Systems and methods for multi-OMIC analysis

Abstract

Disclosed herein are systems and methods for analyzing multi-omic, multianalyte, or multimodal datasets. Systems and methods can include methods for preparing a multi-omic, multianalyte, or multimodal sequencing library. Methods for preparing a multi-omic, multianalyte, or multimodal sequencing library can include ligating an adapter to a methylated DNA fragment. The adapter may comprise a methylated protein-specific oligonucleotide. Systems for analyzing multi-omic, multianalyte, or multimodal datasets can include a multi-omic, multianalyte, or multimodal model.

Description

WSGR Docket No. 68738-702.601SYSTEMS AND METHODS FOR MULTI-OMIC ANALYSISCROSS-REFEREN CE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 754,894, filed February76, 2025, which is incorporated by reference herein in its entirety7.BACKGROUND

[0002] Biological samples such as bodily fluids or tissue samples may be used to diagnose a disease. Biological samples are collected from a subject for diagnostic testing. Biological samples can include whole blood samples or blood derivatives such as plasma used for detection of one or more diseases, for example cancer. Generally, cancers are detected using diagnostics configured to detect at least one biomarker. Biomarker detection and quantification may be used to identify certain information about the disease such as type or subty pe of disease, disease progression, disease risk, and other disease information relating to the subject from whom a biological sample is taken. Biomarker detection can include detecting information about DNA, RNA, proteins, or other biological materials. This information is stored in one or more libraries.SUMMARY

[0003] The present disclosure recognizes that testing and treatment analysis procedures often require a painful and difficult tissue biopsy, which may not be feasible for patients of advanced age or other impairments that render a tissue biopsy risky or not feasible. The present disclosure recognizes that whole blood or blood derivative biological materials from a subject, such as serum, is used to gather information about diseases such as cancer.

[0004] Tools used in whole blood or blood derivative sampling, screening, and diagnostic testing, as well as treatment prediction, may face challenges, for example limitations involving the accuracy and cohesiveness of information gathered from various different sets of biomarkers in these samples and the ease of identifying a subset of biomarkers, such as a small subset of biomarkers, in samples which naturally contain a large amount of various biomarker types and subtypes.

[0005] Further, the generation of libraries for diagnostic sequencing may face challenges, for example the inaccuracies, inefficiencies, and lack of cohesion resulting from the tagging and expansion methods of individually modeled various biomolecule types. Further, multianalyte models, multimodal models, and multi-omic models may be used to increase cohesion, accuracy, and efficiency of library generation. Further, the methods of tagging described hereinWSGR Docket No. 68738-702.601may increase the accuracy, efficiency, and cohesion of libraries, including for difficult-to-detect biomarkers.

[0006] Further, detecting and identifying small amounts of disease such as cancer in a subject for measurable residual disease (MRD) monitoring may face challenges. The present disclosure provides multianalyte models, multimodal models, and multi-omic models, as well as alternative methods and systems for tagging and library generation, that improve the accuracy and efficiency of MRD monitoring and associated library generation for various diseases.

[0007] Disclosed herein in some embodiments are methods, systems, and tools for analyzing biomarkers, generating libraries, and sequencing analysis for use in diagnosing, monitoring, predicting risk, and other diagnostic procedures for various diseases including cancer.

[0008] In an aspect, disclosed herein is a method of preparing a sequencing library comprising ligating an adapter to a methylated DNA fragment, wherein the adapter comprises a methylated protein-specific oligonucleotide.

[0009] In another aspect, disclosed herein is a method of preparing a sequencing library comprising: (a) annealing together an adapter fragment and a methylated ssDNA fragment; and (b) ligating a methylated protein-specific oligonucleotide tag to a site of the adapter fragment, thereby producing a molecular complex.

[0010] In some embodiments, the method further comprises further amplifying the molecular complex. In some embodiments, the method further comprises sequencing the molecular complex to produce sequencing reads. In some embodiments, the method further comprises determining a methylation pattern from the sequencing reads, wherein the methylation pattern comprises methylation information of both the methylated ssDNA and the methylated protein-oligonucleotide tag. In some embodiments, determining the methylation pattern comprises identifying one or more differentially expressed markers or variants. In some embodiments, the one or more differentially expressed markers or variants comprise markers or variants of the ssDNA. In some embodiments, the one or more differentially expressed markers or variants comprises one or more gene mutations of the ssDNA.

[0011] In some embodiments, the method further comprises detecting one or more of the methylated DNA molecules based at least in part on the methylation pattern of the methylated protein-oligonucleotide tag. In some embodiments, the method further comprises detecting one or more of the methylated DNA molecules based at least in part on a combination of the methylation pattern of the methylated protein-oligonucleotide tag and a methylation pattern of the methylated ssDNA. In some embodiments, the method further comprises selectively performing sequencing of the identified methylated DNA molecules. In some embodiments, the method further comprises selectively excluding the identified methylated DNA molecules fromWSGR Docket No. 68738-702.601sequencing. In some embodiments, the method further comprises quantifying the detected one or more methylated DNA molecules.

[0012] In some embodiments, the methylation pattern further comprises a quantitative measure of methylation. In some embodiments, the method further comprises performing error correction of the sequence reads based at least in part on the methylation pattern. In some embodiments, the sequencing comprises whole exome sequencing. In some embodiments, the sequencing comprises whole genome sequencing (WGS) or next generation sequencing (NGS). In some embodiments, the one or more differentially expressed markers or variants comprise single nucleotide variations. In some embodiments, the one or more differentially expressed markers or variants comprise copy number variants. In some embodiments, the one or more differentially expressed markers or variants comprise insertions or deletions (indels).

[0013] In some embodiments, the method further comprises detecting one or more differentially expressed markers or variants in ribonucleic acid (RNA) of the subject. In some embodiments, the one or more differentially expressed markers or variants in the RNA of the subject comprise single nucleotide variants, copy number variants, indels, or expression biomarkers.

[0014] In some embodiments, the method further comprises detecting the one or more differentially expressed markers or variants in a subject comprising one or more epigenetic data characteristics. In some embodiments, the one or more epigenetic data characteristics comprise a comparative methylation indication. In some embodiments, the comparative methylation indication comprises hyper-methylation or hypo-methylation.

[0015] In some embodiments, the method further comprises determining the hyper-methylation or hypo-methylation based at least in part on a dynamic or pre-set normalized methylation value. In some embodiments, the method further comprises detecting the one or more differentially expressed markers or variants in the subject comprising protein abundance characteristics. In some embodiments, the protein abundance characteristics comprise increased protein abundances or decreased protein abundances. In some embodiments, the sequencing reads comprise a multi-omic dataset. In some embodiments, the multi-omic dataset comprises RNA sequencing data, DNA sequencing data, methylation sequencing data, or protein sequencing data, or any combination thereof.

[0016] In some embodiments, the method further comprises generating a multi-omic model, multianalyte model, or multimodal model. In some embodiments, the multi-omic model, multianalyte model, or multimodal model comprises two or more sequencing libraries. In some embodiments, the two or more sequencing libraries comprise a DNA sequencing library, an RNA sequencing library, a methylation sequencing library, or a protein sequencing library, or any combination thereof. In some embodiments, each sequencing library' comprises one or moreWSGR Docket No. 68738-702.601genomic features. In some embodiments, the one or more genomic features comprise the multi-omic dataset associated or grouped with cell type data. In some embodiments, the method further comprises receiving a biological sample. In some embodiments, the biological sample comprises a cell-free sample or a tissue sample.

[0017] In some embodiments, the method further comprises predicting a synthetic tissue fraction using the one or more sequencing libraries. In some embodiments, the method further comprises predicting a synthetic tissue fraction using a methylation library of the methylated DNA molecules, and the comparative methylation indication. In some embodiments, the method further comprises ligating an ssDNA fragment to an oligonucleotide tag, wherein the oligonucleotide tag is isolated from an antibody -oligonucleotide conjugate. In some embodiments, the oligonucleotide tag is methylated.

[0018] In some embodiments, the method further comprises contacting a plurality of antibody -oligonucleotide conjugates with a plurality of proteins. In some embodiments, the method further comprises isolating at least a subset of the plurality of antibody-oligonucleotide conjugates. In some embodiments, the isolated at least the subset of the plurality of antibody-oligonucleotide conjugates are bound to at least a subset of the plurality of proteins. In some embodiments, the oligonucleotides of the antibody-oligonucleotide conjugates are methylated.

[0019] In some embodiments, the method further comprises releasing the methylated oligonucleotides from the antibody-oligonucleotide conjugates. In some embodiments, the method further comprises replicating the plurality of released methylated oligonucleotides. In some embodiments, the method further comprises combining the plurality of released methylated oligonucleotides with similar unmethylated oligonucleotides. In some embodiments, each of the plurality of released methylated oligonucleotides comprise one or more methylation sites. In some embodiments, the released methylated oligonucleotides comprise the methylated oligonucleotide tags.

[0020] In some embodiments, the method further comprises sequencing both the ssDNA fragment and the methylated oligonucleotide tag in the same sequencing run. In some embodiments, the one or more methylation sites comprise a methylation pattern of the methylated oligonucleotide tag.

[0021] In some embodiments, the method further comprises using the methylation pattern to determine one or more characteristics of the methylated oligonucleotide tag, or the ssDNA fragment, or both. In some embodiments, the one or more characteristics comprise one or more pieces of multi -omic data. In some embodiments, the multi-omic data comprises one or more of DNA single nucleotide variations, DNA copy number variations, DNA indels, RNA single nucleotide variations, RNA copy number variations, RNA indels, DNA structural variations,WSGR Docket No. 68738-702.601RNA structural variations, hyper-methylation, hypo-methylation, increased protein abundance, or decreased protein abundance, or any combination thereof. In some embodiments, the ssDNA fragment is methylated.

[0022] In some embodiments, the method further comprises generating a multi-omic library. In some embodiments, the multi-omic library comprises two or more sequencing libraries. In some embodiments, the two or more sequencing libraries comprise the protein library, a DNA sequencing library, an RNA sequencing library, or a methylation sequencing library, or any combination thereof. In some embodiments, each sequencing library comprises one or more genomic features. In some embodiments, the one or more genomic features comprise the multi-omic dataset associated or grouped with cell type data.

[0023] In some embodiments, the multi-omic library comprises a singular multianalyte sequencing library. In some embodiments, the singular multianalyte sequence library comprises two or more of: DNA genomic features, RNA genomic features, methylation genomic features, protein genomic features, or any combination thereof. In some embodiments, each of the genomic features comprise the multi-omic data associated or grouped with cell type data.

[0024] In another aspect, disclosed herein is a method of preparing a multi-omic library, comprising: (a) annealing together an adapter fragment and a methylated ssDNA fragment, thereby producing a first molecular complex; (b) annealing together an adapter fragment to a molecule comprising an RNA fragment, thereby producing a second molecular complex; and (c) ligating a methylated oligonucleotide tag to a site of the adapter fragment of (a), thereby producing a third molecular complex.

[0025] In some embodiments, the method further comprises amplifying the first molecular complex. In some embodiments, the method further comprises amplifying the second molecular complex. In some embodiments, the third molecular complex comprises a subset of amplified molecules of the first molecular complex. In some embodiments, the method further comprises amplify ing the third molecular complex.

[0026] In some embodiments, the method further comprises generating multi-omic data for the third molecular complex. In some embodiments, the method further comprises associating or grouping the multi-omic data with one or more cell types to generate one or more feature matrices. In some embodiments, the method further comprises grouping the one or more feature matrices into one or more genomic feature groups. In some embodiments, the method further comprises providing as input the one or more genomic feature to a multi-omic model, multianalyte model, or multimodal model. In some embodiments, the method further comprises performing multilayer perception on data relating to the one or more genomic feature groups toWSGR Docket No. 68738-702.601generate synthetic tissue fraction values associated with each of the one or more genomic features.

[0027] Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

[0028] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0029] In some embodiments, the method further comprises generating the multi-omic data set from the biological sample simultaneously.

[0030] In some embodiments, the method further comprises performing a multi-omic assay of the biological sample to simultaneously generate the multi-omic data set.

[0031] In some embodiments, the multi-omic assay comprises a genomic assay, a transcriptomic assay, an epigenetic assay, and a proteomic assay.

[0032] In some embodiments, the multi-omic assay simultaneously generates the multi-omic data set comprising multi-omic assay results relating to the RNA sequencing data, the methylation sequencing data, and the protein sequencing data.

[0033] In some embodiments, performing the multi-omic assay of the biological sample comprises performing the genomic assay, the transcriptomic assay, the epigenetic assay, and the proteomic assay in the same sequencing run.

[0034] In some embodiments, the method further comprises preparing the multi-omic library using assay results simultaneously generated by a multi-omic assay of a biological sample.

[0035] In yet another aspect, described are methods for assaying a biological sample of a subject, comprising: (a) performing a multi-omic assay of the biological sample to simultaneously capture genomic, transcriptomic, epigenetic, and proteomic assay results from the biological sample; and (b) generating multi-omic data comprising genomic data, transcriptomic data, epigenetic data, and proteomic data from the simultaneously captured multi-omic assay results.

[0036] In some embodiments, the method further comprises generating a multi-omic library using the multi-omic data.

[0037] In some embodiments, the multi-omic library comprises a plurality of -omic layers.

[0038] In some embodiments, an -omic layer of the plurality of -omic layers corresponds to the genomic data, the transcriptomic data, the epigenetic data, or the proteomic data.WSGR Docket No. 68738-702.601

[0039] In some embodiments, the method further comprises comparing the multi-omic library to one or more reference -omic data sets.

[0040] In some embodiments, the one or more reference -omic data sets comprise a reference genomic data set, a reference transcriptomic data set, a reference epigenetic data set, or a reference proteomic data set, or any combination thereof.

[0041] In some embodiments, the method further comprises determining a variation of the multi-omic data.

[0042] In some embodiments, the variation comprises a genomic sequence variation, a transcriptomic variation, an epigenetic variation, or a proteomic variation, or any combination thereof.

[0043] In some embodiments, the sequence variation comprises a single nucleotide polymorphism (SNP) or a copy number variation (CNV).

[0044] In some embodiments, the epigenetic variation comprises a differential promoter methylation, a differential genome region methylation, or a differential CpG methylation.

[0045] In some embodiments, the proteomic variation comprises a variation in fractional protein content, protein quantity, or protein function, or any combination thereof.

[0046] In some embodiments, the genomic data comprises genomic sequencing data of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0047] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure.Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0048] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGSWSGR Docket No. 68738-702.601

[0049] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0050] FIG. 1A illustrates an example of a portion of exemplary library preparation method operations for multianalyte analysis.

[0051] FIG. IB illustrates an example of a portion of exemplary library preparation method operations for multianalyte analysis.

[0052] FIG. 1C illustrates an example of single-stranded library preparation sequences comprising, for example, DNA or RNA.

[0053] FIG. 2 depicts an example of protein library preparation method operations.

[0054] FIG. 3 shows an example of single-stranded library preparation sequences which can comprise, for example, integrated protein-derived fragments.

[0055] FIG. 4 illustrates an example of a multi-omic analysis method using, for example, analysis software.

[0056] FIG. 5 depicts an example of a machine learning model that is, for example, capable of predicting synthetic tissue fraction values based on multi-omic data.

[0057] FIG. 6 shows an example of a machine learning model configured to. for example, predict synthetic tissue fraction values based on methylation data.

[0058] FIG. 7 illustrates example depictions of training data for a machine learning model involving, for example, variant capture.

[0059] FIG. 8 illustrates an example of a computing device; in this case, a device with one or more processors, memory, storage, and a network interface.

[0060] FIG. 9A illustrates an example of concordance between variant allele frequencies (VAF) of point mutations identified in whole genome sequencing (WGS) alone compared to multi-omic processing.

[0061] FIG. 9B illustrates an example of concordance between copy number variation (CNV) profile of WGS alone compared to multi-omic processing.

[0062] FIG. 9C illustrates an example of CNV profiles for multiple cell lines for WGS alone compared to multi-omic processing.

[0063] FIG. 10A illustrates an example of concordance between gene counts of RNASeq alone compared to multi-omic processing.

[0064] FIG. 10B illustrates an example of multi-omic processing various biomarkers.

[0065] FIG. 10C illustrates an example of gene expression data across multiple cell lines.WSGR Docket No. 68738-702.601

[0066] FIG. 10D illustrates an example of a genomic distribution of transcripts for RNASeq alone compared to multi-omic processing.

[0067] FIG. 11A illustrates an example of concordance between methylation of deep sequencing alone and multi-omic processing.

[0068] FIG. 11B illustrates an example of promoter methylation biomarkers in multiple cell types.

[0069] FIG. 11C illustrates an example of methylation percentage in B cells compared to other tissues.

[0070] FIG. 12A illustrates an example of multi-omic gene expression compared to CNV.

[0071] FIG. 12B illustrates an example of RNA gene expression alone compared to CNV.

[0072] FIG. 13A illustrates an example of VAF correlation between multi-omic processing and DNA sequencing with RNA sequencing (i.e. single-omic sequencing) alone.

[0073] FIG. 13B illustrates an example ratio of DNA VAF to RNA VAF in multi-omic processing compared to single-omic sequencing.

[0074] FIG. 14 illustrates an example of strand bias measures across multiple technical replicates.

[0075] FIG. 15A illustrates an example of a mutational profde of a frequency of variants sequenced via deep sequencing compared to multi-omic processing.

[0076] FIG. 15B illustrates an example of a mutational profde of multi-omic variants in deep sequencing compared to single-omic sequencing.

[0077] FIG. 16A illustrates an example of multi-omic processing for gene expression data compared to gene body methylation data.

[0078] FIG. 16B illustrates an example of independently sequenced gene expression data compared to gene body methylation data.

[0079] FIG. 16C illustrates an example of simultaneous capture of DNA sequence information and methylation information.

[0080] FIG. 17A illustrates an example of protein concentration by sample for various antibodies.

[0081] FIG. 17B illustrates an example of cell lysate sequencing fraction for various antibodies in a five-antibody panel.

[0082] FIG. 17C illustrates an example of replicates of multiple antibodies for multiple different cell lysates.

[0083] FIG. 18A illustrates an example of raw Next Generation Sequencing (NGS) counts for antibody-oligonucleotides for various housekeeping proteins in cancer and non-cancer cell lines.WSGR Docket No. 68738-702.601

[0084] FIG. 18B illustrates an example of fractional protein content for various antibodies in a multi-omic sample and a pure protein sample.

[0085] FIG. 18C illustrates an example of protein measurement over various orders of magnitude.

[0086] FIG. 19A illustrates an example of protein concentration and read count for multi-omic processing.

[0087] FIG. 19B illustrates an example of protein concentration and percentage for multi-omic processing.

[0088] FIG. 19C illustrates an example of protein concentration for detected proteins and percentage for multi-omic processing.

[0089] FIG. 20A illustrates an example of a flow diagram for deconvolution.

[0090] FIG. 20B illustrates an example of multi-omic sequencing reads generated simultaneously from the same biological sample.

[0091] FIG. 20C illustrates an example of base quality of multi-omic sequencing reads generated simultaneously from the same biological sample.

[0092] FIG. 20D illustrates an example of a comparison between TNA libraries from multi-omic data and RNA or DNA only libraries.

[0093] FIG. 21A illustrates an example of CpH retention rate for various positions in a sequencing read of various DNA and RNA samples.

[0094] FIG. 2 IB illustrates an example of CpH average methylation status in deconvoluted RNA reads compared to deconvoluted DNA reads.

[0095] FIG. 22A illustrates an example method workflow for Methylated Total Nucleic Acid (mTNA) sequencing of a tissue sample.

[0096] FIG. 22B illustrates an example of end-state sequencing molecules of mTNA sequencing of a tissue sample.

[0097] FIG. 23A illustrates an example of method workflow for mTNA sequencing of a plasma sample.

[0098] FIG. 23B illustrates an example of end-state sequencing molecules of mTNA sequencing of a plasma sample.

[0099] FIG. 24A illustrates an example of a portion of method workflow for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing (hairy TNA-Seq) of a tissue sample.

[0100] FIG. 24B illustrates an example of a continuing portion of a method workflow for Total hairpin-annealed simultaneously' extracted DNA and RNA sequencing of a tissue sample.WSGR Docket No. 68738-702.601

[0101] FIG. 24C illustrates an example of a continuing portion of a method workflow and endstate sequencing molecules for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing of a tissue sample.

[0102] FIG. 25A illustrates an example of a portion of method workflow for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing of a plasma sample.

[0103] FIG. 25B illustrates an example of a continuing portion of a method workflow for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing of a plasma sample.

[0104] FIG. 25C illustrates an example of a continuing portion of a method workflow and endstate sequencing molecules for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing of a plasma sample.

[0105] FIG. 26A illustrates an example of a portion of a method workflow for Methylated Total Nucleic Acid (mTNA) sequencing and linear protein detection assaying of a tissue sample.

[0106] FIG. 26B illustrates an example of a continuing portion of a method workflow and endstate sequencing molecules for mTNA sequencing and linear protein detection assaying of a tissue sample.

[0107] FIG. 27A illustrates an example of a portion of a method workflow for mTNA sequencing and linear protein detection assaying of a plasma sample.

[0108] FIG. 27B illustrates an example of a continuing portion of a method w orkflow and endstate sequencing molecules for mTNA sequencing and linear protein detection assaying of a plasma sample.

[0109] FIG. 28A illustrates an example of a portion of method workflow for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing and linear protein detection assaying of a tissue sample.

[0110] FIG. 28B illustrates an example of a continuing portion of a method workflow for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing and linear protein detection assaying of a tissue sample.

[0111] FIG. 28C illustrates an example of a continuing portion of a method workflow and endstate sequencing molecules for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing and linear protein detection assaying of a tissue sample.

[0112] FIG. 29A illustrates an example of a portion of method w orkflow for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing and linear protein detection assaying of a plasma sample.

[0113] FIG. 29B illustrates an example of a continuing portion of a method workflow for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing and linear protein detection assaying of a tissue sample.WSGR Docket No. 68738-702.601

[0114] FIG. 29C illustrates an example of a continuing portion of a method workflow and endstate sequencing molecules for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing and linear protein detection assaying of a plasma sample.

[0115] FIG. 30A illustrates an example of a portion of method workflow for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing and hairpin protein detection assaying of a tissue sample.

[0116] FIG. 30B illustrates an example of a continuing portion of a method workflow for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing and hairpin protein detection assaying of a tissue sample.

[0117] FIG. 30C illustrates an example of a continuing portion of a method workflow for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing and hairpin protein detection assaying of a tissue sample.

[0118] FIG. 30D illustrates an example of end-state sequencing molecules for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing and hairpin protein detection assaying of a tissue sample.

[0119] FIG. 31A illustrates an example of a portion of method workflow for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing and hairpin protein detection assaying of a plasma sample.

[0120] FIG. 31B illustrates an example of a continuing portion of a method workflow for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing and hairpin protein detection assaying of a plasma sample.

[0121] FIG. 31C illustrates an example of a continuing portion of a method workflow7for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing and hairpin protein detection assaying of a plasma sample.

[0122] FIG. 31D illustrates an example of end-state sequencing molecules for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing and hairpin protein detection assaying of a plasma sample.

[0123] FIG. 32 illustrates an example of combined multi-analyte pooled DNA and RNA preparation for a multi-omic assay.DETAILED DESCRIPTION

[0124] While preferable embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now7occur to those skilled in the art without departing from the invention. It should be understood that various alternativesWSGR Docket No. 68738-702.601to the embodiments of the invention described herein may be employed in practicing the invention.

[0125] As used herein, the singular forms '‘a,” “an,” and “the” include plural references unless the context clearly dictates otherw ise. Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated.

[0126] As used herein, the phrases “at least one,” “one or more,” and “and / or” are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C,” “at least one of A, B, or C,” “one or more of A, B, and C”, “one or more of A, B, or C” and “A, B, and / or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together. As used herein, the phrase “at most three” can mean less than one, one. two, or three.

[0127] Reference throughout this specification to “some embodiments,” “further embodiments,” or “a particular embodiment,” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrase “in some embodiments.” or “in further embodiments,” or “in a particular embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0128] The terms "subject." "individual," and "patient" may be used interchangeably and refer to humans, as well as non-human mammals (e.g., non-human primates, canines, equines, felines, porcines, bovines, ungulates, lagomorphs, rodents, and the like). In various embodiments, the subject is a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, as an outpatient, or other clinical context. In certain embodiments, the subject may not be under the care or prescription of a physician or other health worker. In some embodiments, the subject may be under the care of a dental professional.

[0129] As used herein, “treatment” or “treating” refers to an approach for obtaining beneficial or desired results with respect to a disease, disorder, or medical condition including, but not limited to, a therapeutic benefit and / or a prophylactic benefit. In certain embodiments, treatment or treating involves administering a therapeutic to a subject. A therapeutic benefit may include the eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit may be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder, such as observing an improvement in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.WSGR Docket No. 68738-702.601

[0130] As used herein, “multimodal” describes or characterizes datasets, models, libraries, or other collections of features comprising or including biological data or information, such as -omics data, for example, and clinical data or information, and information related to or generated by additional modalities, such as imaging modalities.

[0131] As used herein, “multianalyte” describes or characterizes datasets, models, libraries, or other collections of features comprising or including -omics data or information across a plurality of analytes. This -omics data or information can include, for example, genomic data or information, or epigenomic data or information, or both. The -omics data can include, for example, data relating to biological molecules, for example DNA, RNA, or protein, or any combination thereof. The multianalyte data may be generated simultaneously from the same biological sample.

[0132] As used herein, “multi-omic” describes or characterizes datasets, models, libraries, or other collections of features comprising or including multiple types of -omics data or information across a single analyte or across multiple analytes. Multiple types of -omics data can comprise, for example, genomic data or information, or epigenomic data or information pertaining to, for example, DNA, RNA, or proteins. Analytes can comprise, for example, biological molecules such as DNA, RNA, or protein, or any combination thereof. The multi-omic data or multi-omic library may be generated simultaneously from the same biological sample.

[0133] In an aspect, disclosed herein is a method of preparing a sequencing library. In some embodiments, the sequence library may comprise a DNA sequence library. In some embodiments, the sequence library may comprise an RNA sequence library. In some embodiments, the sequence library may comprise a methylation library, for example a DNA methylation, RNA methylation, or protein methylation library’. In some embodiments, the sequence library may comprise a protein library. In some embodiments, the sequencing may comprise next generation sequencing (NGS). In some embodiments, the sequencing may comprise whole genome sequencing (WGS). In some embodiments, each sequencing library may comprise one or more genomic features. In some embodiments, each sequencing library may comprise DNA sequencing features, RNA sequencing features, methylation features, protein features, or any combination thereof. In some embodiments, the one or more genomic features may comprise the multi-omic dataset associated or grouped with cell type data. The multi-omic data and cell type data may comprise one or more matrices. In some cases, cell type data may comprise values associated with each cell type. The values associated with each cell type may comprise one or more cellular deconvolution matrices. In some cases, each cellular deconvolution matrix comprises gene expression data associated with the cell type. In someWSGR Docket No. 68738-702.601embodiments, each cellular deconvolution matrix can be based at least in part on one or more gene expression profile matrices. The WGS or NGS may be performed on DNA or RNA. The WGS or NGS may comprise generating genetic sequencing reads. The genetic sequencing reads may be used to determine one or more genomic sequence variations. The one or more genomic sequence variations may comprise single nucleotide polymorphisms (SNPs), copy number variations (CNVs). as well as other genomic features.

[0134] In some embodiments, the method of preparing a sequencing library may comprise ligating an adapter to a methylated DNA fragment. In some embodiments, the adapter may comprise a full-length adapter. In some embodiments, the adapter may comprise a truncated adapter. In some embodiments, the adapter may comprise an oligonucleotide. In some embodiments, the adapter may comprise a protein-specific oligonucleotide. In some embodiments, the adapter may comprise a methylated protein-specific oligonucleotide. In some embodiments, the adapter may comprise an antisense oligonucleotide. In some embodiments, the adapter may comprise an RNA interference (RNAi) oligonucleotide. In some embodiments, the adapter may comprise splice-switching oligonucleotides (SSOs). In some embodiments, the adapter may comprise an aptamer RNA. In some embodiments, the protein-specific oligonucleotide may comprise an enzy me-derived oligonucleotide. In some embodiments, the protein-specific oligonucleotide may comprise an antibody-derived oligonucleotide. In some embodiments, the protein-specific oligonucleotide may comprise a hormone-derived oligonucleotide. In some embodiments, the protein-specific oligonucleotide may comprise a structural protein-derived oligonucleotide. In some embodiments, the protein-specific oligonucleotide may comprise a transport protein-derived oligonucleotide. In some embodiments, the protein-specific oligonucleotide may comprise a storage protein-derived oligonucleotide. In some embodiments, the protein-specific oligonucleotide may comprise a contractile protein-derived oligonucleotide.

[0135] In some embodiments, the multi-omic library may comprise a singular multianalyte sequencing library. In some embodiments, the singular multianalyte sequence library may comprise two or more of: DNA genomic features. RNA genomic features, methylation genomic features, protein genomic features, or any combination thereof. The singular multianalyte sequence library may comprise DNA genomic features, RNA genomic features, methylation features, and protein features. In some embodiments, each of the genomic features may comprise the multi-omic data associated or grouped with cell type data. The genomic features may comprise DNA sequencing features, RNA sequencing features, methylation features, protein features, or any combination thereof. In some embodiments, the one or more genomic features may comprise the multi-omic dataset associated or grouped with cell t pe data. TheWSGR Docket No. 68738-702.601multi-omic data and cell type data may comprise one or more matrices. In some cases, cell ri pe data may comprise values associated with each cell type. The values associated with each cell ri pe may comprise one or more cellular deconvolution matrices. In some cases, each cellular deconvolution matrix comprises gene expression data associated with the cell type. In some embodiments, each cellular deconvolution matrix can be based at least in part on one or more gene expression profile matrices. The multi-omic data may be generated simultaneously from the same biological sample.

[0136] In another aspect, disclosed herein is a method of preparing a sequencing library. In some embodiments, the method may comprise annealing together an adapter fragment and a methylated ssDNA fragment. In some embodiments, the method may comprise annealing together an adapter fragment and a DNA fragment. In some embodiments, the DNA fragment may comprise a single stranded (ssDNA) fragment. In some embodiments, the DNA fragment may comprise a double-stranded (dsDNA) fragment. In some embodiments, the DNA fragment may comprise a complementary DNA (cDNA) fragment. In some embodiments, the DNA fragment may comprise a cell-free DNA (cfDNA) fragment. In some embodiments, the method may comprise annealing together an RNA fragment and an adapter fragment. In some embodiments, the RNA fragment may comprise a microRNA (miRNA) fragment. In some embodiments, the RNA fragment may comprise a small interfering RNA (siRNA) fragment. In some embodiments, the RNA fragment may comprise a messenger RNA (mRNA) fragment. In some embodiments, the RNA fragment may comprise a transfer RNA (tRNA) fragment. In some embodiments, the RNA fragment may comprise a ribosomal RNA (rRNA) fragment. In some embodiments, the RNA fragment may comprise a small nuclear RNA (snRNA) fragment. In some embodiments, the RNA fragment may comprise a small nucleolar RNA (snoRNA) fragment. In some embodiments, the RNA fragment may comprise a long noncoding RNA (IncRNA) fragment. In some embodiments, the RNA fragment may comprise a pi wi -interacting RNA (piRNA) fragment. In some embodiments, the RNA fragment may comprise an orphan non-coding RNA (oncRNA) fragment. In some embodiments, the adapter may comprise a full-length adapter. In some embodiments, the adapter may comprise a truncated adapter. In some embodiments, the adapter may comprise an oligonucleotide. In some embodiments, the adapter may comprise a protein-specific oligonucleotide. In some embodiments, the adapter may comprise a methylated protein-specific oligonucleotide. In some embodiments, the adapter may comprise an antisense oligonucleotide. In some embodiments, the adapter may comprise an RNA interference (RNAi) oligonucleotide. In some embodiments, the adapter may comprise splice-switching oligonucleotides (SSOs). In some embodiments, the adapter may comprise an aptamer RNA. In some embodiments, the method may comprise ligating a methylated protein-WSGR Docket No. 68738-702.601oligonucleotide tag to a site of the adapter fragment. In some embodiments, the protein-specific oligonucleotide may comprise an enzyme-derived oligonucleotide. In some embodiments, the protein-specific oligonucleotide may comprise an antibody-derived oligonucleotide. In some embodiments, the protein-specific oligonucleotide may comprise a hormone-derived oligonucleotide. In some embodiments, the protein-specific oligonucleotide may comprise a structural protein-derived oligonucleotide. In some embodiments, the protein-specific oligonucleotide may compnse a transport protein-derived oligonucleotide. In some embodiments, the protein-specific oligonucleotide may comprise a storage protein-derived oligonucleotide. In some embodiments, the protein-specific oligonucleotide may comprise a contractile protein-derived oligonucleotide. In some embodiments, the annealed adapter fragment and methylated ssDNA fragment may comprise a molecular complex. In some embodiments, the annealed adapter fragment and methylated DNA fragment may comprise a molecular complex. In some embodiments, the annealed adapter fragment and the DNA fragment may comprise a molecular complex. In some embodiments, the annealed adapter fragment and the dsDNA fragment may comprise a molecular complex. In some embodiments, the annealed adapter fragment and the methylated cfDNA fragment may comprise a molecular complex. In some embodiments, the annealed adapter fragment and the methylated cDNA fragment may comprise a molecular complex. In some embodiments, the annealed adapter fragment and the RNA fragment may comprise a molecular complex. In some embodiments, the annealed adapter fragment and the siRNA fragment may comprise a molecular complex. In some embodiments, the annealed adapter fragment and the miRNA fragment may comprise a molecular complex. In some embodiments, the annealed adapter fragment and the mRNA fragment may comprise a molecular complex. In some embodiments, the annealed adapter fragment and the tRNA fragment may comprise a molecular complex. In some embodiments, the annealed adapter fragment and the rRNA fragment may comprise a molecular complex. In some embodiments, the annealed adapter fragment and the snoRNA fragment may comprise a molecular complex. In some embodiments, the annealed adapter fragment and the snRNA fragment may comprise a molecular complex. In some embodiments, the annealed adapter fragment and the IncRNA fragment may comprise a molecular complex. In some embodiments, the annealed adapter fragment and the piRNA fragment may comprise a molecular complex.

