Methods for analyzing circulating microparticles

Abstract

To provide a method for analyzing a sample containing first circulating fine particles.SOLUTION: Provided is a method for analyzing a sample containing first circulating fine particles, where the first circulating fine particle is a membrane vesicle, the first circulating fine particle contains at least three target molecules, at least two of the target molecules are genome DNA fragments, at least one of the target molecules is a target polypeptide. The method includes a step of measuring a signal corresponding to presence, absence, and / or a level of each of the target molecules, and creating at least two sets of signals informationally connected to the first circulating fine particles.SELECTED DRAWING: Figure 30

Description

[Technical Field]

[0001] The present invention relates to the analysis of cell-free biomolecules (e.g., cell-free nucleic acid molecules and cell-free polypeptides). In particular, it relates to the analysis of cell-free biomolecules contained in or derived from circulating microparticles. Reagents and methods for analyzing biomolecules of circulating microparticles are provided, including reagents and methods for analyzing biomolecules of a single circulating microparticle. [Background technology]

[0002] Circulating cell-free DNA (cfDNA) is typically fragmented (usually ranging in length from 100 to 200 base pairs), and therefore, cfDNA analysis methods have traditionally focused on the biological signals that may be found in these short DNA fragments. For example, tests for fetal chromosomal trisomy (so-called "non-invasive prenatal testing," or NIPT) that assess fetal DNA in maternal circulation either detect single-nucleotide variants within individual molecules or perform "molecular counts" across a large number of sequenced fragments to indirectly infer the presence of large-scale chromosomal abnormalities.

[0003] A wide variety of methods for analyzing circulating cell-free DNA have been previously described. Depending on the specific application area, these assays may use different terminology for a broadly similar set of sample types and technical methods, such as circulating tumor DNA (ctDNA), cell-free fetal DNA (cffDNA), and / or liquid biopsy, or non-invasive prenatal testing. Generally, these methods include experimental protocols for preparing samples of circulating cell-free DNA for sequencing, the sequencing reaction itself, and an informational framework for analyzing the resulting sequences to detect relevant biological signals. These methods include steps of DNA purification and isolation prior to sequencing; that is, subsequent analysis must rely solely on the information contained in the DNA itself. Following sequencing, such methods generally use one or more informational or statistical frameworks to analyze various aspects of the sequence data, such as detecting specific mutations and / or selective enrichment or depletion of specific chromosomal or subchromosomal regions (for example, this may indicate chromosomal aneuploidy in a developing fetus).

[0004] Many of these methods are intended for use in NIPT (e.g., U.S. Patent Nos. 6,258,540B1, 8,296,076B2, 8318,430B2, 8195,415B2, 9447,453B2, and 8442,774B2). The most common non-invasive prenatal testing methods for detecting fetal chromosomal abnormalities (such as trisomy and / or subchromosomal abnormalities such as microdeletions) involve sequencing a large number of molecules of cfDNA, mapping the resulting sequences to the genome (i.e., determining which chromosome and / or which part of a particular chromosome the sequence originates from), then determining the amount of sequence mapped to one or more such chromosomal or subchromosomal regions (e.g., in the form of absolute or relative number of reads), then comparing this to one or more normal or abnormal thresholds or cutoff values ​​and / or performing statistical tests to determine whether the region may be overrepresented in terms of sequence amount (e.g., corresponding to a chromosomal trisomy) and / or whether the region may be underrepresented in terms of sequence amount (e.g., corresponding to a microdeletion).

[0005] Various additional or modified approaches for analyzing cell-free DNA using data from unlinked individual molecules are also described (e.g., WO2016 / 094853A1, US2015 / 344970A1, and US2015 / 0105267A1).

[0006] Despite the wide range of methods available, there is still a need for new and more sensitive methods to analyze cfDNA that enable reliable detection of long-range genetic information (e.g., phasing). For example, in NIPT, fetal cfDNA represents only a small fraction of the total cfDNA of a pregnant individual (the majority of circulating DNA is normal maternal DNA). Therefore, much of the technical challenge in NIPT lies in distinguishing fetal cfDNA from maternal DNA. Similarly, in cancer patients, cfDNA represents only a small fraction of the total circulating DNA. Thus, similar technical challenges exist regarding the use of cfDNA analysis for cancer diagnosis or monitoring.

[0007] Separately, a method enabling the isolation of cell type-specific apoptotic bodies by fluorescence-activated cell sorting (FACS) (Atkin-Smith et al., 2017. Scientific Reports 7, 39846) and a method enabling multiple profiling of protein markers in a single extracellular vesicle (Lee et al., 2018. ACS Nano. 23, 12(1), 494-503) are also described. Detailed description of the invention

[0008] The present invention provides a method for analyzing a sample containing (or derived from) circulating microparticles such as apoptotic bodies. The invention is based on multiparametric measurement of different types of biomolecules contained within or derived from a single circulating microparticle. In particular, the invention enables the measurement of concatenated signals corresponding to the presence, absence, and / or levels of two or more types of target biomolecules within the same circulating microparticle. As shown in Figure 30, signals corresponding to levels of genomic DNA fragments may be generated (e.g., by compartmentalization, barcoding, and sequencing), and signals corresponding to levels of target polypeptides may be generated (e.g., using barcoding affinity probes). Furthermore, signals corresponding to levels of modified nucleotides (e.g., nucleotides containing 5-methylcytosine) may be generated (e.g., by affinity-based enrichment approaches, such as using enrichment probes that specifically or preferentially bind to 5-methylcytosine in genomic DNA fragments). Thus, these measurements and related techniques generate a set of concatenated signals corresponding to the physical and biological state of the circulating microparticles.

