insurance code for different services provided to an individual during a patient visit and a third data table related to one of the services may include multiple columns for additional health insurance codes that correspond to additional information related to the respective services.
  • the second data table and the third data table(s) for a patient visit may indicate one or more dates of service corresponding to the patient visit.
  • the fourth data table may include columns that indicate information about individuals for which information is stored by the integrated data repository .
  • the fourth data table may include columns that indicate information related to at least one of a location of an individual, a gender of an individual, a date of birth of an individual, a date of death of an individual (if applicable), or one or more keys associated with the individual.
  • the fourth data table may include one or more columns related to whether erroneous data has been identified for an individual.
  • a single fourth data table may be generated for respective individuals.
  • the data repository schema may include multiple instances of the fourth data table , such as thousands, tens of thousands, up to hundreds of thousands or more.
  • the fifth data table may include columns that indicate information related to a health insurance company or governmental entity that made payment for one or more services provided to respective individuals.
  • the fifth data table may include one or more payer identifiers.
  • the sixth data table may include columns that include information corresponding to health insurance coverage information for respective individuals.
  • the sixth data table may include columns indicating the presence of medical coverage for an individual, the presence of pharmacy coverage for an individual, and a type of health insurance plan related to the individual, such as health maintenance organization (HMO), preferred provider organization (PPO), and the like.
  • HMO health maintenance organization
  • PPO preferred provider organization
  • the seventh data table may include columns that indicate information related to pharmaceutical treatments obtained by a respective individual.
  • the seventh data table may include one or more columns indicating health insurance codes corresponding to pharmaceutical treatments that are available via a pharmacy.
  • the health insurance codes may correspond to individual pharmaceutical treatments. Additionally, the health insurance codes may indicate a diagnosis of a biological condition with respect to an individual.
  • the seventh data table may also include additional information, such as at least one of dosage amounts, number of days’ supply, quantity dispensed, number of refills authorized, dates of service, or information related to the individual receiving the pharmaceutical treatment.
  • the data repository schema may provide results of analysis of the information stored by the data tables in a more efficient manner than typical data repository schemas.
  • the logical connections between the data tables are arranged to efficiently retrieve data that is related across the different data tables.
  • retrieving data from the integrated data repository from one or more of the data tables to responds to a request for information from the integrated data repository will be less efficient than in situations where the data repository schema is implemented.
  • the architecture may include the data integration and analysis system and the integrated data repository . Additionally, the data integration and analysis system may include at least the data pipeline system and the data analysis system .
  • the data pipeline system may include a number of sets of data processing instructions that are executable to generate respective datasets that may be analyzed by the data analysis system in response to an integrated data repository request to generate data analysis results .
  • the data pipeline system may include first data processing instructions , second data processing instructions , up to Nth data processing instructions .
  • the data processing instructions may be executable by one or more processing units to perform a number of operations to generate respective datasets using information obtained from the integrated data repository .
  • the data processing instructions may include at least one of software code, scripts, API calls, macros, and so forth.
  • the first data processing instructions may be executable to generate a first dataset .
  • the second data processing instructions may be executable to generate a second dataset .
  • the Nth data processing instructions may be executable to generate an Nth dataset .
  • the data pipeline system may cause the data processing instructions , , to be executed to generate the datasets , , .
  • the datasets, may be stored by the integrated data repository or by an additional data repository that is accessible to the data integration and analysis system .
  • At least a portion of the data processing instructions may analyze health insurance codes to generate at least a portion of the datasets.
  • at least a portion of the data processing instructions may analyze genomics data to generate at least a portion of the datasets .
  • the first data processing instructions may be executable to retrieve data from one or more first data tables stored by the integrated data repository .
  • the first data processing instructions may also be executable to retrieve data from one or more specified columns of the one or more first data tables.
  • the first data processing instructions may be executable to identify individuals that have a health insurance code stored in one or more column and row combinations that correspond to one or more diagnosis codes.
  • the first data processing instructions may then be executable to analyze the one or more diagnosis codes to determine a biological condition for which the individuals have been diagnosed.
  • the first data processing instructions may be executable to analyze the one or more diagnosis codes with respect to a library of diagnosis codes that indicates one or more biological conditions that correspond to respective diagnosis codes.
  • the library of diagnosis codes may include hundreds up to thousands of diagnosis codes.
  • the first data processing instructions may also be executable to determine individuals diagnosed with a biological condition by analyzing timing information of the individuals, such as dates of treatment, dates of diagnosis, dates of death, one or more combinations thereof, and the like.
  • the second data processing instructions may be executable to retrieve data from one or more second data tables stored by the integrated data repository .
  • the second data processing instructions may also be executable to retrieve data from one or more specified columns of the one or more second data tables.
  • the second data processing instructions may be executable to identify individuals that have a health insurance code stored in one or more columns and row combinations that correspond to one or more treatment codes.
  • the one or more treatment codes may correspond to treatments obtained from a pharmacy.
  • the one or more treatment codes may correspond to treatments received by a medical procedure, such as an injection or intravenously.
  • the second data processing instructions may be executable to determine one or more treatments that correspond to the respective health insurance codes included in the one or more second data tables by analyzing the health insurance code in relation to a predetermined set of information.
  • the predetermined set of information may include a data library that indicates one or more treatments that correspond to one out of hundreds up to thousands of health insurance codes.
  • the second data processing instructions may generate the second dataset to indicate respective treatments received by a group of individuals.
  • the group of individuals may correspond to the individuals included in the first dataset.
  • the second dataset may be arranged in rows and columns with one or more rows corresponding to a single individual and one or more columns indicating the treatments received by the respective individual.
  • the Nth processing instructions may be executable to generate the Nth dataset by combining information from a number of previously generated datasets, such as the first dataset and the second dataset .
  • the Nth processing instructions may be executable to generate the Nth dataset to retrieve additional information from one or more additional columns of the integrated data repository and incorporate the additional information from the integrated data repository with information obtained from the first dataset and the second dataset .
  • the Nth processing instructions may be executable to identify individuals included in the first dataset that are diagnosed with a biological condition and analyze specified columns of one or more additional data tables of the integrated data repository to determine dates of the treatments indicated in the second dataset that correspond to the individuals included in the first dataset .
  • the Nth processing instructions may be executable to analyze columns of one or more additional data tables of the integrated data repository to determine dosages of treatments indicated in the second dataset received by the individuals included in the first dataset . In this way, the Nth processing instructions may be executable to generate an episodes of care dataset based on information included in a cohort dataset and a treatments dataset.
  • the data analysis system in response to receiving an integrated data repository request, may determine one or more datasets that correspond to the features of the query related to the integrated data repository request . For example, the data analysis system may determine that information included in the first dataset and the second dataset is applicable to responding to the integrated data repository request . In these scenarios, the data analysis system may analyze at least a portion of the data included in the first dataset and the second dataset to generate the data analysis results . In one or more additional examples, the data analysis system may determine different datasets to respond to different queries included in the integrated data repository request in order to generate the data analysis results .
  • the use of specific sets of data processing instructions to generate respective data sets may reduce the number of inputs from users of the data integration and analysis system as well as reduce the computational load, such as the amount of processing resources and memory, utilized to process integrated data repository requests .
  • the data utilized to respond to the integrated data repository request is assembled from the data repository .
  • the data processing instruction to generate the datasets the data needed to respond to various integrated data repository requests has already been assembled and may be accessed by the data analysis system to respond to the integrated data repository request .
  • the computing resources used to respond to the integrated data repository request by implementing the data pipeline system to generate the datasets are less than typical systems that perform an information parsing and collecting process for each integrated data repository request .
  • users of the data integration and analysis system may need to submit multiple integrated data repository request in order to analyze the information that the users are intending to have analyzed either because the ad hoc collection of data to respond to an integrated data repository request in typical systems is inaccurate or because the data analysis system is called upon multiple times to perform an analysis of information in typical systems that may be performed using a single integrated data repository request when the data pipeline system is implemented.
  • the architecture may include the data integration and analysis system , the health insurance claims data repository , and the molecular data repository .
  • the data integration and analysis system may obtain patient information from the molecular data repository .
  • the patient information may include genomics data for individuals having data stored by the molecular data repository .
  • the genomics data may indicate results of one or more nucleic acid sequencing operations that analyze sequences of nucleic acid molecules included in a sample obtained from the individuals with respect to one or more target genomic regions.
  • the sample may be obtained from tissue of one or more individuals.
  • the sample may be obtained from fluid of one or more individuals, such as blood or plasma.
  • the one or more target genomic regions may correspond to genomic regions that correspond to the presence of one or more biological conditions.
  • the target regions may correspond to genomic regions of a reference genome having mutations that are present in individuals in which a biological condition is present.
  • the target regions may correspond to genomic regions of a reference human genome in which one or more mutations are present in individuals in which one or more forms of cancer are present.
  • the patient information may also include information indicating personal information about individuals with data stored by the molecular data repository and information corresponding to the testing and analysis performed on samples provided by individuals.
  • the data integration and analysis system may perform a de-identification process that anonymizes personal information obtained from the molecular data repository .
  • the data integration and analysis system may implement one or more computational techniques as part of the de-identification process to anonymize data related to individuals stored by the molecular data repository such that the de-identified data protects the privacy of the individuals and is in compliance with one or more privacy regulation frameworks.
  • the de-identification process may include, at , accessing tokens.
  • the tokens may comprise an alphanumeric string of characters.
  • the tokens may be generated by the data integration and analysis system.
  • the tokens may be generated by a third-party and obtained by the data integration and analysis system.
  • the tokens may be generated using one or more hash functions in relation to a subset of the patient information .
  • the tokens may be generated using a combination of at least a portion of a first name of the respective individuals, at least a portion of the last name of the respective individuals, at least a portion of a date of birth of the respective individuals, a gender of the individuals, and at least a portion of a location identifier of the respective individuals.
  • the de- identification process may also include, at , generating identifiers for individuals that have data stored by the molecular data repository .
  • the identifiers may be generated by the data integration and analysis system using one or more hash functions that are different from the one or more hash functions used to generate the tokens.
  • the data integration and analysis system may generate an intermediate version of respective identifiers using one or more hash function and then apply one or more salting techniques to the intermediate versions of the identifiers to generate final versions of the identifiers.
  • a salt function includes a function configured to add at least one random bit to each intermediate identifier to generate a respective final identifier.
  • the data integration and analysis system may generate the identifiers at using at least a portion of the information for respective individuals stored by the molecular data repository .