[0137] In some embodiments, the method can further comprise further amplifying the molecular complex. In some embodiments, the molecular complex is amplified to yield between 1000 and 100 million copies of the molecular complex. In some embodiments, the amplification method may comprise one or more of polymerase chain reaction (PCR), Phi29 polymerase amplification, rolling circle amplification (RCA), isothermal multiple strand displacementWSGR Docket No. 68738-702.601amplification (MDA), or any combination thereof. In some embodiments, expansion can be performed using solid-phase reversible immobilization (SPRI) beads. In some embodiments, the method can further comprise sequencing the molecular complex to produce sequencing reads. In some embodiments, the sequencing reads may comprise single read sequencing reads. In some embodiments, the sequencing reads may comprise paired-end sequencing reads. In some embodiments, the sequencing may comprise short-read sequencing. In some embodiments, the sequencing may comprise long-read sequencing. In some embodiments, the method can further comprise determining a methylation pattern from the sequencing reads. In some embodiments, determining the methylation pattern may comprise using one or more of bisulfite sequencing, enzymatic methylation sequencing (EM-seq), high performance liquid chromatography -ultraviolet sequencing (HPLC-UV), liquid chromatography (LC). mass spectrometry (MS), ELISA-based sequencing, nanopore sequencing, luminometric methylation assay (LUMA), or any combination thereof. The sequencing assay may be simultaneously performed with a methylation assay, a proteomics assay, an epigenetic assay, or a transcriptomic assay, or any combination thereof, on the same biological sample. A multi-omic assay may comprise the simultaneous performance of the sequencing assay with a methylation assay, a proteomics assay, an epigenetic assay, or a transcriptomic assay, or any combination thereof.

[0138] In some embodiments, the methylation pattern may comprise methylation information relating to the methylated DNA fragment. In some embodiments, the methylation pattern may comprise methylation information relating to the methylated protein-oligonucleotide tag. In some embodiments, the DNA fragment may comprise a dsDNA, ssDNA, or cDNA fragment. In some embodiments, the methylation pattern may comprise methylation information of both the methylated ssDNA and the methylated protein-oligonucleotide tag. In some embodiments, the methylation pattern may comprise methylation information relating to a methylated RNA fragment. In some embodiments, the methylation pattern may comprise methylation information relating to the methylated protein-oligonucleotide tag. In some embodiments, the RNA fragment may comprise an siRNA, miRNA, snoRNA, snRNA, piRNA, tRNA, or rRNA fragment. In some embodiments, the methylation pattern may comprise methylation information of both the methylated RNA and the methylated protein-oligonucleotide tag. In some embodiments, determining the methylation pattern may comprise identifying one or more differentially expressed markers or variants. In some embodiments, the one or more differentially expressed markers or variants may comprise markers or variants of the ssDNA. In some embodiments, the one or more differentially expressed markers or variants may comprise markers or variants of the DNA fragment. In some embodiments, the one or more differentially expressed markers or variants may comprise markers or variants of the RNA fragment. In some embodiments, the oneWSGR Docket No. 68738-702.601or more differentially expressed markers or variants may comprise one or more gene mutations of the ssDNA. In some embodiments, the one or more differentially expressed markers or variants may comprise one or more gene mutations of the DNA fragment. In some embodiments, the one or more differentially expressed markers or variants may comprise one or more gene mutations of the RNA fragment. In some embodiments, the variants may comprise insertions or deletions (indels), frameshifts, substitutions, or other point mutations or fragment mutations. The methylation assay may be simultaneously performed with a genomic sequencing assay, a proteomics assay, another epigenetic assay, or a transcriptomic assay, or any combination thereof, on the same biological sample. A multi-omic assay may comprise the simultaneous performance of the methylation assay with a genome sequencing assay, a proteomics assay, an epigenetic assay, or a transcriptomic assay, or any combination thereof.

[0139] The one or more differentially expressed markers or variants may be used to determine a presence or absence of a disease. The one or more differentially expressed markers or variants may be used to determine a risk of a subject having a disease. The disease may comprise a cancer. The cancer may comprise one or more of breast cancer, lung cancer, prostate cancer, colorectal cancer, skin cancer, bladder cancer, kidney cancer, liver cancer, leukemia, lymphoma, multiple myeloma, pancreatic cancer, esophageal cancer, stomach cancer, cervical cancer, uterine cancer, ovarian cancer, testicular cancer, brain cancer, thyroid cancer, oral cancer, head and neck cancer, mesothelioma, neuroblastoma, sarcoma, gallbladder cancer, anal cancer, small intestine cancer, carcinoid tumors, soft tissue sarcoma, cutaneous squamous cell carcinoma, basal cell carcinoma, or adrenal cancer, or any combination thereof. The disease may comprise a genetic disease. The genetic disease may comprise one or more of: cystic fibrosis, sickle cell anemia, Huntington's disease, Duchenne muscular dystrophy, hemophilia, Tay-Sachs disease, phenylketonuria (PKU), Down syndrome. Marfan syndrome, Turner syndrome, Klinefelter syndrome, neurofibromatosis, thalassemia, fragile X syndrome, achondroplasia, congenital adrenal hyperplasia, Alport syndrome, Wilson's disease, familial hypercholesterolemia, Rett syndrome, spinal muscular atrophy, Ellis-van Creveld syndrome, osteogenesis imperfecta, myotonic dystrophy, Xeroderma pigmentosum, Bloom syndrome, ataxia-telangiectasia, or von Willebrand disease, or any combination thereof.

[0140] In some embodiments, the method can further comprise detecting one or more of the methylated DNA molecules based at least in part on the methylation pattern of the methylated protein-oligonucleotide tag. In some embodiments, determining the methylation pattern may comprise using one or more of bisulfite sequencing, high performance liquid chromatography -ultraviolet sequencing (HPTC-UV), liquid chromatography (TC), mass spectrometry (MS), ELISA-based sequencing, nanopore sequencing, luminometric methylation assay (LUMA), orWSGR Docket No. 68738-702.601any combination thereof to detect the methylation pattern of the protein-oligonucleotide tags. In some embodiments, the method can further comprise detecting one or more of the methylated DNA molecules based at least in part on a combination of the methylation pattern of the methylated protein-oligonucleotide tag and a methylation pattern of the methylated ssDNA. In some embodiments, the method can further comprise detecting one or more of the methylated DNA molecules based at least in part on a combination of the methylation pattern of the methylated protein-oligonucleotide tag and a methylation pattern of the DNA fragments. In some embodiments, the method can further comprise detecting one or more differentially expressed markers or variants in ribonucleic acid (RNA) of the subject. In some embodiments, the one or more differentially expressed markers or variants in the RNA of the subject may comprise single nucleotide variants, structural variants, copy number variants, indels, or expression biomarkers. In some embodiments, structural variants may comprise inversions, deletions, insertions such as duplications and retroelement insertions, and translocations. The one or more differentially expressed markers or variants may be used to determine a presence or absence of a disease. The one or more differentially expressed markers or variants may be used to determine a risk of a subject having a disease. The disease may comprise a cancer. The cancer may comprise one or more of: breast cancer, lung cancer, prostate cancer, colorectal cancer, skin cancer, bladder cancer, kidney cancer, liver cancer, leukemia, lymphoma, multiple my eloma, pancreatic cancer, esophageal cancer, stomach cancer, cervical cancer, uterine cancer, ovarian cancer, testicular cancer, brain cancer, thyroid cancer, oral cancer, head and neck cancer, mesothelioma, neuroblastoma, sarcoma, gallbladder cancer, anal cancer, small intestine cancer, carcinoid tumors, soft tissue sarcoma, cutaneous squamous cell carcinoma, basal cell carcinoma, or adrenal cancer, or any combination thereof. The disease may comprise a genetic disease. The genetic disease may comprise one or more of: cystic fibrosis, sickle cell anemia, Huntington's disease, Duchenne muscular dystrophy, hemophilia, Tay-Sachs disease, phenylketonuria (PKU), Dow n syndrome, Marfan syndrome, Turner syndrome, Klinefelter syndrome, neurofibromatosis, thalassemia, fragile X syndrome, achondroplasia, congenital adrenal hyperplasia, Alport syndrome, Wilson's disease, familial hypercholesterolemia, Rett syndrome, spinal muscular atrophy, Ellis-van Creveld syndrome, osteogenesis imperfecta, myotonic dystrophy. Xeroderma pigmentosum, Bloom syndrome, ataxia-telangiectasia, or von Willebrand disease, or any combination thereof.

[0141] In some embodiments, the method can further comprise detecting one or more of the methylated RNA molecules based at least in part on a combination of the methylation pattern of the methylated protein-oligonucleotide tag and a methylation pattern of the RNA fragments. In some embodiments, the method can further comprise selectively performing sequencing of theWSGR Docket No. 68738-702.601identified methylated DNA molecules. In some embodiments, the method can further comprise selectively performing sequencing of the identified methylated RNA molecules. In some embodiments, the method can further comprise selectively excluding the identified methylated DNA molecules from sequencing. In some embodiments, the method can further comprise selectively excluding the identified methylated RNA molecules from sequencing. In some embodiments, the method can further comprise quantifying the detected one or more methylated DNA molecules. In some embodiments, the method can further comprise quantifying the detected one or more methylated RNA molecules. In some embodiments, the method can further comprise quantifying the detected one or more DNA molecules annealed to the methylated protein-oligonucleotide tag. In some embodiments, the method can further comprise quantifying the detected one or more RNA molecules annealed to the methylated protein-oligonucleotide tag.

[0142] In some embodiments, the methylation pattern can further comprise a quantitative measure of methylation. In some embodiments, methylation patterns are measured using one or more of bisulfite sequencing, high performance liquid chromatography -ultraviolet sequencing (HPLC-UV), liquid chromatography (LC), mass spectrometry (MS), ELISA-based sequencing, nanopore sequencing, luminometric methylation assay (LUMA), or any combination thereof. In some embodiments, the method can further comprise performing error correction of the sequence reads based at least in part on the methylation pattern. In some embodiments, error correction can include modifying the sequence reads. In some embodiments, modifying the sequence reads may comprise performing structural variations, single nucleotide variations, copy number variations, or indels. In some embodiments, structural variations may comprise inversions, deletions, insertions such as duplications and retroelement insertions, and translocations. In some embodiments, the sequence reads are modified using software. In some embodiments, the software may comprise artificial intelligence (Al) or machine learning (ML) algorithms, models, or modules. In some embodiments, the sequencing may comprise whole exome sequencing. In some embodiments, the sequencing may comprise whole genome sequencing. In some embodiments, the one or more differentially expressed markers or variants may comprise single nucleotide variations. In some embodiments, the one or more differentially expressed markers or variants may comprise copy number variants. In some embodiments, the one or more differentially expressed markers or variants may comprise insertions or deletions (indels). The one or more differentially expressed markers or variants may be used to determine a presence or absence of a disease. The one or more differentially expressed markers or variants may be used to determine a risk of a subject having a disease. The disease may comprise a cancer. The cancer may comprise one or more of: breast cancer, lung cancer, prostate cancer,WSGR Docket No. 68738-702.601colorectal cancer, skin cancer, bladder cancer, kidney cancer, liver cancer, leukemia, lymphoma, multiple myeloma, pancreatic cancer, esophageal cancer, stomach cancer, cervical cancer, uterine cancer, ovarian cancer, testicular cancer, brain cancer, thyroid cancer, oral cancer, head and neck cancer, mesothelioma, neuroblastoma, sarcoma, gallbladder cancer, anal cancer, small intestine cancer, carcinoid tumors, soft tissue sarcoma, cutaneous squamous cell carcinoma, basal cell carcinoma, or adrenal cancer, or any combination thereof. The disease may comprise a genetic disease. The genetic disease may comprise one or more of: cystic fibrosis, sickle cell anemia. Huntington's disease, Duchenne muscular dystrophy, hemophilia, Tay-Sachs disease, phenylketonuria (PKU), Down syndrome, Marfan syndrome, Turner syndrome, Klinefelter syndrome, neurofibromatosis, thalassemia, fragile X syndrome, achondroplasia, congenital adrenal hyperplasia, Alport syndrome. Wilson's disease, familial hypercholesterolemia, Rett syndrome, spinal muscular atrophy, Ellis-van Creveld syndrome, osteogenesis imperfecta, myotonic dystrophy, Xeroderma pigmentosum, Bloom syndrome, ataxia-telangiectasia, or von Willebrand disease, or any combination thereof.

[0143] In some embodiments, multiple types of biomarkers and analytes can be combined for analysis by the AI / ML models. The combined biomarkers or analytes can be in silico combinations of biomarkers or analytes. The in silico combination of biomarkers or analytes can be generated, modified, or both, by the model. The combined biomarkers or analytes can be generated in vitro as a mixture of biomarkers or analytes. The in vitro mixture of biomarkers or analytes can be diluted. In some embodiments, the multi-omic model, multianalyte model, or multimodal model may comprise one or more AI / ML models. The multi-omic model, multianalyte model, or multimodal model can be configured to receive as input the in silico or in vitro combinations of biomarkers or analytes.

[0144] In some embodiments, the method can further comprise detecting the one or more differentially expressed markers or variants in a subject may comprise one or more epigenetic data characteristics. In some embodiments, the differentially expressed markers or variants may comprise one or more of DNA SNVs, DNA CNVs, DNA indels, DNA expression, RNA SNVs, RNA CNVs, RNA indels, RNA expression, increased protein abundances, decreased protein abundances, or any combination thereof. In some embodiments, the one or more epigenetic characteristics may comprise hypomethylation, hypermethylation, epigenetic SNVs, epigenetic CNVs, epigenetic indels, or any combination thereof. In some embodiments, the one or more epigenetic data characteristics may comprise a comparative methylation indication. In some embodiments, the comparative methylation indication may comprise hyper-methylation or hypomethylation.WSGR Docket No. 68738-702.601

[0145] In some embodiments, the method can further comprise determining the hypermethylation or hypo-methylation based at least in part on a dynamic or pre-set normalized methylation value. In some embodiments, the normalized methylation value is based at least in part on a known average methylation value. In some embodiments, the normalized methylation value is personalized to the subject or patient. In some embodiments, the normalized methylation value is dynamically generated for one or more diseases. In some embodiments, the normalized methylation value is selected from a series of profiles similar in some aspects to the patient’s profile. In some embodiments, the method can further comprise detecting the one or more differentially expressed markers or variants in the subject may comprise protein abundance characteristics. In some embodiments, the protein abundance characteristics may comprise increased protein abundances or decreased protein abundances. In some embodiments, the protein abundance characteristics may comprise maintained or unchanged protein abundance. In some embodiments, the protein characteristics may comprise protein ty pe characteristics. In some embodiments, the protein characteristics may comprise protein concentration characteristics, protein percentage characteristics, protein ratio characteristics, protein configuration characteristics, or other protein information. In some embodiments, the sequencing reads may comprise a multi-omic dataset. In some embodiments, the multi-omic dataset may comprise RNA sequencing data, methylation sequencing data, epigenetic data, or protein sequencing data, or any combination thereof. In some embodiments, the multi-omic dataset may comprise DNA information, RNA information, epigenetic information, protein information, or any combination thereof. The epigenetic assay may be simultaneously performed with a methylation assay, a proteomics assay, a genomic sequencing assay, or a transcriptomic assay, or any combination thereof, on the same biological sample. A multi-omic assay may comprise the simultaneous performance of the epigenetic assay with a methylation assay, a proteomics assay, a genomic sequencing assay, or a transcriptomic assay, or any combination thereof. The one or more differentially expressed markers or variants may be used to determine a presence or absence of a disease. The one or more differentially expressed markers or variants may be used to determine a risk of a subject having a disease. The disease may comprise a cancer. The cancer may comprise one or more of: breast cancer, lung cancer, prostate cancer, colorectal cancer, skin cancer, bladder cancer, kidney cancer, liver cancer, leukemia, lymphoma, multiple myeloma, pancreatic cancer, esophageal cancer, stomach cancer, cervical cancer, uterine cancer, ovarian cancer, testicular cancer, brain cancer, thyroid cancer, oral cancer, head and neck cancer, mesothelioma, neuroblastoma, sarcoma, gallbladder cancer, anal cancer, small intestine cancer, carcinoid tumors, soft tissue sarcoma, cutaneous squamous cell carcinoma, basal cell carcinoma, or adrenal cancer, or any combination thereof. The disease may comprise aWSGR Docket No. 68738-702.601genetic disease. The genetic disease may comprise one or more of: cystic fibrosis, sickle cell anemia, Huntington's disease, Duchenne muscular dystrophy, hemophilia, Tay-Sachs disease, phenylketonuria (PKU), Down syndrome, Marfan syndrome, Turner syndrome, Klinefelter syndrome, neurofibromatosis, thalassemia, fragile X syndrome, achondroplasia, congenital adrenal hyperplasia, Alport syndrome, Wilson's disease, familial hypercholesterolemia, Rett syndrome, spinal muscular atrophy, Ellis-van Creveld syndrome, osteogenesis imperfecta, myotonic dystrophy. Xeroderma pigmentosum. Bloom syndrome, ataxia-telangiectasia, or von Willebrand disease, or any combination thereof.

[0146] In some embodiments, the method can further comprise generating a multi-omic model, multianalyte model, or multimodal model. In some embodiments, the multi-omic model, multianalyte model, or multimodal model may comprise two or more sequencing libraries. In some embodiments, the multi-omic model, multianalyte model, or multimodal model may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty -two, twenty -three, twenty -four, twenty -five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one. thirty-two, thirty-three, thirty-four, thirty-five, thirty-six. thirty-seven, thirty-eight, thirty-nine, forty, forty-one, forty-two, forty-three, forty-four, forty-five, forty-six, forty-seven, forty-eight, forty-nine, fifty, fifty -one, fifty -two, fifty-three, fifty-four, fifty-five, fifty-six, fifty-seven, fiftyeight, fifty -nine, sixty, sixty -one, sixty-two, sixty-three, sixty-four, sixty-five, sixty-six, sixtyseven, sixty-eight, sixty-nine, seventy, seventy-one, seventy-two, seventy-three, seventy-four, seventy -five, seventy -six, seventy-seven, seventy-eight, seventy-nine, eighty, eighty-one, eighty-two, eighty-three, eighty-four, eighty-five, eighty-six, eighty-seven, eighty-eight, eighty-nine, ninety, ninety-one, ninety-two, ninety-three, ninety -four, ninety-five, ninety-six, ninety-seven, ninety-eight, ninety-nine, one hundred, or more than one hundred sequencing libraries. In some embodiments, the two or more sequencing libraries may comprise a DNA sequencing library, an RNA sequencing library, a methylation library, or a protein library, or any combination thereof. In some embodiments, the two or more libraries may comprise a DNA library, an RNA library, a methylation library, and a protein library. In some embodiments, the DNA library may comprise a cDNA 11 brary . a genomic DNA library, or both. In some embodiments, the two or more libraries may comprise lipid libraries. In some embodiments, the two or more libraries may comprise small molecule libraries.

[0147] In some embodiments, the method can further comprise receiving a biological sample. In some embodiments, the biological sample may comprise a tissue sample. In some embodiments, the biological sample may comprise a fluid sample. In some embodiments, the fluid sample may comprise a bodily fluid sample. In some embodiments, the bodily fluid sample may comprise aWSGR Docket No. 68738-702.601blood sample, a saliva sample, a urine sample, a stool sample, a sputum sample, a sweat sample, a vomitus sample, a tears sample, a mucosal sample, a secretion sample, a breast milk sample, a seminal fluid sample, or another type of bodily fluid, or any combination thereof. In some embodiments, the biological sample may comprise a whole blood sample. In some embodiments, the biological sample may comprise a sample derived from whole blood such as plasma, platelets, red blood cells, or white blood cells. In some embodiments, the biological sample may comprise a cell-free sample or a tissue sample. In some embodiments, the cell-free sample may comprise a saliva sample, a urine sample, a pleural effusion sample, a bronchial lavage sample, a bronchial aspirate sample, a breast milk sample, an amniotic fluid sample, a colostrum sample, a tear sample, a seminal fluid sample, a peritoneal fluid sample, a pleural effusion sample, a nipple aspirate fluid sample, a breath sample, or a stool sample, or any combination thereof. In some embodiments, the method can further comprise predicting a synthetic tissue fraction using the one or more sequencing libraries.

[0148] In some embodiments, the method can further comprise predicting a synthetic tissue fraction. In some embodiments, the method can further comprise predicting a synthetic tissue fraction using a methylation library. In some embodiments, the method can further comprise predicting a synthetic tissue fraction using a methylation library of the methylated DNA molecules. In some embodiments, the method can further comprise predicting a synthetic tissue fraction using a methylation library of the methylated oligonucleotide tag. In some embodiments, the method can further comprise predicting a synthetic tissue fraction using a methylation library of the methylated RNA molecules. In some embodiments, the method can further comprise predicting a synthetic tissue fraction using a methylation library of the methylated DNA molecules and the comparative methylation indication. In some embodiments, the method can further comprise predicting a synthetic tissue fraction using a methylation library of the methylated oligonucleotide tag and the comparative methylation indication. In some embodiments, the method can further comprise predicting a synthetic tissue fraction using a methylation library of the methylated RNA molecules and the comparative methylation indication.

[0149] In some embodiments, the method can further comprise predicting a genetic variation tissue fraction. In some embodiments, the method can further comprise predicting a genetic variation tissue fraction using a methylation library of the methylated DNA molecules. In some embodiments, the method can further comprise predicting a genetic variation tissue fraction using a methylation library of the methylated oligonucleotide tag. In some embodiments, the method can further comprise predicting a genetic variation tissue fraction using a methylation library of the methylated RNA molecules. In some embodiments, the method can furtherWSGR Docket No. 68738-702.601comprise predicting a genetic variation tissue fraction using a methylation I i brary of the methylated DNA molecules and the comparative methylation indication. In some embodiments, the method can further comprise predicting a genetic variation tissue fraction using a methylation library of the methylated oligonucleotide tag and the comparative methylation indication. In some embodiments, the method can further comprise predicting a genetic variation tissue fraction using a methylation library of the methylated RNA molecules and the comparative methylation indication.

[0150] In some embodiments, the method can further comprise ligating a DNA fragment to an oligonucleotide tag. In some embodiments, the method can further comprise ligating an ssDNA fragment to an oligonucleotide tag. In some embodiments, the method can further comprise ligating a dsDNA fragment to an oligonucleotide tag. In some embodiments, the method can further comprise ligating a cDNA fragment to an oligonucleotide tag. In some embodiments, the method can further comprise ligating an RNA fragment to an oligonucleotide tag. In some embodiments, the method can further comprise ligating an siRNA fragment to an oligonucleotide tag. In some embodiments, the method can further comprise ligating an miRNA fragment to an oligonucleotide tag. In some embodiments, the method can further comprise ligating an rnRNA fragment to an oligonucleotide tag. In some embodiments, the method can further comprise ligating an snRNA fragment to an oligonucleotide tag. In some embodiments, the method can further comprise ligating an snoRNA fragment to an oligonucleotide tag. In some embodiments, the method can further comprise ligating a tRNA fragment to an oligonucleotide tag. In some embodiments, the method can further comprise ligating an rRNA fragment to an oligonucleotide tag. In some embodiments, the oligonucleotide tag is isolated from an antibody-oligonucleotide conjugate. In some embodiments, the oligonucleotide tag can be isolated from a lipid-oligonucleotide conjugate. In some embodiments, the oligonucleotide tag can be isolated from a small molecule-oligonucleotide conjugate. In some embodiments, the oligonucleotide tag can be isolated from a radionucleotide-oligonucleotide conjugate. In some embodiments, the oligonucleotide tag can be isolated from a viral envelope-oligonucleotide conjugate. In some embodiments, the oligonucleotide tag can be isolated from a nanobody conjugate. In some embodiments, the nanobody conjugate may comprise a single-domain antibody (sdAb), a VHH antibody, or a camelid antibody, or any combination thereof. In some embodiments, the oligonucleotide tag can be methylated. In some embodiments, the oligonucleotide tag can be unmethylated.

[0151] In some embodiments, the method can further comprise contacting a plurality of antibody-oligonucleotide conjugates with a plurality of proteins. In some embodiments, the plurality of proteins may comprise enzymes, transport proteins, antibodies, signaling proteins,WSGR Docket No. 68738-702.601structural proteins, ER proteins, or any combination thereof. In some embodiments, the method can further comprise isolating at least a subset of the plurality of antibody-oligonucleotide conjugates. In some embodiments, the isolated subset of the plurality of antibody-oligonucleotide conjugates may comprise about less than 1%, about 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%. 54%. 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or about more than 99% of the plurality of antibody-oligonucleotide conjugates. In some embodiments, the isolated at least the subset of the plurality of antibody-oligonucleotide conjugates is bound to at least a subset of the plurality7of proteins. In some embodiments, the subset of the plurality of antibody-oligonucleotide conjugates is bound to about less than 1%, about 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%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%, 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or about more than 99% of the plurality of proteins. In some embodiments, the oligonucleotides of the antibody-oligonucleotide conjugates is methylated. In some embodiments, the oligonucleotides of the antibody-oligonucleotide conjugates is unmethylated.

[0152] In some embodiments, the method can further comprise releasing the methylated oligonucleotides from the antibody-oligonucleotide conjugates. In some embodiments, the method can further comprise releasing a subset of the methylated oligonucleotides from the antibody-oligonucleotide conjugates. In some embodiments, the method can further comprise releasing substantially all of the methylated oligonucleotides from the antibody-oligonucleotide conjugates.

[0153] In some embodiments, the method can further comprise replicating the plurality of released methylated oligonucleotides. In some embodiments, the plurality of released methylated oligonucleotides can be replicated between about 10-fold and 100 million-fold. In some embodiments, the methylated oligonucleotides can be replicated before release as part of the antibody-oligonucleotide conjugates. In some embodiments, the methylated oligonucleotides can be replicated after release from the antibody-oligonucleotide conjugates.WSGR Docket No. 68738-702.601

[0154] In some embodiments, the method can further comprise combining the plurality of released methylated oligonucleotides with similar unmethylated oligonucleotides. In some embodiments, the method can further comprise purifying the released methylated oligonucleotides. In some embodiments, purifying the released methylated oligonucleotides may comprise binding the released methylated oligonucleotides to one or more magnetic beads. In some embodiments, each of the plurality of released methylated oligonucleotides may comprise one or more methylation sites. In some embodiments, each of the plurality of released methylated oligonucleotides may comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty7, or more than twenty' methylation sites. In some embodiments, the released methylated oligonucleotides may comprise the methylated oligonucleotide tags. In some embodiments, each of the released methylated oligonucleotides may comprise at least a portion of a methylated oligonucleotide tag.

[0155] In some embodiments, the method can further comprise sequencing both the DNA fragment and the methylated oligonucleotide tag in the same sequencing run. In some embodiments, the method can further comprise sequencing both the ssDNA fragment and the methylated oligonucleotide tag in the same sequencing run. In some embodiments, the method can further comprise sequencing both the RNA fragment and the methylated oligonucleotide tag in the same sequencing run. In some embodiments, the method can further comprise sequencing both the dsDNA fragment and the methylated oligonucleotide tag in the same sequencing run. In some embodiments, the method can further comprise sequencing both the cDNA fragment and the methylated oligonucleotide tag in the same sequencing run. In some embodiments, the method can further comprise sequencing both the mRNA fragment and the methylated oligonucleotide tag in the same sequencing run. In some embodiments, the method can further comprise sequencing both the miRNA fragment and the methylated oligonucleotide tag in the same sequencing run. In some embodiments, the method can further comprise sequencing both the siRNA fragment and the methylated oligonucleotide tag in the same sequencing run. In some embodiments, the method can further comprise sequencing both the snoRNA fragment and the methylated oligonucleotide tag in the same sequencing run. In some embodiments, the method can further comprise sequencing both the tRNA fragment and the methylated oligonucleotide tag in the same sequencing run. In some embodiments, the method can further comprise sequencing both the rRNA fragment and the methylated oligonucleotide tag in the same sequencing run. In some embodiments, the method can further comprise sequencing both the piRNA fragment and the methylated oligonucleotide tag in the same sequencing run. In some embodiments, the method can further comprise sequencing both the snRNA fragment and the methylated oligonucleotide tag in the same sequencing run. In some embodiments, the one orWSGR Docket No. 68738-702.601more methylation sites may comprise a methylation pattern of the methylated oligonucleotide tag. In some embodiments, the methylation pattern is detected by using one or more of bisulfite sequencing, high performance liquid chromatography-ultraviolet sequencing (HPLC-UV), liquid chromatography (LC), mass spectrometry (MS), ELISA-based sequencing, nanopore sequencing, luminometric methylation assay (LUMA), or any combination thereof. In some embodiments, the methylation pattern is detected by using computer software, some embodiments, the methylation pattern is detected by using Al / ML algorithms or modules.

[0156] In some embodiments, the method can further comprise using the methylation pattern to determine one or more characteristics of the methylated oligonucleotide tag, or the DNA fragment, or both. In some embodiments, the method can further comprise using the methylation pattern to determine one or more characteristics of the methylated oligonucleotide tag, or the RNA fragment, or both. In some embodiments, the method can further comprise using the methylation pattern to determine one or more characteristics of the methylated oligonucleotide tag, or the ssDNA fragment, or both. In some embodiments, the one or more characteristics may comprise one or more pieces of multi-omic data. In some embodiments, the multi-omic data may comprise one or more of DNA single nucleotide variations, DNA copy number variations, DNA indels, RNA single nucleotide variations, RNA copy number variations, RNA indels, DNA structural variations, RNA structural variations, hyper-methylation, hypo-methylation, increased protein abundance, or decreased protein abundance, or any combination thereof. In some embodiments, structural variations may comprise inversions, deletions, insertions such as duplications and retroelement insertions, and translocations. In some embodiments, the hypomethylation and hyper-methylation can be determined relative to a normalized methylation value. In some embodiments, the normalized methylation value can be a predetermined set value, or a dynamic value. In some embodiments, the normalized methylation value is based at least in part on a known average methylation value. In some embodiments, the normalized methylation value can be personalized to the subject or patient. In some embodiments, the normalized methylation value can be dynamically generated for one or more diseases. In some embodiments, the normalized methylation value can be selected from a series of profiles similar in some aspects to the patient’s profile. In some embodiments, the method can further comprise detecting the one or more differentially expressed markers or variants in the subject comprising protein abundance characteristics. In some embodiments, the protein abundance characteristics may comprise increased protein abundances or decreased protein abundances. In some embodiments, the protein abundance characteristics may comprise maintained or unchanged protein abundance. In some embodiments, the protein characteristics may comprise protein type characteristics. In some embodiments, the protein characteristics may comprise proteinWSGR Docket No. 68738-702.601concentration characteristics, protein percentage characteristics, protein ratio characteristics, protein configuration characteristics, or other protein information. The proteomics assay may be simultaneously performed with a methylation assay, a genome sequencing assay, an epigenetic assay, or a transcriptomic assay, or any combination thereof, on the same biological sample. A multi-omic assay may comprise the simultaneous performance of the proteomics assay with a methylation assay, a genome sequencing assay, an epigenetic assay, or a transcriptomic assay, or any combination thereof. The one or more differentially expressed markers or variants may be used to determine a presence or absence of a disease. The one or more differentially expressed markers or variants may be used to determine a risk of a subject having a disease. The disease may comprise a cancer. The cancer may comprise one or more of: breast cancer, lung cancer, prostate cancer, colorectal cancer, skin cancer, bladder cancer, kidney cancer, liver cancer, leukemia, lymphoma, multiple myeloma, pancreatic cancer, esophageal cancer, stomach cancer, cervical cancer, uterine cancer, ovarian cancer, testicular cancer, brain cancer, thyroid cancer, oral cancer, head and neck cancer, mesothelioma, neuroblastoma, sarcoma, gallbladder cancer, anal cancer, small intestine cancer, carcinoid tumors, soft tissue sarcoma, cutaneous squamous cell carcinoma, basal cell carcinoma, or adrenal cancer, or any combination thereof. The disease may comprise a genetic disease. The genetic disease may comprise one or more of: cystic fibrosis, sickle cell anemia, Huntington's disease, Duchenne muscular dystrophy, hemophilia, Tay-Sachs disease, phenylketonuria (PKU), Down syndrome. Marfan syndrome, Turner syndrome, Klinefelter syndrome, neurofibromatosis, thalassemia, fragile X syndrome, achondroplasia, congenital adrenal hyperplasia, Alport syndrome, Wilson's disease, familial hypercholesterolemia, Rett syndrome, spinal muscular atrophy, Ellis-van Creveld syndrome, osteogenesis imperfecta, myotonic dystrophy, Xeroderma pigmentosum, Bloom syndrome, ataxia-telangiectasia, or von Willebrand disease, or any combination thereof.