  • the data integration and analysis system may generate a token file .
  • the token file may include first tokens accessed at operation for respective individuals having data stored by the molecular data repository .
  • the token file may have a data structure that includes a number of columns that include information for respective individuals.
  • the data structure may include a column indicating respective identifiers generated by the data integration and analysis system and columns indicating one or more first tokens associated with the respective identifiers.
  • the data integration and analysis system may send the token file to a health insurance claims data management system that is coupled to the health insurance claims data repository .
  • the health insurance claims data management system may analyze the first tokens with respect to corresponding second tokens .
  • the second tokens may be accessed by or generated by the health insurance claims data management system .
  • the second tokens may be generated using a same or similar subset of information for individuals having data stored in the health insurance claims data repository as the subset of the patient information .
  • the second tokens may be generated using a combination of at least a portion of a first name of the respective individuals, at least a portion of the last name of the respective individuals, at least a portion of a date of birth of the respective individuals, a gender of the individuals, and at least a portion of a location identifier of the respective individuals.
  • the health insurance claims data management system may retrieve health insurance claims data from the health insurance claims data repository for individuals associated with respective second tokens that match corresponding first tokens .
  • a first token may match a second token when the data of the first token has at least a threshold amount of similarity with respect to the data of the second token .
  • a first token may match a second token when the data of the first token is the same as the data of the second token .
  • the health insurance claims data management system may generate modified health insurance claims data .
  • the health insurance claims data management system may send the modified health insurance claims data to the data integration and analysis system .
  • the modified health insurance claims data may be formatted according to a data structure .
  • the data structure may include a column that includes a subset of the second tokens that correspond to the first tokens and a number of columns that include the health insurance claims data.
  • the data integration and analysis system may integrate genomics data and health insurance claims data of individuals that are common to both the molecular data repository and the health insurance claims data repository .
  • the data integration and analysis system may determine individuals that are common to both the molecular data repository and the health insurance claims data repository by determining genomics data and health insurance claims data corresponding to common tokens.
  • the data integration and analysis system may determine that a first token related to a portion of the genomics data corresponds to a second token related to a portion of the health insurance claims data by determining a measure of similarity between the first token and the second token .
  • the data integration and analysis system may store the corresponding portion of the genomics data and the corresponding portion of the health insurance claims data in relation to the identifier of the individual in an integrated data repository, such as an integrated data repository .
  • the implementation of the architecture may implement a cryptographic protocol that enables de-identified information from disparate data repositories to be integrated into a single data repository. In this way, the security of the data stored by the integrated data repository is increased. Additionally, the cryptographic protocol implemented by the architecture may enable more efficient retrieval and accurate analysis of information stored by the integrated data repository than in situations where the cryptographic protocol of the architecture is not utilized.
  • the data integration and analysis system may match information stored by disparate data repositories that correspond to a same individual. Without implementing the cryptographic protocol of the architecture, the probability of incorrectly attributing information from one data repository to one or more individuals increases, which decreases the accuracy of results provided by the data integration and analysis system in response to integrated data repository requests sent to the data integration and analysis system .
  • the integrated data repository may store health insurance claims data and genomics data for a group of individuals .
  • the integrated data repository may store information obtained from health insurance claims records of the group of individuals .
  • the integrated data repository may store information obtained from multiple health insurance claim records .
  • the information stored by the integrated data repository may include and/or be derived from thousands, tens of thousands, hundreds of thousands, up to millions of health insurance claims records for a number of individuals.
  • each health insurance claim record may include multiple columns.
  • the integrated data repository may be generated through the analysis of millions of columns of health insurance claims data.
  • health insurance claims data may be organized according to a structured data format
  • health insurance claims data is typically arranged to be viewed by health insurance providers, patients, and healthcare providers in order to show financial information and insurance code information related to services provided to individuals by healthcare providers.
  • health insurance claims data is not easily analyzed to gain insights that may be available in relation to characteristics of individuals in which a biological condition is present and that may aid in the treatment of the individuals with respect to the biological condition.
  • the integrated data repository may be generated and organized by analyzing and modifying raw health insurance claims data in a manner that enables the data stored by the integrated data repository to be further analyzed to determine trends, characteristics, features, and/or insights with respect to individuals in which one or more biological conditions may be present.
  • health insurance codes may be stored in the integrated data repository in such a way that at least one of medical procedures, biological conditions, treatments, dosages, manufacturers of medications, distributors of medications, or diagnoses may be determined for a given individual based on health insurance claims data for the individual.
  • the data integration and analysis system may generate and implement one or more tables that indicate correlations between health insurance claims data and various treatments, symptoms, or biological conditions that correspond to the health insurance claims data.
  • the integrated data repository may be generated using genomics data records of the group of individuals .
  • the large amounts of health insurance claims data may be matched with genomics data for the group of individuals to generate the integrated data repository .
  • the data integration and analysis system may determine correlations between the presence of one or more biomarkers that are present in the genomics data records with other characteristics of individuals that are indicated by the health insurance claims data records that existing systems are typically unable to determine. For example, the data integration and analysis system may determine one or more genomic characteristics of individuals that correspond to treatments received by individuals, timing of treatments, dosages of treatments, diagnoses of individuals, smoking status, presence of one or more biological conditions, presence of one or more symptoms of a biological condition, one or more combinations thereof, and the like.
  • cohorts of individuals that may benefit from one or more treatments may be identified that would not have been identified in existing systems.
  • the processes and techniques implemented to integrate the health insurance claims records and the genomics claims records in order to generate the integrated data repository may be complex and implement efficiency-enhancing techniques, systems, and processes in order to minimize the amount of computing resources used to generate the integrated data repository .
  • the data pipeline system may access information stored by the integrated data repository to generate datasets that include a number of additional data records that include information related to at least a portion of the group of individuals .
  • the additional data record includes information indicating whether individuals are included in a cohort of individuals in which lung cancer is present.
  • the data pipeline system may execute a plurality of different sets of data processing instructions to determine a cohort of the group of individuals in which lung cancer is present.
  • the additional data record may indicate information used to determine a status of an individual with respect to lung cancer, such as one or more transaction insurance identifier, one or more international classification of diseases (ICD) codes, and one or more health insurance transaction dates.
  • ICD international classification of diseases
  • the additional data record may include a column indicating a confidence level of the status of the individual with respect to the presence of lung cancer.
  • the additional data record may include a column indicating a confidence level of the status of the individual with respect to the presence of lung cancer.
  • the computing architecture may include a medical records data repository .
  • the medical records data repository may store medical records data from a number of individuals.
  • the medical records data may include imaging information, laboratory test results, diagnostic test information, clinical observations, dental health information, notes of healthcare practitioners, medical history forms, diagnostic request forms, medical procedure order forms, medical information charts, one or more combinations thereof, and so forth.
  • the medical records data repository may store information obtained from one or more healthcare practitioners that is related to the individual.
  • the computing architecture may perform operation that includes obtaining data packages from the medical records data repository .
  • the data packages may be obtained in response to one or more requests sent to the medical records data repository for medical records that correspond to one or more individuals.
  • the data packages may be obtained by the computing architecture using one or more application programming interface (API) calls.
  • API application programming interface
  • a first data package, a second data package , up to an Nth data package may be obtained using the computing architecture .
  • the individual data packages, may correspond medical records of a respective individual.
  • the first data package may include medical records of a first individual
  • the second data package may include medical records of a second individual
  • the Nth data package may include medical records of a third individual.
  • Individual data packages may include a number of components.
  • individual data packages may include individual components that correspond to medical records from different healthcare providers.
  • the individual data packages may include individual components that correspond to different parts of medical records that correspond to one or more healthcare providers.
  • the second data package may include a first component , a second component , up to an Nth component .
  • the first component may include a first portion of medical records of an individual
  • the second component may include a second portion of medical records of an individual
  • the Nth component may include a third portion of medical records of an individual.
  • the first component may correspond to medical records of a first healthcare provider for the individual
  • the second component may correspond to medical records of a second healthcare provider for the individual
  • the third component may correspond to medical records of a third healthcare provider for the individual.
  • the first component may include a first section of medical records of the individual, such as one or more forms related to a diagnostic test or procedure
  • the second component may include a second section of medical records of the individual, such as a pathology report of the individual.
  • the computing architecture may preprocess individual data packages to identify a corpus of information to be analyzed.
  • the preprocessing of data packages obtained from the medical records data repository may include transforming the data included in the data packages.
  • preprocessing the data packages may include transforming at least a portion of the data obtained from the medical records data repository to machine encoded information.
  • preprocessing the data packages may include performing one or more optical character recognition (OCR) operations with respect to at least a portion of the data packages obtained from the medical records data repository.
  • OCR optical character recognition
  • the data packages may be subjected to a number of operations, such as one or more parsing operations to identify one or more characters or strings of characters or one or more editing operations that are unable to be performed with respect to at least a portion of the data packages obtained from the medical records data repository .
  • the preprocessing of individual data packages may include determining information included in individual data packages that is to be excluded from further analysis by the computing architecture.
  • one or more components of individual data packages may be excluded from a corpus of information to be analyzed.
  • the computing architecture may determine that the first component is to be excluded from further analysis by the computing architecture .
  • the computing architecture may analyze the components , , and/or with respect to one or more keywords to identify at least one of the components , , and/or to exclude from further analysis by the computing architecture .
  • the computing architecture may parse the components , , and/or to identify one or more keywords and in response to identifying the one or more keywords in a component , , and/or , the computing architecture may determine to exclude the respective component , , and/or from further analysis by the computing architecture .
  • the computing architecture may determine that the first component of the second data package is a test requisition form for one or more diagnostic procedures or tests. In these scenarios, the computing architecture may determine that the first component is to be excluded from further analysis by the computing architecture .
  • the computing architecture may determine that at least one of the second component and/or correspond to one or more pathology reports for an individual based on one or more keywords included in at least one of the second component or the Nth component . In these instances, the computing architecture may determine that at least a portion of the second component and/or at least a portion of the Nth component is to be included in the corpus of information to be further analyzed by the computing architecture . [0233] In addition, a subset of the components of individual data packages obtained from the medical records data repository may be included in the corpus of information . In various examples, one or more additional operations may be performed to narrow the corpus of information. For example, one or more queries may be applied to a subset of information obtained from the medical records data repository.
  • the one or more queries may extract information from the one or more data packages that satisfy the one or more queries.
  • the one or more queries may be a group of queries that are applied to individual components of a data package.
  • the group of queries may determine information to be included in the corpus of information and additional information that is to be excluded from the corpus of information .
  • one or more sections of at least one component of a data package may be excluded from the corpus of information.
  • the computing architecture may then cause one or more queries to be implemented with respect to at least one the second component or the Nth component .
  • the one or more queries may determine that a section of the second component , such as a section that indicates family history for one or more biological conditions, is to be excluded from the corpus of information .
  • the one or more queries may be directed to identifying a number of keywords and/or combinations of keywords included in at least one of the second component or the Nth component .
  • the computing architecture may exclude from the corpus of information one or more portions of the individual components of the data packages that include one or more keywords or combinations of keywords. In one or more additional examples, the computing architecture may exclude from the corpus of information a number of words, a number of characters, and/or a number of symbols following one or more keywords that are included in one or more portions of the individual components of the data packages.
  • the computing architecture may analyze the corpus of information to determine characteristics of individuals.
  • the computing architecture may analyze the corpus of information to determine individuals that have one or more phenotypes.
  • the computing architecture may analyze the corpus of information to determine one or more biomarkers that are indicative of a biological condition.
  • the computing architecture may analyze the corpus of information to determine individuals having one or more genetic characteristics.
  • the one or more genetic characteristics may include at least one of one or more variants of a genomic region that correspond to a biological condition.
  • the one or more genetic characteristics may correspond to one or more variants of a genomic region that correspond to a type of cancer.
  • the one or more biomarkers may correspond to levels of an analyte being outside of a specified range.
  • the computing architecture may analyze the corpus of information to determine individuals having levels of one or more proteins and/or levels of one or more small molecules present that are indicative of a biological condition. In these scenarios, the computing architecture may analyze results of laboratory tests to determine levels of analytes of individuals. In one or more additional examples, the computing architecture may analyze the corpus of information to determine individuals in which one or more symptoms are present that are indicative of a biological condition. In one or more further examples, the computing architecture may analyze imaging information included in the corpus of information to determine individuals in which one or more biomarkers are present.
  • the computing architecture may implement one or more machine learning techniques to analyze the corpus of information .
  • the computing architecture may implement one or more artificial neural networks, such as at least one of one or more convolutional neural networks or one or more residual neural networks to analyze the corpus of information .
  • the computing architecture may also implement at least one of one or more random forests techniques, one or more hidden Markov models, or one or more support vector machines to analyze the corpus of information .
  • the computing architecture may analyze the corpus of information by performing one or more queries with respect to the corpus of information .
  • the one or more queries may correspond to one or more keywords and/or combinations of keywords.
  • the one or more keywords and/or combinations of keywords may correspond to at least one of characters or symbols that correspond to one or more biological conditions.
  • a keyword may correspond to characters related to a mutation of a genomic region, such as HER2.
  • one or more criteria may be associated with combinations of keyworks.
  • a criterion that corresponds to a combination of keywords may include a number of words being present within a specified distance of one another in a portion of the corpus of information for an individual, such as the words fatigue, blood pressure, and swelling occurring within characters of one another.
  • the computing architecture may parse the corpus of information for the one or more keywords and/or combinations of keywords.
  • the computing architecture in response to determining that the one or more keywords and/or combinations of keywords are present in accordance with one or more criteria, the computing architecture may determine that a biological condition is present with respect to a given individual.
  • the one or more queries may be image-based and the computing architecture may analyze images included in the corpus of information with respect to template images.
  • the template images may be generated based on analyzing a number of images in which a biological condition is present and aggregating the number of images into a template image.
  • the computing architecture may analyze images included in the corpus of information with respect to one or more template images to determine a measure of similarity between the images included in the corpus of information and the template images. In situations where the measure of similarity for an individual is at least a threshold value, the computing architecture may determine that a characteristic of a biological condition is present in the individual.
  • the computing architecture may, at operation , generate data structures that store data for individuals having the one or more characteristics.
  • the computing architecture may generate data tables that indicate individuals having an individual characteristics and/or individuals having a group of characteristics.
  • the computing architecture may generate a first data table and a second data table .
  • the first data table may indicate individuals having one or more first characteristics and the second data table may indicate individuals having one or more second characteristics.
  • the first data table may indicate individuals having one or more first biomarkers for a biological condition and the second data table may indicate individual having one or more second biomarkers for the biological condition.
  • the one or more first biomarkers may correspond to one or more first genomic variants that are associated with the biological condition and the one or more second biomarkers may correspond to one or more second genomic variants that are associated with the biological condition.
  • the data tables may indicate whether or not the one or more characteristics associated with the individual data tables , are present with respect to individual individuals.
  • the first data table may include a first indication for individuals in which one or more first genomic variants are present and a second indication for individuals in which the one or more first genomic variants are not present.
  • the first data table may indicate smoking status of individuals and the second data table may indicate whether or not individual individuals have received one or more treatments for a biological condition.
  • the first data table and the second data table may have rows that correspond to individual individuals.
  • an individual identifier may be present in individual rows.
  • the individual identifier may include at least one of alphanumeric characters or symbols that correspond to an individual.
  • the individual identifier may be present in a data package that corresponds to an individual.
  • Columns of the first data table and the second data table may indicate a status of individual individuals with respect to one or more characteristics.
  • the columns of the data tables may include an identifier that includes at least one of alphanumeric characters or symbols that indicate the presence or absence of one or more characteristics for a given individual.
  • the computing architecture may generate more data tables or fewer data tables.
  • the computing architecture may store the data structures in an additional data repository.
  • the computing architecture may store at least the first data tale and/or the second data table in an intermediate data repository .
  • the first data table and the second data table may be temporarily stored in the intermediate data repository .
  • the first data table and the second data table may be stored in the intermediate data repository before being added to the integrated data repository .
  • the integrated data repository may be periodically generated and/or updated. In these scenarios, data structures generated by the computing architecture based on analyzing the corpus of information may be stored in the intermediate data repository until a time when the integrated data repository is to be at least one of generated or updated.
  • the computing architecture may perform one or more de-identification processes at operation .
  • the data structures stored by the intermediate data repository may be de- identified in order to preserve the privacy of individuals.
  • the one or more de-identification processes may include applying one or more electronically implemented cryptographic techniques to information of individuals included in the data structures stored by the intermediate data repository.
  • the computing architecture may generate tokens that correspond to individual individuals that have information stored in data structures of the intermediate data repository .
  • the tokens may be generated by applying one or more hash functions to information related to individual individuals.
  • the one or more de-identification processes may include applying a salt function to information corresponding to individual individuals to generate tokens for the individual individuals.
  • the one or more cryptographic techniques applied to de-identify the data structures stored by the intermediate data repository may be the same or similar to those applied to information obtained from the health insurance claims data repository.
  • the computing architecture may store the de-identified data structures in conjunction with the integrated data repository .
  • the information stored in the intermediate data repository for a given individual may be stored in conjunction with additional information about the given individual in the integrated data repository .
  • the integrated data repository may store information for a given individual obtained from at least two of the molecular data repository , obtained from the health insurance claims data repository , and obtained from the intermediate data repository .
  • information about a given individual obtained from a number of disparate data repositories may be stored in the integrated data repository.
  • information about individuals that is obtained from the different data repositories may be analyzed together rather than analyzed separately as with many existing systems.
  • the information stored by the intermediate data repository may be used to validate one or more determinations made by the data integration and analysis system .
  • the data integration and analysis system may analyze information obtained from the health insurance claims data repository and the molecular data repository to determine characteristics of individuals. The data integration and analysis system may then analyze information obtained from the intermediate data repository to determine whether the predicted characteristics identified from the information obtained from the health insurance claims data repository and from the molecular data repository correspond to the characteristics for the same individuals with respect to information stored by the intermediate data repository .
  • the one or more cryptographic techniques applied to de-identify the data structures stored by the intermediate data repository may utilize the same or similar information that was used to generate at least one of the first tokens or the second tokens.
  • the operation may implement one or more cryptographic techniques using a combination of at least a portion of a first name of the respective individuals, at least a portion of the last name of the respective individuals, at least a portion of a date of birth of the respective individuals, a gender of the individuals, and at least a portion of a location identifier of the respective individuals to deidentify the data structures of the intermediate data repository.
  • the data structures of the integrated data repository and the data structures of the intermediate data repository that are associated with a same individual may not be stored in a manner such that the information stored by the integrated data repository and the information stored by the intermediate data repository may be retrieved together for a given individual, which may lead to inaccurate information being provided by the data integration and analysis system .
  • the absence of a specified cryptographic protocol as described herein may also lead to the use of more computing resources to determine the information stored in the integrated data repository from other data sources and the information stored by the intermediate data repository that correspond to a given individual.
  • the process may include generating a data file that includes tokens generated using a first hash function.
  • Individual tokens may correspond to a respective individual of a group of individuals having data stored by a molecular data repository.
  • an individual having data stored by the molecular data repository may be associated with one or more tokens.
  • the tokens may be generated by applying one or more first hash functions to a subset of information corresponding to the group of individuals stored by the genomics data repository.
  • individual tokens may be generated by applying one or more first hash functions to one or more combinations of at least a portion of a first name of a respective individual of the group of individuals, at least a portion of a second name of a respective individual of the group of individuals, a location identifier of a respective individual of the group of individuals, a gender of a respective individual of the group of individuals, and a date of birth of a respective individual of the group of individuals.
  • the tokens may be generated by a data integration and analysis system that is coupled to the genomics data repository.
  • the tokens may be generated by a third-party system and accessed by a data integration and analysis system coupled to the molecular data repository.
  • the process may also include, at operation , sending the data file to a health insurance claims data management system.
  • the health insurance claims data management system may match the tokens included in the data file with second tokens accessed by the health insurance data management system and generated based on information stored by a health insurance claims data repository.
  • the process may include obtaining, from the health insurance claims data management system, in response to the data file, first data corresponding to the group of individuals, where the first data includes health insurance claims data.
  • affirmative consent is obtained from the members of the group of individuals for their data to be transferred from the health insurance claims data management system.
  • the data is transferred in an anonymized format, such that the data may not be traced back to an individual member.
  • the health insurance claims data management system may be coupled to a health insurance claims data repository that stores health insurance claims information for a number of individuals.
  • the health insurance claims data management system may analyze the tokens of the data file with respect to additional tokens generated by the health insurance claims data management system.