[0157] In some embodiments, the sequencing reads may comprise a multi-omic dataset. In some embodiments, each sequencing read may comprise DNA sequencing data and methylation data. In some embodiments, each sequencing read may comprise RNA sequencing data and methylation data. In some embodiments, each sequencing read may comprise DNA sequencing data, methylation data, and protein data. In some embodiments, each sequencing read may comprise RNA sequencing data, methylation data, and protein data. In some cases, methylation data may comprise methylation sequencing data, methylation pattern data, or both. In some embodiments, the multi-omic dataset may comprise RNA sequencing data, methylation sequencing data, epigenetic data, or protein sequencing data, or any combination thereof. In some embodiments, the multi-omic dataset may comprise DNA information, RNA information, epigenetic information, protein information, or any combination thereof. In some embodiments,WSGR Docket No. 68738-702.601the DNA fragment is methylated or unmethylated. In some embodiments, the RNA fragment is methylated or unmethylated. In some embodiments, the ssDNA fragment is methylated or unmethylated. The DNA information, RNA information, epigenetic information, and protein information may be generated simultaneously from a biological sample. The one or more differentially expressed markers or variants may be used to determine a presence or absence of a disease. The one or more differentially expressed markers or variants may be used to determine a risk of a subject having a disease. The disease may comprise a cancer. The cancer may comprise one or more of breast cancer, lung cancer, prostate cancer, colorectal cancer, skin cancer, bladder cancer, kidney cancer, liver cancer, leukemia, lymphoma, multiple myeloma, pancreatic cancer, esophageal cancer, stomach cancer, cervical cancer, uterine cancer, ovarian cancer, testicular cancer, brain cancer, thyroid cancer, oral cancer, head and neck cancer, mesothelioma, neuroblastoma, sarcoma, gallbladder cancer, anal cancer, small intestine cancer, carcinoid tumors, soft tissue sarcoma, cutaneous squamous cell carcinoma, basal cell carcinoma, or adrenal cancer, or any combination thereof. The disease may comprise a genetic disease. The genetic disease may comprise one or more of cystic fibrosis, sickle cell anemia, Huntington's disease, Duchenne muscular dystrophy, hemophilia, Tay-Sachs disease, phenylketonuria (PKU), Down syndrome, Marfan syndrome, Turner syndrome, Klinefelter syndrome, neurofibromatosis, thalassemia, fragile X syndrome, achondroplasia, congenital adrenal hyperplasia, Alport syndrome, Wilson's disease, familial hypercholesterolemia, Rett syndrome, spinal muscular atrophy, Ellis-van Creveld syndrome, osteogenesis imperfecta, myotonic dystrophy, Xeroderma pigmentosum, Bloom syndrome, ataxia-telangiectasia, or von Willebrand disease, or any combination thereof.

[0158] In some embodiments, the method can further comprise generating a multi-omic library'. In some embodiments, the multi-omic library may comprise two or more sequencing libraries. In some embodiments, the two or more sequencing libraries may comprise the protein library, a DNA sequencing library, an RNA sequencing library, or a methylation sequencing library, or any combination thereof. In some embodiments, the multi-omic model, multianalyte model, or multimodal model may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty -one, twenty-two, twenty -three, twenty-four, twenty -five, twenty -six, twenty-seven, twenty-eight, twenty -nine, thirty7, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty seven, thirty-eight, thirty-nine, forty, forty-one, forty-two, forty-three, forty-four, forty-five, forty-six. forty -seven, forty-eight, forty-nine, fifty, fifty-one, fifty-two, fifty-three, fifty-four, fifty-five, fifty-six, fifty-seven, fifty-eight, fifty-nine, sixty, sixty-one, sixty -two, sixty-three, sixty-four, sixty-five, sixty-six, sixty -seven, sixty-eight, sixty-nine, seventy, seventy-one,WSGR Docket No. 68738-702.601seventy-two, seventy-three, seventy-four, seventy-five, seventy -six, seventy-seven, seventyeight, seventy-nine, eighty, eighty-one, eighty-two, eighty-three, eighty-four, eighty-five, eighty-six, eighty -seven, eighty-eight, eighty-nine, ninety', ninety-one, ninety-two, ninety -three, ninety-four, ninety-five, ninety-six, ninety-seven, ninety-eight, ninety -nine, one hundred, or more than one hundred sequencing libraries. In some embodiments, the two or more sequencing libraries may comprise a DNA sequencing library’, an RNA sequencing library, a methylation library, or a protein library, or any combination thereof. In some embodiments, the two or more libraries may comprise a DNA library, an RNA library, a methylation library, and a protein library'. In some embodiments, the DNA library' may comprise a cDNA library', a genomic DNA library', or both. In some embodiments, the two or more libraries may comprise lipid libraries. In some embodiments, the two or more libraries may comprise small molecule libraries. The multi-omic library may comprise a DNA sequencing library’, an RNA sequencing library, a methylation library', and a protein library generated simultaneously. The multi-omic library may be generated simultaneously from a biological sample. The one or more differentially expressed markers or variants may be used to determine a presence or absence of a disease. The one or more differentially expressed markers or variants may be used to determine a risk of a subject having a disease. The disease may comprise a cancer. The cancer may comprise one or more of: breast cancer, lung cancer, prostate cancer, colorectal cancer, skin cancer, bladder cancer, kidney¬ cancer, liver cancer, leukemia, lymphoma, multiple myeloma, pancreatic cancer, esophageal cancer, stomach cancer, cervical cancer, uterine cancer, ovarian cancer, testicular cancer, brain cancer, thyroid cancer, oral cancer, head and neck cancer, mesothelioma, neuroblastoma, sarcoma, gallbladder cancer, anal cancer, small intestine cancer, carcinoid tumors, soft tissue sarcoma, cutaneous squamous cell carcinoma, basal cell carcinoma, or adrenal cancer, or any combination thereof. The disease may comprise a genetic disease. The genetic disease may’ comprise one or more of: cystic fibrosis, sickle cell anemia, Huntington's disease, Duchenne muscular dystrophy, hemophilia, Tay-Sachs disease, phenylketonuria (PKU), Down syndrome, Marfan syndrome, Turner syndrome, Klinefelter syndrome, neurofibromatosis, thalassemia, fragile X syndrome, achondroplasia, congenital adrenal hyperplasia, Alport syndrome, Wilson's disease, familial hypercholesterolemia, Rett syndrome, spinal muscular atrophy, Ellis-van Creveld syndrome, osteogenesis imperfecta, myotonic dystrophy, Xeroderma pigmentosum, Bloom syndrome, ataxia-telangiectasia, or von Willebrand disease, or any combination thereof.

[0159] Disclosed herein in yet another embodiment is a method of preparing a multi-omic library. In some embodiments, the method of preparing a multi-omic library may comprise annealing together an adapter fragment and a DNA fragment. In some embodiments, the DNA fragment can be a methylated DNA fragment. In some embodiments, the DNA fragment can beWSGR Docket No. 68738-702.601a methylated ssDNA fragment. In some embodiments, the annealed adapter fragment and methylated ssDNA fragment may comprise a first molecular complex. In some embodiments, the annealed adapter fragment and methylated DNA fragment may comprise a first molecular complex. In some embodiments, the annealed adapter fragment and the DNA fragment may comprise a first molecular complex. In some embodiments, the annealed adapter fragment and the dsDNA fragment may comprise a first molecular complex. In some embodiments, the annealed adapter fragment and the methylated cfDNA fragment may comprise a first molecular complex. In some embodiments, the annealed adapter fragment and the methylated cDNA fragment may comprise a first molecular complex. In some embodiments, annealing together an adapter fragment with a DNA fragment can produce a first molecular complex.

[0160] In some embodiments, the method of preparing a multi-omic library may comprise annealing together an adapter fragment and an RNA fragment, thereby producing a second molecular complex. In some embodiments, the annealed adapter fragment and the RNA fragment may comprise a molecular complex. In some embodiments, the annealed adapter fragment and the siRNA fragment may comprise a second molecular complex. In some embodiments, the annealed adapter fragment and the miRNA fragment may comprise a second molecular complex. In some embodiments, the annealed adapter fragment and the mRNA fragment may comprise a second molecular complex. In some embodiments, the annealed adapter fragment and the tRNA fragment may comprise a second molecular complex. In some embodiments, the annealed adapter fragment and the rRNA fragment may comprise a second molecular complex. In some embodiments, the annealed adapter fragment and the snoRNA fragment may comprise a second molecular complex. In some embodiments, the annealed adapter fragment and the snRNA fragment may comprise a second molecular complex. In some embodiments, the annealed adapter fragment and the IncRNA fragment may comprise a second molecular complex. In some embodiments, the annealed adapter fragment and the piRNA fragment may comprise a second molecular complex.

[0161] In some embodiments, the method of preparing a multi-omic I i brary may comprise ligating a methylated oligonucleotide tag to a site of an adapter fragment. In some embodiments, the method of preparing a multi-omic library may comprise ligating an oligonucleotide tag to a site of an adapter fragment. In some embodiments, the method of preparing a multi-omic I ibrary may comprise ligating an oligonucleotide tag to a site of an adapter fragment can produce a third molecular complex. In some embodiments, the method of preparing a multi-omic library may comprise ligating a methylated oligonucleotide tag to a site of an adapter fragment can produce a third molecular complex.WSGR Docket No. 68738-702.601

[0162] In some embodiments, the method can further comprise amplifying the first molecular complex. In some embodiments, the method can further comprise amplifying the second molecular complex. In some embodiments, the method can further comprise amplifying the third molecular complex. In some embodiments, the third molecular complex may comprise a subset of amplified molecules of the first molecular complex. In some embodiments, the third molecular complex may comprise a subset of amplified molecules of the second molecular complex. In some embodiments, the method can further comprise amplifying the first molecular complex, the second molecular complex, and the third molecular complex. In some embodiments, the method can further comprise generating multi-omic data for the third molecular complex. The multi-omic data may comprise one or more of DNA single nucleotide variations, DNA copy number variations, DNA indels, RNA single nucleotide variations, RNA copy number variations, RNA indels, DNA structural variations, RNA structural variations, hyper-methylation, hypo-methylation, increased protein abundance, or decreased protein abundance, or any combination thereof. In some embodiments, structural variations may comprise inversions, deletions, insertions such as duplications and retroelement insertions, and translocations. The multi-omic data may be generated simultaneously from the same biological sample. The multi-omic data may be used to determine a presence or absence of a disease. The multi-omic data may be used to determine a risk of a subject having a disease. The disease may comprise a cancer. The cancer may comprise one or more of: breast cancer, lung cancer, prostate cancer, colorectal cancer, skin cancer, bladder cancer, kidney cancer, liver cancer, leukemia, lymphoma, multiple myeloma, pancreatic cancer, esophageal cancer, stomach cancer, cervical cancer, uterine cancer, ovarian cancer, testicular cancer, brain cancer, thyroid cancer, oral cancer, head and neck cancer, mesothelioma, neuroblastoma, sarcoma, gallbladder cancer, anal cancer, small intestine cancer, carcinoid tumors, soft tissue sarcoma, cutaneous squamous cell carcinoma, basal cell carcinoma, or adrenal cancer, or any combination thereof. The disease may comprise a genetic disease. The genetic disease may comprise one or more of: cystic fibrosis, sickle cell anemia, Huntington's disease, Duchenne muscular dystrophy, hemophilia, Tay-Sachs disease, phenylketonuria (PKU). Down syndrome, Marfan syndrome, Turner syndrome, Klinefelter syndrome, neurofibromatosis, thalassemia, fragile X syndrome, achondroplasia, congenital adrenal hyperplasia, Alport syndrome, Wilson's disease, familial hypercholesterolemia, Rett syndrome, spinal muscular atrophy, Ellis-van Creveld syndrome, osteogenesis imperfecta, myotonic dystrophy, Xeroderma pigmentosum, Bloom syndrome, ataxia-telangiectasia, or von Willebrand disease, or any combination thereof.

[0163] In some embodiments, the method can further comprise associating or grouping the multi-omic data with one or more cell types to generate one or more feature matrices. In someWSGR Docket No. 68738-702.601embodiments, the method can further comprise grouping the one or more feature matrices into one or more genomic feature groups. The one or more genomic feature groups may comprise DNA sequencing features, RNA sequencing features, methylation features, protein features, or any combination thereof. In some embodiments, the one or more genomic features may comprise the multi-omic dataset associated or grouped with cell type data. The multi-omic data and cell type data may comprise one or more matrices. In some cases, cell type data may comprise values associated with each cell type. The values associated with each cell type may comprise one or more cellular deconvolution matrices. In some cases, each cellular deconvolution matrix comprises gene expression data associated with the cell type. In some embodiments, each cellular deconvolution matrix can be based at least in part on one or more gene expression profde matrices. In some embodiments, the method can further comprise providing as input the one or more genomic feature to a multi-omic model, multianalyte model, or multimodal model. In some embodiments, the method can further comprise performing multilayer perception (MLP) on data relating to the one or more genomic feature groups to generate synthetic tissue fraction values associated with each of the one or more genomic features.

[0164] In some embodiments, the method can further comprise generating a multimodal library. The multimodal library may comprise biological data, such as -omics data, and data from one or more additional modalities. In some embodiments, the one or more additional modalities may comprise testing modalities such as imaging modalities. The imaging modalities may comprise imaging genetics. In some embodiments, the imaging data may comprise one or more of: MRI imaging data, ultrasound imaging data, X-ray imaging data, PET scan imaging data, fluoroscopy imaging data, CT scan imaging data, or any combination thereof. In some cases, MRI imaging data may comprise fMRI imaging data.

[0165] In some embodiments, the method may further comprise generating the multi-omic data set from the biological sample simultaneously. In some embodiments, the method may further comprise performing a multi-omic assay of the biological sample to simultaneously generate the multi-omic data set. The multi-omic assay may comprise a genomic assay, a transcriptomic assay, an epigenetic assay, and a proteomic assay. The multi-omic assay may simultaneously generate the multi-omic data set comprising multi-omic assay results relating to the RNA sequencing data, the methylation sequencing data, and the protein sequencing data. Performing the multi-omic assay of the biological sample may comprise performing the genomic assay, the transcriptomic assay, the epigenetic assay, and the proteomic assay in the same sequencing run. In some embodiments, the method may further comprise preparing the multi-omic library using assay results simultaneously generated by a multi-omic assay of a biological sample.WSGR Docket No. 68738-702.601

[0166] In yet another aspect, described are methods for assaying a biological sample of a subject, comprising: (a) performing a multi-omic assay of the biological sample to simultaneously capture genomic, transcriptomic, epigenetic, and proteomic assay results from the biological sample; and (b) generating multi-omic data comprising genomic data, transcriptomic data, epigenetic data, and proteomic data from the simultaneously captured multi-omic assay results.

[0167] In some embodiments, the method may further comprise generating a multi-omic library using the multi-omic data. The multi-omic library may comprise a plurality of -omic layers. The -omic layer of the plurality' of -omic layers may correspond to the genomic data, the transcriptomic data, the epigenetic data, or the proteomic data. In some embodiments, the method may further comprise comparing the multi-omic library to one or more reference -omic data sets. The one or more reference -omic data sets may comprise a reference genomic data set, a reference transcriptomic data set, a reference epigenetic data set, or a reference proteomic data set, or any combination thereof.

[0168] In some embodiments, the method may further comprise determining a variation of the multi-omic data. The variation may comprise a genomic sequence variation, a transcriptomic variation, an epigenetic variation, or a proteomic variation, or any combination thereof. The sequence variation may comprise a single nucleotide polymorphism (SNP) or a copy number variation (CNV). The epigenetic variation may comprise a differential promoter methylation, a differential genome region methylation, or a differential CpG methylation. The proteomic variation may comprise a variation in fractional protein content, protein quantity, or protein function, or any combination thereof. The genomic data may comprise genomic sequencing data of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0169] The multi-omic data may be used to determine a presence or absence of a disease. The multi-omic data may be used to determine a risk of a subject having a disease. The disease may comprise a cancer. The cancer may comprise one or more of: breast cancer, lung cancer, prostate cancer, colorectal cancer, skin cancer, bladder cancer, kidney cancer, liver cancer, leukemia, lymphoma, multiple myeloma, pancreatic cancer, esophageal cancer, stomach cancer, cervical cancer, uterine cancer, ovarian cancer, testicular cancer, brain cancer, thyroid cancer, oral cancer, head and neck cancer, mesothelioma, neuroblastoma, sarcoma, gallbladder cancer, anal cancer, small intestine cancer, carcinoid tumors, soft tissue sarcoma, cutaneous squamous cell carcinoma, basal cell carcinoma, or adrenal cancer, or any combination thereof. The disease may comprise a genetic disease. The genetic disease may comprise one or more of: cystic fibrosis, sickle cell anemia, Huntington's disease, Duchenne muscular dystrophy, hemophilia, Tay-Sachs disease, phenylketonuria (PKU), Down syndrome, Marfan syndrome, Turner syndrome,WSGR Docket No. 68738-702.601Klinefelter syndrome, neurofibromatosis, thalassemia, fragile X syndrome, achondroplasia, congenital adrenal hyperplasia, Alport syndrome, Wilson's disease, familial hypercholesterolemia, Rett syndrome, spinal muscular atrophy, Ellis-van Creveld syndrome, osteogenesis imperfecta, myotonic dystrophy, Xeroderma pigmentosum, Bloom syndrome, ataxia-telangiectasia, or von Willebrand disease, or any combination thereof.Multi-omic Data Analysis

[0170] As illustrated in, for example FIG. 4, various information types 400 associated with a sample, such as a sample tumor 401 may be extracted and analyzed. DNA data may be extracted 402 which includes, for example, information about DNA SNVs 403, DNA CNVs 404, DNA indels 405. and DNA expression 406. RNA data can also be extracted 407 from the sample tumor 401. Extracted RNA data 407 can include RNA SNVs 408, RNA CNVs 409, RNA indels 410, and RNA expression data 411. Epigenetic data can also be extracted 412 from a sample such as a sample tumor 401 including epigenetic data relating to SNVs 413, epigenetic data relating to CNVs 414, epigenetic data relating to indels 415, epigenetic data relating to hypermethylation 416, and epigenetic data relating to hypomethylation 417. Protein data can also be extracted 418 from a sample such as a sample tumor 401. Extracted protein data 418 can include, for example, increased protein abundances 419 and decreased protein abundances 420.As shown in, for example FIG. 4, extracted data such as extracted DNA data 402, extracted RNA data 407, extracted epigenetic data 412, and extracted protein data 418 is combined with a post-treatment plasma sampling 421 for mapping. The extracted data and the post-treatment plasma data, as illustrated in FIG. 4, is combined so that the extracted tumor sample data 401 is mapped onto the post-treatment plasma sample 421 for processing 422. This mapping 422 allows the post-treatment plasma 421 data to be associated with extracted data for analysis instead of requiring independent data collection and analysis of biomarkers in the sampled posttreatment plasma 421.

[0171] Referring to FIG. 5, an exemplary multi-omic, multianaly te, or multimodal model is shown. The exemplary multi-omic model, multianalyte model, or multimodal model 500 can receive data relating to a biological sample 501 and can partition or otherwise divide the data of the biological sample 501 into one or more data groups corresponding to each biomarker or analyte type. For example, as illustrated in FIG. 5, biological sample data 501 is grouped into DNA features 502, RNA features 503. methylation features 504, and protein features 505.AI / ML architecture such as, for example, multilayer perception (MLP) 509 transformer architecture 507 or convolutional neural networks 508 is used to predict a synthetic tissue fraction of the biological sample for type of the combined biomarkers or analytes. Multiple typesWSGR Docket No. 68738-702.601of biomarkers and analytes are combined. The combined biomarkers or analytes can be, for example, in silico combinations of biomarkers or analytes. The combined biomarkers or analytes can be, for example, generated in vitro as a mixture of biomarkers or analytes. The in vitro mixture of biomarkers or analytes can be diluted, for example. As shown in, for example FIG. 5, the synthetic tissue fraction prediction is output as a matrix or data list. As illustrated in FIG. 5, the grouped feature data is further parsed or separated into feature subgroups 506 based on one or more additional features. AI / ML architecture such as neural networks, for example multilayer perception (MLP) 509 is applied to the feature subgroups 506 to predict a synthetic tissue fraction. As shown in, for example FIG. 5, the synthetic tissue fraction prediction is output as a matrix or data list.

[0172] As illustrated in FIG. 6, for example, models are used for each type of extraction data. For example, as shown in FIG. 6, a cell type deconvolution model is used to analyze methylation data, as a methylation model 600. Data associated with a biological sample 601 is input into the methylation model 600. A subset of data of the biological sample 600 is selected, for example methylation data to produce methylation feature data 602. AI / ML architecture, for example transformer architecture 604 or convolutional neural networks 605 is applied to a plurality of methylation feature data 602 groups to predict synthetic fraction of the biological sample 601 based on methylation data, for example methylation data alone. As shown in FIG. 6, for example, methylation feature data 602 is further divided into feature subgroups 603, and multilayer perception (MLP) 606 is applied to predict synthetic fraction from subgroups 603 of methylation data. The AI / ML architecture, for example the transformer architecture 604, the convolutional neural network 605, or the MLP 606 is used to analyze and determine a predictive relationship between the methylation data features 602 and the synthetic fraction, for example in a subset of genomic regions that is preselected or dynamic.

[0173] As illustrated in FIG. 7, AI / ML models are trained on multi-omic, multianalyte, or multimodal information 700 found at the site of a genomic variant. A reference genome 701 is used to compare captured variant information with reference information. As illustrated in FIG.7, one or more variants captured by DNA sequencing 702 is compared to multi-omic, multianalyte, or multimodal information at a reference site or reference area of a reference genome 701. As illustrated in FIG. 7, one or more variants captured by RNA sequencing 703 is compared to multi-omic, multianalyte, or multimodal information at a reference site or reference area of a reference genome 701. As illustrated in FIG. 7, one or more variants captured by methylation mapping 704 is compared to multi-omic. multianalyte, or multimodal information at a mapped reference site or mapped reference area of a reference genome 701.WSGR Docket No. 68738-702.601

[0174] Analysis of the multi-omic assay may comprise identifying sequence variations using a genome sequencing assay portion of the multi-omic assay. The sequence variations may comprise single nucleotide polymorphisms (SNVs), copy number variations (CNVs), and other genomic features. As illustrated in FIGs. 9A-C, variant allele frequencies (VAF) may be more consistent in multi-omic assay analysis than using WGS alone. FIG. 9A shows concordance between VAF of mutations in WGS of DNA alone compared to multi-omic sequencing. FIG.9B shows concordance between CNVs of WGS of DNA alone compared to multi-omic sequencing. FIG. 9C shows CNV profdes across multiple cell lines for lung cancer, liver cancer, melanoma, and normal cells.

[0175] The methods may further comprise performing quantification of gene expression counts and cell type interpretation based on expression profiles of the multi-omic data. As illustrated in FIGs. 10A-D, RNA gene expression may be more consistent in multi-omic assay analysis than using RNA sequencing alone. FIG. 10A shows concordance between RNA gene expression counts in RNA sequencing alone compared to multi-omic sequencing. FIG. 10B shows amounts of transcripts identified in markers for various melanoma tissue cell types and markers for B cell types. FIG. 10C shows tissue-specific gene expression of various biomarkers across multiple cell lines. FIG. 10D shows genomic distribution of transcripts for RNA sequencing alone compared to multi-omic sequencing.

[0176] The multi-omic assay may comprise performing whole genome bisulfite sequencing. Analysis of assay results of the bisulfite sequencing assay component of the multi-omic assay may comprise identifying differential promoter methylation, genome region methylation, and CpG level methylation identification. As illustrated in FIGs. 11A-C, methylation data may be more consistent and accurate in multi-omic assay analysis as compared to DNA methylation analysis alone. FIG. 11A shows genomic region methylation concordance of DNA methylation assays alone compared to multi-omic assay analysis for gene bodies, gene promoters, and CpG islands. FIG. 11B shows an example of data relating to average promoter methylation in a tissue at promoters of genes expressed in a cell type A and a cell ty pe B. FIG. 11C shows an example of data relating to methylation percentage in regions hypomethylated in B cells for blood, colon tissue, liver tissue, lung tissue, and melanoma tissue.

[0177] The multi-omic assay may be used to generate multi-omic data simultaneously from the same biological sample. The simultaneous generation of multi-omic data from the same biological sample may improve detection of multi-omic biomarker variations. The improved detection may comprise reduced error, reduced bias, and reduced noise in multi-omic data, as DNA and RNA may be simultaneously measured from the same biological sample. The same biological sample may comprise the same population of cells or the same subset of cells of aWSGR Docket No. 68738-702.601population of cells. The same biological sample may comprise the same tissue. FIGs. 12A-B show gene expression corresponding to CNVs. FIG. 12A shows example data relating to gene expression corresponding to CNVs for a multi-omic assay. FIG. 12B shows example data relating to gene expression corresponding to CNVs for single-omic DNA and RNA assays alone.

[0178] In some cases, a multi-omic, multianalyte, or multimodal model can be trained based at least in part on multi-omic, multianalyte, or multimodal information associated with one or more time points. The multi-omic, multianalyte, or multimodal information can be longitudinal information. In some cases, DNA features, RNA features, methylation features, and protein features can be combined and associated with a first time point. The combined features are associated with a first time point because they are captured at the first time point. The same types of DNA features, RNA features, methylation features, and protein features can be combined and associated with a second time point. The combined features are associated with a second time point because they are captured at the second time point. The combined features of the first time point and the combined features of the second time point can be used as input to train the multi-omic, multianalyte, or multimodal model on longitudinal multi-omic, multianalyte, or multimodal data. The training data may comprise combined features of the same cell ty pes collected or measured at two or more different time points.

[0179] The multi-omic model, multianalyte model, or multimodal model can determine one or more synthetic tumor fractionation values of combined multi-omic data based at least in part on longitudinal multi-omic data, for example longitudinal multi-omic training data or reference data. The multi-omic, multianalyte, or multimodal model can be configured to match one or more input groups of DNA features, RNA features, methylation features, or protein features, or any combination thereof, with one or more corresponding reference feature corresponding to a first time point. The multi-omic model, multianalyte model, or multimodal model can be configured to match one or more input groups of DNA features, RNA features, methylation features, or protein features, or any combination thereof, with one or more corresponding reference feature corresponding to a second time point. The multi-omic, multianalyte, or multimodal model can be configured to determine one or more characteristics, such as one or more tumor fractionation values, of the input multi-omic data based at least in part on the reference multi-omic data corresponding to the first time point, the multi-omic data corresponding to the second time point, or both. The multi-omic, multianalyte, or multimodal model can be configured to determine one or more characteristics, such as one or more tumor fractionation values, of the input multi-omic data based at least in part on changes betweenWSGR Docket No. 68738-702.601corresponding reference multi-omic data at a first time point and corresponding reference multi-omic data at a second time point.

[0180] In some embodiments, the method comprises simultaneously capturing two or more types of multi-omic data. The multi-omic data types may comprise proteomic information, genomic information, transcriptomic information, and epigenetic information from a biological sample. The multi-omic data may be captured from a single biological sample. The two or more types of multi-omic data may be captured from the sample biological sample. The two or more multi-omic data types may be captured from the same biological sample simultaneously. In some embodiments, the method comprises simultaneously capturing genomic, transcriptomic, epigenetic, and proteomic information from the same biological sample. These measurements may be used in analysis of multi-omic information. The multi-omic information may be analyzed to reduce or eliminate errors associated with sequencing separate -omic data. The errors reduced or eliminated may comprise reducing or eliminating batch effects associated with the individual sequencing of each -omic layer. The errors reduced or eliminated may comprise reducing or eliminating noise associated with the individual sequencing of each -omic layer. The errors reduced or eliminated may comprise reducing or eliminating batch effects and technical noise associated with the individual sequencing of each -omic layer. This improved data quality along with the information being captured from the same set of cells may be used to determine a complex biological relationship between the -omic layers. Errors comprising noise or batch effects, or both, may be reduced by about between 1% and 100%. Batch effects may be reduced by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more than 10%. Batch effects may be reduced by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or more than 99%. Batch effects may be eliminated. Batch effects may be eliminated for one or more layers or types of -omic information. Batch effects may be reduced or eliminated for 1, 2, 3, 4. 5, or more than 5 layers or types of -omic information. Batch effects may be reduced or eliminated for all layers or types of -omic information. Technical noise may be reduced by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more than 10%. Technical noise may be reduced by about 20%. 30%. 40%, 50%, 60%, 70%, 80%, 90%, 99%, or more than 99%. Technical noise may be eliminated. Batch effects may be eliminated for one or more layers or types of -omic information. Technical noise may be reduced or eliminated for 1, 2, 3, 4, 5, or more than 5 layers or types of -omic information. Technical noise may be reduced or eliminated for all layers or types of -omic information.

[0181] Variant Allele Frequency (VAF) in mutations found in both DNA and RNA may be approximately the same ( i.e. loglO(ratio) = loglO(l) = 0 ). Deviations from this expected ratio may be caused at least in part by technical noise. The technical noise may comprise sequencingWSGR Docket No. 68738-702.601artifacts or library' preparation artifacts. FIG. 13A shows VAF correlation between amulti-omic assay and single-omic DNA and RNA assays alone. FIG. 13B shows a ratio of DNA VAF to RNA VAF for a multi-omic assay as compared to a single-omic sequencing assay.

[0182] Reads containing true point mutations may be present in both forward and reverse strands of DNA in about equal quantities. Deviation from a ratio of 1.0 [loglO(l) = 0] (Equal # of Forward / Reverse Mutated Reads) may be attributable at least in part to sequencing errors or other technical errors. FIG. 14 illustrates a comparison of multi-omic strand bias and single-omic strand bias, illustrating a consistent reduction of strand bias for multi-omic assay analysis across technical replicates.

[0183] Genome-wide mutational concordance may be analyzed by comparing mutational profiles of variants across types of mutations. Mutational profiles of SNVs may be determined by quantifying a number of a particular mutation type (e g. OT) determined in selected flanking genomic nucleotides. FIG. 15A shows a mutational profile of multi-omic variants in deep sequencing as compared to multi-omic sequencing as described herein (TrinitySeq). FIG. 15B shows a mutational profile of multiomic variants in deep sequencing as compared to multi-omic sequencing as described herein (TrinitySeq). FIG. 16A shows a gene expression profile as compared to a gene body methylation profile for multi-omic assay analysis. FIG. 16B shows a gene expression profile as compared to a gene body profile for independently sequenced single-omic assays. FIG. 16C shows methylation information percentage with simultaneous capture of DNA and methylation information in multi-omic assay analysis, illustrating capture of methylation information while preserving the original DNA sequence. This illustrates increased accuracy for variant identification in multi-omic assay analysis as compared to single-omic sequencing analysis.

[0184] In some embodiments, methylated protein barcode sequences of multi-omic assays may be integrated into TNA libraries of multi-omic libraries that are directly quantified via qPCR assays or next generation sequencing (NGS). FIG. 17A illustrates protein concentration analysis of an FIT-29 cell lysate with five barcoded antibodies in a colorectal cancer sample. The antibodies may comprise annexin, CD51, cofilin, enolase, and tubulin. Barcodes were analyzed by qPCR. The negative control was a no protein (PBS only) sample tested with the same antibody panel comprising annexin, CD51, cofilin, enolase, and tubulin. FIG. 17B illustrates an example of data relating to multiple different cell lysates analyzed with a five antibody panel comprising annexin, CD51, cofilin, enolase, and tubulin. Antibody barcodes were measured by qPCR (x-axis) and short read DNA sequencing (y-axis). FIG. 17C illustrates example data relating to a reproducibility study where multiple different cell lysates were analyzed with a fiveWSGR Docket No. 68738-702.601antibody panel comprising annexin, CD51, cofilin, enolase, and tubulin. Antibody barcodes were measured by NGS (y-axis) across 8 replicates.

[0185] As illustrated in FIG. 18A, raw NGS counts for antibody-oligo barcodes for five housekeeping proteins Annexin V, Cofilin I, Integrin oV5 (CD51), Tubulin A, and Vimentin were measured in six cancer and non-cancer cell lines. The cell lines comprised B cells, Calu3, Colo829, Hepa RG, HepG2, and HT29, as well as an orthogonal protein control. As illustrated in FIG. 18B, fractional protein content was measured by multi-omic assay analysis as described herein (TrinitySeq) using six different protein detection antibodies comprising Annexin V, Cofilin I, Integrin oV5 (CD51), Tubulin A, and Vimentin. Two samples were tested, one complex biological sample (human cell line HT29), one control pure protein sample (ovalbumin alone). FIG. 18C illustrates an example of data relating to protein measurement in multi-omic assay analysis as described herein (TrinitySeq) using a titration of test protein (ovalbumin) over four orders of magnitude. FIGs. 19A-D illustrate examples of data relating to multi-omic assay analysis protein measurement titration quantifications based on extracted barcode tags. FIG. 19A illustrates an example of data for absolute read counts. FIG. 19B illustrates an example of data for relative proportion of protein concentration. FIG. 19C illustrates an example of data for relative proportion of protein concentration for detected proteins only.