  • the additional tokens may be generated based on a same set of information used to generate the tokens included in the data file. However, an individual’s identity may not be determined based on a token.
  • the health insurance claims data management system may match tokens included in the data file with additional tokens generated based on information stored by the health insurance claims data repository to determine individuals having information stored by the health insurance claims data repository that also have information stored by the genomics data repository.
  • the technology disclosed herein complies with legal and best practice privacy standards, such as HIPAA and GDPR.
  • the process may include generating a number of identifiers using a second hash function that is different from the first hash function.
  • individual identifiers may correspond to one or more tokens related to a respective individual of the group of individuals.
  • the identifiers may be unique with respect to a given individual of the group of individuals and are de-identified.
  • the identifiers may be generated using information stored by the genomics data repository for the group of individuals that is different from the information stored by the genomics data repository used to generate the tokens.
  • intermediate identifiers may be generated by applying the second hash function to information of the respective groups of individuals and final versions of the identifiers may be generated by applying one or more salting techniques to the intermediate identifiers.
  • Information stored by the genomics data repository for respective individuals may be stored in association with the identifiers such that at least a portion of the information for given individuals stored by the genomics data repository may be accessed using respective identifiers of the given individuals.
  • the process may include, at operation , obtaining, using the number of identifiers, second data from the molecular data repository for the group of individuals, and, at operation , the process may include determining respective portions of the first data that correspond to respective portions of the second data for the group of individuals. For example, for a given individual, the first data corresponding to health insurance claims data for the given individual may be identified in addition to second data corresponding to molecular data of the given individual, such as genomics data. In this way, for a given individual, both health insurance claims data and molecular data may be identified.
  • the process may include, at operation , generating an integrated data repository that stores the respective portions of the first data and the respective portions of the second data in relation to respective identifiers of the number of identifiers.
  • the integrated data repository may store health insurance claims data and genomics claims data for a given individual in association with an identifier that may be used to access the health insurance claims data and the genomics claims data for the given individual.
  • the information stored by the integrated data repository may be organized according to a data repository schema.
  • the integrated data repository may store health insurance claims data and genomics data for the group of individuals in a number of data tables. In one or more examples, information stored by the number of data tables may be linked.
  • the data repository schema may include a first data table that stores genomics data of the group of individuals.
  • the first data table may store information corresponding to a panel used to generate genomics data, mutations of genomic regions, types of mutations, copy numbers of genomic regions, coverage data indicating numbers of nucleic acid molecules identified in a sample having one or more mutations, testing dates, and patient information.
  • the data repository schema may also include a second data that stores data related to one or more patient visits by individuals to one or more healthcare providers and a third data table that stores information corresponding to respective services provided to individuals with respect to one or more patient visits to one or more healthcare providers indicated by the second data table. Additionally, the data repository schema may include a fourth data table that stores personal information of the group of individuals and a fifth data table that stores information related to a health insurance company or governmental entity that made payment for services provided to the group of individuals. Further, the data repository schema may include a sixth data table storing information corresponding to health insurance coverage information for the group of individuals, such as a type of health insurance plan related to the group of individuals. The data repository schema may also include a seventh data table that stores information related to pharmaceutical treatments obtained by the group of individuals.
  • the integrated data repository may also store medical records that correspond to at least a portion of the group of individuals.
  • the medical records may be obtained from one or more data repositories storing the medical records.
  • One or more optical character recognition (OCR) operations may be performed with respect to the medical records.
  • OCR optical character recognition
  • the medical records may be analyzed to determine one or more portions of the additional information to remove to produce a corpus of information.
  • the corpus of information may be analyzed to determine a portion of the subset of the additional group of individuals that correspond to one or more biomarkers.
  • One or more data structures may be generated from the corpus of information that store identifiers of the portion of the subset of the additional group of individuals and that store an indication that the portion of the subset of the additional group of individuals corresponds to the one or more biomarkers.
  • the one or more data structures may be stored by an intermediate data repository.
  • One or more de-identification operations may be performed with respect to the identifiers of the portion of the subset of the additional group of individuals before modifying the integrated data repository to store at least a portion of the additional information of the medical records of the portion of the subset of the additional group of individuals in relation to the number of identifiers.
  • the information stored by the integrated data repository may be added to the integrated data repository.
  • the de-identified medical records information may be added to the integrated data repository in addition to or in lieu of the health insurance claims data.
  • the one or more data structures storing the de- identified medical records information with respect to the biomarker data may have one or more logical connections with other data structures stored in the integrated data repository.
  • the one or more data structures storing the de-identified medical records information with respect to the biomarker data may have one or more logical connections with at least one of the first data table may store information corresponding to a panel used to generate genomics data, mutations of genomic regions, types of mutations, copy numbers of genomic regions, coverage data indicating numbers of nucleic acid molecules identified in a sample having one or more mutations, testing dates, and patient information, the second data that stores data related to one or more patient visits by individuals to one or more healthcare providers, the a third data table that stores information corresponding to respective services provided to individuals with respect to one or more patient visits to one or more healthcare providers indicated by the second data table, the fourth data table that stores personal information of the group of individuals, the fifth data table that stores information related to a health insurance company or governmental entity that made payment for services provided to the group of individuals, the sixth data table storing information corresponding to health insurance coverage information for the group of individuals, such as a type of health insurance plan related to the group of individuals, or the seventh data table that stores information corresponding
  • the medical records data may be added to the integrated data repository by generating a data file including the first tokens generated using a first hash function.
  • Individual first tokens may correspond to a respective individual of a group of individuals having data stored by a molecular data repository.
  • the data file may be sent to a medical records data management system and medical records data corresponding to the group of individuals may be obtained from the medical records data management system in response to the data file.
  • a number of identifiers may be generated using a second hash function that is different from the first hash function. Each identifier may correspond to one or more tokens related to each individual of the group of individuals.
  • second data may be obtained from the molecular data repository for the group of individuals.
  • respective portions of the first data may be determined to correspond to respective portions of the second data for the group of individuals.
  • the integrated data repository may be generated that stores the respective portions of the first data and the respective portions of the second data in relation to respective identifiers of the number of identifiers.
  • a request may be received to determine data with respect to a number of individuals having data stored in the integrated data repository.
  • the request includes one or more search criteria.
  • a subset of the number of individuals having one or more characteristics that correspond to the one or more search criteria may be determined and information of the subset of the number of individuals may be analyzed to determine a measure of significance of a characteristic of the one or more characteristics with respect to a biological condition.
  • one or more genomic mutations may be determined to be present in the subset of the number of individuals and a plurality of treatments provided to the subset of the number of individuals may also be determined.
  • respective survival rates for the subset of the number of individuals may be determined, such as real -world survival rates.
  • the measure of significance may correspond to survival rate with respect to a treatment of the plurality of treatments and a genomic mutation of the one or more genomic mutations. Based on measure of significance, an effectiveness of the treatment for the subset of the number of individuals may be determined.
  • individuals in subset of the number of individuals that have not received the treatment may be determined.
  • One or more therapeutically effective amounts of the treatment may be administered to the individuals in the subset of the number of individuals that have not received the treatment.
  • the process may include, at operation , determining a first set of data processing instructions that are executable in relation to the first data stored by an integrated data repository.
  • the integrated data repository may store health insurance claims data and molecular data for a common group of individuals.
  • the first set of data processing instructions may be included in a plurality of sets of data processing instructions that are part of a data processing pipeline. Each of the sets of data processing instructions of the data processing pipeline may be executed to generate a respective analytics ready dataset.
  • the process may also include, at operation , causing the first set of data processing instructions to be executed to generate a first dataset.
  • the first dataset may indicate a subset of the group of individuals in which a biological condition is present.
  • the first set of data processing instructions may be executed to analyze data stored by the integrated data repository to identify a cohort of individuals in which the biological condition is present.
  • the biological condition may include a cancer.
  • the first set of data processing instructions may be executed to analyze data stored by the integrated data repository to identify a cohort of individuals in which lung cancer is present.
  • the data processing pipeline may include multiple sets of data processing instructions to identify cohorts of individuals in which different biological conditions are present.
  • the first set of data processing instructions may be executed to analyze at least one of health insurance claims data or molecular data to determine a cohort of individuals in which the biological condition is present. For example, the first set of data processing instructions may be executed to identify individuals having one or more health insurance codes present in health insurance claims data to determine a group of individuals in which the biological condition is present. Additionally, the first set of data processing instructions may be executed to identify individuals in which one or more mutations are present in a genomic region of nucleic acid molecules derived from samples obtained from the individuals to determine a group of individuals in which the biological condition is present.
  • the process may include causing the second set of data processing instructions to be executed to generate a second dataset indicating one or more treatments provided to a second subset of the group of individuals.
  • the second dataset may indicate a subset of the group of individuals that have received one or more treatments.
  • the one or more treatments may be provided to individuals in which one or more biological conditions are present.
  • the second set of data processing instructions may be executed to analyze data stored by the integrated data repository to identify a cohort of individuals that received the one or more treatments.
  • the second set of data processing instructions may be executed to analyze at least one health insurance claims data or genomics data to determine a cohort of individuals that received the one or more treatments.
  • the second set of data processing instructions may be executed to identify individuals having one or more health insurance codes present in health insurance claims data to determine a group of individuals that received the one or more treatments.
  • information included in at least one of the first dataset or the second dataset may be analyzed to determine an amount of influence of a mutation of a genomic region on a survival rate of individuals in which the biological condition is present. Additionally, information included in the first dataset and the second dataset may be analyzed to determine an amount of impact of one or more treatments with respect to individuals in which the biological condition is present and in which one or more genomic mutations are also present.
  • the instructions transform the general, non-programmed machine into a particular machine programmed to carry out the described and illustrated functions in the manner described.
  • the machine operates as a standalone device or may be coupled (e.g., networked) to other machines.
  • the machine may operate in the capacity of a server machine or a client machine in a server-client network environment, or as a peer machine in a peer-to-peer (or distributed) network environment.
  • a circuit may be implemented mechanically or electronically.
  • a circuit may comprise dedicated circuitry or logic that is specifically configured to perform one or more techniques such as discussed above, such as including a special-purpose processor, a field programmable gate array (FPGA) or an application-specific integrated circuit (ASIC).