[0186] Multi-omic library construction methods as described herein capture information on multiple -omics channels with high quality’ and reproducibility across targets. Custom deconvolution tags may be used to identify cDNA species in the final library. The custom deconvolution tags may be derived from RNA. The custom deconvolution tags may be able to separate the cDNA species from the DNA sequencing reads and methylation reads in the multi-omic library. Methylated protein quantification barcodes may be attached to reads with molecular identifiers to achieve a high accuracy in detection of targets. FIG. 20A illustrates a flow diagram relating to splitting DNA and RNA channels based on methylated random octamer RNA tags, then subsequently extracting protein quantification barcodes from the deconvoluted DNA and RNA reads. FIG. 20B illustrates an average base qualify of NGS reads generated simultaneously from the same biological sample using multi-omic assay analysis as described herein. FIG. 20C illustrates a number of reads generated simultaneously from the same biological sample using multi-omic assay analysis as described herein. FIG. 20D illustrates an example of data relating to accuracy of RNA deconvolution based on sequence tags. The example data relates to comparing true TNA libraries from multi-omic sequencing as described herein comprising about 15% to 20% barcodes species, against libraries with only RNA or only DNA single-omic data libraries. The example data illustrates a high rate of RNA sequence tagsWSGR Docket No. 68738-702.601in the RNA-only single-omic libraries. This supports high sensitivity of this deconvolution method in multi-omic assays as described herein.

[0187] FIG. 21A illustrates an example of data relating to CpH artificial methylation in cDNA species shown across sequencing reads. The CpH artificial methylation may comprise methylating CTPs during cDNA synthesis. The CpH retention rate may relate to the fraction of CpH events that are not converted by methyl conversion process because of methyl protection. Methylated CTP included during cDNA synthesis may be a secondary control for DNA and RNA deconvolution. FIG. 21B illustrates data relating to an average methylation status of CpH sites in deconvoluted RNA reads as compared to DNA reads.

[0188] DNA and genomic DNA (gDNA) may be pooled from a single sample for assaying and analysis. FIG. 32 illustrates an example of combined multi-analyte pooled DNA and RNA preparation for a multi-omic assay.Computing Systems

[0189] Referring to FIG. 8, a block diagram is shown depicting an exemplary machine that includes a computer system 800 (e.g., a processing or computing system) within which a set of instructions can execute for causing a device to perform or execute any one or more of the aspects and / or methodologies of the present disclosure. The components in FIG. 8 are examples only and do not limit the scope of use or functionality of any hardware, softw are, embedded logic component, or a combination of two or more such components implementing particular embodiments.

[0190] Computer system 800 may include one or more processors 801, a memory' 803, and a storage 808 that communicate with each other, and with other components, via a bus 840. The bus 840 may also link a display 832, one or more input devices 833 (which may, for example, include a keypad, a keyboard, a mouse, a stylus, etc.), one or more output devices 134, one or more storage devices 835, and various tangible storage media 836. All of these elements may interface directly or via one or more interfaces or adaptors to the bus 840. For instance, the various tangible storage media 836 can interface with the bus 840 via storage medium interface 826. Computer system 800 may have any suitable physical form, including but not limited to one or more integrated circuits (ICs), printed circuit boards (PCBs), mobile handheld devices (such as mobile telephones or PDAs), laptop or notebook computers, distributed computer systems, computing grids, or servers.

[0191] Computer system 800 includes one or more processor(s) 801 (e.g., central processing units (CPUs) or general-purpose graphics processing units (GPGPUs)) that carry' out functions. Processor(s) 801 optionally contains a cache memory unit 802 for temporary local storage ofWSGR Docket No. 68738-702.601instructions, data, or computer addresses. Processor(s) 801 are configured to assist in execution of computer readable instructions. Computer system 800 may provide functionality’ for the components depicted in FIG. 8 as a result of the processor(s) 801 executing non-transitory, processor-executable instructions embodied in one or more tangible computer-readable storage media, such as memory' 803, storage 808, storage devices 835, and / or storage medium 836. The computer-readable media may store software that implements particular embodiments, and processor(s) 801 may execute the software. Memory 803 may read the software from one or more other computer-readable media (such as mass storage device(s) 835, 836) or from one or more other sources through a suitable interface, such as network interface 820. The software may cause processor(s) 801 to carry out one or more processes or one or more operations of one or more processes described or illustrated herein. Carrying out such processes or operations may include defining data structures stored in memory 803 and modifying the data structures as directed by the software.

[0192] The memory 803 may include various components (e.g., machine readable media) including, but not limited to. a random access memory component (e.g., RAM 804) (e.g., static RAM (SRAM), dynamic RAM (DRAM), ferroelectric random access memory (FRAM), phasechange random access memory (PRAM), etc.), a read-only memory component (e.g., ROM 805), and any combinations thereof. ROM 805 may act to communicate data and instructions unidirectionally to processors) 801, and RAM 804 may act to communicate data and instructions bidirectionally with processors) 801. ROM 805 and RAM 804 may include any suitable tangible computer-readable media described below. In one example, a basic input / output system 806 (BIOS), including basic routines that help to transfer information between elements within computer system 800, such as during start-up. may be stored in the memory 803.

[0193] Fixed storage 808 is connected bidirectionally to processor(s) 801, optionally through storage control unit 807. Fixed storage 808 provides additional data storage capacity and may also include any suitable tangible computer-readable media described herein. Storage 808 may¬ be used to store operating system 809. executable(s) 810, data 811, applications 812 (application programs), and the like. Storage 808 can also include an optical disk drive, a solid-state memory device (e.g., flash-based systems), or a combination of any of the above. Information in storage 808 may, in appropriate cases, be incorporated as virtual memory- in memory- 803.

[0194] In one example, storage device(s) 835 may be removably interfaced with computer system 800 (e.g.. via an external port connector (not shown)) via a storage device interface 825. Particularly, storage device(s) 835 and an associated machine-readable medium may provide non-volatile and / or volatile storage of machine-readable instructions, data structures, programWSGR Docket No. 68738-702.601modules, and / or other data for the computer system 800. In one example, software may reside, completely or partially, within a machine-readable medium on storage device(s) 835. In another example, software may reside, completely or partially, within processor(s) 801.

[0195] Bus 840 connects a wide variety of subsystems. Herein, reference to a bus may encompass one or more digital signal lines serving a common function, where appropriate. Bus 840 may be any of several types of bus structures including, but not limited to, a memory’ bus, a memory controller, a peripheral bus, a local bus, and any combinations thereof, using any of a variety of bus architectures. As an example and not by way of limitation, such architectures include an Industry' Standard Architecture (ISA) bus, an Enhanced ISA (EISA) bus, a Micro Channel Architecture (MCA) bus, a Video Electronics Standards Association local bus (VLB), a Peripheral Component Interconnect (PCI) bus, a PCI-Express (PCI-X) bus. an Accelerated Graphics Port (AGP) bus, HyperTransport (HTX) bus, serial advanced technology attachment (SATA) bus, and any combinations thereof.

[0196] Computer system 800 may also include an input device 833. In one example, a user of computer system 80 may enter commands and / or other information into computer system 800 via input device(s) 833. Examples of an input device(s) 833 include, but are not limited to, an alpha-numeric input device (e.g., a keyboard), a pointing device (e.g., a mouse or touchpad), a touchpad, a touch screen, a multi-touch screen, a joystick, a stylus, a gamepad, an audio input device (e.g., a microphone, a voice response system, etc.), an optical scanner, a video or still image capture device (e.g., a camera), and any combinations thereof. In some embodiments, the input device is a Kinect, Leap Motion, or the like. Input device(s) 833 may be interfaced to bus 840 via any of a variety’ of input interfaces 823 (e.g., input interface 823) including, but not limited to, serial, parallel, game port, USB, FIREWIRE, THUNDERBOLT, or any combination of the above.

[0197] In particular embodiments, when computer system 800 is connected to network 830, computer system 800 may communicate with other devices, specifically mobile devices and enterprise systems, distributed computing systems, cloud storage systems, cloud computing systems, and the like, connected to network 830. Communications to and from computer system 800 may be sent through network interface 820. For example, network interface 820 may receive incoming communications (such as requests or responses from other devices) in the form of one or more packets (such as Internet Protocol (IP) packets) from network 130, and computer system 800 may store the incoming communications in memory’ 803 for processing. Computer system 800 may similarly store outgoing communications (such as requests or responses to other devices) in the form of one or more packets in memory 803 and communicated to network 830WSGR Docket No. 68738-702.601from network interface 820. Processor(s) 801 may access these communication packets stored in memory 803 for processing.

[0198] Examples of the network interface 820 include, but are not limited to, a network interface card, a modem, and any combination thereof. Examples of a network 830 or network segment 830 include, but are not limited to, a distributed computing sy stem, a cloud computing system, a wide area network (WAN) (e.g., the Internet, an enterprise network), a local area network (LAN) (e.g., a network associated with an office, a building, a campus or other relatively small geographic space), a telephone network, a direct connection between two computing devices, a peer-to-peer network, and any combinations thereof. A network, such as network 830, may employ a wired and / or a wireless mode of communication. In general, any network topology may be used.

[0199] Information and data is displayed through a display 832. Examples of a display 832 include, but are not limited to, a cathode ray tube (CRT), a liquid crystal display (LCD), a thin film transistor liquid crystal display (TFT-LCD), an organic liquid cry stal display (OLED) such as a passive-matrix OLED (PMOLED) or active-matrix OLED (AMOLED) display, a plasma display, and any combinations thereof. The display 832 can interface to the processor(s) 801, memory 803, and fixed storage 808, as well as other devices, such as input device(s) 833, via the bus 840. The display 832 is linked to the bus 840 via a video interface 822, and transport of data between the display 832 and the bus 840 is controlled via the graphics control 821. In some embodiments, the display is a video projector. In some embodiments, the display is a headmounted display (HMD) such as a VR headset. Examples of suitable VR headsets include HTC Vive, Oculus Rift, Samsung Gear VR, Microsoft HoloLens, Razer OSVR, FOVE VR, Zeiss VR One, Avegant Glyph, Freefly VR headset, and the like. In still further embodiments, the display is a combination of devices such as those disclosed herein.

[0200] In addition to a display 832, computer system 800 may include one or more other peripheral output devices 834 including, but not limited to, an audio speaker, a printer, a storage device, and any combinations thereof. Such peripheral output devices may be connected to the bus 840 via an output interface 824. Examples of an output interface 824 include, but are not limited to, a serial port, a parallel connection, a USB port, a FIREWIRE port, a THUNDERBOLT port, and any combinations thereof.

[0201] In addition or as an alternative, computer system 800 may provide functionality as a result of logic hardwired or otherwise embodied in a circuit, which may operate in place of or together with software to execute one or more processes or one or more operations of one or more processes described or illustrated herein. Reference to software in this disclosure may encompass logic, and reference to logic may encompass software. Moreover, reference to aWSGR Docket No. 68738-702.601computer-readable medium may encompass a circuit (such as an IC) storing software for execution, a circuit embodying logic for execution, or both, where appropriate. The present disclosure encompasses any suitable combination of hardware, software, or both.

[0202] Various illustrative logical blocks, modules, circuits, and algorithm operations described in connection with the embodiments disclosed herein may be implemented as electronic hardware, computer software, or combinations of both. To clearly illustrate this interchangeability of hardware and software, vanous illustrative components, blocks, modules, circuits, and operations have been described above generally in terms of their functionality.

[0203] The various illustrative logical blocks, modules, and circuits described in connection with the embodiments disclosed herein may be implemented or performed with a general purpose processor, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA) or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. A general purpose processor may be a microprocessor, but in the alternative, the processor may be any conventional processor, controller, microcontroller, or state machine. A processor may also be implemented as a combination of computing devices, e.g., a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration.

[0204] The operations of a method or algorithm described in connection with the embodiments disclosed herein may be embodied directly in hardware, in a software module executed by one or more processor(s), or in a combination of the two. A software module may reside in RAM memory, flash memory, ROM memory, EPROM memory', EEPROM memory, registers, hard disk, a removable disk, a CD-ROM. or any other form of storage medium known in the art. An exemplary storage medium is coupled to the processor such the processor can read information from, and write information to, the storage medium. In the alternative, the storage medium may be integral to the processor. The processor and the storage medium may reside in an ASIC. The ASIC may reside in a user terminal. In the alternative, the processor and the storage medium may reside as discrete components in a user terminal.

[0205] Examples of suitable computing devices include cloud computing platforms, distributed computing platforms, server clusters, server computers, desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, and netpad computers.

[0206] In some embodiments, the computing device includes an operating system configured to perform executable instructions. The operating system is, for example, software, including programs and data, which manages the device’s hardware and provides services for execution ofWSGR Docket No. 68738-702.601applications. Various suitable server operating systems include FreeBSD, OpenBSD, NetBSD®, Linux, Apple® Mac OS X Server®, Oracle® Solaris®, Windows Server®, and Novell® NetWare®. Various suitable personal computer operating systems include Microsoft® Windows®, Apple® Mac OS X®, UNIX®, and UNIX-like operating systems such as GNU / Linux®. In some embodiments, the operating system is provided by cloud computing. Various suitable mobile smartphone operating systems include Nokia® Symbian® OS, Apple® iOS®, Research in Motion® BlackBerry OS®, Google® Android®, Microsoft® Windows Phone® OS, Microsoft® Windows Mobile® OS, Linux®, and Palm® WebOS®.Non-transitory Computer Readable Storage Medium

[0207] In some embodiments, the platforms, systems, media, and methods disclosed herein include one or more non-transitory computer readable storage media encoded with a program including instructions executable by the operating system of an optionally networked computing device. In further embodiments, a computer readable storage medium is a tangible component of a computing device. In still further embodiments, a computer readable storage medium is optionally removable from a computing device. Examples of computer readable storage media include CD-ROMs, DVDs, flash memory devices, solid state memory, magnetic disk drives, magnetic tape drives, optical disk drives, distributed computing systems including cloud computing systems and services, and the like. The program and instructions are permanently, substantially permanently, semi-permanently, or non-transitorily encoded on the media.Computer Programs

[0208] In some embodiments, the platforms, systems, media, and methods disclosed herein include at least one computer program, or use of the same. A computer program includes a sequence of instructions, executable by one or more processor(s) of the computing device’s CPU, written to perform a specified task. Computer readable instructions may be implemented as program modules, such as functions, objects, Application Programming Interfaces (APIs), computing data structures, and the like, which perform particular tasks or implement particular abstract data types. A computer program may be written in various versions of various languages.

[0209] The functionality' of the computer readable instructions may be combined or distributed as desired in various environments. In some embodiments, a computer program comprises one sequence of instructions. In some embodiments, a computer program comprises a plurality of sequences of instructions. In some embodiments, a computer program is provided from one location. In other embodiments, a computer program is provided from a plurality' of locations. InWSGR Docket No. 68738-702.601various embodiments, a computer program includes one or more software modules. In various embodiments, a computer program includes, in part or in whole, one or more web applications, one or more mobile applications, one or more standalone applications, one or more web browser plug-ins, extensions, add-ins, or add-ons, or combinations thereof.Software Modules

[0210] In some embodiments, the platforms, systems, media, and methods disclosed herein include software, server, and / or database modules, or use of the same. In view of the disclosure provided herein, software modules are created by various techniques using various machines, software, and languages. The software modules disclosed herein are implemented in a multitude of ways. In various embodiments, a software module comprises a file, a section of code, a programming object, a programming structure, a distributed computing resource, a cloud computing resource, or combinations thereof. In further various embodiments, a software module comprises a plurality of files, a plurality of sections of code, a plurality of programming objects, a plurality of programming structures, a plurality of distributed computing resources, a plurality of cloud computing resources, or combinations thereof. Examples of software modules include a web application, a mobile application, a standalone application, and a distributed or cloud computing application. In some embodiments, software modules are in one computer program or application. In other embodiments, software modules are in more than one computer program or application. In some embodiments, software modules are hosted on one machine. In other embodiments, software modules are hosted on more than one machine. In further embodiments, software modules are hosted on a distributed computing platform such as a cloud computing platform. In some embodiments, software modules are hosted on one or more machines in one location. In other embodiments, software modules are hosted on one or more machines in more than one location.Databases

[0211] In some embodiments, the platforms, systems, media, and methods disclosed herein include one or more databases, or use of the same. Various databases are suitable for storage and retrieval of information, for example customer incident data. Examples of suitable databases include relational databases, non-relational databases, object oriented databases, object databases, entity-relationship model databases, associative databases, XML databases, document oriented databases, and graph databases. Further examples include SQL, PostgreSQL, MySQL, Oracle, DB2, Sybase, and MongoDB. In some embodiments, a database is Internet-based. In further embodiments, a database is web-based. In still further embodiments, a database is cloudWSGR Docket No. 68738-702.601computing-based. In a particular embodiment, a database is a distributed database. In other embodiments, a database is based on one or more local computer storage devices.

[0212] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.EXAMPLESExample 1; Methylated Total Nucleic Acid Sequencing (mTNA-Seq) Library Prep

[0213] An example of a method for mTNA library prep is illustrated in FIGs. 1A-1C. As illustrated in exemplary FIG. 1A, for example, mTNA library preparation 100 first comprised performing ribosomal DNA depletion on an RNA sample 101 to produce r-RNA depleted RNA 102. To perform ribosomal DNA depletion, for example, in a PCR tube (e.g. Ribo-zero) rRNA removal solution was combined with a sample 101 at a volume proportional to the amount of RNA in the sample 101. For example 2pL of rRNA removal solution was used. For example, the volume of the RNase-free water was adjusted in the total RNA sample to a final volume of lOpL. Reaction buffer (e.g. Ribo-zero) was then added, for example, at a volume of 8pL. The solution was mixed gently, for example, by pipetting up and down.

[0214] The rRNA depletion was then continued by performing incubation. The incubation included, for example, placing the sample 101 in a thermocycler to incubate at 65°C for about five minutes. After about five minutes of incubation, the sample 101 was then put on ice for about five minutes.

[0215] The rRNA depletion was then continued in the example shown in FIG. 1A by adding 40pL of magnetic beads to the RNA mixture. The magnetic beads were washed, for example, prior to being added with magnetic beads wash solution. The RNA mixture was then incubatedWSGR Docket No. 68738-702.601for about 15 minutes at room temperature, for example to facilitate rRNA hybridization to the beads. During incubation the RNA mixture was gently mixed by inversion or vortexing briefly about even' 2-3 minutes, for example.

[0216] The rRNA depletion was then continued, for example by magnetic separation. The RNA mixture was placed on a magnetic stand for about 5 minutes, for example, or until the sample was clear. The beads pelleted, for example at the side of the tube. The supernatant was transferred to a new RNase-free tube, for example, the supernatant containing rRNA-depleted RNA.

[0217] The rRNA depletion was then continued with cleanup of the rRNA-depleted RNA, as two volumes, for example about lOOpL of RNA binding buffer was added to the supernatant from, for example, the RNA clean and concentrator kit. The mixture was mixed thoroughly by, for example, pipetting. The mixture was then loaded onto the RNA clean and concentrator column. The loaded column was then centrifuged at about 13,000 rpm for about 1 minute, for example. The column was then washed with, for example, about 400pL of about 80% ethanol twice. The column was, for example, centrifuged after each wash. The rRNA-depleted RNA was then eluted using about 20uL of RNase-free water. The column was eluted by, for example, centrifuging the column at about 13,000 rpm for about 1 minute into a clean RNase-free tube. The elute was the rRNA-depleted RNA sample 102.

[0218] The rRNA depletion was then continued by quantify ing the rRNA-depleted RNA sample 102 using a Qubit™ RNA HS assay. The quality of the rRNA depletion in the rRNA-depleted RNA sample 102 was then assessed by, for example, running an aliquot of the rRNA depletion sample on a bioanalyzer, for example an Agilent Bioanalyzer™ or TapeStation™. Reduction or absence of rRNA peaks, for example, indicated successful depletion of the rRNA-depleted RNA sample 102. The rRNA-depleted RNA sample 102 was then stored at about -80°C.

[0219] Next, the nucleic acid fragmentation was performed on the rRNA-depleted RNA sample 102 to produce the fragmented rRNA-depleted RNA sample 103. The fragmentation may be performed by sonification. The sonification may be performed using a Covaris™ ultrasonicator.

[0220] Next, for example as illustrated in FIG. 1A, RNA adapter ligation was performed on the fragmented rRNA-depleted RNA sample 103 using RNA ligase to produce a ligated RNA mixture 104. Heat denaturation was performed by, for example, combining about lOpL to about 15pL of the fragmented rRNA-depleted RNA sample 103 with about l uL of a 3' ligation adapter in a microcentrifuge tube. In some cases, between IpL and about 25 pL of the fragmented rRNA-depleted RNA sample 103 can be combined with about IpL of a 3' ligation adapter in a microcentrifuge tube. Next, for example, incubation was performed on the mixture at about 65°C for about five minutes to denature secondary RNA structures.WSGR Docket No. 68738-702.601

[0221] The ligation reaction, for example, was then performed by adding about lOpL of the 3' ligation buffer to the denatured RNA adapter mixture. Then, for example, about 3pL of the 3' ligation enzyme mix was added to the reaction. The reaction mixture was then incubated at about 25°C for about 1 hour, for example. A heated lid was used and set to about 50°C for condensation prevention. The ligated RNA mixture 104 was produced.

[0222] Next, for example illustrated in FIG. 1A, cDNA synthesis from RNA was then performed on the ligated RNA mixture 104 to produce the cDNA sample 105. The reaction setup was performed by adding about 8pL of first-strand reaction buffer to the ligated RNA mixture 104. Then about IpL of murine RNase inhibitor was added to the reaction mix for degradation protection. Next, for example, about IpL of ProtoScript II™ reverse transcriptase was added to reverse the transcription process. The reaction mix was then incubated at about 42°C for about 1 hour to facilitate reverse transcription. A heated lid was used and set to about 50°C for condensation prevention. The SPRI bead clean-up was then performed. The cDNA sample 105 was produced from these operations.

[0223] Next, for example illustrated in FIG. 1A, end repair and hairpin adapter ligation was performed on the cDNA sample 105 to produce the hairpin adapter ligation sample 106. To perform the end repair reaction setup, about 3.5pL of NEBNext™ End Prep reaction buffer (NEB, catalog no. E7647) and about 1.5pL of NEBNext™ End Prep enz me mix were added to the cDNA sample 105 and to a spike-in control mixture. The DNA enzyme mix and the spike-in control mixture were then incubated, for example at 20°C for about 30 minutes, followed by a second incubation at about 65°C for about 30 minutes.

[0224] Next, hairpin ligation was performed by adding about 3.75pL of NEBNext™ Adapter 1 to the reaction mix. Adapter 1 was methylated at all cytosines, for example. Additionally, about 0.5pL of ligation enhancer and about 15pL of NEBNext™ ligation master mix (NEB, catalog no. E7647) were added to the mixture along with Adapter 1. The mixture was incubated at about 20°C for about 15 minutes and SPRI bead clean-up was performed to produce the hairpin adapter ligation sample 106.

[0225] Next, as for example illustrated in FIG. IB, strand separation and synthesis was performed on the hairpin ligation sample 106 to produce the separated ssDNA strand sample 112. The hairpin ligation double stranded cDNA of the hairpin ligation sample 106 were prepared by, for example, adding to the hairpin ligation sample 106 about 2pL of 1 OX NEB Buffer™ (NEB, catalog no. B7004S), about 2pL of dNTPs of about lOmM each (Thermo Fisher Scientific, catalog no. R0192). about 2pL of Klenow (exo-)™ (Thermo Fisher Scientific, catalog no. P7010-LC), and about 2pL of T4 PNK (Thermo Fisher Scientific, catalog no. EK0032). TheWSGR Docket No. 68738-702.601mix was then incubated at about 37°C for about 30 minutes, for example. The DNA was denatured by incubating the mix at about 95°C for about 2 minutes.

[0226] As illustrated in FIG. IB, strand synthesis was then performed to create single-hairpin ligated DNA sample 113. The reaction was then cooled at, for example about -0.1°C per second for reannealing. This created single hairpin synthesized DNA strands in the ligated DNA sample 113

[0227] Next, as illustrated in FIG. IB, PCR adapter ligation was performed on the ligated DNA sample 113 to produce the PCR adapter ligation sample 114. PCR adapter ligation was performed, for example, by adding to the ligated DNA sample 113 about 2.5pL of Adapter 2 with methylated cytosines, along with about 0.5pL of ligation enhancer, about 15pL of ligation master mix (NEB, catalog no. E7647). and about 12pL of nuclease-free water (Sigma, catalog no. W4502). The sample was then incubated at about 20°C for about 15 minutes and SPRI bead clean-up was performed. The PCR adapter ligation sample 114 was produced.

[0228] Next, as illustrated in FIG. IB, TET2 oxidation was performed for protection of methylated cytosols in the PCR adapter ligation sample 114 to produce the protected methylated sample 115. TET2 oxidation was performed by adding to the PCR adapter ligation sample 114, for example, about lOpL of reconstituted TET25x supplement buffer (10 mM a-Ketoglutarate, 0.25 M Tris-HCl pH 8.0, 10 mM ATP), about IpL of UDP glucose (Thermo Fisher Scientific, catalog no. EO0831), about IpL of T4 beta-glucosyltransferase (Thermo Fisher Scientific, catalog no. EO0831), about IpL of lOOmM DTT, about 2pL of TET2, and about 5pLof 500mM FE(II) sulfate hexahydrate (diluted 1:1250 with nuclease-free water). The reaction mix was then incubated at about 37°C for about 60 minutes and SPRI bead clean-up was performed. The protected methylated sample 115 was produced.

[0229] Next, APOBEC deamination was performed on the protected methylated sample 115 as illustrated in, for example FIG. IB to produce double stranded DNA 116. The APOBEC-A3A reaction mix was used in APOBEC deamination and was produced by adding to the protected methylated sample 115 about 12.75pL of nuclease-free water, about 17.5pL of 4x APOBEC buffer (200 mM BisTris pH 6.1, 0.4% Tween), about 1.75pL of lOOmM ATP (Sigma, catalog no. A6559), about 3.5pL lOOmM MgCh (Sigma, catalog no. M1028), about 2pL of APOBEC-A3A, and about 2.5pL of UvrD helicase. The reaction mix was then incubated at about 37°C for about 90 minutes to facilitate double stranded DNA unwinding. The double stranded DNA sample 116 was produced. The unwinding of the double stranded DNA sample 116 resulted in the TNA Library strands shown in, for example, FIG. 1C.

[0230] The TNA Library strands of FIG. 1C, for example, are strands of unwound single-strand DNA or RNA that are each include methylated sites, an unwound hairpin loop fragment, one orWSGR Docket No. 68738-702.601more adapter fragments, and PCR primers. The unwound ligated ssDNA strand includes the unwound ssDNA 121, the unwound annealed hairpin loop fragment 122, a first adapter fragment 123, and PCR primers 125. The unwound ligated RNA strand includes the RNA 126, a first adapter fragment 123, a second adapter fragment 124, and PCR primers 125.

[0231] To amplify the ligated single stranded DNA and RNA as illustrated in FIG. 1C, PCR amplification was performed. The PCR reaction mix was prepared by adding to a sample of the strands illustrated in FIG. 1C about 5pL of Abclonal™ Unique Dual Index Primers for Illumina (Abclonal no. RK21624_SetA) and about 25pL of 2X KAPA HiFi U+ Polymerase (KK2802). PCR cycling was performed for 6 cycles for about 80ng of gDNA, about 7 cycles for lOng of cfDNA, and about 8 cycles for about 2ng of cfDNA. PCR was performed on the various samples using a PCR program. The PCR program included an initial denaturation at about 98°C for about 30 seconds. Next, the second denaturation was performed at about 98°C for about 10 seconds. Next, annealing was performed at about 62°C for about 30 seconds, followed by the extension at about 65°C for about 60 seconds. The final extension was then performed at about 65°C for about 5 minutes. SPRI bead clean-up was performed.

[0232] The libraries were quantified, for example, using TapeStation™ D5000 reagents and library quantification methods disclosed herein. Sequencing was performed using methods disclosed herein.Example 2: Protocol for Preparation of Methylated Total Nucleic Acid (mTNA-Seq) Libraries in Tissue Samples

[0233] Step 1: Poly(A) Selection of RNAs

[0234] Protocol:

[0235] Dilute the total RNA with nuclease-free water to a final volume of 50 pl in a nuclease-free 0.2 ml PCR tube and keep on ice.

[0236] Add 40 pl NEBNext Oligo d(T)25 beads per reaction to a 1.5 ml tube.

[0237] Place the tube on the magnetic rack at room temperature for 2 minutes or until the solution is clear.

[0238] Remove and discard all of the supernatant from the tube. Take care not to disturb the beads.

[0239] Remove the tube from the magnetic rack.

[0240] Add 50 pl NEBNext RNA Binding Buffer (2X) to the beads and wash by pipetting up and down 6 times.

[0241] Place the tube on the magnet and incubate at room temperature until the solution is clear (~2 minutes).

[0242] Remove and discard the supernatant from the tube. Take care not to disturb the beads.WSGR Docket No. 68738-702.601

[0243] Remove the tube from the magnet and add 50 p.1 NEBNext RNA Binding Buffer (2X) to the beads and mix by pipetting up and down until beads are homogenous.

[0244] Add 50 pl beads to each RNA sample from Step 1. Mix thoroughly by pipetting up and down 6 times.

[0245] Heat the sample to denature the RNA and to facilitate binding of the poly (A) RNA to the beads. Place in a thermal cycler, with the heated lid set to > 90°C, and run: 2 min at 80°C, 5 min at 25°C, Hold at 25°C.

[0246] Remove tubes from the thermal cycler when the temperature reaches hold at 25°C.

[0247] Place the tubes on the magnetic rack at room temperature for 2 minutes or until the solution is clear.

[0248] Remove and discard all of the supernatant. Take care not to disturb the beads.

[0249] While still on the magnet rinse the beads by gently adding 200 pl of NEBNext Wash Buffer to the tubes to remove unbound RNA.

[0250] Remove and discard all of the supernatant from each tube. Take care not to disturb the beads.

[0251] Remove the tubes from the magnetic rack.

[0252] Table: Master Mix for Poly(A) Selection of RNAsNEBNext RNA Binding Buffer (2X) 50 plTotal Volume i 100 pl

[0253] Add the above mix to each tube containing mRNA bound beads. Mix thoroughly by gently pipetting up and down 6 times.

[0254] Place the tubes in a thermal cycler, with the heated lid set to > 90°C, and run: 2 min at 80°C, 5 min at 25°C, Hold at 25°C.

[0255] Remove the tubes from the thermal cycler when the temperature reaches hold at 25°C.

[0256] Place the tubes on the magnetic stand at room temperature for 2 minutes or until the solution is clear.

[0257] Remove and discard all of the supernatant from each tube.

[0258] While still on the magnet, rinse the beads by gently adding 200 pl of NEBNext Wash Buffer.

[0259] Remove and discard all of the supernatant from the tubes.

[0260] Remove the tubes from the magnetic rack.WSGR Docket No. 68738-702.601

[0261] Elute the mRNA from the beads by adding 17 gil of the NEBNext Tris Buffer, mix by pipetting 6 times and incubate at 80°C for 2 min, then cool to 25°C. Place tubes on magnetic rack for 2 minutes.

[0262] Collect the purified mRNA by transferring 15 pl of the supernatant to a clean nuclease-free PCR tube.

[0263] Step 2: cDNA Synthesis (Two-Step Process)

[0264] Step 2A: First Strand cDNA Synthesis

[0265] Protocol:

[0266] Prepare the reaction as described below:

[0267] Table: Master Mix for First Strand cDNA SynthesisComponent Volume

[0268] Take samples to the thermocycler and run: 25°C for 10 min, 37°C for 120 min, 4°C hold.

[0269] Remove samples from thermocycler and prepare the next step.

[0270] Step 2B: RNase H and Second Strand cDNA Synthesis

[0271] Prepare the RNase H reaction:

[0272] Table: Master Mix for RNase H TreatmentComponent VolumeFirst-strand cDNA from Step 2A : 25.0 pl10X RNase H buffer i 3.0 plRNase H 0.5 plNuclease Free Water : 1.5 plWSGR Docket No. 68738-702.601Total Volume 30.0 pl

[0273] Incubate at 37°C for 20 minutes.

[0274] Perform a clean up using SPRIselect beads at a ratio of 1.5x according to the protocol below:

[0275] Add 45 pL of beads to each sample

[0276] Mix well by pipetting or vortexing

[0277] Incubate at room temperature for 5 minutes

[0278] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0279] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[0280] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[0281] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[0282] Let the beads dry at room temperature

[0283] Add 22 pL of nuclease free water per sample and mix well by pipetting or vortexing

[0284] Incubate at room temperature for 5 minutes

[0285] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0286] Carefully remove 20 pL of supernatant and transfer to a new clean tube

[0287] For the 2nd strand synthesis, prepare the reaction:

[0288] Table: Master Mix for 2nd Strand SynthesisComponent VolumeEluted first-strand cDNA i 20.0 plWSGR Docket No. 68738-702.601

[0289] Take samples to the thermocycler and run: 25°C for 5 min, 37°C for 60 min, 65°C for 10 min.

[0290] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[0291] Add 50 pL of beads to each sample

[0292] Mix well by pipetting or vortexing

[0293] Incubate at room temperature for 5 minutes

[0294] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0295] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[0296] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[0297] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[0298] Let the beads dry at room temperature

[0299] Add 17 pL of nuclease free water per sample and mix well by pipetting or vortexing

[0300] Incubate at room temperature for 5 minutes

[0301] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0302] Carefully remove 15 pL of supernatant and transfer to a new clean tube

[0303] Keep the samples at 4°C or -20°C until next step.

[0304] Step 3: DNA Fragmentation

[0305] Protocol:

[0306] Dilute the isolated DNA with TE lx pH 8.0 to a final volume of 25 pl in a nuclease-free 0.2 ml PCRtube.

[0307] Prepare the fragmentation reaction:

[0308] Table: Master Mix for DNA Fragmentation

[0309] Take samples to the thermocycler and run: 37°C for 35 min, 65°C for 15 min, 4°C hold.WSGR Docket No. 68738-702.601

[0310] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[0311] Add 30 pL of beads to each sample

[0312] Mix well by pipetting or vortexing

[0313] Incubate at room temperature for 5 minutes

[0314] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0315] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[0316] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[0317] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[0318] Let the beads dry at room temperature

[0319] Add 17 pL of nuclease free water per sample and mix well by pipetting or vortexing

[0320] Incubate at room temperature for 5 minutes

[0321] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0322] Carefully remove 15 pL of supernatant and transfer to a new clean tube

[0323] Keep the samples at 4°C or -20°C until next step.

[0324] Step 4: End Repair and dA-Tailing

[0325] Protocol:

[0326] Mix 12.5 pL of cDNA from Step 2 and 12.5 pL of fragmented DNA from Step 3 to generate TNA sample.

[0327] Prepare the reaction:

[0328] Table: Master Mix for End Repair and dA-TailingComponent;Volume

[0329] Take samples to the thermocycler and run: 20°C for 30 min, 65°C for 30 min, 4°C hold.

[0330] Move straight to next step.

[0331] Step 5: Stubby Adapter Ligation

[0332] Protocol:WSGR Docket No. 68738-702.601

[0333] Prepare the reaction:

[0334] Table: Master Mix for Stubby Adapter LigationComponent Volume

[0336] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[0337] Add 47 pL of beads to each sample

[0338] Mix well by pipetting or vortexing

[0339] Incubate at room temperature for 5 minutes

[0340] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0341] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[0342] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[0343] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[0344] Let the beads dry at room temperature

[0345] Add 22 pL of nuclease free water per sample and mix well by pipetting or vortexing

[0346] Incubate at room temperature for 5 minutes

[0347] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0348] Carefully remove 20 pL of supernatant and transfer to a new clean tube

[0349] Proceed to the next step.

[0350] Step 6: Bisulfite Conversion

[0351] Protocol:

[0352] Prepare the reaction by adding 180 pL SuperMethyl Fast Conversion Reagent to 20 pL of the sample from Step 5.

[0353] Mix samples gently by low speed vortex for 5 seconds or pipette.

[0354] Briefly spin down.WSGR Docket No. 68738-702.601

[0355] Take samples to the thermocycler and run: 98°C for 7 min, 4°C hold.

[0356] Add 500 pL Binding Buffer and 10 pL Purification Beads to a low bind tube and mix thoroughly.

[0357] Transfer samples to bead mixture.

[0358] Mix samples gently by low speed vortex for 5 seconds or pipette.

[0359] Incubate for 10 min at room temperature with continuous rotation.

[0360] Briefly spin down.

[0361] Place samples on magnet for 5 minutes.

[0362] Remove and discard the supernatant. Take care not to disturb the beads.

[0363] Remove tube from magnet.

[0364] Add 400 pL Fast Wash Buffer and resuspend the beads.

[0365] Mix samples gently by low speed vortex for 5 seconds or pipette.

[0366] Briefly spin down.

[0367] Place samples on magnet for 3 minutes.

[0368] Remove and discard the supernatant. Take care not to disturb the beads.

[0369] Remove tube from magnet.

[0370] Add 200 pL Fast Desulphonation Buffer.

[0371] Mix samples gently by low speed vortex for 5 seconds or pipette.

[0372] Incubate for 10 min at room temperature with continuous rotation.

[0373] Briefly spin down.

[0374] Place samples on magnet for 3 minutes.

[0375] Remove and discard the supernatant. Take care not to disturb the beads.

[0376] Remove tube from magnet.

[0377] Add 400 pL Fast Wash Buffer and resuspend the beads.

[0378] Mix samples gently by low speed vortex for 5 seconds or pipette.

[0379] Briefly spin down.

[0380] Place samples on magnet for 3 minutes.

[0381] Remove and discard the supernatant. Take care not to disturb the beads.

[0382] Repeat wash lx.

[0383] Remove and discard the supernatant, making sure to leave no residue.

[0384] With tubes uncapped, incubate at 55°C for 5-10 min to completely dry the beads.

[0385] Add 15 pL Fast Elution Buffer.

[0386] Mix samples gently by low speed vortex for 5 seconds or pipette.

[0387] Briefly spin down.

[0388] Place samples on magnet for 1 -3 minutes or until the liquid is clear.WSGR Docket No. 68738-702.601

[0389] Transfer 10 pL of supernatant to anew clean tube.

[0390] Proceed to the next step.

[0391] Step 7: Final Library Amplification

[0392] Protocol:

[0393] Prepare the reaction:

[0394] Table: Master Mix for Library PCRComponent VolumSamples from Step 6 ; 10 pL12.5 pLIndex Primers 2.5 pLTotal Volume 25 pL

[0395] Take samples to the thermocycler and run the program below:

[0396] Table: Cycling Conditions for PCRbelow:

[0398] Add 20 pL of beads to each sample

[0399] Mix well by pipetting or vortexing

[0400] Incubate at room temperature for 5 minutes

[0401] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0402] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[0403] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[0404] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.WSGR Docket No. 68738-702.601

[0405] Let the beads dry at room temperature

[0406] Add 17 pL of nuclease free water per sample and mix well by pipetting or vortexing

[0407] Incubate at room temperature for 5 minutes

[0408] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0409] Carefully remove 15 pL of supernatant and transfer to a new clean tube

[0410] FIG. 22A shows an example of a workflow for Methylated Total Nucleic Acid Sequencing (mTNA-Seq) library preparation for a tissue sample. The extraction utilizes lysates to extract the DNA and RNA from the tissue for multi-omic analysis. FIG. 22B shows an example of end-state sequencing molecules that are RNA-derived and DNA-derived for a tissue sample.Example 3: Protocol for Preparation of Methylated Total Nucleic Acid (mTNA-Seq) Libraries in Plasma Samples

[0411] Step 1: Poly(A) Selection of RNAs

[0412] Protocol:

[0413] Dilute the total RNA with nuclease-free water to a final volume of 50 pl in a nuclease-free 0.2 ml PCR tube and keep on ice.

[0414] Add 40 pl NEBNext Oligo d(T)25 beads per reaction to a 1.5 ml tube.

[0415] Place the tube on the magnetic rack at room temperature for 2 minutes or until the solution is clear.

[0416] Remove and discard all of the supernatant from the tube. Take care not to disturb the beads.

[0417] Remove the tube from the magnetic rack.

[0418] Add 50 pl NEBNext RNA Binding Buffer (2X) to the beads and wash by pipetting up and down 6 times.

[0419] Place the tube on the magnet and incubate at room temperature until the solution is clear (~2 minutes).

[0420] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[0421] Remove the tube from the magnet and add 50 pl NEBNext RNA Binding Buffer (2X) to the beads and mix by pipetting up and down until beads are homogenous.

[0422] Add 50 pl beads to each RNA sample from Step 1. Mix thoroughly by pipetting up and down 6 times.

[0423] Heat the sample to denature the RNA and to facilitate binding of the poly(A) RNA to the beads. Place in a thermal cycler, with the heated lid set to > 90°C, and run: 2 min at 80°C, 5 min at 25°C, Hold at 25°C.WSGR Docket No. 68738-702.601

[0424] Remove tubes from the thermal cycler when the temperature reaches hold at 25°C.

[0425] Place the tubes on the magnetic rack at room temperature for 2 minutes or until the solution is clear.

[0426] Remove and discard all of the supernatant. Take care not to disturb the beads.

[0427] While still on the magnet rinse the beads by gently adding 200 pl of NEBNext Wash Buffer to the tubes to remove unbound RNA.

[0428] Remove and discard all of the supernatant from each tube. Take care not to disturb the beads.

[0429] Remove the tubes from the magnetic rack.

[0430] Table: Master Mix for Poly(A) Selection of RNAs

[0431] Add the above mix to each tube containing mRNA bound beads. Mix thoroughly by gently pipetting up and down 6 times.

[0432] Place the tubes in a thermal cycler, with the heated lid set to > 90°C, and run: 2 min at 80°C, 5 min at 25°C, Hold at 25°C.

[0433] Remove the tubes from the thermal cycler when the temperature reaches hold at 25°C.

[0434] Place the tubes on the magnetic stand at room temperature for 2 minutes or until the solution is clear.

[0435] Remove and discard all of the supernatant from each tube.

[0436] While still on the magnet, rinse the beads by gently adding 200 pl of NEBNext Wash Buffer.

[0437] Remove and discard all of the supernatant from the tubes.

[0438] Remove the tubes from the magnetic rack.

[0439] Elute the mRNA from the beads by adding 17 pl of the NEBNext Tris Buffer, mix by pipetting 6 times and incubate at 80°C for 2 min, then cool to 25°C. Place tubes on magnetic rack for 2 minutes.

[0440] Collect the purified mRNA by transferring 15 pl of the supernatant to a clean nuclease-free PCR tube.

[0441] Step 2: cDNA Synthesis (Two-Step Process)

[0442] Step 2A: First Strand cDNA SynthesisWSGR Docket No. 68738-702.601

[0443] Protocol:

[0444] Prepare the reaction as described below:

[0445] Table: Master Mix for First Strand cDNA Synthesis

[0446] Take samples to the thermocycler and run: 25°C for 10 min, 37°C for 120 min. 4°C hold.

[0447] Remove samples from thermocycler and prepare the next step.

[0448] Step 2B: RNase H and Second Strand cDNA Synthesis

[0449] Prepare the RNase H reaction:

[0450] Table: Master Mix for RNase H TreatmentComponent VolumeFirst-strand cDNA from Step 2A ; 25.0 pl1 OX RNase H buffer 3.0 plRNase H 0.5 plNuclease Free Water 1.5 plTotal Volume 30.0 pl

[0451] Incubate at 37°C for 20 minutes.

[0452] Perform a clean up using SPRIselect beads at a ratio of 1.5x according to the protocol below:

[0453] Add 45 pL of beads to each sample

[0454] Mix well by pipetting or vortexing

[0455] Incubate at room temperature for 5 minutesWSGR Docket No. 68738-702.601

[0456] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0457] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[0458] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[0459] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[0460] Let the beads dry at room temperature

[0461] Add 22 pL of nuclease free water per sample and mix well by pipetting or vortexing

[0462] Incubate at room temperature for 5 minutes

[0463] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0464] Carefully remove 20 pL of supernatant and transfer to a new clean tube

[0465] For the 2nd strand synthesis, prepare the reaction:

[0466] Table: Master Mix for 2nd Strand Synthesis

[0467] Take samples to the thermocycler and run: 25°C for 5 min, 37°C for 60 min, 65°C for 10 min.

[0468] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[0469] Add 50 pL of beads to each sample

[0470] Mix well by pipetting or vortexing

[0471] Incubate at room temperature for 5 minutes

[0472] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnetWSGR Docket No. 68738-702.601

[0473] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[0474] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[0475] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[0476] Let the beads dry at room temperature

[0477] Add 17 pL of nuclease free water per sample and mix well by pipetting or vortexing

[0478] Incubate at room temperature for 5 minutes

[0479] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0480] Carefully remove 15 pL of supernatant and transfer to a new clean tube

[0481] Keep the samples at 4°C or -20°C until next step.

[0482] Step 3: End Repair and dA-Tailing

[0483] Protocol:

[0484] Mix 12.5 pL of cDNA from Step 2 and 12.5 pL of DNA sample (plasma cfDNA does not require fragmentation) to generate TNA sample.

[0485] Prepare the reaction:

[0486] Table: Master Mix for End Repair and dA-Tailing

[0490] Protocol:

[0491] Prepare the reaction:

[0492] Table: Master Mix for Stubby Adapter LigationWSGR Docket No. 68738-702.601NEBNext Ligation Enhancer : 0.5 pLNEBNext Ultra II Ligation MM 15 pLTotal Volume : 47.0 pl.

[0493] Incubate samples at 20°C for 1 hour.

[0494] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[0495] Add 47 pL of beads to each sample

[0496] Mix well by pipetting or vortexing

[0497] Incubate at room temperature for 5 minutes

[0498] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0499] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[0500] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[0501] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[0502] Let the beads dry at room temperature

[0503] Add 22 pL of nuclease free water per sample and mix well by pipetting or vortexing

[0504] Incubate at room temperature for 5 minutes

[0505] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0506] Carefully remove 20 pL of supernatant and transfer to a new clean tube

[0507] Proceed to the next step.

[0508] Step 5: Bisulfite Conversion

[0509] Protocol:

[0510] Prepare the reaction by adding 180 pL SuperMethyl Fast Conversion Reagent to 20 pL of the sample from Step 4.

[0511] Mix samples gently by low speed vortex for 5 seconds or pipette.

[0512] Briefly spin down.

[0513] Take samples to the thermocycler and run: 98°C for 7 min, 4°C hold.

[0514] Add 500 pL Binding Buffer and 10 pL Purification Beads to a low bind tube and mix thoroughly.

[0515] Transfer samples to bead mixture.

[0516] Mix samples gently by low speed vortex for 5 seconds or pipette.

[0517] Incubate for 10 min at room temperature with continuous rotation.WSGR Docket No. 68738-702.601

[0518] Briefly spin down.

[0519] Place samples on magnet for 5 minutes.

[0520] Remove and discard the supernatant. Take care not to disturb the beads.

[0521] Remove tube from magnet.

[0522] Add 400 pL Fast Wash Buffer and resuspend the beads.

[0523] Mix samples gently by low speed vortex for 5 seconds or pipette.

[0524] Briefly spin down.

[0525] Place samples on magnet for 3 minutes.

[0526] Remove and discard the supernatant. Take care not to disturb the beads.

[0527] Remove tube from magnet.

[0528] Add 200 pL Fast Desulphonation Buffer.

[0529] Mix samples gently by low speed vortex for 5 seconds or pipette.

[0530] Incubate for 10 min at room temperature with continuous rotation.

[0531] Briefly spin down.

[0532] Place samples on magnet for 3 minutes.

[0533] Remove and discard the supernatant. Take care not to disturb the beads.

[0534] Remove tube from magnet.

[0535] Add 400 pL Fast Wash Buffer and resuspend the beads.

[0536] Mix samples gently by low speed vortex for 5 seconds or pipette.

[0537] Briefly spin down.

[0538] Place samples on magnet for 3 minutes.

[0539] Remove and discard the supernatant. Take care not to disturb the beads.

[0540] Repeat wash lx.

[0541] Remove and discard the supernatant, making sure to leave no residue.

[0542] With tubes uncapped, incubate at 55°C for 5-10 min to completely dry the beads.

[0543] Add 15 pL Fast Elution Buffer.

[0544] Mix samples gently by low speed vortex for 5 seconds or pipette.

[0545] Briefly spin down.

[0546] Place samples on magnet for 1-3 minutes or until the liquid is clear.

[0547] Transfer 10 pL of supernatant to anew clean tube.

[0548] Proceed to the next step.

[0549] Step 6: Final Library Amplification

[0550] Protocol:

[0551] Prepare the reaction:

[0552] Table: Master Mix for Library PCRWSGR Docket No. 68738-702.601

[0555] Perform a clean up using SPRIselect beads at a ratio of 0.8x according to the protocol below:

[0556] Add 20 pL of beads to each sample

[0557] Mix well by pipetting or vortexing

[0558] Incubate at room temperature for 5 minutes

[0559] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0560] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[0561] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[0562] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[0563] Let the beads dry at room temperature

[0564] Add 17 pL of nuclease free water per sample and mix well by pipetting or vortexing

[0565] Incubate at room temperature for 5 minutes

[0566] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0567] Carefully remove 15 pL of supernatant and transfer to a new clean tubeWSGR Docket No. 68738-702.601

[0568] FIG. 23A shows an example of a workflow for Methylated Total Nucleic Acid Sequencing (mTNA-Seq) library preparation for a plasma sample. The extraction comprises extracting cfDNA and cfRNA from the plasma sample for multi-omic analysis. FIG. 23B shows an example of end-state sequencing molecules that are RNA-derived and DNA-derived for a plasma sample.Example 4: Methylated Total Nucleic Acid Sequencing (mTNA-Seq) Protein Library Prep

[0569] To prepare the protein libraries for mTNA-Seq, as illustrated in for example FIG. 2, antibody-oligonucleotide conjugates were designed, methylated, and conjugated according to methods disclosed herein.

[0570] Next, antibody-oligo conjugates were incubated with proteins as shown in FIG. 2 in the incubation mix 201. The protein sample was diluted to the desired concentration, for example, using an incubation buffer (PBS with 0.05% Tween-20). The antibody-oligo conjugate probes were diluted in the incubation buffer for example, to ensure an excess of probes relative to an expected protein concentration to form the incubation mix 201. The diluted protein sample was then combined with the antibody-oligo conjugate probes in a microcentrifuge tube. The incubation mix 201 was incubated at room temperature at about, for example, 4°C for about 1 to 2 hours with gentle agitation, to facilitate binding between the antibody-oligo probes and the proteins. In some embodiments, washing is performed using gentle washes with an incubation buffer to remove unbound proteins.

[0571] Next, as illustrated in FIG. 2, spin column filtration was performed to separate bound antibody-oligo conjugates from unbound antibody-oligo conjugates. The incubation mix 201 was added to a spin column having a molecular weight limit that retained the protein-antibody-oligo conjugates complexes 202 while allowing free antibody-oligo conjugates to pass through. The column was centrifuged and the flow-through was collected. The retained sample was the bound antibody-oligo conjugates complexes 202.

[0572] Next, the oligonucleotides were released from the antibody-oligo conjugates complexes 202 as shown, for example, in FIG. 2 producing released oligonucleotides 203 that were then purified. The oligonucleotides were released by adding Dithiothreitol (DTT) at a concentration of, for example, about between 10 mM and 50mM in a buffer to the antibody-oligo complexes sample 202 to reduce disulfide bonds. The mix was then incubated at about 37°C for about 30 to 60 minutes, to release the oligonucleotides. Next, the released oligonucleotides 203 were purified by binding the released oligonucleotides to magnetic beads. The oligonucleotides bound to the beads were then washed with a wash buffer to remove remaining proteins and contaminants. The purified oligonucleotides were then eluted from the beads into a cleanWSGR Docket No. 68738-702.601microcentrifuge tube using an elution buffer. The released and purified oligonucleotides 203 were prepared for PCR by annealing PCR primers to create the oligonucleotide library preparation sample 205, which was then replicated with PCR for example, to generate the oligonucleotide PCR sample 204.

[0573] As show n in FIG. 3, the oligonucleotide PCR strands are deaminated, for example, and unwound to be ligated to ssDNA or RNA fragments, or the unwound hairpin fragments thereof. As illustrated in FIG. 3, the oligonucleotide PCR strands 307 were methylated and annealed to PCR primers 303 as described in the exemplary protein library prep method. As illustrated in FIG. 3, the oligonucleotide PCR strands 307 were annealed to an adapter fragment 306. The adapter fragment was annealed to an unwound hairpin fragment 305 which is annealed to an RNA adapter fragment 302 or to an ssDNA fragment 304 or an RNA fragment 301.

[0574] The protein library may be linearized using linearization and methyl conversion. The linearization and methyl conversion may be performed using a bisulfite or enzymatic methyl kit. Lysates may be used to co-extract genomicDNA, totalRNA, and protein from a single tissue sample.Example 5: Preparation of Methylated Total Nucleic Acid Sequencing (mTNA-Seq) Linear Protein libraries in Tissue Samples

[0575] Step 1: Lysate Biotinylation + Cleanup

[0576] Description: Biotinylation of lysate proteins for streptavidin bead capture.

[0577] Protocol:

[0578] If unknown, measure the protein concentration using the A660 protein quantification kit.

[0579] Calculate amount of biotin to add to the protein mixture.

[0580] Table: Biotinylation Reaction Setup

[0581] Note: If the final reaction concentration of biotin is > 16 pM, two sequential Zeba purifications are recommended. If concentration is > 320 pM, three sequential Zeba purifications are recommended.

[0582] Step 2: Streptavidin Bead Preparation

[0583] Description: Initial wash and preparation of Streptavidin beads.

[0584] Protocol:WSGR Docket No. 68738-702.601

[0585] Remove beads from 4°C. vortex, and allow time to equilibrate to room temperature.

[0586] Pipette the required amount of beads into PCR strip tubes. This protocol typically uses 25 pL beads (1 EQ).

[0587] Vortex briefly if needed.

[0588] Wash 2x with 1 EQ PBS:

[0589] Place on magnet for 1 minute until solution is clear.

[0590] Aspirate out supernatant.

[0591] Remove from magnet.

[0592] Pipette 1 EQ volume of PBS into beads and resuspend.

[0593] Notes:

[0594] Ensure binding capacity of beads is greater than input protein concentration.

[0595] Bead capacity is approximately 120 ng / pL.

[0596] Step 3: Lysate Incubation

[0597] Description: Incubation of lysate with streptavidin beads.

[0598] Protocol:

[0599] Place samples on magnetic tube rack.

[0600] Aspirate out supernatant.

[0601] Pipette 1 EQ of lysate into its corresponding sample tube and resuspend.

[0602] Incubate at room temperature for 1 hour.

[0603] Wash 3x with 1 EQ PBS + 1% NP40:

[0604] Place on magnetic rack for 1 minute until solution is clear.

[0605] Aspirate out supernatant.

[0606] Remove from magnet.

[0607] Pipette 1 EQ volume of PBS + 1% NP40 into beads and resuspend.

[0608] Note: Typical protocol is to leave at room temperature for 1 hour with a brief gentle vortex at approximately 30 minutes to resuspend beads.

[0609] Step 4: Probe-Target Complex Formation

[0610] Description: Incubation of AOC (or other probes) with lysate on streptavidin beads.

[0611] Protocol:

[0612] Place samples on magnetic tube rack.

[0613] Aspirate out supernatant.

[0614] Pipette 1 EQ of probe cocktail into its corresponding sample tube and resuspend.

[0615] Incubate at room temperature for 1 hour.

[0616] Wash 6x with 4 EQ PBS + 1% NP40.

[0617] Wash lx with 4 EQ PBS (no NP40).WSGR Docket No. 68738-702.601

[0618] Wash lx with 2 EQ PBS (no NP40).

[0619] Wash lx with 1 EQ PBS (no NP40).

[0620] Transfer the beads in PBS to a new set of PCR strip tubes.

[0621] Note: The bead transfer step at the end is critical for minimizing non-specific binding.

[0622] Step 5: On-Bead Amplification of Oligo Conjugate

[0623] PCR Setup:

[0624] 5 pL bead mixture (from previous step)

[0625] 5 pL 12.5 pM mixture of integration amplification primers (MO166 and MO167)

[0626] 15 pL nuclease free water

[0627] 25 pL NEBNext Ultra II Q5U Master Mix

[0628] Cycling Conditions:

[0629] 98°C x 30 sec (initial denaturation)

[0630] 12 cycles of: 98°C x 10 sec, 66°C x 15 sec, 72°C x 10 sec

[0631] 4°C Hold

[0632] Protocol:

[0633] Perform above PCR.

[0634] Clean and concentrate with the Zymo Oligo Clean and Concentrator kit-5.

[0635] Elute in 15 pL nuclease free water.

[0636] QC: Run amplified product on a 4% agarose gel with ultra low bp DNA ladder. Expect a single band at approximately 70 bp.

[0637] Step 6: Restriction Digestion

[0638] Reaction Setup:

[0639] 2 pL NEB lOx CutSmart Buffer

[0640] 5 pL template DNA (from previous step)

[0641] 0.5 pL NEB Xcml restriction enzyme

[0642] 0.5 pL NEB AhdI restriction enzyme

[0643] 11 pL nuclease free water

[0644] Reaction Conditions:

[0645] 37°C for 1 hour

[0646] 4°C Hold

[0647] Manually add in 0.5 pL NEB SphI and PstI restriction enzy mes

[0648] 37°C for 1 hour

[0649] 4°C Hold

[0650] Protocol:

[0651] Perform above restriction digest.WSGR Docket No. 68738-702.601

[0652] Clean and concentrate with the Zymo Oligo Clean and Concentrator kit-5.

[0653] Elute in 15 pL nuclease free water.

[0654] QC: Run digested product on a 4% agarose E-gel. Expect complete digestion of the original band (~70 bp) and target band at approximately 35 bp.

[0655] Step 7: Custom Y-Shaped Adapter Preparation

[0656] Reaction Setup:

[0657] Table: Adapter Annealing Reaction

[0658] Reaction Conditions:

[0659] Heat to 85°C for 1 minute in thermocycler.

[0660] Slow ramp to 25°C at a rate of 0.2°C per second.

[0661] Note: Annealed adapters can be stored long term at -20°C.

[0662] Method Steps - TNA Processing

[0663] Step 8: Poly(A) Selection of RNAs

[0664] Protocol:

[0665] Dilute the total RNA with nuclease-free water to a final volume of 50 pl in a nuclease-free 0.2 ml PCR tube and keep on ice.

[0666] Add 40 pl NEBNext Oligo d(T)25 beads per reaction to a 1.5 ml tube.

[0667] Place the tube on the magnetic rack at room temperature for 2 minutes or until the solution is clear.

[0668] Remove and discard all of the supernatant from the tube. Take care not to disturb the beads.

[0669] Remove the tube from the magnetic rack.

[0670] Add 50 pl NEBNext RNA Binding Buffer (2X) to the beads and wash by pipetting up and down 6 times.

[0671] Place the tube on the magnet and incubate at room temperature until the solution is clear (~2 minutes).

[0672] Remove and discard the supernatant from the tube. Take care not to disturb the beads.WSGR Docket No. 68738-702.601

[0673] Remove the tube from the magnet and add 50 p.1 NEBNext RNA Binding Buffer (2X) to the beads and mix by pipetting up and down until beads are homogenous.

[0674] Add 50 pl beads to each RNA sample from Step 1. Mix thoroughly by pipetting up and down 6 times.

[0675] Heat the sample to denature the RNA and to facilitate binding of the poly(A) RNA to the beads. Place in a thermal cycler, with the heated lid set to > 90°C, and run: 2 min at 80°C, 5 min at 25°C, Hold at 25°C.

[0676] Remove tubes from the thermal cycler when the temperature reaches hold at 25°C.

[0677] Place the tubes on the magnetic rack at room temperature for 2 minutes or until the solution is clear.

[0678] Remove and discard all of the supernatant. Take care not to disturb the beads.

[0679] While still on the magnet rinse the beads by gently adding 200 pl of NEBNext Wash Buffer to the tubes to remove unbound RNA.

[0680] Remove and discard all of the supernatant from each tube. Take care not to disturb the beads.

[0681] Remove the tubes from the magnetic rack.

[0682] Table: Master Mix for Poly(A) Selection of RNAsNEBNext Tris Buffer 50 plNEBNext RNA Binding Buffer (2X) 50 plTotal Volume 100 pl

[0683] Add the above mix to each tube containing mRNA bound beads. Mix thoroughly by gently pipetting up and down 6 times.

[0684] Place the tubes in a thermal cycler, with the heated lid set to > 90°C, and run: 2 min at 80°C, 5 mm at 25°C. Hold at 25°C.

[0685] Remove the tubes from the thermal cycler when the temperature reaches hold at 25°C.

[0686] Place the tubes on the magnetic stand at room temperature for 2 minutes or until the solution is clear.

[0687] Remove and discard all of the supernatant from each tube.

[0688] While still on the magnet, rinse the beads by gently adding 200 pl of NEBNext Wash Buffer.

[0689] Remove and discard all of the supernatant from the tubes.

[0690] Remove the tubes from the magnetic rack.WSGR Docket No. 68738-702.601

[0691] Elute the mRNA from the beads by adding 17 gil of the NEBNext Tris Buffer, mix by pipetting 6 times and incubate at 80°C for 2 min, then cool to 25°C. Place tubes on magnetic rack for 2 minutes.

[0692] Collect the purified mRNA by transferring 15 pl of the supernatant to a clean nuclease-free PCR tube.

[0693] Step 9: cDNA Synthesis (Two-Step Process)

[0694] Step 9A: First Strand cDNA Synthesis

[0695] Protocol:

[0696] Prepare the reaction as described below:

[0697] Table: Master Mix for First Strand cDNA SynthesisWSGR Docket No. 68738-702.601

[0703] Incubate at 37°C for 20 minutes.

[0704] Perform a clean up using SPRIselect beads at a ratio of 1.5x according to the protocol below:

[0705] Add 45 pL of beads to each sample

[0706] Mix well by pipetting or vortexing

[0707] Incubate at room temperature for 5 minutes

[0708] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0709] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[0710] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[0711] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[0712] Let the beads dry at room temperature

[0713] Add 22 pL of nuclease free water per sample and mix well by pipetting or vortexing

[0714] Incubate at room temperature for 5 minutes

[0715] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0716] Carefully remove 20 pL of supernatant and transfer to a new clean tube

[0717] For the 2nd strand synthesis, prepare the reaction:

[0718] Table: Master Mix for 2nd Strand Synthesis

[0719] Take samples to the thermocycler and run: 25°C for 5 min, 37°C for 60 min, 65°C for 10 min.WSGR Docket No. 68738-702.601

[0720] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[0721] Add 50 pL of beads to each sample

[0722] Mix well by pipetting or vortexing

[0723] Incubate at room temperature for 5 minutes

[0724] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0725] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[0726] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[0727] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[0728] Let the beads dry at room temperature

[0729] Add 17 pL of nuclease free water per sample and mix well by pipetting or vortexing

[0730] Incubate at room temperature for 5 minutes

[0731] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0732] Carefully remove 15 pL of supernatant and transfer to a new clean tube

[0733] Keep the samples at 4°C or -20°C until next step.

[0734] Step 10: DNA Fragmentation

[0735] Protocol:

[0736] Dilute the isolated DNA with TE lx pH 8.0 to a final volume of 25 pl in a nuclease-free 0.2 ml PCRtube.

[0737] Prepare the fragmentation reaction:

[0738] Table: Master Mix for DNA Fragmentation

[0739] Take samples to the thermocycler and run: 37°C for 35 min, 65°C for 15 min, 4°C hold.

[0740] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[0741] Add 30 pL of beads to each sampleWSGR Docket No. 68738-702.601

[0742] Mix well by pipetting or vortexing

[0743] Incubate at room temperature for 5 minutes

[0744] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0745] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[0746] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[0747] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[0748] Let the beads dry' at room temperature

[0749] Add 17 pL of nuclease free water per sample and mix well by pipetting or vortexing

[0750] Incubate at room temperature for 5 minutes

[0751] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0752] Carefully remove 15 pL of supernatant and transfer to a new clean tube

[0753] Keep the samples at 4°C or -20°C until next step.

[0754] Step 11: End Repair and dA-Tailing

[0755] Protocol:

[0756] Mix 12.5 pL of cDNA from Step 9 and 12.5 pL of fragmented DNA from Step 10 to generate TNA sample.

[0757] Prepare the reaction:

[0758] Table: Master Mix for End Repair and dA-TailingComponent VolumeTNA Sample 25.0 pL NEBNext Ultra II End Prep Buffer 3.5 pLNEBNext Ultra II End Prep Enzyme Mix 1.5 pLT otal V olume 30.0 pL

[0759] Take samples to the thermocycler and run: 20°C for 30 min, 65°C for 30 min, 4°C hold.

[0760] Move straight to next step.

[0761] Step 12: Ligation to TNA Library

[0762] Reaction Setup:

[0763] Table: Ligation ReactionComponent VolumeWSGR Docket No. 68738-702.601

[0764] Reaction Conditions: 20°C for duration of reaction (overnight).

[0765] Protocol:

[0766] Set up above reaction and incubate at 20°C.

[0767] After 4-6 hours, spike in 1 pL of Stubby xGen Adapter (15 pM).

[0768] Leave reaction overnight (12-18 hours).