  • a circuit may comprise programmable logic (e.g., circuitry, as encompassed within a general-purpose processor or other programmable processor) that may be temporarily configured (e.g., by software) to perform the certain operations. It will be appreciated that the decision to implement a circuit mechanically (e.g., in dedicated and permanently configured circuitry), or in temporarily configured circuitry (e.g., configured by software) may be driven by cost and time considerations.
  • circuits may provide information to, and receive information from, other circuits.
  • the circuits may be regarded as being communicatively coupled to one or more other circuits.
  • communications may be achieved through signal transmission (e.g., over appropriate circuits and buses) that connect the circuits.
  • communications between such circuits may be achieved, for example, through the storage and retrieval of information in memory structures to which the multiple circuits have access.
  • one circuit may perform an operation and store the output of that operation in a memory device to which it is communicatively coupled.
  • a further circuit may then, at a later time, access the memory device to retrieve and process the stored output.
  • circuits may be configured to initiate or receive communications with input or output devices and may operate on a resource (e.g., a collection of information).
  • the methods described herein may be at least partially processor implemented. For example, at least some or all of the operations of a method may be performed by one or processors or processor-implemented circuits. The performance of certain of the operations may be distributed among the one or more processors, not only residing within a single machine, but deployed across a number of machines. In an example, the processor or processors may be located in a single location (e.g., within a home environment, an office environment or as a server farm), while in other examples the processors may be distributed across a number of locations.
  • Example implementations may be implemented in digital electronic circuitry, in computer hardware, in firmware, in software, or in any combination thereof.
  • Example implementations may be implemented using a computer program product (e.g., a computer program, tangibly embodied in an information carrier or in a machine readable medium, for execution by, or to control the operation of, data processing apparatus such as a programmable processor, a computer, or multiple computers).
  • a computer program product e.g., a computer program, tangibly embodied in an information carrier or in a machine readable medium, for execution by, or to control the operation of, data processing apparatus such as a programmable processor, a computer, or multiple computers.
  • operations may be performed by one or more programmable processors executing a computer program to perform functions by operating on input data and generating output.
  • Examples of method operations may also be performed by, and example apparatus may be implemented as, special purpose logic circuitry (e.g., a field programmable gate array (FPGA) or an application-specific integrated circuit (ASIC)).
  • FPGA field programmable gate array
  • ASIC application-specific integrated circuit
  • the computing system may include clients and servers.
  • a client and server are generally remote from each other and generally interact through a communication network.
  • the relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship with each other.
  • both hardware and software architectures require consideration.
  • the choice of whether to implement certain functionality in permanently configured hardware e.g., an ASIC
  • temporarily configured hardware e.g., a combination of software and a programmable processor
  • a combination of permanently and temporarily configured hardware may be a design choice.
  • hardware e.g., computing device
  • software architectures that may be deployed in example implementations.
  • the computing device may operate in the capacity of either a server or a client machine in server-client network environments.
  • computing device may act as a peer machine in peer-to-peer (or other distributed) network environments.
  • the computing device may be a personal computer (PC), a tablet PC, a set-top box (STB), a mobile telephone, a web appliance, a network router, switch or bridge, or any machine capable of executing instructions (sequential or otherwise) specifying actions to be taken (e.g., performed) by the computing device .
  • PC personal computer
  • PDA set-top box
  • STB set-top box
  • mobile telephone a web appliance
  • network router switch or bridge
  • Example computing device may include a processor (e.g., a central processing unit CPU), a graphics processing unit (GPU) or both), a main memory and a static memory , some or all of which may communicate with each other via a bus .
  • the computing device may further include a display unit , an alphanumeric input device (e.g., a keyboard), and a user interface (UI) navigation device (e.g., a mouse).
  • the display unit, input device and UI navigation device may be a touch screen display.
  • the computing device may additionally include a storage device (e.g., drive unit) , a signal generation device (e.g., a speaker), a network interface device , and one or more sensors , such as a global positioning system (GPS) sensor, compass, accelerometer, or another sensor.
  • a storage device e.g., drive unit
  • a signal generation device e.g., a speaker
  • a network interface device e.g., a Wi-Fi
  • sensors such as a global positioning system (GPS) sensor, compass, accelerometer, or another sensor.
  • GPS global positioning system
  • the storage device may include a machine readable medium on which is stored one or more sets of data structures or instructions (e.g., software) embodying or utilized by any one or more of the methodologies or functions described herein.
  • the instructions may also reside, completely or at least partially, within the main memory , within static memory , or within the processor during execution thereof by the computing device .
  • one or any combination of the processor, the main memory , the static memory , or the storage device may constitute machine readable media.
  • machine readable medium may include a single medium or multiple media (e.g., a centralized or distributed database, and/or associated caches and servers) that configured to store the one or more instructions .
  • the term “machine readable medium” may also be taken to include any tangible medium that is capable of storing, encoding, or carrying instructions for execution by the machine and that cause the machine to perform any one or more of the methodologies of the present disclosure or that is capable of storing, encoding or carrying data structures utilized by or associated with such instructions.
  • the term “machine readable medium” may accordingly be taken to include, but not be limited to, solid-state memories, and optical and magnetic media. Specific examples of machine-readable media may include non-volatile memory, including, by way of example, semiconductor memory devices (e.g., Electrically Programmable Read-Only Memory
  • the instructions may further be transmitted or received over a communications network 828 using a transmission medium via the network interface device 822 utilizing any one of a number of transfer protocols (e.g., frame relay, IP, TCP, UDP, HTTP, etc.).
  • Example communication networks may include a local area network (LAN), a wide area network (WAN), a packet data network (e.g., the Internet), mobile telephone networks (e.g., cellular networks), Plain Old Telephone (POTS) networks, and wireless data networks (e.g., IEEE 802.11 standards family known as Wi-Fi®, IEEE 802.16 standards family known as WiMax®), peer-to-peer (P2P) networks, among others.
  • the term “transmission medium” shall be taken to include any intangible medium that is capable of storing, encoding or carrying instructions for execution by the machine, and includes digital or analog communications signals or other intangible medium to facilitate communication of such software.
  • a component may refer to a device, physical entity, or logic having boundaries defined by function or subroutine calls, branch points, APIs, or other technologies that provide for the partitioning or modularization of particular processing or control functions. Components may be combined via their interfaces with other components to carry out a machine process.
  • a component may be a packaged functional hardware unit designed for use with other components and a part of a program that usually performs a particular function of related functions. Components may constitute either software components (e.g., code embodied on a machine-readable medium) or hardware components.
  • a "hardware component” is a tangible unit capable of performing certain operations and may be configured or arranged in a certain physical manner.
  • one or more computer systems e.g., a standalone computer system, a client computer system, or a server computer system
  • one or more hardware components of a computer system e.g., a processor or a group of processors
  • software e.g., an application or application portion
  • the present methods can be used to diagnose presence of conditions, in a subject, to characterize conditions, monitor response to treatment of a condition, effect prognosis risk of developing a condition or subsequent course of a condition.
  • the present disclosure can also be useful in determining the efficacy of a particular treatment option.
  • Successful treatment options may increase the amount of nucleic acids, such as cell free nucleic acids, detected in subject's blood if the treatment is successful as diseased and dysfunctional die and shed DNA or otherwise exhibit chronic and acute signs of inflammation. In other examples, this may not occur.
  • certain treatment options may be correlated with genetic profiles of disease types and sub-types over time. This correlation may be useful in selecting a therapy.
  • the methods and systems disclosed herein may be used to identify customized or targeted therapies to treat a given disease or condition in patients based on the classification of a nucleic acid variant as being of somatic or germline origin.
  • the disease under consideration is a type of cancer.
  • the methods of the disclosure may be used to characterize the heterogeneity of an abnormal condition in a subject.
  • Such methods can include, e.g., generating a genomic and epigenomic profile of extracellular polynucleotides derived from the subject, wherein the genetic profile includes a plurality of data that can characterize malfunctions and abnormalities associated with the heart muscle and valve tissues (e.g., hypertrophy), the decreased supply of blood flow and oxygen supply to the heart are often secondary symptoms of debilitation and/or deterioration of the blood now and supply system caused by physical and biochemical stresses.
  • cardiovascular diseases that are directly affected by these types of stresses include atherosclerosis, coronary artery disease, peripheral vascular disease and peripheral artery disease, along with various cardias and arrythmias which may represent other forms of disease and dysfunction.
  • the present methods can be used to generate our profile, fingerprint or set of data that is a summation of genetic information derived from different cells in a heterogeneous disease. This set of data may comprise copy number variation, epigenetic variation, and mutation analyses alone or in combination.
  • the present methods can be used to diagnose, prognose, monitor or observe cancers, or other diseases.
  • the methods herein do not involve the diagnosing, prognosing or monitoring a fetus and as such are not directed to non-invasive prenatal testing.
  • these methodologies may be employed in a pregnant subject to diagnose, prognose, monitor or observe cancers or other diseases in an unborn subject whose DNA and other polynucleotides may co-circulate with maternal molecules.
  • Non-limiting examples of other genetic-based diseases, disorders, or conditions that are optionally evaluated using the methods and systems disclosed herein include achondroplasia, alpha- 1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, Charcot-Marie-Tooth (CMT), cri du chat, Crohn's disease, cystic fibrosis, Dercum disease, down syndrome, Duane syndrome, Duchenne muscular dystrophy, Factor V Leiden thrombophilia, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, Klinefelter syndrome, Marfan syndrome, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland anomaly, porphyria, progeria, retinitis pigmentosa
  • the methods disclosed herein relate to identifying and administering customized therapies to patients given the status of a nucleic acid variant as being of somatic or germline origin.
  • essentially any cancer therapy e.g., surgical therapy, radiation therapy, chemotherapy, and/or the like
  • customized therapies include at least one immunotherapy (or an immunotherapeutic agent).
  • Immunotherapy refers generally to methods of enhancing an immune response against a given cancer type.
  • immunotherapy refers to methods of enhancing a T cell response against a tumor or cancer.
  • the status of a nucleic acid variant from a sample from a subject as being of somatic or germline origin may be compared with a database of comparator results from a reference population to identify customized or targeted therapies for that subject.
  • the reference population includes patients with the same cancer or disease type as the test subject and/or patients who are receiving, or who have received, the same therapy as the test subject.
  • a customized or targeted therapy (or therapies) may be identified when the nucleic variant and the comparator results satisfy certain classification criteria (e.g., are a substantial or an approximate match).
  • the customized therapies described herein are typically administered parenterally (e.g., intravenously or subcutaneously).
  • Pharmaceutical compositions containing an immunotherapeutic agent are typically administered intravenously.
  • Certain therapeutic agents are administered orally.
  • customized therapies e.g., immunotherapeutic agents, etc.
  • kits including the compositions as described herein.
  • the kits can be useful in performing the methods as described herein.
  • a kit includes a first reagent for partitioning a sample into a plurality of subsamples as described herein, such as any of the partitioning reagents described elsewhere herein.
  • Kits may further comprise a plurality of oligonucleotide probes that selectively hybridize to least 5, 6, 7, 8, 9, 10, 20, 30, 40 or all genes selected from the group consisting of ALK, APC, BRAF, CDKN2A, EGFR, ERBB2, FBXW7, KRAS, MYC, NOTCH1, NRAS, PIK3CA, PTEN, RBI, TP53, MET, AR, ABL1, AKT1, ATM, CDH1, CSFIR, CTNNB1, ERBB4, EZH2, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, MLH1, MPL, NPM1, PDGFRA, PROC, PTPN11, RET,SMAD4, SMARCB1, SMO, SRC, STK11, VHL, TERT, CCND1, CDK4, CDKN2B
  • the number genes to which the oligonucleotide probes can selectively hybridize can vary.
  • the number of genes can comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54.
  • the kit can include a container that includes the plurality of oligonucleotide probes and instructions for performing any of the methods described herein.
  • the kit can comprise at least 4, 5, 6, 7, or 8 different library adaptors having distinct molecular barcodes and identical sample barcodes.
  • the library adaptors may not be sequencing adaptors.
  • the library adaptors do not include flow cell sequences or sequences that permit the formation of hairpin loops for sequencing.
  • the different variations and combinations of molecular barcodes and sample barcodes are described throughout and are applicable to the kit.
  • the adaptors are not sequencing adaptors.
  • the adaptors provided with the kit can also comprise sequencing adaptors.
  • a sequencing adaptor can comprise a sequence hybridizing to one or more sequencing primers.
  • a sequencing adaptor can further comprise a sequence hybridizing to a solid support, e.g., a flow cell sequence.
  • a sequencing adaptor can be a flow cell adaptor.
  • the sequencing adaptors can be attached to one or both ends of a polynucleotide fragment.
  • the kit can comprise at least 8 different library adaptors having distinct molecular barcodes and identical sample barcodes.
  • the library adaptors may not be sequencing adaptors.
  • the kit can further include a sequencing adaptor having a first sequence that selectively hybridizes to the library adaptors and a second sequence that selectively hybridizes to a flow cell sequence.
  • a sequencing adaptor can be hairpin shaped.
  • the hairpin shaped adaptor can comprise a complementary double stranded portion and a loop portion, where the double stranded portion can be attached ⁇ e.g., ligated) to a double-stranded polynucleotide.
  • Hairpin shaped sequencing adaptors can be attached to both ends of a polynucleotide fragment to generate a circular molecule, which can be sequenced multiple times.
  • a sequencing adaptor can be up to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
  • the sequencing adaptor can comprise 20-30, 20-
  • a sequencing adaptor can comprise one or more barcodes.
  • a sequencing adaptor can comprise a sample barcode.
  • the sample barcode can comprise a pre-determined sequence.
  • the sample barcodes can be used to identify the source of the polynucleotides.
  • the sample barcode can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, or more (or any length as described throughout) nucleic acid bases, e.g., at least 8 bases.
  • the barcode can be contiguous or non-contiguous sequences, as described above.
  • the library adaptors can be blunt ended and Y-shaped and can be less than or equal to 40 nucleic acid bases in length. Other variations of the can be found throughout and are applicable to the kit.
  • a biomarker is a genetic variant. Biomarkers may be determined using any of several resources or methods. A biomarker may have been previously discovered or may be discovered de novo using experimental or epidemiological techniques. Detection of a biomarker may be indicative of a disease when the biomarker is highly correlated to the disease. Detection of a biomarker may be indicative of cancer when a biomarker in a region or gene occur with a frequency that is greater than a frequency for a given background population or dataset.
  • the biomarker may affect splicing, may change the amino acid coded by a particular codon, may result in a frameshift, or may result in a premature stop codon.
  • the biomarker may result in a conservative substitution of an amino acid.
  • One or more biomarkers may result in a conservative substitution of an amino acid.
  • One or more biomarkers may result in a nonconservative substitution of an amino acid.
  • the frequency of a biomarker may be as low as 0.001%.
  • the frequency of a biomarker may be as low as 0.005%.
  • the frequency of a biomarker may be as low as 0.01%.
  • the frequency of a biomarker may be as low as 0.02%.
  • the frequency of a biomarker may be as low as 0.03%.
  • the frequency of a biomarker may be as low as 0.05%.
  • the frequency of a biomarker may be as low as 0.1%.
  • the frequency of a biomarker may be as low as 1%.
  • No single biomarker may be present in more than 50%, of subjects having the cancer.
  • No single biomarker may be present in more than 40%, of subjects having the cancer.
  • No single biomarker may be present in more than 30%, of subjects having the cancer.
  • No single biomarker may be present in more than 20%, of subjects having the cancer.
  • No single biomarker may be present in more than 10%, of subjects having the cancer.
  • No single biomarker may be present in more than 5%, of subjects having the cancer.
  • a single biomarker may be present in 0.001% to 50% of subjects having cancer.
  • a single biomarker may be present in 0.01% to 50% of subjects having cancer.
  • a single biomarker may be present in 0.01% to 30% of subjects having cancer.
  • a single biomarker may be present in 0.01% to 20% of subjects having cancer.
  • a single biomarker may be present in 0.01% to 10% of subjects having cancer.
  • a single biomarker may be present in 0.1% to 10% of subjects having cancer.
  • a single biomarker may be present in 0.1% to 5% of subjects having cancer.
  • a biomarker from a subject is in at least one exon in the panel.
  • One or more cancers selected from the group including ovarian cancer, pancreatic cancer, breast cancer, colorectal cancer, non-small cell lung carcinoma (squamous cell or adenocarcinoma), or any other cancer each exhibit a biomarker in at least one exon in the panel.
  • Each of at least 3 of the cancers may exhibit a biomarker in at least one exon in the panel.
  • Each of at least 4 of the cancers may exhibit a biomarker in at least one exon in the panel.
  • Each of at least 5 of the cancers may exhibit a biomarker in at least one exon in the panel.
  • Each of at least 8 of the cancers may exhibit a biomarker in at least one exon in the panel.
  • Each of at least 10 of the cancers may exhibit a biomarker in at least one exon in the panel.
  • All of the cancers may exhibit a biomarker in at least one exon in the panel.
  • a biomarker from a subject is in at least one exon or gene in the panel. At least 85% of subjects having a cancer may exhibit a biomarker in at least one exon or gene in the panel. At least 90%, of subjects having a cancer may exhibit a biomarker in at least one exon or gene in the panel. At least 92% of subjects having a cancer may exhibit a biomarker in at least one exon or gene in the panel. At least 95% of subjects having a cancer may exhibit a biomarker in at least one exon or gene in the panel. At least 96% of subjects having a cancer may exhibit a biomarker in at least one exon or gene in the panel.
  • At least 97% of subjects having a cancer may exhibit a biomarker in at least one exon or gene in the panel. At least 98% of subjects having a cancer may exhibit a biomarker in at least one exon or gene in the panel. At least 99% of subjects having a cancer may exhibit a biomarker in at least one exon or gene in the panel. At least 99.5% of subjects having a cancer may exhibit a biomarker in at least one exon or gene in the panel.
  • a biomarker from a subject is in at least one region in the panel. At least 85% of subjects having a cancer may exhibit a biomarker in at least one region in the panel. At least 90%, of subjects having a cancer may exhibit a biomarker in at least one region in the panel. At least 92% of subjects having a cancer may exhibit a biomarker in at least one region in the panel. At least 95% of subjects having a cancer may exhibit a biomarker in at least one region in the panel. At least 96% of subjects having a cancer may exhibit a biomarker in at least one region in the panel. At least 97% of subjects having a cancer may exhibit a biomarker in at least one region in the panel.
  • At least 98% of subjects having a cancer may exhibit a biomarker in at least one region in the panel. At least 99% of subjects having a cancer may exhibit a biomarker in at least one region in the panel. At least 99.5% of subjects having a cancer may exhibit a biomarker in at least one region in the panel.
  • Detection may be performed with a high sensitivity and/or a high specificity.
  • Sensitivity can refer to a measure of the proportion of positives that are correctly identified as such.
  • sensitivity refers to the percentage of all existing biomarkers that are detected.
  • sensitivity refers to the percentage of sick people who are correctly identified as having certain disease.
  • Specificity can refer to a measure of the proportion of negatives that are correctly identified as such.
  • specificity refers to the proportion of unaltered bases which are correctly identified.
  • specificity refers to the percentage of healthy people who are correctly identified as not having certain disease.
  • Detection may be performed with a sensitivity of at least 95%, 97%, 98%, 99%, 99.5%, or 99.9% and/or a specificity of at least 80%, 90%, 95%, 97%, 98% or 99%. Detection may be performed with a sensitivity of at least 90%, 95%, 97%, 98%, 99%, 99.5%, 99.6%, 99.98%, 99.9% or 99.95%.
  • Detection may be performed with a specificity of at least 90%, 95%, 97%, 98%, 99%, 99.5%, 99.6%, 99.98%, 99.9% or 99.95%. Detection may be performed with a specificity of at least 70% and a sensitivity of at least 70%, a specificity of at least 75% and a sensitivity of at least 75%, a specificity of at least 80% and a sensitivity of at least 80%, a specificity of at least 85% and a sensitivity of at least 85%, a specificity of at least 90% and a sensitivity of at least 90%, a specificity of at least 95% and a sensitivity of at least 95%, a specificity of at least 96% and a sensitivity of at least 96%, a specificity of at least 97% and a sensitivity of at least 97%, a specificity of at least 98% and a sensitivity of at least 98%, a specificity of at least 99% and a sensitivity of at least 99%,
  • the methods can detect a biomarker at a sensitivity of sensitivity of about 80% or greater. In some cases, the methods can detect a biomarker at a sensitivity of sensitivity of about 95% or greater. In some cases, the methods can detect a biomarker at a sensitivity of sensitivity of about 80% or greater, and a sensitivity of sensitivity of about 95% or greater.