[0769] Perform a clean up using SPRIselect beads at a ratio of 0.8x according to the protocol below:

[0770] Add 18 pL of beads to each sample

[0771] Mix well by pipetting or vortexing

[0772] Incubate at room temperature for 5 minutes

[0773] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0774] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[0775] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[0776] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[0777] Let the beads dry at room temperature

[0778] Add 17 pL of nuclease free water per sample and mix well by pipetting or vortexing

[0779] Incubate at room temperature for 5 minutes

[0780] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0781] Carefully remove 15 pL of supernatant and transfer to a new clean tube

[0782] Step 13: Bisulfite Conversion

[0783] Protocol:

[0784] Prepare the reaction by adding 180 pL SuperMethyl Fast Conversion Reagent to 20 pL of the sample from Step 12.

[0785] Mix samples gently by low speed vortex for 5 seconds or pipette.WSGR Docket No. 68738-702.601

[0786] Briefly spin down.

[0787] Take samples to the thermocycler and run: 98°C for 7 min, 4°C hold.

[0788] Add 500 pL Binding Buffer and 10 pL Purification Beads to a low bind tube and mix thoroughly.

[0789] Transfer samples to bead mixture.

[0790] Mix samples gently by low speed vortex for 5 seconds or pipette.

[0791] Incubate for 10 min at room temperature with continuous rotation.

[0792] Briefly spin down.

[0793] Place samples on magnet for 5 minutes.

[0794] Remove and discard the supernatant. Take care not to disturb the beads.

[0795] Remove tube from magnet.

[0796] Add 400 pL Fast Wash Buffer and resuspend the beads.

[0797] Mix samples gently by low speed vortex for 5 seconds or pipette.

[0798] Briefly spin down.

[0799] Place samples on magnet for 3 minutes.

[0800] Remove and discard the supernatant. Take care not to disturb the beads.

[0801] Remove tube from magnet.

[0802] Add 200 pL Fast Desulphonation Buffer.

[0803] Mix samples gently by low speed vortex for 5 seconds or pipette.

[0804] Incubate for 10 min at room temperature with continuous rotation.

[0805] Briefly spin down.

[0806] Place samples on magnet for 3 minutes.

[0807] Remove and discard the supernatant. Take care not to disturb the beads.

[0808] Remove tube from magnet.

[0809] Add 400 pL Fast Wash Buffer and resuspend the beads.

[0810] Mix samples gently by low- speed vortex for 5 seconds or pipette.

[0811] Briefly spin down.

[0812] Place samples on magnet for 3 minutes.

[0813] Remove and discard the supernatant. Take care not to disturb the beads.

[0814] Repeat wash lx.

[0815] Remove and discard the supernatant, making sure to leave no residue.

[0816] With tubes uncapped, incubate at 55°C for 5-10 min to completely dry the beads.

[0817] Add 15 pL Fast Elution Buffer.

[0818] Mix samples gently by low- speed vortex for 5 seconds or pipette.

[0819] Briefly spin down.WSGR Docket No. 68738-702.601

[0820] Place samples on magnet for 1 -3 minutes or until the liquid is clear.

[0821] Transfer 10 pL of supernatant to anew clean tube.

[0822] Proceed to the next step.

[0823] Step 14: Final Library Amplification

[0824] Protocol:

[0825] Prepare the reaction:

[0826] Table: Master Mix for Library PCR

[0827] Take samples to the thermocycler and run the program below:

[0828] Table: Cycling Conditions for PCR

[0829] Perform a clean up using SPRIselect beads at a ratio of 0.8x according to the protocol below:

[0830] Add 20 pL of beads to each sample

[0831] Mix well by pipetting or vortexing

[0832] Incubate at room temperature for 5 minutes

[0833] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0834] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[0835] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each washWSGR Docket No. 68738-702.601

[0836] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[0837] Let the beads dry at room temperature

[0838] Add 17 pL of nuclease free water per sample and mix well by pipetting or vortexing

[0839] Incubate at room temperature for 5 minutes

[0840] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0841] Carefully remove 15 pL of supernatant and transfer to a new clean tube

[0842] FIG. 26A illustrates an example of a portion of a method workflow for Methylated Total Nucleic Acid (mTNA) sequencing and linear protein detection assaying of a tissue sample.

[0843] FIG. 26B illustrates an example of a continuing portion of a method workflow and endstate sequencing molecules for mTNA sequencing and linear protein detection assaying of a tissue sample. In an example, cfDNA, cfRNA, and protein may be co-extracted from a plasma sample. The cfDNA, cfRNA, and protein may be co-extracted from the same plasma sample sequentially.Example 6: Preparation of Methylated Total Nucleic Acid Sequencing (mTNA-Seq) Linear Protein libraries in Plasma Samples

[0844] Step 1: Lysate Biotinylation + Cleanup

[0845] Description: Biotinylation of lysate proteins for streptavidin bead capture.

[0846] Protocol:

[0847] If unknown, measure the protein concentration using the A660 protein quantification kit.

[0848] Calculate amount of biotin to add to the protein mixture.

[0849] Table: Biotinylation Reaction Setup

[0850] Note: If the final reaction concentration of biotin is > 16 pM, two sequential Zeba purifications are recommended. If concentration is > 320 pM. three sequential Zeba purifications are recommended.

[0851] Step 2: Streptavidin Bead Preparation

[0852] Description: Initial wash and preparation of Streptavidin beads.

[0853] Protocol:

[0854] Remove beads from 4°C. vortex, and allow time to equilibrate to room temperature.WSGR Docket No. 68738-702.601

[0855] Pipette the required amount of beads into PCR strip tubes. This protocol typically uses 25 pL beads (1 EQ).

[0856] V ortex briefly if needed.

[0857] Wash 2x with 1 EQ PBS:

[0858] Place on magnet for 1 minute until solution is clear.

[0859] Aspirate out supernatant.

[0860] Remove from magnet.

[0861] Pipette 1 EQ volume of PBS into beads and resuspend.

[0862] Notes:

[0863] Ensure binding capacity of beads is greater than input protein concentration.

[0864] Bead capacity is approximately 120 ng / pL.

[0865] Step 3: Lysate Incubation

[0866] Description: Incubation of lysate with streptavidin beads.

[0867] Protocol:

[0868] Place samples on magnetic tube rack.

[0869] Aspirate out supernatant.

[0870] Pipette 1 EQ of lysate into its corresponding sample tube and resuspend.

[0871] Incubate at room temperature for 1 hour.

[0872] Wash 3x with 1 EQ PBS + 1% NP40:

[0873] Place on magnetic rack for 1 minute until solution is clear.

[0874] Aspirate out supernatant.

[0875] Remove from magnet.

[0876] Pipette 1 EQ volume of PBS + 1% NP40 into beads and resuspend.

[0877] Note: Typical protocol is to leave at room temperature for 1 hour with a brief gentle vortex at approximately 30 minutes to resuspend beads.

[0878] Step 4: Probe-Target Complex Formation

[0879] Description: Incubation of AOC (or other probes) with lysate on streptavidin beads.

[0880] Protocol:

[0881] Place samples on magnetic tube rack.

[0882] Aspirate out supernatant.

[0883] Pipette 1 EQ of probe cocktail into its corresponding sample tube and resuspend.

[0884] Incubate at room temperature for 1 hour.

[0885] Wash 6x with 4 EQ PBS + 1% NP40.

[0886] Wash lx with 4 EQ PBS (no NP40).

[0887] Wash lx with 2 EQ PBS (no NP40).WSGR Docket No. 68738-702.601

[0888] Wash lx with 1 EQ PBS (no NP40).

[0889] Transfer the beads in PBS to a new set of PCR strip tubes.

[0890] Note: The bead transfer step at the end is critical for minimizing non-specific binding.

[0891] Step 5: On-Bead Amplification of Oligo Conjugate

[0892] PCR Setup:

[0893] 5 pL bead mixture (from previous step)

[0894] 5 pL 12.5 pM mixture of integration amplification primers (MO166 and MO167)

[0895] 15 pL nuclease free water

[0896] 25 pL NEBNext Ultra II Q5U Master Mix

[0897] Cycling Conditions:

[0898] 98°C x 30 sec (initial denaturation)

[0899] 12 cycles of: 98°C x 10 sec, 66°C x 15 sec, 72°C x 10 sec

[0900] 4°C Hold

[0901] Protocol.

[0902] Perform above PCR.

[0903] Clean and concentrate with the Zymo Oligo Clean and Concentrator kit-5.

[0904] Elute in 15 pL nuclease free water.

[0905] QC: Run amplified product on a 4% agarose gel with ultra low bp DNA ladder. Expect a single band at approximately 70 bp.

[0906] Step 6: Restriction Digestion

[0907] Reaction Setup:

[0908] 2 pL NEB lOx CutSmart Buffer

[0909] 5 pL template DNA (from previous step)

[0910] 0.5 pL NEB Xcml restriction enzyme

[0911] 0.5 pL NEB AhdI restriction enzyme

[0912] 11 pL nuclease free water

[0913] Reaction Conditions:

[0914] 37°C for 1 hour

[0915] 4°C Hold

[0916] Manually add in 0.5 pL NEB SphI and PstI restriction enzymes

[0917] 37°C for 1 hour

[0918] 4°C Hold

[0919] Protocol:

[0920] Perform above restriction digest.

[0921] Clean and concentrate with the Zymo Oligo Clean and Concentrator kit-5.WSGR Docket No. 68738-702.601

[0922] Elute in 15 pL nuclease free water.

[0923] QC: Run digested product on a 4% agarose E-gel. Expect complete digestion of the original band (—70 bp) and target band at approximately 35 bp.

[0924] Step 7: Custom Y-Shaped Adapter Preparation

[0925] Reaction Setup:

[0926] Table: Adapter Annealing Reaction

[0927] Reaction Conditions:

[0928] Heat to 85°C for 1 minute in thermocycler.

[0929] Slow ramp to 25°C at a rate of 0.2°C per second.

[0930] Note: Annealed adapters can be stored long term at -20°C.

[0931] Method Steps - TNA Processing

[0932] Step 8: Poly(A) Selection of RNAs

[0933] Protocol:

[0934] Dilute the total RNA with nuclease-free water to a final volume of 50 pl in a nuclease-free 0.2 ml PCR tube and keep on ice.

[0935] Add 40 pl NEBNext Oligo d(T)25 beads per reaction to a 1.5 ml tube.

[0936] Place the tube on the magnetic rack at room temperature for 2 minutes or until the solution is clear.

[0937] Remove and discard all of the supernatant from the tube. Take care not to disturb the beads.

[0938] Remove the tube from the magnetic rack.

[0939] Add 50 pl NEBNext RNA Binding Buffer (2X) to the beads and wash by pipetting up and down 6 times.

[0940] Place the tube on the magnet and incubate at room temperature until the solution is clear (~2 minutes).

[0941] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[0942] Remove the tube from the magnet and add 50 pl NEBNext RNA Binding Buffer (2X) to the beads and mix by pipetting up and down until beads are homogenous.WSGR Docket No. 68738-702.601

[0943] Add 50 pl beads to each RNA sample from Step 1. Mix thoroughly by pipetting up and down 6 times.

[0944] Heat the sample to denature the RNA and to facilitate binding of the poly(A) RNA to the beads. Place in a thermal cycler, with the heated lid set to > 90°C, and run: 2 min at 80°C, 5 min at 25°C, Hold at 25°C.

[0945] Remove tubes from the thermal cycler when the temperature reaches hold at 25°C.

[0946] Place the tubes on the magnetic rack at room temperature for 2 minutes or until the solution is clear.

[0947] Remove and discard all of the supernatant. Take care not to disturb the beads.

[0948] While still on the magnet rinse the beads by gently adding 200 pl of NEBNext Wash Buffer to the tubes to remove unbound RNA.

[0949] Remove and discard all of the supernatant from each tube. Take care not to disturb the beads.

[0950] Remove the tubes from the magnetic rack.

[0951] Table: Master Mix for Poly(A) Selection of RNAsNEBNext Tris Buffer 50 plNEBNext RNA Binding Buffer (2X) 50 plTotal Volume 100 pl

[0952] Add the above mix to each tube containing mRNA bound beads. Mix thoroughly by gently pipetting up and down 6 times.

[0953] Place the tubes in a thermal cycler, with the heated lid set to > 90°C, and run: 2 min at 80°C, 5 min at 25°C, Hold at 25°C.

[0954] Remove the tubes from the thermal cycler when the temperature reaches hold at 25°C.

[0955] Place the tubes on the magnetic stand at room temperature for 2 minutes or until the solution is clear.

[0956] Remove and discard all of the supernatant from each tube.

[0957] While still on the magnet, rinse the beads by gently adding 200 pl of NEBNext Wash Buffer.

[0958] Remove and discard all of the supernatant from the tubes.

[0959] Remove the tubes from the magnetic rack.WSGR Docket No. 68738-702.601

[0960] Elute the mRNA from the beads by adding 17 gil of the NEBNext Tris Buffer, mix by pipetting 6 times and incubate at 80°C for 2 min, then cool to 25°C. Place tubes on magnetic rack for 2 minutes.

[0961] Collect the purified mRNA by transferring 15 pl of the supernatant to a clean nuclease-free PCR tube.

[0962] Step 9: cDNA Synthesis (Two-Step Process)

[0963] Step 9A: First Strand cDNA Synthesis

[0964] Protocol:

[0965] Prepare the reaction as described below:

[0966] Table: Master Mix for First Strand cDNA SynthesisWSGR Docket No. 68738-702.601

[0972] Incubate at 37°C for 20 minutes.

[0973] Perform a clean up using SPRIselect beads at a ratio of 1.5x according to the protocol below:

[0974] Add 45 pL of beads to each sample

[0975] Mix well by pipetting or vortexing

[0976] Incubate at room temperature for 5 minutes

[0977] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0978] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[0979] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[0980] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[0981] Let the beads dry at room temperature

[0982] Add 22 pL of nuclease free water per sample and mix well by pipetting or vortexing

[0983] Incubate at room temperature for 5 minutes

[0984] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0985] Carefully remove 20 pL of supernatant and transfer to a new clean tube

[0986] For the 2nd strand synthesis, prepare the reaction:

[0987] Table: Master Mix for 2nd Strand Synthesis

[0988] Take samples to the thermocycler and run: 25°C for 5 min, 37°C for 60 min, 65°C for 10 min.WSGR Docket No. 68738-702.601

[0989] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[0990] Add 50 pL of beads to each sample

[0991] Mix well by pipetting or vortexing

[0992] Incubate at room temperature for 5 minutes

[0993] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[0994] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[0995] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[0996] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[0997] Let the beads dry at room temperature

[0998] Add 17 pL of nuclease free water per sample and mix well by pipetting or vortexing

[0999] Incubate at room temperature for 5 minutes

[1000] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1001] Carefully remove 15 pL of supernatant and transfer to a new clean tube

[1002] Keep the samples at 4°C or -20°C until next step.

[1003] Step 10: End Repair and dA-Tailing

[1004] Protocol:

[1005] Mix 12.5 pL of cDNA from Step 9 and 12.5 pL of DNA sample (plasma cfDNA does not require fragmentation) to generate TNA sample.

[1006] Prepare the reaction:

[1007] Table: Master Mix for End Repair and dA-Tailing

[1008] Take samples to the thermocycler and run: 20°C for 30 min, 65°C for 30 min, 4°C hold.

[1009] Move straight to next step.

[1010] Step 11: Ligation to TNA Library

[1011] Reaction Setup:WSGR Docket No. 68738-702.601

[1012] Table: Ligation Reaction

[1013] Reaction Conditions: 20°C for duration of reaction (overnight).

[1014] Protocol:

[1015] Set up above reaction and incubate at 20°C.

[1016] After 4-6 hours, spike in 1 pL of Stubby xGen Adapter (15 pM).

[1017] Leave reaction overnight (12-18 hours).

[1018] Perform a clean up using SPRIselect beads at a ratio of 0.8x according to the protocol below:

[1019] Add 18 pL of beads to each sample

[1020] Mix well by pipetting or vortexing

[1021] Incubate at room temperature for 5 minutes

[1022] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1023] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1024] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each w ash

[1025] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1026] Let the beads dry at room temperature

[1027] Add 17 pL of nuclease free water per sample and mix w ell by pipetting or vortexing

[1028] Incubate at room temperature for 5 minutes

[1029] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1030] Carefully remove 15 pL of supernatant and transfer to a new clean tube

[1031] Step 12: Bisulfite Conversion

[1032] Protocol:WSGR Docket No. 68738-702.601

[1033] Prepare the reaction by adding 180 pL SuperMethyl Fast Conversion Reagent to 20 pL of the sample from Step 11.

[1034] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1035] Briefly spin down.

[1036] Take samples to the thermocycler and run: 98°C for 7 min, 4°C hold.

[1037] Add 500 pL Binding Buffer and 10 pL Purification Beads to a low bind tube and mix thoroughly.

[1038] Transfer samples to bead mixture.

[1039] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1040] Incubate for 10 min at room temperature with continuous rotation.

[1041] Briefly spin down.

[1042] Place samples on magnet for 5 minutes.

[1043] Remove and discard the supernatant. Take care not to disturb the beads.

[1044] Remove tube from magnet.

[1045] Add 400 pL Fast Wash Buffer and resuspend the beads.

[1046] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1047] Briefly spin down.

[1048] Place samples on magnet for 3 minutes.

[1049] Remove and discard the supernatant. Take care not to disturb the beads.

[1050] Remove tube from magnet.

[1051] Add 200 pL Fast Desulphonation Buffer.

[1052] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1053] Incubate for 10 min at room temperature with continuous rotation.

[1054] Briefly spin down.

[1055] Place samples on magnet for 3 minutes.

[1056] Remove and discard the supernatant. Take care not to disturb the beads.

[1057] Remove tube from magnet.

[1058] Add 400 pL Fast Wash Buffer and resuspend the beads.

[1059] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1060] Briefly spin down.

[1061] Place samples on magnet for 3 minutes.

[1062] Remove and discard the supernatant. Take care not to disturb the beads.

[1063] Repeat wash lx.

[1064] Remove and discard the supernatant, making sure to leave no residue.

[1065] With tubes uncapped, incubate at 55°C for 5-10 min to completely dry the beads.WSGR Docket No. 68738-702.601

[1066] Add 15 pL Fast Elution Buffer.

[1067] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1068] Briefly spin down.

[1069] Place samples on magnet for 1-3 minutes or until the liquid is clear.

[1070] Transfer 10 pL of supernatant to anew clean tube.

[1071] Proceed to the next step.

[1072] Step 13: Final Library Amplification

[1073] Protocol:

[1074] Prepare the reaction:

[1075] Table: Master Mix for Library PCRSamples from Step 12 10 pL

[1078] Perform a clean up using SPRIselect beads at a ratio of 0.8x according to the protocol below:

[1079] Add 20 pL of beads to each sample

[1080] Mix well by pipetting or vortexing

[1081] Incubate at room temperature for 5 minutes

[1082] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnetWSGR Docket No. 68738-702.601

[1083] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1084] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[1085] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1086] Let the beads dry at room temperature

[1087] Add 17 pL of nuclease free water per sample and mix well by pipetting or vortexing

[1088] Incubate at room temperature for 5 minutes

[1089] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1090] Carefully remove 15 pL of supernatant and transfer to a new clean tube

[1091] FIG. 27A illustrates an example of a portion of a method workflow for mTNA sequencing and linear protein detection assaying of a plasma sample. FIG. 27B illustrates an example of a continuing portion of a method workflow and end-state sequencing molecules for mTNA sequencing and linear protein detection assaying of a plasma sample.Example 7: hairy TNA-Seq Library Preparation

[1092] The following example method was used prepare libraries of total nucleic acids with bisulfite methyl treatment with hairpin / stemloop methods to detect SNPs on single reads for simultaneous multi-omic assay and analysis of DNA sequencing, RNA sequencing, and methylation sequencing from the same sample.

[1093] Poly(A) selection of RNAs was performed. Next, cDNA synthesis was performed in two steps. First, a first strand cDNA synthesis was performed. Next, an RNase H Treatment and second strand cDNA synthesis w ere performed. . DNA fragmentation was then performed to fragment the DNA molecules. The fragmented DNA molecules w ere pooled with the cDNA molecules. End repair and dA-Tailing was then performed on the pooled fragmented DNA and cDNA molecules. Hairpin adapter ligation was then performed to anneal a hairpin adapter to at least a portion of the pooled fragmented DNA molecules and cDNA molecules. Exonuclease treatment w as then applied to the ligated hairpin pooled DNA and cDNA molecules. A USER treatment was then performed on the exonuclease-treated ligated hairpin pooled DNA and cDNA molecules to remove the hairpin adapters. The treated strands of pooled DNA and cDNA molecules were then filled with methyl dCTP. Stubby adapters w ere then ligated to the treated pooled DNA and cDNA molecules. Bisulfite conversion and linearization was then performed on the treated pooled DNA and cDNA molecules. PCR amplification was then performed on the treated pooled DNA and cDNA molecules. The amplified molecules were indexed in the multi-omic library.Example 8: hairy Total Nucleic Acid (TNA-Seq) Library Preparation in Tissue SamplesWSGR Docket No. 68738-702.601

[1094] Step 1: Poly(A) Selection of RNAs

[1095] Protocol:

[1096] Dilute the total RNA with nuclease-free water to a final volume of 50 pl in a nuclease-free 0.2 ml PCR tube and keep on ice.

[1097] Add 40 pl NEBNext Oligo d(T)25 beads per reaction to a 1.5 ml tube.

[1098] Place the tube on the magnetic rack at room temperature for 2 minutes or until the solution is clear.

[1099] Remove and discard all of the supernatant from the tube. Take care not to disturb the beads.

[1100] Remove the tube from the magnetic rack.

[1101] Add 50 pl NEBNext RNA Binding Buffer (2X) to the beads and wash by pipetting up and down 6 times.

[1102] Place the tube on the magnet and incubate at room temperature until the solution is clear (~2 minutes).

[1103] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1104] Remove the tube from the magnet and add 50 pl NEBNext RNA Binding Buffer (2X) to the beads and mix by pipetting up and down until beads are homogenous.

[1105] Add 50 pl beads to each RNA sample from Step 1. Mix thoroughly by pipetting up and down 6 times.

[1106] Heat the sample to denature the RNA and to facilitate binding of the poly(A) RNA to the beads. Place in a thermal cycler, with the heated lid set to > 90°C, and run: 2 min at 80°C, 5 min at 25°C, Hold at 25°C.

[1107] Remove tubes from the thermal cycler when the temperature reaches hold at 25°C.

[1108] Place the tubes on the magnetic rack at room temperature for 2 minutes or until the solution is clear.

[1109] Remove and discard all of the supernatant. Take care not to disturb the beads.

[1110] While still on the magnet rinse the beads by gently adding 200 pl of NEBNext Wash Buffer to the tubes to remove unbound RNA.[Hill Remove and discard all of the supernatant from each tube. Take care not to disturb the beads.[1H2] Remove the tubes from the magnetic rack.[1H3] Table: Master Mix for Poly(A) Selection of RNAsComponent VolumNEBNext Tris Buffer 50 plWSGR Docket No. 68738-702.601NEBNext RNA Binding Buffer (2X) 50 plTotal Volume 100 pl[1H4] Add the above mix to each tube containing mRNA bound beads. Mix thoroughly by gently pipetting up and down 6 times.[1H5] Place the tubes in a thermal cycler, with the heated lid set to > 90°C, and run: 2 min at 80°C, 5 mm at 25°C, Hold at 25°C.[1H6] Remove the tubes from the thermal cycler when the temperature reaches hold at 25°C.

[1117] Place the tubes on the magnetic stand at room temperature for 2 minutes or until the solution is clear.

[1118] Remove and discard all of the supernatant from each tube.[1H9] While still on the magnet, rinse the beads by gently adding 200 pl of NEBNext Wash Buffer.

[1120] Remove and discard all of the supernatant from the tubes.

[1121] Remove the tubes from the magnetic rack.

[1122] Elute the mRNA from the beads by adding 17 pl of the NEBNext Tris Buffer, mix by pipetting 6 times and incubate at 80°C for 2 min, then cool to 25°C. Place tubes on magnetic rack for 2 minutes.[H23] Collect the purified mRNA by transferring 15 pl of the supernatant to a clean nuclease- free PCR tube.

[1124] Step 2: cDNA Synthesis (Two-Step Process)

[1125] Step 2A: First Strand cDNA Synthesis

[1126] Protocol:

[1127] Prepare the reaction as described below:

[1128] Table: Master Mix for First Strand cDNA SynthesisMultiscribe RT 1.5 plRNase Inhibitor 1.0 plNNSR Primers (50 pM) 2.5 plWSGR Docket No. 68738-702.601Total Volume 25.0 pl

[1129] Take samples to the thermocycler and run: 25°C for 10 min, 37°C for 120 min, 4°C hold.

[1130] Remove samples from thermocycler and prepare the next step.

[1131] Step 2B: RNase H and Second Strand cDNA Synthesis

[1132] Prepare the RNase H reaction:

[1133] Table: Master Mix for RNase H Treatment[H34] Incubate at 37°C for 20 minutes.

[1135] Perform a clean up using SPRIselect beads at a ratio of 1.5x according to the protocol below:

[1136] Add 45 pL of beads to each sample

[1137] Mix well by pipetting or vortexing

[1138] Incubate at room temperature for 5 minutes

[1139] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1140] Remove and discard the supernatant from the tube. Take care not to disturb the beads.[H41] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each w ash[H42] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1143] Let the beads dry at room temperature

[1144] Add 22 pL of nuclease free water per sample and mix well by pipetting or vortexing [H45] Incubate at room temperature for 5 minutes

[1146] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1147] Carefully remove 20 pL of supernatant and transfer to a new clean tube

[1148] For the 2nd strand synthesis, prepare the reaction:

[1149] Table: Master Mix for 2nd Strand Sy nthesisWSGR Docket No. 68738-702.601>

[1150] Take samples to the thermocycler and run: 25°C for 5 min, 37°C for 60 min, 65°C for 10 min.

[1151] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:[H52] Add 50 pL of beads to each sample

[1153] Mix well by pipetting or vortexing[H54] Incubate at room temperature for 5 minutes

[1155] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1156] Remove and discard the supernatant from the tube. Take care not to disturb the beads.[H57] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each w ash

[1158] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1159] Let the beads dry at room temperature

[1160] Add 17 pL of nuclease free water per sample and mix w ell by pipetting or vortexing

[1161] Incubate at room temperature for 5 minutes

[1162] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1163] Carefully remove 15 pL of supernatant and transfer to a new clean tube

[1164] Keep the samples at 4°C or -20°C until next step.

[1165] Step 3: DNA Fragmentation

[1166] Protocol:

[1167] Dilute the isolated DNA with TE lx pH 8.0 to a final volume of 25 pl in a nuclease-free 0.2 ml PCRtube.WSGR Docket No. 68738-702.601

[1168] Prepare the fragmentation reaction:

[1169] Table: Master Mix for DNA FragmentationTotal Volume 30.0 pL

[1170] Take samples to the thermocycler and run: 37°C for 35 min, 65°C for 15 min, 4°C hold.

[1171] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[1172] Add 30 pL of beads to each sample[H73] Mix well by pipetting or vortexing

[1174] Incubate at room temperature for 5 minutes

[1175] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1176] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1177] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[1178] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1179] Let the beads dry' at room temperature

[1180] Add 17 pL of nuclease free water per sample and mix w ell by pipetting or vortexing

[1181] Incubate at room temperature for 5 minutes

[1182] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1183] Carefully remove 15 pL of supernatant and transfer to a new' clean tube

[1184] Keep the samples at 4°C or -20°C until next step.

[1185] Step 4: End Repair and dA-Tailing

[1186] Protocol:

[1187] Mix 12.5 pL of cDNA from Step 2 and 12.5 pL of fragmented DNA from Step 3 to generate TNA sample.

[1188] Prepare the reaction:

[1189] Table: Master Mix for End Repair and dA-TailingWSGR Docket No. 68738-702.601Total Volume 30.0 pL

[1190] Take samples to the thermocycler and run: 20°C for 30 min, 65°C for 30 min, 4°C hold.[H91] Move straight to next step.

[1192] Step 5: Hairpin Adapter Ligation

[1193] Protocol:

[1194] Prepare the hairpin for ligation by reannealing:

[1195] Dilute the hairpin adapter to 20 pM in IX annealing buffer.

[1196] Incubate at 80°C for 2 min then ramp temperature to 25°C at a rate of 0.1°C per second.

[1197] Prepare the reaction:

[1198] Table: Master Mix for Hairpin Ligation

[1199] Incubate samples at 16°C overnight.

[1200] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[1201] Add 50 pL of beads to each sample

[1202] Mix well by pipetting or vortexing

[1203] Incubate at room temperature for 5 minutes

[1204] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1205] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1206] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each washWSGR Docket No. 68738-702.601

[1207] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1208] Let the beads dry at room temperature

[1209] Add 22 pL of nuclease free water per sample and mix well by pipetting or vortexing

[1210] Incubate at room temperature for 5 minutes

[1211] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1212] Carefully remove 20 pL of supernatant and transfer to a new clean tube

[1213] Proceed to the next step.

[1214] Step 6: Exonuclease Treatment

[1215] Protocol:

[1216] Prepare the reaction:

[1217] Table: Master Mix for Exonuclease TreatmentExoIII (lOOU / pl) 1.25 pLExoVII (lOU / pl) 1.25 pL1 OX NEB Cutsmart buffer 2.5 pLTotal Volume 25.0 pL

[1218] Incubate samples at 37°C for 1 hour.

[1219] Clean up samples using Oligo Clean & Concentrator:

[1220] Add 25 pl of water to bring volume to 50 pl.

[1221] Add 100 pl Oligo Binding Buffer to 50 pl sample.

[1222] Add 200 pl ethanol (95-100%) and mix well.

[1223] Transfer to Zymo-Spin IC Column and centrifuge at 12,000 x g for 30 seconds.

[1224] Discard flow-through.

[1225] Add 750 pl DNA Wash Buffer and centrifuge at 12,000 x g for 1 minute.

[1226] Transfer column to nuclease-free tube.

[1227] Add 23 pl water and centrifuge at 12,000 x g for 1 minute.

[1228] Proceed to the next step.

[1229] Step 7: USER Treatment

[1230] Protocol:

[1231] Prepare the reaction:

[1232] Table: Master Mix for USER TreatmentWSGR Docket No. 68738-702.601ota oume . p

[1233] Incubate samples at 37°C for 1 hour.

[1234] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[1235] Add 25 pL of beads to each sample

[1236] Mix well by pipetting or vortexing

[1237] Incubate at room temperature for 5 minutes

[1238] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1239] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1240] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each w ash

[1241] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1242] Let the beads dry at room temperature

[1243] Add 20 pL of nuclease free water per sample and mix well by pipetting or vortexing

[1244] Incubate at room temperature for 5 minutes

[1245] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1246] Carefully remove 18 pL of supernatant and transfer to a new clean tube

[1247] Proceed to the next step.

[1248] Step 8: Strand Filling

[1249] Protocol:

[1250] Prepare the reaction:

[1251] Table: Master Mix for Strand FillingWSGR Docket No. 68738-702.601MgSO4 (100 mM) 1.5 pL methyl-dCTP (lOmM) 1.0 pL dNTPs (No dCTP, lOmM) 1.0 pL Bst 2.0 DNA Polymerase (8,000 U / ml) 1.0 pL Total Volume 25.0 pL

[1252] Incubate samples at 65°C for 1 hour.

[1253] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[1254] Add 25 pL of beads to each sample

[1255] Mix well by pipeting or vortexing

[1256] Incubate at room temperature for 5 minutes

[1257] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1258] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1259] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[1260] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1261] Let the beads dry at room temperature

[1262] Add 32 pL of nuclease free water per sample and mix well by pipeting or vortexing

[1263] Incubate at room temperature for 5 minutes

[1264] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1265] Carefully remove 30 pL of supernatant and transfer to a new clean tube

[1266] Proceed to the next step.

[1267] Step 9: Stubby Adapter Ligation

[1268] Protocol:

[1269] Prepare the reaction:

[1270] Table: Master Mix for Stubby Adapter LigationWSGR Docket No. 68738-702.601NEBNext Ultra II Ligation MM 15 pLTotal Volume 47.0 pL

[1271] Incubate samples at 20°C for 1 hour.

[1272] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[1273] Add 47 pL of beads to each sample

[1274] Mix well by pipetting or vortexing

[1275] Incubate at room temperature for 5 minutes

[1276] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1277] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1278] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[1279] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1280] Let the beads dry at room temperature

[1281] Add 22 pL of nuclease free water per sample and mix well by pipetting or vortexing

[1282] Incubate at room temperature for 5 minutes

[1283] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1284] Carefully remove 20 pL of supernatant and transfer to a new clean tube

[1285] Proceed to the next step.

[1286] Step 10: Bisulfite Conversion

[1287] Protocol:

[1288] Prepare the reaction by adding 180 pL SuperMethyl Fast Conversion Reagent to 20 pL of the sample from Step 9.

[1289] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1290] Briefly spin down.

[1291] Take samples to the thermocycler and run: 98°C for 7 min, 4°C hold.

[1292] Add 500 pL Binding Buffer and 10 pL Purification Beads to a low bind tube and mix thoroughly.

[1293] Transfer samples to bead mixture.

[1294] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1295] Incubate for 10 min at room temperature with continuous rotation.

[1296] Briefly spin down.WSGR Docket No. 68738-702.601

[1297] Place samples on magnet for 5 minutes.

[1298] Remove and discard the supernatant. Take care not to disturb the beads.

[1299] Remove tube from magnet.

[1300] Add 400 pL Fast Wash Buffer and resuspend the beads.

[1301] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1302] Briefly spin down.

[1303] Place samples on magnet for 3 minutes.

[1304] Remove and discard the supernatant. Take care not to disturb the beads.

[1305] Remove tube from magnet.

[1306] Add 200 pL Fast Desulphonation Buffer.

[1307] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1308] Incubate for 10 min at room temperature with continuous rotation.

[1309] Briefly spin down.

[1310] Place samples on magnet for 3 minutes.

[1311] Remove and discard the supernatant. Take care not to disturb the beads.

[1312] Remove tube from magnet.

[1313] Add 400 pL Fast Wash Buffer and resuspend the beads.

[1314] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1315] Briefly spin down.

[1316] Place samples on magnet for 3 minutes.

[1317] Remove and discard the supernatant. Take care not to disturb the beads.

[1318] Repeat wash lx.

[1319] Remove and discard the supernatant, making sure to leave no residue.

[1320] With tubes uncapped, incubate at 55°C for 5-10 min to completely dry the beads.

[1321] Add 15 pL Fast Elution Buffer.

[1322] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1323] Briefly spin down.

[1324] Place samples on magnet for 1-3 minutes or until the liquid is clear.

[1325] Transfer 10 pL of supernatant to anew clean tube.

[1326] Proceed to the next step.

[1327] Step 11: Final Library Amplification

[1328] Protocol:

[1329] Prepare the reaction:

[1330] Table: Master Mix for Library PCRWSGR Docket No. 68738-702.601

[1331] Take samples to the thermocycler and run the program below:

[1332] Table: Cycling Conditions for PCR

[1333] Perform a clean up using SPRIselect beads at a ratio of 0.8x according to the protocol below:

[1334] Add 20 pL of beads to each sample

[1335] Mix well by pipetting or vortexing

[1336] Incubate at room temperature for 5 minutes

[1337] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1338] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1339] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[1340] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1341] Let the beads dry at room temperature

[1342] Add 17 pL of nuclease free water per sample and mix well by pipetting or vortexing

[1343] Incubate at room temperature for 5 minutes

[1344] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1345] Carefully remove 15 pL of supernatant and transfer to a new clean tubeWSGR Docket No. 68738-702.601

[1346] The example method is illustrated for a tissue sample in FIGs. 24A-C.Example 9: hairy Total Nucleic Acid (TNA-Seq) Library Preparation in Plasma Samples

[1347] Protocol: Step 1: Poly(A) Selection of RNAs

[1348] Dilute the total RNA with nuclease-free water to a final volume of 50 pl in a nuclease- free 0.2 ml PCR tube and keep on ice.

[1349] Add 40 pl NEBNext Oligo d(T)25 beads per reaction to a 1.5 ml tube.

[1350] Place the tube on the magnetic rack at room temperature for 2 minutes or until the solution is clear.

[1351] Remove and discard all of the supernatant from the tube. Take care not to disturb the beads.

[1352] Remove the tube from the magnetic rack.

[1353] Add 50 pl NEBNext RNA Binding Buffer (2X) to the beads and wash by pipetting up and down 6 times.

[1354] Place the tube on the magnet and incubate at room temperature until the solution is clear (~2 minutes).

[1355] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1356] Remove the tube from the magnet and add 50 pl NEBNext RNA Binding Buffer (2X) to the beads and mix by pipetting up and down until beads are homogenous.

[1357] Add 50 pl beads to each RNA sample from Step 1. Mix thoroughly by pipetting up and down 6 times.

[1358] Heat the sample to denature the RNA and to facilitate binding of the poly(A) RNA to the beads. Place in a thermal cycler, with the heated lid set to > 90°C, and run: 2 min at 80°C, 5 min at 25°C, Hold at 25°C.

[1359] Remove tubes from the thermal cycler when the temperature reaches hold at 25°C.

[1360] Place the tubes on the magnetic rack at room temperature for 2 minutes or until the solution is clear.

[1361] Remove and discard all of the supernatant. Take care not to disturb the beads.

[1362] While still on the magnet rinse the beads by gently adding 200 pl of NEBNext Wash Buffer to the tubes to remove unbound RNA.

[1363] Remove and discard all of the supernatant from each tube. Take care not to disturb the beads.

[1364] Remove the tubes from the magnetic rack.

[1365] Table: Master Mix for Poly(A) Selection of RNAsWSGR Docket No. 68738-702.601NEBNext Tris Buffer 50 plNEBNext RNA Binding Buffer (2X) 50 plTotal Volume 100 pl

[1366] Add the above mix to each tube containing mRNA bound beads. Mix thoroughly by gently pipetting up and down 6 times.

[1367] Place the tubes in a thermal cycler, with the heated lid set to > 90°C, and run: 2 min at 80°C, 5 mm at 25°C. Hold at 25°C.

[1368] Remove the tubes from the thermal cycler when the temperature reaches hold at 25°C.

[1369] Place the tubes on the magnetic stand at room temperature for 2 minutes or until the solution is clear.

[1370] Remove and discard all of the supernatant from each tube.

[1371] While still on the magnet, rinse the beads by gently adding 200 pl of NEBNext Wash Buffer.

[1372] Remove and discard all of the supernatant from the tubes.

[1373] Remove the tubes from the magnetic rack.

[1374] Elute the mRNA from the beads by adding 17 pl of the NEBNext Tris Buffer, mix by pipetting 6 times and incubate at 80°C for 2 min, then cool to 25°C. Place tubes on magnetic rack for 2 minutes.

[1375] Collect the purified mRNA by transferring 15 pl of the supernatant to a clean nuclease- free PCR tube.

[1376] Step 2: cDNA Synthesis (Two-Step Process)

[1377] Step 2A: First Strand cDNA Synthesis

[1378] Protocol:

[1379] Prepare the reaction as described below:

[1380] Table: Master Mix for First Strand cDNA SynthesisRNase Inhibitor 1.0 plWSGR Docket No. 68738-702.601NNSR Primers (50 pM) 2.5 plTotal Volume 25.0 pl

[1381] Take samples to the thermocycler and run: 25°C for 10 min, 37°C for 120 min, 4°C hold.

[1382] Remove samples from thermocycler and prepare the next step.

[1383] Step 2B: RNase H and Second Strand cDNA Synthesis

[1384] Prepare the RNase H reaction:

[1385] Table: Master Mix for RNase H TreatmentTotal Volume 30.0 pl

[1386] Incubate at 37°C for 20 minutes.

[1387] Perform a clean up using SPRIselect beads at a ratio of 1.5x according to the protocol below:

[1388] Add 45 pL of beads to each sample

[1389] Mix well by pipetting or vortexing

[1390] Incubate at room temperature for 5 minutes

[1391] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1392] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1393] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[1394] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1395] Let the beads dry at room temperature

[1396] Add 22 pL of nuclease free water per sample and mix well by pipetting or vortexing

[1397] Incubate at room temperature for 5 minutes

[1398] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1399] Carefully remove 20 pL of supernatant and transfer to a new clean tube

[1400] For the 2nd strand synthesis, prepare the reaction:- Ill -WSGR Docket No. 68738-702.601

[1401] Table: Master Mix for 2nd Strand Synthesis

[1402] Take samples to the thermocycler and run: 25°C for 5 min, 37°C for 60 min, 65°C for 10 min.

[1403] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[1404] Add 50 pL of beads to each sample

[1405] Mix well by pipetting or vortexing

[1406] Incubate at room temperature for 5 minutes

[1407] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1408] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1409] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[1410] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1411] Let the beads dry at room temperature

[1412] Add 17 pL of nuclease free water per sample and mix well by pipetting or vortexing

[1413] Incubate at room temperature for 5 minutes

[1414] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1415] Carefully remove 15 pL of supernatant and transfer to a new clean tube

[1416] Keep the samples at 4°C or -20°C until next step.

[1417] Step 3: End Repair and dA-Tailing

[1418] Protocol:WSGR Docket No. 68738-702.601

[1419] Mix 12.5 pL of cDNA from Step 2 and 12.5 pL of DNA sample (plasma cfDNA does not require fragmentation) to generate TNA sample.

[1420] Prepare the reaction:

[1421] Table: Master Mix for End Repair and dA-Tailing

[1422] Take samples to the thermocycler and run: 20°C for 30 min, 65°C for 30 min, 4°C hold.

[1423] Move straight to next step.

[1424] Step 4: Hairpin Adapter Ligation

[1425] Protocol:

[1426] Prepare the hairpin for ligation by reannealing:

[1427] Dilute the hairpin adapter to 20 pM in IX annealing buffer.

[1428] Incubate at 80°C for 2 min then ramp temperature to 25°C at a rate of 0.1°C per second.

[1429] Prepare the reaction:

[1430] Table: Master Mix for Hairpin Ligation

[1431] Incubate samples at 16°C overnight.

[1432] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[1433] Add 50 pL of beads to each sample

[1434] Mix well by pipetting or vortexing

[1435] Incubate at room temperature for 5 minutesWSGR Docket No. 68738-702.601

[1436] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1437] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1438] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[1439] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1440] Let the beads dry at room temperature

[1441] Add 22 pL of nuclease free water per sample and mix well by pipetting or vortexing

[1442] Incubate at room temperature for 5 minutes

[1443] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1444] Carefully remove 20 pL of supernatant and transfer to a new clean tube

[1445] Proceed to the next step.

[1446] Step 5: Exonuclease Treatment

[1447] Protocol:

[1448] Prepare the reaction:

[1449] Table: Master Mix for Exonuclease Treatment

[1450] Incubate samples at 37°C for 1 hour.

[1451] Clean up samples using Oligo Clean & Concentrator:

[1452] Add 25 pl of water to bring volume to 50 pl.

[1453] Add 100 pl Oligo Binding Buffer to 50 pl sample.

[1454] Add 200 pl ethanol (95-100%) and mix well.

[1455] Transfer to Zymo-Spin IC Column and centrifuge at 12,000 x g for 30 seconds.

[1456] Discard flow-through.

[1457] Add 750 pl DNA Wash Buffer and centrifuge at 12,000 x g for 1 minute.

[1458] Transfer column to nuclease-free tube.

[1459] Add 23 pl water and centrifuge at 12,000 x g for 1 minute.WSGR Docket No. 68738-702.601

[1460] Proceed to the next step.

[1461] Step 6: USER Treatment

[1462] Protocol:

[1463] Prepare the reaction:

[1464] Table: Master Mix for USER Treatment

[1465] Incubate samples at 37°C for 1 hour.

[1466] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[1467] Add 25 pL of beads to each sample

[1468] Mix well by pipetting or vortexing

[1469] Incubate at room temperature for 5 minutes

[1470] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1471] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1472] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[1473] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1474] Let the beads dry at room temperature

[1475] Add 20 pL of nuclease free water per sample and mix well by pipetting or vortexing

[1476] Incubate at room temperature for 5 minutes

[1477] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1478] Carefully remove 18 pL of supernatant and transfer to a new clean tube

[1479] Proceed to the next step.

[1480] Step 7: Strand Filling

[1481] Protocol:

[1482] Prepare the reaction:WSGR Docket No. 68738-702.601

[1483] Table: Master Mix for Strand Filling

[1484] Incubate samples at 65°C for 1 hour.

[1485] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[1486] Add 25 pL of beads to each sample

[1487] Mix well by pipetting or vortexing

[1488] Incubate at room temperature for 5 minutes

[1489] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1490] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1491] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each w ash

[1492] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1493] Let the beads dry at room temperature

[1494] Add 32 pL of nuclease free water per sample and mix w ell by pipetting or vortexing

[1495] Incubate at room temperature for 5 minutes

[1496] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1497] Carefully remove 30 pL of supernatant and transfer to a new clean tube

[1498] Proceed to the next step.

[1499] Step 8: Stubby Adapter Ligation

[1500] Protocol:

[1501] Prepare the reaction:

[1502] Table: Master Mix for Stubby Adapter LigationWSGR Docket No. 68738-702.601ota oume . p

[1503] Incubate samples at 20°C for 1 hour.

[1504] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[1505] Add 47 pL of beads to each sample

[1506] Mix well by pipetting or vortexing

[1507] Incubate at room temperature for 5 minutes

[1508] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1509] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1510] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each w ash

[1511] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1512] Let the beads dry at room temperature

[1513] Add 22 pL of nuclease free water per sample and mix well by pipetting or vortexing

[1514] Incubate at room temperature for 5 minutes

[1515] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1516] Carefully remove 20 pL of supernatant and transfer to a new clean tube

[1517] Proceed to the next step.

[1518] Step 9: Bisulfite Conversion

[1519] Protocol:

[1520] Prepare the reaction by adding 180 pL SuperMethyl Fast Conversion Reagent to 20 pL of the sample from Step 8.

[1521] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1522] Briefly spin down.

[1523] Take samples to the thermocycler and run: 98°C for 7 min, 4°C hold.WSGR Docket No. 68738-702.601

[1524] Add 500 pL Binding Buffer and 10 pL Purification Beads to a low bind tube and mix thoroughly.

[1525] Transfer samples to bead mixture.

[1526] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1527] Incubate for 10 min at room temperature with continuous rotation.

[1528] Briefly spin down.

[1529] Place samples on magnet for 5 minutes.

[1530] Remove and discard the supernatant. Take care not to disturb the beads.

[1531] Remove tube from magnet.

[1532] Add 400 pL Fast Wash Buffer and resuspend the beads.

[1533] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1534] Briefly spin down.

[1535] Place samples on magnet for 3 minutes.

[1536] Remove and discard the supernatant. Take care not to disturb the beads.

[1537] Remove tube from magnet.

[1538] Add 200 pL Fast Desulphonation Buffer.

[1539] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1540] Incubate for 10 min at room temperature with continuous rotation.

[1541] Briefly spin down.

[1542] Place samples on magnet for 3 minutes.

[1543] Remove and discard the supernatant. Take care not to disturb the beads.

[1544] Remove tube from magnet.

[1545] Add 400 pL Fast Wash Buffer and resuspend the beads.

[1546] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1547] Briefly spin down.

[1548] Place samples on magnet for 3 minutes.

[1549] Remove and discard the supernatant. Take care not to disturb the beads.

[1550] Repeat wash lx.

[1551] Remove and discard the supernatant, making sure to leave no residue.

[1552] With tubes uncapped, incubate at 55°C for 5-10 min to completely dry the beads.

[1553] Add 15 pL Fast Elution Buffer.

[1554] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1555] Briefly spin down.

[1556] Place samples on magnet for 1 -3 minutes or until the liquid is clear.

[1557] Transfer 10 pL of supernatant to anew clean tube.WSGR Docket No. 68738-702.601

[1558] Proceed to the next step.

[1559] Step 10: Final Library Amplification

[1560] Protocol:

[1561] Prepare the reaction:

[1562] Table: Master Mix for Library PCRbelow:

[1566] Add 20 pL of beads to each sample

[1567] Mix well by pipetting or vortexing

[1568] Incubate at room temperature for 5 minutes

[1569] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1570] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1571] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[1572] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1573] Let the beads dry at room temperatureWSGR Docket No. 68738-702.601

[1574] Add 17 pL of nuclease free water per sample and mix well by pipeting or vortexing

[1575] Incubate at room temperature for 5 minutes

[1576] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1577] Carefully remove 15 pL of supernatant and transfer to a new clean tube

[1578] The example method is illustrated for a plasma sample in FIGs.25A-C.Example 10: hairy TNA-Seq and protein Library Preparation

[1579] The following example method was used prepare libraries of total nucleic acids and proteins with bisulfite methyl treatment with hairpin / stemloop methods to detect SNPs and protein biomarkers on single reads for simultaneous multi-omic assaying and analysis of DNA sequencing, RNA sequencing, methylation sequencing, and proteomic assaying from the same sample.

[1580] Lysate biotinylation and cleanup was first performed for streptavidin bead capture of proteins. Streptavidin beads were washed and prepared for binding to proteins. Next, the streptavidin beads were incubated with lysate. Probes such as AOC were incubated with lysate on the streptavidin beads to generate a probe-target complex formation. PCR was performed for on-bead amplification of oligonucleotide conjugates. Restriction digestion was performed. Custom Y-shaped adapters were prepared for annealing to the restricted protein molecules. Poly(A) selection of RNAs was then performed. Next, cDNA synthesis was performed in two steps. A first strand cDNA synthesis was performed, then a second strand cDNA synthesis was performed using an RNase enzyme. DNA fragmentation was then performed to fragment the DNA molecules. The fragmented DNA molecules were pooled with the cDNA molecules. End repair and dA-Tailing was then performed on the pooled fragmented DNA and cDNA molecules. Hairpin adapter ligation was then performed to anneal a hairpin adapter to at least a portion of the pooled fragmented DNA molecules and cDNA molecules. Exonuclease treatment was then applied to the ligated hairpin pooled DNA and cDNA molecules. A USER treatment was then performed on the exonuclease-treated ligated hairpin pooled DNA and cDNA molecules to remove the hairpin adapters. The treated strands of pooled DNA and cDNA molecules were then filled with methyl dCTP. Stubby adapters were then ligated to the treated pooled DNA and cDNA molecules. Bisulfite conversion and linearization was then performed on the treated pooled DNA and cDNA molecules. PCR amplification was then performed on the treated pooled DNA and cDNA molecules and the restricted protein molecules annealed to the Y-shaped adapters. The amplified molecules were indexed in the multi-omic library.Example 11: hairy TNA-Seq and Linear protein Library Preparation in Tissue SamplesWSGR Docket No. 68738-702.601

[1581] The restricted protein molecules may be linearized. Lysates may be used to extract proteins, DNA molecules, and RNA molecules from a single tissue sample.

[1582] Step 1: Lysate Biotinylation + Cleanup

[1583] Description: Biotinylation of lysate proteins for streptavidin bead capture.

[1584] Protocol:

[1585] If unknow n, measure the protein concentration using the A660 protein quantification kit.

[1586] Calculate amount of biotin to add to the protein mixture.

[1587] Table: Biotinylation Reaction Setup

[1588] Note: If the final reaction concentration of biotin is > 16 pM, two sequential Zeba purifications are recommended. If concentration is > 320 pM, three sequential Zeba purifications are recommended.

[1589] Step 2: Streptavidin Bead Preparation

[1590] Description: Initial wash and preparation of Streptavidin beads.

[1591] Protocol:

[1592] Remove beads from 4°C, vortex, and allow time to equilibrate to room temperature.

[1593] Pipette the required amount of beads into PCR strip tubes. This protocol typically uses 25 pL beads (1 EQ).

[1594] Vortex briefly if needed.

[1595] Wash 2x with 1 EQ PBS:

[1596] Place on magnet for 1 minute until solution is clear.

[1597] Aspirate out supernatant.

[1598] Remove from magnet.

[1599] Pipette 1 EQ volume of PBS into beads and resuspend.

[1600] Notes:

[1601] Ensure binding capacity of beads is greater than input protein concentration.

[1602] Bead capacity is approximately 120 ng / pL.

[1603] Step 3: Lysate Incubation

[1604] Description: Incubation of lysate with streptavidin beads.WSGR Docket No. 68738-702.601

[1605] Protocol:

[1606] Place samples on magnetic tube rack.

[1607] Aspirate out supernatant.

[1608] Pipette 1 EQ of lysate into its corresponding sample tube and resuspend.

[1609] Incubate at room temperature for 1 hour.

[1610] Wash 3x with 1 EQ PBS + 1% NP40:11611] Place on magnetic rack for 1 minute until solution is clear.

[1612] Aspirate out supernatant.

[1613] Remove from magnet.

[1614] Pipette 1 EQ volume of PBS + 1% NP40 into beads and resuspend.

[1615] Note: Typical protocol is to leave at room temperature for 1 hour with a brief gentle vortex at approximately 30 minutes to resuspend beads.

[1616] Step 4: Probe-Target Complex Formation

[1617] Description: Incubation of AOC (or other probes) with lysate on streptavidin beads.

[1618] Protocol:

[1619] Place samples on magnetic tube rack.

[1620] Aspirate out supernatant.

[1621] Pipette 1 EQ of probe cocktail into its corresponding sample tube and resuspend.

[1622] Incubate at room temperature for 1 hour.

[1623] Wash 6x with 4 EQ PBS + 1% NP40.

[1624] Wash lx with 4 EQ PBS (no NP40).

[1625] Wash lx with 2 EQ PBS (no NP40).

[1626] Wash lx with 1 EQ PBS (no NP40).

[1627] Transfer the beads in PBS to a new set of PCR strip tubes.

[1628] Note: The bead transfer step at the end is critical for minimizing non-specific binding.

[1629] Step 5: On-Bead Amplification of Oligo Conjugate

[1630] PCR Setup:

[1631] 5 pL bead mixture (from previous step)

[1632] 5 pL 12.5 pM mixture of integration amplification primers (MO166 and MO167)

[1633] 15 pL nuclease free water

[1634] 25 pL NEBNext Ultra II Q5U Master Mix

[1635] Cycling Conditions:

[1636] 98°C x 30 sec (initial denaturation)

[1637] 12 cycles of: 98°C x 10 sec, 66°C x 15 sec, 72°C x 10 sec

[1638] 4°C HoldWSGR Docket No. 68738-702.601

[1639] Protocol:

[1640] Perform above PCR.

[1641] Clean and concentrate with the Zymo Oligo Clean and Concentrator kit-5.

[1642] Elute in 15 pL nuclease free water.

[1643] QC: Run amplified product on a 4% agarose gel with ultra low bp DNA ladder. Expect a single band at approximately 70 bp.

[1644] Step 6: Restriction Digestion

[1645] Reaction Setup:

[1646] 2 pL NEB lOx CutSmart Buffer

[1647] 5 pL template DNA (from previous step)

[1648] 0.5 pL NEB Xcml restriction enzyme

[1649] 0.5 pL NEB AhdI restriction enzyme

[1650] 11 pL nuclease free water

[1651] Reaction Conditions:

[1652] 37°C for 1 hour

[1653] 4°C Hold

[1654] Manually add in 0.5 pL NEB SphI and PstI restriction enzymes

[1655] 37°C for 1 hour

[1656] 4°C Hold

[1657] Protocol:

[1658] Perform above restriction digest.

[1659] Clean and concentrate with the Zymo Oligo Clean and Concentrator kit-5.

[1660] Elute in 15 pL nuclease free water.

[1661] QC: Run digested product on a 4% agarose E-gel. Expect complete digestion of the original band (~70 bp) and target band at approximately 35 bp.

[1662] Step 7: Custom Y-Shaped Adapter Preparation

[1663] Reaction Setup:

[1664] Table: Adapter Annealing ReactionComponent : VolumeWSGR Docket No. 68738-702.601

[1665] Reaction Conditions:

[1666] Heat to 85°C for 1 minute in thermocycler.

[1667] Slow ramp to 25°C at a rate of 0.2°C per second.

[1668] Note: Annealed adapters can be stored long term at -20°C.

[1669] Method Steps - TNA Processing

[1670] Step 8: Poly(A) Selection of RNAs

[1671] Protocol:

[1672] Dilute the total RNA with nuclease-free water to a final volume of 50 pl in a nuclease-free 0.2 ml PCR tube and keep on ice.

[1673] Add 40 pl NEBNext Oligo d(T)25 beads per reaction to a 1.5 ml tube.

[1674] Place the tube on the magnetic rack at room temperature for 2 minutes or until the solution is clear.

[1675] Remove and discard all of the supernatant from the tube. Take care not to disturb the beads.

[1676] Remove the tube from the magnetic rack.

[1677] Add 50 pl NEBNext RNA Binding Buffer (2X) to the beads and wash by pipetting up and down 6 times.

[1678] Place the tube on the magnet and incubate at room temperature until the solution is clear (~2 minutes).

[1679] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1680] Remove the tube from the magnet and add 50 pl NEBNext RNA Binding Buffer (2X) to the beads and mix by pipetting up and dow n until beads are homogenous.

[1681] Add 50 pl beads to each RNA sample from Step 1. Mix thoroughly by pipetting up and down 6 times.

[1682] Heat the sample to denature the RNA and to facilitate binding of the poly(A) RNA to the beads. Place in a thermal cycler, with the heated lid set to > 90°C, and run: 2 min at 80°C, 5 min at 25°C, Hold at 25°C.

[1683] Remove tubes from the thermal cycler when the temperature reaches hold at 25°C.

[1684] Place the tubes on the magnetic rack at room temperature for 2 minutes or until the solution is clear.

[1685] Remove and discard all of the supernatant. Take care not to disturb the beads.

[1686] While still on the magnet rinse the beads by gently adding 200 pl of NEBNext Wash Buffer to the tubes to remove unbound RNA.

[1687] Remove and discard all of the supernatant from each tube. Take care not to disturb the beads.WSGR Docket No. 68738-702.601

[1688] Remove the tubes from the magnetic rack.

[1689] Table: Master Mix for Poly(A) Selection of RNAsComponent Volum

[1690] Add the above mix to each tube containing mRNA bound beads. Mix thoroughly by gently pipetting up and down 6 times.

[1691] Place the tubes in a thermal cycler, with the heated lid set to > 90°C, and run: 2 min at 80°C, 5 min at 25°C. Hold at 25°C.

[1692] Remove the tubes from the thermal cycler when the temperature reaches hold at 25°C.

[1693] Place the tubes on the magnetic stand at room temperature for 2 minutes or until the solution is clear.

[1694] Remove and discard all of the supernatant from each tube.

[1695] While still on the magnet, rinse the beads by gently adding 200 pl of NEBNext Wash Buffer.

[1696] Remove and discard all of the supernatant from the tubes.

[1697] Remove the tubes from the magnetic rack.

[1698] Elute the mRNA from the beads by adding 17 pl of the NEBNext Tris Buffer, mix by pipetting 6 times and incubate at 80°C for 2 min, then cool to 25°C. Place tubes on magnetic rack for 2 minutes.

[1699] Collect the purified mRNA by transferring 15 pl of the supernatant to a clean nuclease-free PCR tube.

[1700] Step 9: cDNA Synthesis (Two-Step Process)

[1701] Step 9A: First Strand cDNA Synthesis

[1702] Protocol:

[1703] Prepare the reaction as described below:

[1704] Table: Master Mix for First Strand cDNA SynthesisWSGR Docket No. 68738-702.601

[1705] Take samples to the thermocycler and run: 25°C for 10 min, 37°C for 120 min, 4°C hold.

[1706] Remove samples from thermocycler and prepare the next step.

[1707] Step 9B: RNase H and Second Strand cDNA Synthesis

[1708] Prepare the RNase H reaction:

[1709] Table: Master Mix for RNase H TreatmentC ompon ent VolumeFirst-strand cDNA from Step 2A : 25.0 pl1 OX RNase H buffer 3.0 plRNase H 0.5 plNuclease Free Water 1.5 plTotal Volume 30.0 pl

[1710] Incubate at 37°C for 20 minutes.

[1711] Perform a clean up using SPRIselect beads at a ratio of 1.5x according to the protocol below:

[1712] Add 45 pL of beads to each sample

[1713] Mix well by pipetting or vortexing

[1714] Incubate at room temperature for 5 minutes

[1715] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1716] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1717] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[1718] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1719] Let the beads dry at room temperature

[1720] Add 22 pL of nuclease free water per sample and mix well by pipetting or vortexing

[1721] Incubate at room temperature for 5 minutesWSGR Docket No. 68738-702.601

[1722] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1723] Carefully remove 20 pL of supernatant and transfer to a new clean tube

[1724] For the 2nd strand synthesis, prepare the reaction:

[1726] Take samples to the thermocycler and run: 25°C for 5 min, 37°C for 60 min, 65°C for 10 min.

[1727] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[1728] Add 50 pL of beads to each sample

[1729] Mix well by pipetting or vortexing

[1730] Incubate at room temperature for 5 minutes

[1731] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1732] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1733] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[1734] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1735] Let the beads dry at room temperature

[1736] Add 17 pL of nuclease free water per sample and mix well by pipetting or vortexing

[1737] Incubate at room temperature for 5 minutes

[1738] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1739] Carefully remove 15 pL of supernatant and transfer to a new clean tubeWSGR Docket No. 68738-702.601

[1740] Keep the samples at 4°C or -20°C until next step.

[1741] Step 10: DNA Fragmentation

[1742] Protocol:

[1743] Dilute the isolated DNA with TE lx pH 8.0 to a final volume of 25 pl in a nuclease-free 0.2 ml PCRtube.

[1744] Prepare the fragmentation reaction:

[1745] Table: Master Mix for DNA Fragmentationbelow:

[1748] Add 30 pL of beads to each sample

[1749] Mix well by pipetting or vortexing

[1750] Incubate at room temperature for 5 minutes

[1751] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1752] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1753] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[1754] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1755] Let the beads dry at room temperature

[1756] Add 17 pL of nuclease free water per sample and mix well by pipetting or vortexing

[1757] Incubate at room temperature for 5 minutes

[1758] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1759] Carefully remove 15 pL of supernatant and transfer to a new clean tube

[1760] Keep the samples at 4°C or -20°C until next step.

[1761] Step 11: End Repair and dA-Tailing

[1762] Protocol:WSGR Docket No. 68738-702.601

[1763] Mix 12.5 pL of cDNA from Step 9 and 12.5 pL of fragmented DNA from Step 10 to generate TNA sample.

[1764] Prepare the reaction:

[1765] Table: Master Mix for End Repair and dA-Tailing

[1766] Take samples to the thermocycler and run: 20°C for 30 min, 65°C for 30 min, 4°C hold.

[1767] Move straight to next step.

[1768] Step 12: Hairpin Adapter Ligation

[1769] Protocol:

[1770] Prepare the hairpin for ligation by reannealing:

[1771] Dilute the hairpin adapter to 20 pM in IX annealing buffer.

[1772] Incubate at 80°C for 2 min then ramp temperature to 25°C at a rate of 0.1°C per second.

[1773] Prepare the reaction:

[1774] Table: Master Mix for Hairpin Ligation

[1776] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[1777] Add 50 pL of beads to each sample

[1778] Mix well by pipetting or vortexing

[1779] Incubate at room temperature for 5 minutesWSGR Docket No. 68738-702.601

[1780] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1781] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1782] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[1783] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1784] Let the beads dry at room temperature

[1785] Add 22 pL of nuclease free water per sample and mix well by pipetting or vortexing

[1786] Incubate at room temperature for 5 minutes

[1787] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1788] Carefully remove 20 pL of supernatant and transfer to a new clean tube

[1789] Proceed to the next step.

[1790] Step 13: Exonuclease Treatment

[1791] Protocol:

[1792] Prepare the reaction:

[1793] Table: Master Mix for Exonuclease Treatment

[1794] Incubate samples at 37°C for 1 hour.

[1795] Clean up samples using Oligo Clean & Concentrator:

[1796] Add 25 pl of water to bring volume to 50 pl.

[1797] Add 100 pl Oligo Binding Buffer to 50 pl sample.

[1798] Add 200 pl ethanol (95-100%) and mix well.

[1799] Transfer to Zymo-Spin IC Column and centrifuge at 12,000 x g for 30 seconds.

[1800] Discard flow-through.

[1801] Add 750 pl DNA Wash Buffer and centrifuge at 12,000 x g for 1 minute.

[1802] Transfer column to nuclease-free tube.WSGR Docket No. 68738-702.601

[1803] Add 23 pl water and centrifuge at 12,000 x g for 1 minute.

[1804] Proceed to the next step.

[1805] Step 14: USER Treatment

[1806] Protocol:

[1807] Prepare the reaction:

[1808] Table: Master Mix for USER TreatmentComponentVolume

[1810] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[1811] Add 25 pL of beads to each sample

[1812] Mix well by pipetting or vortexing

[1813] Incubate at room temperature for 5 minutes

[1814] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1815] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1816] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[1817] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1818] Let the beads dry at room temperature

[1819] Add 20 pL of nuclease free water per sample and mix well by pipetting or vortexing

[1820] Incubate at room temperature for 5 minutes

[1821] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1822] Carefully remove 18 pL of supernatant and transfer to a new clean tube

[1823] Proceed to the next step.

[1824] Step 15: Strand Filling

[1825] Protocol:WSGR Docket No. 68738-702.601

[1826] Prepare the reaction:

[1827] Table: Master Mix for Strand FillingComponent VolumeSample from Step 14 : 18.0 pL1 OX Isothermal Amplification Buffer 2.5 pLMgSO I ( lOO mM) 1.5 pLmethyl-dCTP (lOmM) ; 1.0 pLdNTPs (No dCTP, 10mM) H.O pLBst 2.0 DNA Polymerase (8,000 U / ml) i 1.0 pLTotal Volume : 25.0 pL

[1828] Incubate samples at 65°C for 1 hour.

[1829] Perform a clean up using SPRlselect beads at a ratio of lx according to the protocol below:

[1830] Add 25 pL of beads to each sample

[1831] Mix well by pipetting or vortexing

[1832] Incubate at room temperature for 5 minutes

[1833] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1834] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1835] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[1836] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1837] Let the beads dry at room temperature

[1838] Add 32 pL of nuclease free water per sample and mix well by pipetting or vortexing

[1839] Incubate at room temperature for 5 minutes

[1840] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1841] Carefully remove 30 pL of supernatant and transfer to a new clean tube

[1842] Proceed to the next step.