  • Detection may be highly accurate. Accuracy may apply to the identification of biomarkers in cell free DNA, and/or to the diagnosis of cancer. Statistical tools, such as covariate analysis described above, may be used to increase and/or measure accuracy.
  • the methods can detect a biomarker at an accuracy of at least 80%, 90%, 95%, 97%, 98% or 99%, 99.5%, 99.6%, 99.98%, 99.9%, or 99.95%. In some cases, the methods can detect a biomarker at an accuracy of at least 95% or greater.
  • Genes included in the panel for sequencing can include the fully transcribed region, the promoter region, enhancer regions, regulatory elements, and/or downstream sequence. To further increase the likelihood of detecting tumor indicating mutations only exons may be included in the panel.
  • the panel can comprise all exons of a selected gene, or only one or more of the exons of a selected gene.
  • the panel may comprise of exons from each of a plurality of different genes.
  • the panel may comprise at least one exon from each of the plurality of different genes.
  • a panel of exons from each of a plurality of different genes is selected such that a determined proportion of subjects having a cancer exhibit a genetic variant in at least one exon in the panel of exons.
  • At least one full exon from each different gene in a panel of genes may be sequenced.
  • the sequenced panel may comprise exons from a plurality of genes.
  • the panel may comprise exons from 2 to 100 different genes, from 2 to 70 genes, from 2 to 50 genes, from 2 to 30 genes, from 2 to 15 genes, or from 2 to 10 genes.
  • a selected panel may comprise a varying number of exons.
  • the panel may comprise from 2 to 3000 exons.
  • the panel may comprise from 2 to 1000 exons.
  • the panel may comprise from 2 to 500 exons.
  • the panel may comprise from 2 to 100 exons.
  • the panel may comprise from 2 to 50 exons.
  • the panel may comprise no more than 300 exons.
  • the panel may comprise no more than 200 exons.
  • the panel may comprise no more than 100 exons.
  • the panel may comprise no more than 50 exons.
  • the panel may comprise no more than 40 exons.
  • the panel may comprise no more than 30 exons.
  • the panel may comprise no more than 25 exons.
  • the panel may comprise no more than 20 exons.
  • the panel may comprise no more than 15 exons.
  • the panel may comprise no more than 10 exons.
  • the panel may comprise no more than 9 exons.
  • the panel may comprise no more than 8 exons.
  • the panel may comprise no more than 7 exons.
  • the panel may comprise one or more exons from a plurality of different genes.
  • the panel may comprise one or more exons from each of a proportion of the plurality of different genes.
  • the panel may comprise at least two exons from each of at least 25%, 50%, 75% or 90% of the different genes.
  • the panel may comprise at least three exons from each of at least 25%, 50%, 75% or 90% of the different genes.
  • the panel may comprise at least four exons from each of at least 25%, 50%, 75% or 90% of the different genes.
  • the sequencing panel can be at least lOkb, 12 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb , 110 kb, 120 kb, 130 kb, 140 kb, or 150 kb in size.
  • the sequencing panel may be less than 100 kb, 90 kb, 80 kb, 70 kb, 60 kb, or 50 kb in size.
  • the panel selected for sequencing can comprise at least 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 80, or 100 regions.
  • the regions in the panel are selected that the size of the regions are relatively small.
  • the regions in the panel have a size of about 10 kb or less, about 8 kb or less, about 6 kb or less, about 5 kb or less, about 4 kb or less, about 3 kb or less, about 2.5 kb or less, about 2 kb or less, about 1.5 kb or less, or about 1 kb or less or less.
  • the regions in the panel have a size from about 0.5 kb to about 10 kb, from about 0.5 kb to about 6 kb, from about 1 kb to about 11 kb, from about 1 kb to about 15 kb, from about 1 kb to about 20 kb, from about 0.1 kb to about 10 kb, or from about 0.2 kb to about 1 kb.
  • the regions in the panel can have a size from about 0.1 kb to about 5 kb.
  • the panel selected herein can allow for deep sequencing that is sufficient to detect low- frequency genetic variants (e.g., in cell-free nucleic acid molecules obtained from a sample).
  • An amount of genetic variants in a sample may be referred to in terms of the minor allele frequency for a given genetic variant.
  • the minor allele frequency may refer to the frequency at which minor alleles (e.g., not the most common allele) occurs in a given population of nucleic acids, such as a sample. Genetic variants at a low minor allele frequency may have a relatively low frequency of presence in a sample.
  • the panel allows for detection of genetic variants at a minor allele frequency of at least 0.0001%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5%.
  • the panel can allow for detection of genetic variants at a minor allele frequency of 0.001% or greater.
  • the panel can allow for detection of genetic variants at a minor allele frequency of 0.01% or greater.
  • the panel can allow for detection of genetic variant present in a sample at a frequency of as low as 0.0001%, 0.001%, 0.005%, 0.01%, 0.025%, 0.05%, 0.075%, 0.1%, 0.25%, 0.5%, 0.75%, or 1.0%.
  • the regions in the panel can be selected so that they comprise sequences differentially transcribed across one or more tissues.
  • the regions can comprise sequences transcribed in certain tissues at a higher level compared to other tissues.
  • the regions can comprise sequences transcribed in certain tissues but not in other tissues.
  • the regions in the panel can be selected to detect (diagnose) a cancer with a desired positive predictive value.
  • Positive predictive value can be increased by increasing sensitivity (e.g., chance of an actual positive being detected) and/or specificity (e.g., chance of not mistaking an actual negative for a positive).
  • regions in the panel can be selected to detect the one or more genetic variant with a positive predictive value of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.
  • the regions in the panel can be selected to detect the one or more genetic variant with a positive predictive value of 100%.
  • the regions in the panel can be selected to detect (diagnose) a cancer with a desired accuracy.
  • accuracy may refer to the ability of a test to discriminate between a disease condition (e.g., cancer) and health.
  • Accuracy may be can be quantified using measures such as sensitivity and specificity, predictive values, likelihood ratios, the area under the ROC curve, Youden’s index and/or diagnostic odds ratio.
  • Accuracy may presented as a percentage, which refers to a ratio between the number of tests giving a correct result and the total number of tests performed.
  • the regions in the panel can be selected to detect cancer with an accuracy of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.
  • the regions in the panel can be selected to detect cancer with an accuracy of 100%.
  • a panel may be selected such that when one or more regions or genes in the panel are removed, specificity is appreciably decreased. Removal of one region from the panel may result in a decrease in specificity of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more.
  • a panel may be selected such that the addition of one or more regions or genes to the panel does not appreciably increase the specificity of the panel, e.g., does not increase the specificity by more than 1%, 2%, 5%, 10%, 15%, or 20%.
  • a panel may be of a size such that when one or more regions or genes in the panel are removed, this appreciably decreases sensitivity, e.g., sensitivity is decreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more.
  • a panel may be selected such that the addition of one or more regions or genes to the panel does not appreciably increase the sensitivity of the panel, e.g., does not increase the sensitivity by more than 1%, 2%, 5%, 10%, 15%, or 20%.
  • a panel may be of a size such that when one or more regions or genes in the panel are removed, accuracy is appreciably decreased, e.g., accuracy is decreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more.
  • a panel may be of a size such that when one or more regions or genes the panel are removed, positive predictive value is appreciably decreased, e.g., positive predictive value is decreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more.
  • a panel may be selected such that the addition of one or more regions or genes to the panel does not appreciably increase the positive predictive value of the panel, e.g., does not increase the positive predictive value by more than 1%, 2%, 5%, 10%, 15%, or 20%
  • a panel may be selected to be highly sensitive and detect low frequency genetic variants. For instance, a panel may be selected such that a genetic variant or biomarker present in a sample at a frequency as low as 0.01%, 0.05%, or 0.001% may be detected at a sensitivity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%. Regions in a panel may be selected to detect a biomarker present at a frequency of 1% or less in a sample with a sensitivity of 70% or greater.
  • a panel may be selected to detect a biomarker at a frequency in a sample as low as 0.1% with a sensitivity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.
  • a panel may be selected to detect a biomarker at a frequency in a sample as low as 0.01% with a sensitivity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.
  • a panel may be selected to detect a biomarker at a frequency in a sample as low as 0.001% with a sensitivity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.
  • a panel may be selected to be highly specific and detect low frequency genetic variants. For instance, a panel may be selected such that a genetic variant or biomarker present in a sample at a frequency as low as 0.01%, 0.05%, or 0.001% may be detected at a specificity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%. Regions in a panel may be selected to detect a biomarker present at a frequency of 1% or less in a sample with a specificity of 70% or greater.
  • a panel may be selected to detect a biomarker at a frequency in a sample as low as 0.1% with a specificity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.
  • a panel may be selected to detect a biomarker at a frequency in a sample as low as 0.01% with a specificity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.
  • a panel may be selected to detect a biomarker at a frequency in a sample as low as 0.001% with a specificity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.
  • a panel may be selected to be highly accurate and detect low frequency genetic variants.
  • a panel may be selected such that a genetic variant or biomarker present in a sample at a frequency as low as 0.01%, 0.05%, or 0.001% may be detected at an accuracy of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.
  • Regions in a panel may be selected to detect a biomarker present at a frequency of 1% or less in a sample with an accuracy of 70% or greater.
  • a panel may be selected to detect a biomarker at a frequency in a sample as low as 0.1% with an accuracy of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.
  • a panel may be selected to detect a biomarker at a frequency in a sample as low as 0.01% with an accuracy of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.
  • a panel may be selected to detect a biomarker at a frequency in a sample as low as 0.001% with an accuracy of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.
  • a panel may be selected to be highly predictive and detect low frequency genetic variants.
  • a panel may be selected such that a genetic variant or biomarker present in a sample at a frequency as low as 0.01%, 0.05%, or 0.001% may have a positive predictive value of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.
  • the concentration of probes or baits used in the panel may be increased (2 to 6 ng/pL) to capture more nucleic acid molecule within a sample.
  • the concentration of probes or baits used in the panel may be at least 2 ng/pL, 3 ng/ pL, 4 ng/ pL, 5 ng/pL, 6 ng/pL, or greater.
  • the concentration of probes may be about 2 ng/pL to about 3 ng/pL, about 2 ng/pL to about 4 ng/pL, about 2 ng/pL to about 5 ng/pL, about 2 ng/pL to about 6 ng/pL.
  • the concentration of probes or baits used in the panel may be 2 ng/pL or more to 6 ng/pL or less. In some instances this may allow for more molecules within a biological to be analyzed thereby enabling lower frequency alleles to be detected.