[1843] Step 16: Ligation to TNA Library

[1844] Reaction Setup:

[1845] Table: Ligation ReactionWSGR Docket No. 68738-702.601NEBNext Ligation Enhancer 0.25 pLNEBNext Ultra II Ligation MM : 7.25 pLTotal Volume23.0 pL

[1846] Reaction Conditions: 20°C for duration of reaction (overnight).

[1847] Protocol:

[1848] Set up above reaction and incubate at 20°C.

[1849] After 4-6 hours, spike in 1 pL of Stubby xGen Adapter (15 pM).

[1850] Leave reaction overnight (12-18 hours).

[1851] Perform a clean up using SPRIselect beads at a ratio of 0.8x according to the protocol below:

[1852] Add 18 pL of beads to each sample

[1853] Mix well by pipetting or vortexing

[1854] Incubate at room temperature for 5 minutes

[1855] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1856] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1857] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[1858] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1859] Let the beads dry at room temperature

[1860] Add 22 pL of nuclease free water per sample and mix well by pipetting or vortexing

[1861] Incubate at room temperature for 5 minutes

[1862] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1863] Carefully remove 20 pL of supernatant and transfer to a new clean tube

[1864] Step 17: Bisulfite Conversion

[1865] Protocol:WSGR Docket No. 68738-702.601

[1866] Prepare the reaction by adding 180 pL SuperMethyl Fast Conversion Reagent to 20 pL of the sample from Step 16.

[1867] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1868] Briefly spin down.

[1869] Take samples to the thermocycler and run: 98°C for 7 min, 4°C hold.

[1870] Add 500 pL Binding Buffer and 10 pL Purification Beads to a low bind tube and mix thoroughly.

[1871] Transfer samples to bead mixture.

[1872] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1873] Incubate for 10 min at room temperature with continuous rotation.

[1874] Briefly spin down.

[1875] Place samples on magnet for 5 minutes.

[1876] Remove and discard the supernatant. Take care not to disturb the beads.

[1877] Remove tube from magnet.

[1878] Add 400 pL Fast Wash Buffer and resuspend the beads.

[1879] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1880] Briefly spin down.

[1881] Place samples on magnet for 3 minutes.

[1882] Remove and discard the supernatant. Take care not to disturb the beads.

[1883] Remove tube from magnet.

[1884] Add 200 pL Fast Desulphonation Buffer.

[1885] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1886] Incubate for 10 min at room temperature with continuous rotation.

[1887] Briefly spin down.

[1888] Place samples on magnet for 3 minutes.

[1889] Remove and discard the supernatant. Take care not to disturb the beads.

[1890] Remove tube from magnet.

[1891] Add 400 pL Fast Wash Buffer and resuspend the beads.

[1892] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1893] Briefly spin down.

[1894] Place samples on magnet for 3 minutes.

[1895] Remove and discard the supernatant. Take care not to disturb the beads.

[1896] Repeat wash lx.

[1897] Remove and discard the supernatant, making sure to leave no residue.

[1898] With tubes uncapped, incubate at 55°C for 5-10 min to completely dry the beads.WSGR Docket No. 68738-702.601

[1899] Add 15 pL Fast Elution Buffer.

[1900] Mix samples gently by low speed vortex for 5 seconds or pipette.

[1901] Briefly spin down.

[1902] Place samples on magnet for 1-3 minutes or until the liquid is clear.

[1903] Transfer 10 pL of supernatant to anew clean tube.

[1904] Proceed to the next step.

[1905] Step 18: Final Library Amplification

[1906] Protocol:

[1907] Prepare the reaction:

[1908] Table: Master Mix for Library PCRbelow:

[1912] Add 20 pL of beads to each sample

[1913] Mix well by pipetting or vortexing

[1914] Incubate at room temperature for 5 minutes

[1915] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnetWSGR Docket No. 68738-702.601

[1916] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[1917] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[1918] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[1919] Let the beads dry at room temperature

[1920] Add 17 pL of nuclease free water per sample and mix well by pipetting or vortexing

[1921] Incubate at room temperature for 5 minutes

[1922] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[1923] Carefully remove 15 pL of supernatant and transfer to a new clean tube

[1924] FIG.28A illustrates an example of a portion of method workflow for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing and linear protein detection assaying of a tissue sample. FIG. 28B illustrates an example of a continuing portion of a method workflow- for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing and linear protein detection assaying of a tissue sample. FIG. 28C illustrates an example of a continuing portion of a method workflow and end-state sequencing molecules for Total hairpin-annealed simultaneously extracted DNA and RNA sequencing and linear protein detection assaying of a tissue sample.Example 12: hairy TNA-Seq and Linear protein Library Preparation in Plasma Samples

[1925] Proteins, cfDNA, and cfRNA may be extracted simultaneously from a single plasma sample.

[1926] Step 1: Lysate Biotinylation + Cleanup

[1927] Description: Biotinylation of lysate proteins for streptavidin bead capture.

[1928] Protocol:

[1929] If unknown, measure the protein concentration using the A660 protein quantification kit.

[1930] Calculate amount of biotin to add to the protein mixture.

[1931] Table: Biotinylation Reaction SetupWSGR Docket No. 68738-702.601

[1932] Note: If the final reaction concentration of biotin is > 16 pM, two sequential Zeba purifications are recommended. If concentration is > 320 pM, three sequential Zeba purifications are recommended.

[1933] Step 2: Streptavidin Bead Preparation

[1934] Description: Initial wash and preparation of Streptavidin beads.

[1935] Protocol:

[1936] Remove beads from 4°C vortex, and allow time to equilibrate to room temperature.

[1937] Pipette the required amount of beads into PCR strip tubes. This protocol typically uses 25 pL beads (1 EQ).

[1938] Vortex briefly if needed.

[1939] Wash 2x with 1 EQ PBS:

[1940] Place on magnet for 1 minute until solution is clear.

[1941] Aspirate out supernatant.

[1942] Remove from magnet.

[1943] Pipette 1 EQ volume of PBS into beads and resuspend.

[1944] Notes:

[1945] Ensure binding capacity of beads is greater than input protein concentration.

[1946] Bead capacity is approximately 120 ng / pL.

[1947] Step 3: Lysate Incubation

[1948] Description: Incubation of lysate with streptavidin beads.

[1949] Protocol:

[1950] Place samples on magnetic tube rack.

[1951] Aspirate out supernatant.

[1952] Pipette 1 EQ of lysate into its corresponding sample tube and resuspend.

[1953] Incubate at room temperature for 1 hour.

[1954] Wash 3x with 1 EQ PBS + 1% NP40:

[1955] Place on magnetic rack for 1 minute until solution is clear.

[1956] Aspirate out supernatant.

[1957] Remove from magnet.

[1958] Pipette 1 EQ volume of PBS + 1% NP40 into beads and resuspend.

[1959] Note: Typical protocol is to leave at room temperature for 1 hour with a brief gentle vortex at approximately 30 minutes to resuspend beads.

[1960] Step 4: Probe-Target Complex Formation

[1961] Description: Incubation of AOC (or other probes) with lysate on streptavidin beads.

[1962] Protocol:WSGR Docket No. 68738-702.601

[1963] Place samples on magnetic tube rack.

[1964] Aspirate out supernatant.

[1965] Pipette 1 EQ of probe cocktail into its corresponding sample tube and resuspend.

[1966] Incubate at room temperature for 1 hour.

[1967] Wash 6x with 4 EQ PBS + 1% NP40.

[1968] Wash lx with 4 EQ PBS (no NP40).

[1969] Wash lx with 2 EQ PBS (no NP40).

[1970] Wash lx with 1 EQ PBS (no NP40).

[1971] Transfer the beads in PBS to a new set of PCR strip tubes.

[1972] Note: The bead transfer step at the end is critical for minimizing non-specific binding.

[1973] Step 5: On-Bead Amplification of Oligo Conjugate

[1974] PCR Setup:

[1975] 5 pL bead mixture (from previous step)

[1976] 5 pL 12.5 pM mixture of integration amplification primers (MO166 and MO167)

[1977] 15 pL nuclease free water

[1978] 25 pL NEBNext Ultra II Q5U Master Mix

[1979] Cycling Conditions:

[1980] 98°C x 30 sec (initial denaturation)

[1981] 12 cycles of: 98°C x 10 sec, 66°C x 15 sec, 72°C x 10 sec

[1982] 4°C Hold

[1983] Protocol:

[1984] Perform above PCR.

[1985] Clean and concentrate with the Zymo Oligo Clean and Concentrator kit-5.

[1986] Elute in 15 pL nuclease free water.

[1987] QC: Run amplified product on a 4% agarose gel with ultra low bp DNA ladder. Expect a single band at approximately 70 bp.

[1988] Step 6: Restriction Digestion

[1989] Reaction Setup:

[1990] 2 pL NEB lOx CutSmart Buffer

[1991] 5 pL template DNA (from previous step)

[1992] 0.5 pL NEB Xcml restriction enzy me

[1993] 0.5 pL NEB AhdI restriction enzyme

[1994] 11 pL nuclease free water

[1995] Reaction Conditions:

[1996] 37°C for 1 hourWSGR Docket No. 68738-702.601

[1997] 4°C Hold

[1998] Manually add in 0.5 pL NEB SphI and PstI restriction enzymes

[1999] 37°C for 1 hour

[2000] 4°C Hold

[2001] Protocol:

[2002] Perform above restriction digest.

[2003] Clean and concentrate with the Zymo Oligo Clean and Concentrator kit-5.

[2004] Elute in 15 pL nuclease free water.

[2005] QC: Run digested product on a 4% agarose E-gel. Expect complete digestion of the original band (~70 bp) and target band at approximately 35 bp.

[2006] Step 7: Custom Y-Shaped Adapter Preparation

[2007] Reaction Setup:

[2008] Table: Adapter Annealing ReactionL

[2009] Reaction Conditions:

[2010] Heat to 85°C for 1 minute in thermocycler.

[2011] Slow ramp to 25°C at a rate of 0.2°C per second.

[2012] Note: Annealed adapters can be stored long term at -20°C.

[2013] Method Steps - TNA Processing

[2014] Step 8: Poly(A) Selection of RNAs

[2015] Protocol:

[2016] Dilute the total RNA with nuclease-free water to a final volume of 50 pl in a nuclease-free 0.2 ml PCR tube and keep on ice.

[2017] Add 40 pl NEBNext Oligo d(T)25 beads per reaction to a 1.5 ml tube.

[2018] Place the tube on the magnetic rack at room temperature for 2 minutes or until the solution is clear.

[2019] Remove and discard all of the supernatant from the tube. Take care not to disturb the beads.

[2020] Remove the tube from the magnetic rack.WSGR Docket No. 68738-702.601

[2021] Add 50 pl NEBNext RNA Binding Buffer (2X) to the beads and wash by pipetting up and down 6 times.

[2022] Place the tube on the magnet and incubate at room temperature until the solution is clear (~2 minutes).

[2023] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[2024] Remove the tube from the magnet and add 50 pl NEBNext RNA Binding Buffer (2X) to the beads and mix by pipetting up and down until beads are homogenous.

[2025] Add 50 pl beads to each RNA sample from Step 1. Mix thoroughly by pipetting up and down 6 times.

[2026] Heat the sample to denature the RNA and to facilitate binding of the poly (A) RNA to the beads. Place in a thermal cycler, with the heated lid set to > 90°C, and run: 2 min at 80°C, 5 min at 25°C, Hold at 25°C.

[2027] Remove tubes from the thermal cycler when the temperature reaches hold at 25°C.

[2028] Place the tubes on the magnetic rack at room temperature for 2 minutes or until the solution is clear.

[2029] Remove and discard all of the supernatant. Take care not to disturb the beads.

[2030] While still on the magnet rinse the beads by gently adding 200 pl of NEBNext Wash Buffer to the tubes to remove unbound RNA.

[2031] Remove and discard all of the supernatant from each tube. Take care not to disturb the beads.

[2032] Remove the tubes from the magnetic rack.

[2033] Table: Master Mix for Poly(A) Selection of RNAsComponent: Volume

[2034] Add the above mix to each tube containing mRNA bound beads. Mix thoroughly by gently pipetting up and down 6 times.

[2035] Place the tubes in a thermal cycler, with the heated lid set to > 90°C, and run: 2 min at 80°C, 5 min at 25°C, Hold at 25°C.

[2036] Remove the tubes from the thermal cycler when the temperature reaches hold at 25°C.

[2037] Place the tubes on the magnetic stand at room temperature for 2 minutes or until the solution is clear.WSGR Docket No. 68738-702.601

[2038] Remove and discard all of the supernatant from each tube.

[2039] While still on the magnet, rinse the beads by gently adding 200 pl of NEBNext Wash Buffer.

[2040] Remove and discard all of the supernatant from the tubes.

[2041] Remove the tubes from the magnetic rack.

[2042] Elute the mRNA from the beads by adding 17 pl of the NEBNext Tris Buffer, mix by pipetting 6 times and incubate at 80°C for 2 min, then cool to 25°C. Place tubes on magnetic rack for 2 minutes.

[2043] Collect the purified mRNA by transferring 15 pl of the supernatant to a clean nuclease- free PCR tube.

[2044] Step 9: cDNA Synthesis (Two-Step Process)

[2045] Step 9A: First Strand cDNA Synthesis

[2046] Protocol:

[2047] Prepare the reaction as described below:

[2048] Table: Master Mix for First Strand cDNA Synthesis

[2049] Take samples to the thermocycler and run: 25°C for 10 min, 37°C for 120 min, 4°C hold.

[2050] Remove samples from thermocycler and prepare the next step.

[2051] Step 9B: RNase H and Second Strand cDNA Synthesis

[2052] Prepare the RNase H reaction:

[2053] Table: Master Mix for RNase H TreatmentWSGR Docket No. 68738-702.601

[2054] Incubate at 37°C for 20 minutes.

[2055] Perform a clean up using SPRIselect beads at a ratio of 1.5x according to the protocol below:

[2056] Add 45 pL of beads to each sample

[2057] Mix well by pipetting or vortexing

[2058] Incubate at room temperature for 5 minutes

[2059] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[2060] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[2061] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[2062] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[2063] Let the beads dry at room temperature

[2064] Add 22 pL of nuclease free water per sample and mix well by pipetting or vortexing

[2065] Incubate at room temperature for 5 minutes

[2066] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[2067] Carefully remove 20 pL of supernatant and transfer to a new clean tube

[2068] For the 2nd strand synthesis, prepare the reaction:

[2069] Table: Master Mix for 2nd Strand SynthesisKlenow Fragment (3'— >5' exo-) (5 U / pL) 4.0 plNuclease Free Water 14.5 pl50.0 plWSGR Docket No. 68738-702.601

[2070] Take samples to the thermocycler and run: 25°C for 5 min, 37°C for 60 min, 65°C for 10 min.

[2071] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[2072] Add 50 pL of beads to each sample

[2073] Mix well by pipetting or vortexing

[2074] Incubate at room temperature for 5 minutes

[2075] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[2076] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[2077] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[2078] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[2079] Let the beads dry at room temperature

[2080] Add 17 pL of nuclease free water per sample and mix well by pipetting or vortexing

[2081] Incubate at room temperature for 5 minutes

[2082] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[2083] Carefully remove 15 pL of supernatant and transfer to a new clean tube

[2084] Keep the samples at 4°C or -20°C until next step.

[2085] Step 10: End Repair and dA-Tailing

[2086] Protocol:

[2087] Mix 12.5 pL of cDNA from Step 9 and 12.5 pL of DNA sample (plasma cfDNA does not require fragmentation) to generate TNA sample.

[2088] Prepare the reaction:

[2089] Table: Master Mix for End Repair and dA-Tailing

[2090] Take samples to the thermocycler and run: 20°C for 30 min, 65°C for 30 min, 4°C hold.

[2091] Move straight to next step.WSGR Docket No. 68738-702.601

[2092] Step 11: Hairpin Adapter Ligation

[2093] Protocol:

[2094] Prepare the hairpin for ligation by reannealing:

[2095] Dilute the hairpin adapter to 20 pM in IX annealing buffer.

[2096] Incubate at 80°C for 2 min then ramp temperature to 25°C at a rate of 0.1°C per second.

[2097] Prepare the reaction:

[2098] Table: Master Mix for Hairpin Ligation

[2100] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[2101] Add 50 pL of beads to each sample

[2102] Mix well by pipetting or vortexing

[2103] Incubate at room temperature for 5 minutes

[2104] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[2105] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[2106] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[2107] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[2108] Let the beads dry at room temperature

[2109] Add 22 pL of nuclease free water per sample and mix well by pipetting or vortexing

[2110] Incubate at room temperature for 5 minutes[21H] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[2112] Carefully remove 20 pL of supernatant and transfer to a new clean tube[2H3] Proceed to the next step.

[2114] Step 12: Exonuclease TreatmentWSGR Docket No. 68738-702.601

[2115] Protocol:

[2116] Prepare the reaction:

[2117] Table: Master Mix for Exonuclease TreatmentComponent ; VolumeLigated Samples from Step 1 1 : 20.0 pL

[2118] Incubate samples at 37°C for 1 hour.

[2119] Clean up samples using Oligo Clean & Concentrator:

[2120] Add 25 pl of water to bring volume to 50 pl.

[2121] Add 100 pl Oligo Binding Buffer to 50 pl sample.

[2122] Add 200 pl ethanol (95-100%) and mix well.

[2123] Transfer to Zymo-Spin IC Column and centrifuge at 12,000 x g for 30 seconds.

[2124] Discard flow-through.

[2125] Add 750 pl DNA Wash Buffer and centrifuge at 12,000 x g for 1 minute.

[2126] Transfer column to nuclease-free tube.

[2127] Add 23 pl water and centrifuge at 12,000 x g for 1 minute.

[2128] Proceed to the next step.

[2129] Step 13: USER Treatment

[2130] Protocol:

[2131] Prepare the reaction:

[2132] Table: Master Mix for USER TreatmentComponent VolumeSample from Step 12 20.0 pL

[2133] Incubate samples at 37°C for 1 hour.WSGR Docket No. 68738-702.601

[2134] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[2135] Add 25 pL of beads to each sample

[2136] Mix well by pipetting or vortexing

[2137] Incubate at room temperature for 5 minutes

[2138] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[2139] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[2140] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[2141] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[2142] Let the beads dry at room temperature

[2143] Add 20 pL of nuclease free water per sample and mix well by pipetting or vortexing

[2144] Incubate at room temperature for 5 minutes

[2145] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[2146] Carefully remove 18 pL of supernatant and transfer to a new clean tube

[2147] Proceed to the next step.

[2148] Step 14: Strand Filling

[2149] Protocol:

[2150] Prepare the reaction:

[2151] Table: Master Mix for Strand FillingComponent VolumeSample from Step 13 : 18.0 pL1 OX Isothermal Amplification Buffer 2.5 pL MgS04 (100 mM)1.5 pLmethyl-dCTP ( lOmM) i 1.0 pLdNTPs (No dCTP, lOmM) H.O pLBst 2.0 DNA Polymerase (8,000 U / ml) i 1.0 pLTotal Volume 25.0 pL

[2152] Incubate samples at 65°C for 1 hour.WSGR Docket No. 68738-702.601

[2153] Perform a clean up using SPRIselect beads at a ratio of lx according to the protocol below:

[2154] Add 25 pL of beads to each sample

[2155] Mix well by pipetting or vortexing

[2156] Incubate at room temperature for 5 minutes

[2157] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[2158] Remove and discard the supernatant from the tube. Take care not to disturb the beads.

[2159] Wash the beads twice with freshly prepared 80% Ethanol waiting 30 seconds between each wash

[2160] Remove and discard all ethanol from the tubes. Take care to not disturb the beads.

[2161] Let the beads dry at room temperature

[2162] Add 32 pL of nuclease free water per sample and mix well by pipetting or vortexing

[2163] Incubate at room temperature for 5 minutes

[2164] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[2165] Carefully remove 30 pL of supernatant and transfer to a new clean tube

[2166] Proceed to the next step.

[2167] Step 15: Ligation to TNA Library

[2168] Reaction Setup:

[2169] Table: Ligation ReactionC ompon en t Vol um

[2170] Reaction Conditions: 20°C for duration of reaction (overnight).

[2171] Protocol:

[2172] Set up above reaction and incubate at 20°C.

[2173] After 4-6 hours, spike in 1 pL of Stubby xGen Adapter (15 pM).WSGR Docket No. 68738-702.601

[2174] Leave reaction overnight (12-18 hours).

[2175] Perform a clean up using SPRIselect beads at a ratio of 0.8x according to the protocol below:

[2176] Add 18 pL of beads to each sample

[2177] Mix well by pipetting or vortexing

[2178] Incubate at room temperature for 5 minutes

[2179] Place samples on magnet for 5 minutes or until the liquid is clear and beads are pelleted against the magnet

[2180] Remove and discard the supernatant from the t...

Claims

1. WSGR Docket No. 68738-702.601CLAIMSWhat is claimed is;1. A method of preparing a sequencing library comprising ligating an adapter to a methylated deoxyribonucleic acid (DNA) fragment, wherein the adapter comprises a methylated proteinspecific oligonucleotide.

2. A method of preparing a sequencing library comprising:(a) annealing together an adapter fragment and a methylated single-stranded DNA (ssDNA fragment; and(b) ligating a methylated protein-specific oligonucleotide tag to a site of the adapter fragment, thereby producing a molecular complex.

3. The method of claim 2, further amplifying the molecular complex.

4. The method of claim 2 or 3, further comprising sequencing the molecular complex to produce sequencing reads.

5. The method of claim 4, further comprising determining a methylation pattern from the sequencing reads, wherein the methylation pattern comprises methylation information of both the methylated ssDNA and the methylated protein-oligonucleotide tag.

6. The method of claim 5, wherein determining the methylation pattern comprises identifying one or more differentially expressed markers or variants.

7. The method of claim 6, wherein the one or more differentially expressed markers or variants comprise markers or variants of the ssDNA.

8. The method of claim 6, wherein the one or more differentially expressed markers or variants comprises one or more gene mutations of the ssDNA.

9. The method of claim 5, further comprising detecting one or more of the methylated DNA molecules based at least in part on the methylation pattern of the methylated protein- oligonucleotide tag.WSGR Docket No. 68738-702.60110. The method of claim 5, further comprising detecting one or more of the methylated DNA molecules based at least in part on a combination of the methylation pattern of the methylated protein-oligonucleotide tag and a methylation pattern of the methylated ssDNA.

11. The method of claims 9 or 10, further comprising selectively performing sequencing of the identified methylated DNA molecules.

12. The method of claims 9 or 10, further comprising selectively excluding the identified methylated DNA molecules from sequencing.

13. The method of claims 9 or 10, further comprising quantifying the detected one or more methylated DNA molecules.

14. The method of any one of claims 5-13, wherein the methylation pattern further comprises a quantitative measure of methylation.

15. The method of any one of claims 5-14, further comprising performing error correction of the sequence reads based at least in part on the methylation pattern.

16. The method of any one of claims 4-15, wherein the sequencing comprises whole exome sequencing.

17. The method of any one of claims 6-16, wherein the one or more differentially expressed markers or variants comprise single nucleotide variations.

18. The method of any one of claims 6-16, wherein the one or more differentially expressed markers or variants comprise copy number variants.

19. The method of any one of claims 6-16, wherein the one or more differentially expressed markers or variants comprise structural variants such as insertions or deletions (indels).

20. The method of any one of claims 1-19, further comprising detecting one or more differentially expressed markers or variants in ribonucleic acid (RNA) of the subject.

21. The method of claim 20, wherein the one or more differentially expressed markers or variants in the RNA of the subject comprise single nucleotide variants, copy number variants, indels, or expression biomarkers.WSGR Docket No. 68738-702.60122. The method of any one of claims 1-21, further comprising detecting the one or more differentially expressed markers or variants in a subject comprising one or more epigenetic data characteristics.

23. The method of claim 22, wherein the one or more epigenetic data characteristics comprise a comparative methylation indication.

24. The method of claim 23, wherein the comparative methylation indication comprises hypermethylation or hypo-methylation.

25. The method of claim 24, further comprising determining the hyper-methylation or hypomethylation based at least in part on a dynamic or pre-set normalized methylation value.

26. The method of any one of claims 6-25, further comprising detecting the one or more differentially expressed markers or variants in the subject comprising protein abundance characteristics.

27. The method of claim 26, wherein the protein abundance characteristics comprise increased protein abundances or decreased protein abundances.

28. The method of any one of claims 4-27, wherein the sequencing reads comprise a multi-omic dataset.

29. The method of claim 28, wherein the multi-omic dataset comprises RNA sequencing data, methylation sequencing data, or protein sequencing data, or any combination thereof.

30. The method of any one of claims 1-29, further comprising generating a multi-omic model, multianalyte model, or multimodal model.

31. The method of claim 30, wherein the multi-omic model, multianalyte model, or multimodal model comprises two or more sequencing libraries.

32. The method of claim 31, wherein the two or more sequencing libraries comprise a DNA sequencing library, an RNA sequencing library, a methylation sequencing library, or a protein sequencing library, or any combination thereof.

33. The method of claim 32, wherein each sequencing library comprises one or more genomic features.WSGR Docket No. 68738-702.60134. The method of claim 33, wherein the one or more genomic features comprise the multi-omic dataset associated or grouped with cell type data.

35. The method of any one of claims 1-34, further comprising receiving a biological sample.

36. The method of claim 35. wherein the biological sample comprises a cell-free sample or a tissue sample.

37. The method of claim 36, further comprising predicting a synthetic tissue fraction using the two or more sequencing libraries.

38. The method of claim 37, further comprising predicting a synthetic tissue fraction using a methylation library of the methylated DNA molecules, and the comparative methylation indication.

39. A method for preparing a protein library, comprising ligating an ssDNA fragment to an oligonucleotide tag, wherein the oligonucleotide tag is isolated from an antibody- oligonucleotide conjugate.

40. The method of claim 39, wherein the oligonucleotide tag is methylated.

41. The method of claim 39 or 40, further comprising contacting a plurality of antibody - oligonucleotide conjugates with a lurality of proteins.

42. The method of any one of claims 39-41, further comprising isolating at least a subset of the plurality of antibody-oligonucleotide conjugates.

43. The method of claim 42, wherein the isolated at least the subset of the plurality of antibody- oligonucleotide conjugates are bound to at least a subset of the plurality' of proteins.

44. The method of claim 43, wherein the oligonucleotides of the antibody-oligonucleotide conjugates are methylated.

45. The method of any one of claims 42-44. further comprising releasing the methylated oligonucleotides from the antibody-oligonucleotide conjugates.

46. The method of claim 45, further comprising replicating the plurality of released methylated oligonucleotides.WSGR Docket No. 68738-702.60147. The method of claim 45 or 46, further comprising combining the plurality of released methylated oligonucleotides with similar unmethylated oligonucleotides.

48. The method of any one of claims 45-47, wherein each of the plurality' of released methylated oligonucleotides comprise one or more methylation sites.

49. The method of claim 48, wherein the released methylated oligonucleotides comprise the methylated oligonucleotide tags.

50. The method of claim 49, further comprising sequencing both the ssDNA fragment and the methylated oligonucleotide tag in the same sequencing run.

51. The method of any one of claims 48-50, wherein the one or more methylation sites comprise a methylation pattern of the methylated oligonucleotide tag.

52. The method of claim 51, further comprising using the methylation pattern to determine one or more characteristics of the methylated oligonucleotide tag, or the ssDNA fragment, or both.

53. The method of claim 52, wherein the one or more characteristics comprise one or more pieces of multi-omic data.

54. The method of claim 53, wherein the one or more pieces of multi-omic data comprise longitudinal multi-omic data, time-series multi-omic data, or both.

55. The method of claim 53 or 54, wherein the multi-omic data comprises one or more of DNA single nucleotide variations, DNA copy number variations, DNA indels, RNA single nucleotide variations, RNA copy number variations, RNA indels, DNA structural variations, RNA structural variations, hyper-methylation, hypo-methylation, increased protein abundance, or decreased protein abundance, or any combination thereof.

56. The method of any of claims 39-55, wherein the ssDNA fragment is methylated.

57. The method of any one of claims 39-56, further comprising generating a multi-omic library.

58. The method of claim 57, wherein the multi-omic library' comprises two or more sequencing libraries.WSGR Docket No. 68738-702.60159. The method of claim 58, wherein the two or more sequencing libraries comprise the protein library, a DNA sequencing library, an RNA sequencing library, or a methylation sequencing library, or any combination thereof.

60. The method of claim 57, wherein the multi-omic library comprises a singular multianalyte sequencing library.

61. The method of claim 60, wherein the singular multianalyte sequence library comprises two or more of: DNA genomic features, RNA genomic features, methylation genomic features, protein genomic features, or any combination thereof.

62. The method of claim 61, wherein each of the genomic features comprise the multi-omic data associated or grouped with cell ty pe data.

63. A method of preparing a multi-omic library, comprising:(a) annealing together an adapter fragment and a methylated ssDNA fragment, thereby producing a first molecular complex;(b) annealing together an adapter fragment and an RNA fragment, thereby producing a second molecular complex; and(c) ligating a methylated oligonucleotide tag to a site of the adapter fragment of (a), thereby producing a third molecular complex.

64. The method of claim 63, further comprising amplifying the first molecular complex.

65. The method of claim 63 or 64, further comprising amplifying the second molecular complex.

66. The method of claim 63. wherein the third molecular complex comprises a subset of amplified molecules of the first molecular complex.

67. The method of any one of claims 63-66, further comprising amplifying the third molecular complex.

68. The method of claim 63, further comprising generating multi-omic data for the third molecular complex.

69. The method of claim 68, further comprising associating or grouping the multi-omic data with one or more cell types to generate one or more feature matrices.WSGR Docket No. 68738-702.60170. The method of claim 69, further comprising grouping the one or more feature matrices into one or more genomic feature groups.

71. The method of claim 70, further comprising providing as input the one or more genomic features to a multi-omic model, multianalyte model, or multimodal model.

72. The method of claim 71, further comprising performing multilayer perception (MLP) on data relating to the one or more genomic feature groups to generate synthetic tissue fraction values associated with each of the one or more genomic features.

73. The method of any one of claims 35-38, further comprising generating the multi-omic data set from the biological sample simultaneously.

74. The method of claim 73, further comprising performing a multi-omic assay of the biological sample to simultaneously generate the multi-omic data set.

75. The method of claim 74, wherein the multi-omic assay comprises a genomic assay, a transcriptomic assay, an epigenetic assay, and a proteomic assay.

76. The method of claim 75, wherein the multi-omic assay simultaneously generates the multi- omic data set comprising multi-omic assay results relating to the RNA sequencing data, the methylation sequencing data, and the protein sequencing data.

77. The method of any one of claims 53-62, further comprising generating the multi-omic data from a biological sample simultaneously.

78. The method of claim 77, further comprising performing a multi-omic assay of the biological sample to generate the multi-omic data simultaneously.

79. The method of claim 78, wherein the multi-omic assay comprises a genomic assay, a transcriptomic assay, an epigenetic assay, and a proteomic assay.

80. The method of claim 79, wherein performing the multi-omic assay of the biological sample comprises performing the genomic assay, the transcriptomic assay, the epigenetic assay, and the proteomic assay in the same sequencing run.

81. The method of any one of claims 63-72, further comprising preparing the multi-omic library using assay results simultaneously generated by a multi-omic assay of a biological sample.

82. A method for assaying a biological sample of a subject, comprising:WSGR Docket No. 68738-702.601(a) performing a multi-omic assay of the biological sample to simultaneously capture genomic, transcriptomic. epigenetic, and proteomic assay results from the biological sample:(b) generating multi-omic data comprising genomic data, transcriptomic data, epigenetic data, and proteomic data from the simultaneously captured multi-omic assay results.

83. The method of claim 82, further comprising generating a multi-omic library using the multi- omic data.

84. The method of claim 83. wherein the multi-omic library comprises a plurality of -omic layers.

85. The method of claim 84, wherein an -omic layer of the plurality of -omic layers corresponds to the genomic data, the transcriptomic data, the epigenetic data, or the proteomic data.

86. The method of claim 85, further comprising comparing the multi-omic library7to one or more reference -omic data sets.

87. The method of claim 86, wherein the one or more reference -omic data sets comprise a reference genomic data set, a reference transcriptomic data set, a reference epigenetic data set, or a reference proteomic data set, or any combination thereof.

88. The method of claim 87, further comprising determining a variation of the multi-omic data.

89. The method of claim 88, wherein the variation comprises a genomic sequence variation, a transcriptomic variation, an epigenetic variation, or a proteomic variation, or any combination thereof.

90. The method of claim 89, wherein the sequence variation comprises a single nucleotide polymorphism (SNP) or a copy number variation (CNV).

91. The method of claim 89, wherein the epigenetic variation comprises a differential promoter methylation, a differential genome region methylation, or a differential CpG methylation.

92. The method of claim 89, wherein the proteomic variation comprises a variation in fractional protein content, protein quantity, or protein function, or any combination thereof.

93. The method of any one of claims 82-92, wherein the genomic data comprises genomic sequencing data of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

Images