  • Genetic analysis includes detection of nucleotide sequence variants and copy number variations. Genetic variants can be determined by sequencing.
  • the sequencing method can be massively parallel sequencing, that is, simultaneously (or in rapid succession) sequencing any of at least 100,000, 1 million, 10 million, 100 million, or 1 billion polynucleotide molecules.
  • Sequencing methods may include, but are not limited to: high-throughput sequencing, pyrosequencing, sequencing-by-synthesis, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing-by-ligation, sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression (Helicos), Next-generation sequencing, Single Molecule Sequencing by Synthesis (SMSS)(Helicos), massively-parallel sequencing, Clonal Single Molecule Array (Solexa), shotgun sequencing, Maxam-Gilbert or Sanger sequencing, primer walking, sequencing using PacBio, SOLiD, Ion Torrent, or Nanopore platforms and any other sequencing methods known in the art.
  • Sequencing can be made more efficient by performing sequence capture, that is, the enrichment of a sample for target sequences of interest, e.g., sequences including the KRAS and/or EGFR genes or portions of them containing sequence variant biomarkers. Sequence capture can be performed using immobilized probes that hybridize to the targets of interest.
  • Cell free DNA can include small amounts of tumor DNA mixed with germline DNA. Sequencing methods that increase sensitivity and specificity of detecting tumor DNA, and, in particular, genetic sequence variants and copy number variation, can be useful in the methods of this invention. Such methods are described in, for example, in WO 2014/039556.
  • One method includes high efficiency tagging of DNA molecules in the sample, e.g., tagging at least any of 50%, 75% or 90% of the polynucleotides in a sample. This increases the likelihood that a low-abundance target molecule in a sample will be tagged and subsequently sequenced, and significantly increases sensitivity of detection of target molecules.
  • Another method involves molecular tracking, which identifies sequence reads that have been redundantly generated from an original parent molecule, and assigns the most likely identity of a base at each locus or position in the parent molecule. This significantly increases specificity of detection by reducing noise generated by amplification and sequencing errors, which reduces frequency of false positives.
  • Methods of the present disclosure can be used to detect genetic variation in non-uniquely tagged initial starting genetic material (e.g., rare DNA) at a concentration that is less than 5%, 1%, 0.5%, 0.1%, 0.05%, or 0.01%, at a specificity of at least 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or 99.99999%.
  • Sequence reads of tagged polynucleotides can be subsequently tracked to generate consensus sequences for polynucleotides with an error rate of no more than 2%, 1%, 0.1%, or 0.01%.
  • a gene of interest may be amplified using primers that recognize the gene of interest.
  • the primers may hybridize to a gene upstream and/or downstream of a particular region of interest (e.g., upstream of a mutation site).
  • a detection probe may be hybridized to the amplification product. Detection probes may specifically hybridize to a wildtype sequence or to a mutated/variant sequence. Detection probes may be labeled with a detectable label (e.g., with a fluorophore). Detection of a wild-type or mutant sequence may be performed by detecting the detectable label (e.g., fluorescence imaging).
  • a gene of interest may be compared with a reference gene. Differences in copy number between the gene of interest and the reference gene may indicate amplification or deletion/truncation of a gene.
  • platforms suitable to perform the methods described herein include digital PCR platforms such as e.g., Fluidigm Digital Array.
  • Fig. 1 The Inventors collected methylation and genomic information from a genomic and epigenomic assay (Fig. 1) test taken prior to documented cardiac event for patients on trastuzumab that ultimately developed either heart failure and/or severe myocarditis (case group). This data was compared to methylation and genomic information from patients on trastuzumab that did not develop heart failure and/or severe myocarditis (control group) (Figs. 2, 3).
  • Genomic and epigenetic data was compared between case and controls groups to identify biomarkers that were statistically more prevalent in either the case or control groups.
  • biomarkers that were statistically more prevalent in either the case or control groups.
  • These analyses identified novel genomic and epigenomic biomarkers that are useful for determining whether a patient is at high risk of developing heart disease including AF and HF as well as increased risk of poor cardiac remodeling and myocardial interstitial fibrosis following myocardial infarction.
  • These biomarkers are also novel targets for drugs in order to decrease patient risk of heart disease and improve patient outcome after myocardial infarction or injury.
  • Case and control cohorts were determined via the de-identified real world evidence database.
  • Case cohort consisted of female patients with record of breast cancer who had blood collection for Guardant test while on trastuzumab with no prior record of heart failure or severe myocardis and subsequently had heart failure or severe myocardis.
  • Control cohort was built to match case cohort with the exception that control cohort had no record of heart failure or severe myocardis after the Guardant blood collection.
  • cfDNA was isolated from plasma of respective patients and run on genomic and epigenomic assay for profiling genomic and epigenomic alterations/loci. Significant difference in the frequency of alterations/regions between cases and controls was determined by two broadly different methods. Thereafter, one can use statistics to determine methylation regions that are more active in either case or control
  • Genomic and epigenetic biomarkers identified here could be used for more sensitive or more convenient diagnosis of heart disease risk than currently possible. Patients being genotyped or monitored for cancer disease and receiving cancer treatments known to increase risk of heart disease could be simultaneously be monitored for the development of heart disease before it happens.
  • Closed-loop capability consists of multiple analytical approaches, including validation of historical diagnostic results using RWE, optimization and development of new bioinformatic algorithms based on RWE insights and development and validation of new products via running historical samples on the new test and validating their analytical and clinical performance using RWE. This includes ascertaining biomarker status, and clinical diagnoses, as well as their outcomes summarized in variables such as real world time to next treatment (rwTTNT), and real world survival (rwOS).
  • rwTTNT real world time to next treatment
  • rwOS real world survival
  • TGFBI functions similar to periostin when overexpressed and can support greater cardiac growth response and repair after injury.
  • Periostin is thought to be a target of the approved drug valsartan for improving cardiac remodeling after heart injury. Signal at this region is not correlated with sample-level TF, consistent with heart disease associated signal (Fig. 6)
  • Example 9 - CGB7 hypermethylation calls more frequent in case (positives) than controls.
  • Example 10 - C0X6B1 hypermethylation calls more frequent in case than controls
  • COX6B1 is implicated in hypertropic cardiomyopathy due to a missense p.R20C mutation in the COX6B1 gene
  • Regions/peak normalized counts demonstrated correlation with TF for some samples. Additionally, some samples have either low TF of region is not hyper methylated (samples removed from regression lines of each group of samples), whereas negative samples show no correlation with TF (hypermethylation signal coming from normal cells). In contrast, positive samples show 2 clusters: one similar to negative samples and one correlated with TF. As one example, methylation signal: chrl : 167789527-167789647 (ADCY10) is shown as well as methylation signal: chrl9:49, 866, 910-49, 867, 251 (DKKL1) and methylation signal: chr2: 119611083-119611424.
  • Processing of methylation peaks, including for each targeted region was as follows: First, use normalized peak counts, thereafter, project samples onto the 2 dimensional space.
  • a pseudo chi square test can use the posterior probabilities for each data point for belonging to each of the two mixture fit lines, followed by determining a psuedo-chisq test statistic for sample category and regression line fit. This is performed by determining an “expected” number of negative observations in the first mixture population, which is the mixed combined proportion of first population times the number of negative observations, with same calculation for full 2x2 table of sample type crossed with two mixture populations.
  • selected regions final LR (lasso) model selected predictors (30).
  • selected regions top 150 (lowest) regions p-values pseudo chisq test ( ⁇ 0.001).
  • One approach includes enrichment tests of genes that are methylated more (or less) frequently that expected by chance in the samples from patients positive for heart failure versus the patient negative for heart failure. Gene methylation that distinguishes between case and control is not confounded by presence of tumor signal (quantified by Tumor methylation score percentage).
  • plots show raw counts of methylated molecules (x-axis) for select genes in case (patient develops heart failure, orange) versus control (patient does not develop heart failure, blue) versus y-axis which displays the tumor content of the sample.
  • Many implicated genes such as those shown below have signal that differentiate case versus control and do not have significant association with the presence of tumor (potential confounding factor when evaluating likelihood of developing heart failure in patients with cancer. Only a subset of genes are shown here.
  • a 3-step approach can include the following:
  • Step 1 For each targeted region: Use normalized peak counts
  • Step 2 Region p-value: pseudo chi square test (neg vs pos): use the posterior probabilities for each data point for belonging to each of the two mixture fit lines calculate a psuedo-chisq test statistic for sample category and regression line fit: the “expected” number of negative observations in the first mixture population is just the mixing proportion of first population times the number of negative observations. . .
  • Step 3 Cardiac AE prediction using posterior probabilities trained from cardiac AE samples
  • the model built from the discovery cohort has significant performance in predicting whether patients will later develop heart failure or decline in left ejection fraction when used to predict in an unrelated validation cohort.
  • analysis included breast cancer patients except for requirement on prior treatment history; both HER2 targeted treatment and non-HER2 targeted sub-cohorts (Fig. 19). This includes:
  • HER2 treatment -ve (HR therapies only): 86 patients without documented HER2 treatment and on hormone therapy with 90 days of blood collection.
  • HER2 treatment +ve HR +ve (sequential HR then HER2 therapies): 66 patients with documented HER2 treatment within 90 days of blood collection and hormone therapy within 183 days of blood collection.
  • HER2 treatment +ve HR -ve 41 patients with documented HER2 treatment within 365 days of blood collection and absence of hormone therapy.

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Abstract

L'invention concerne des procédés, des compositions et des dispositifs destinés à être utilisés dans le diagnostic et le traitement d'une maladie, notamment d'une maladie cardiovasculaire, et une telle maladie et un tel dysfonctionnement liés à l'administration d'une thérapie anticancéreuse. Les procédés comprennent le séquençage d'un panel de régions dans des molécules d'acide nucléique acellulaire et la détection d'un ou plusieurs biomarqueurs qui sont révélateurs d'une maladie cardiovasculaire et d'un dysfonctionnement.
PCT/US2024/052026 2023-10-18 2024-10-18 Biomarqueurs génomiques et de méthylation permettant de déterminer le risque de maladie cardiaque d'un patient et de nouvelles cibles de médicament génomique et épigénomique pour diminuer le risque de maladie cardiaque et/ou améliorer le résultat pour un patient après un infarctus du myocarde ou une lésion cardiaque Pending WO2025085784A1 (fr)

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