[0009] The multiparametric methods provided herein add an additional layer of information to the prior inventions provided by the inventors in PCT / GB2017 / 053820, PCT / GB2017 / 053812, and PCT / GB2017 / 053816.

[0010] In PCT / GB2017 / 053820, the inventors previously provided a method for analyzing nucleic acid fragments in circulating microparticles (or microparticles derived from blood). The invention is based on a linked fragment approach in which nucleic acid fragments from a single microparticle are linked together. This linkage allows for the generation of a set of linked sequence reads (i.e., a set of linked signals) corresponding to the sequences of fragments from a single microparticle.

[0011] The linked fragment approach provides highly sensitive cfDNA analysis and also enables the detection of long-range genetic information. This approach is based on combining insights. Firstly, this method utilizes the insight that individual circulating microparticles (e.g., individual circulating apoptotic bodies) contain a number of fragments of genomic DNA generated from the same individual cell (at a certain location in the body) that underwent apoptosis. Secondly, some of these fragments of genomic DNA within individual microparticles preferentially contain sequences from one or more specific chromosomal regions. Thus, cumulatively, such circulating microparticles function as data-rich and multifunctional “molecular stethoscopes” to observe potentially very complex genetic events occurring in a limited somatic cell tissue space somewhere in the body. Importantly, since most of these microparticles enter circulation before clearance or metabolism, they can be detected non-invasively. This invention describes experimental and beneficial methods using these “stethoscopes,” i.e., sets of linked fragments and linked sequence reads (either single individual microparticles or, in many embodiments, complex samples containing a large number of single circulating microparticles) to perform analytical and diagnostic tasks.

[0012] This invention advances the concept of a “molecular stethoscope” by utilizing data provided by, for example, the co-localization of non-nucleic acid molecules (e.g., target polypeptides) and nucleic acid molecules (e.g., fragments of genomic DNA) in a single circulating microparticle. This advance is based on the discovery that many biomolecules (e.g., nucleic acid molecules and polypeptides) present in the circulation are biophysically retained within circulating microparticles, rather than being specific and freely diffusing in the blood. This invention utilizes this rich source of information by measuring signals corresponding to the presence, absence, and / or levels of multiple target biomolecules in the circulating microparticle to generate a set of (informationally) linked signals from the circulating microparticle. Furthermore, by including one or more signals corresponding to one or more target biomolecules characteristic of a particular cell or tissue type in this set, the cellular origin of a particular set of linked signals derived from a single circulating microparticle can be determined. This provides a “cellular context” for the set of linked signals and offers a far richer source of information than currently available methods. In doing so, this invention provides an analytical method with high accuracy, sensitivity, and precision. Such a method is clearly applicable to cancer diagnosis and monitoring, as well as a wide range of diagnostic and monitoring applications such as NIPT.

[0013] The inventors have previously provided reagents and methods related to barcoding. In WO2016 / 207639, the inventors provide a wide range of reagents, kits, and methods for molecular barcoding, including a multimer barcoding reagent. In PCT / GB2017 / 053812, the inventors provide further methods and reagents for molecular barcoding. In PCT / GB2017 / 053816, the inventors provide reagents and methods for molecular barcoding of nucleic acids in single cells.

[0014] The entire contents of WO2016 / 207639, PCT / GB2017 / 053812, PCT / GB2017 / 053816, and PCT / GB2017 / 053820 are incorporated herein by reference.

[0015] The present invention provides a method for analyzing a sample containing circulating microparticles or a sample derived from circulating microparticles, wherein the circulating microparticles contain at least two target molecules, and at least two target molecules are biomolecules, and the method comprises measuring signals corresponding to the presence, absence, and / or level of each of the target molecules to generate at least two (informationally) coupled signals for the circulating microparticles, wherein at least one of the coupled signals corresponds to the presence, absence, and / or level of a first biomolecule in the sample, and at least one of the coupled signals corresponds to the presence, absence, and / or level of a second biomolecule in the sample.

[0016] The present invention provides a method for analyzing a sample containing circulating microparticles or a sample derived from circulating microparticles, wherein the circulating microparticles contain at least two target molecules, and at least two target molecules are biomolecules, and the method comprises measuring signals corresponding to the presence, absence, and / or level of each of the target molecules to generate a single signal for the circulating microparticles, the single signal corresponding to the presence, absence, and / or level of biomolecules in the sample.

[0017] The first biomolecule may be a fragment of a target nucleic acid (e.g., a fragment of genomic DNA), and the second biomolecule may be a target (or predefined) non-nucleic acid biomolecule (e.g., a target polypeptide). Optionally, the fragment of the target nucleic acid may contain at least one modified nucleotide or nucleic acid base.

[0018] The target molecule may comprise at least one, preferably at least two, fragments of a target nucleic acid (e.g., genomic DNA).

[0019] The first biomolecule may be a polypeptide, and the second target biomolecule may be a fragment of a target nucleic acid (e.g., genomic DNA) containing epigenetic modifications (e.g., 5-hydroxymethylcytosine DNA or 5-methylcytosine DNA).

[0020] The first biomolecule could be 5-hydroxymethylcytosine DNA, and the second target biomolecule could be a fragment of RNA.

[0021] The first biomolecule could be 5-methylcytosine DNA, and the second target biomolecule could be a fragment of RNA.

[0022] The first biomolecule may be 5-hydroxymethylcytosine DNA, and the second target biomolecule may be a biomolecule selected from biomolecule group 1.

[0023] The first biomolecule may be 5-methylcytosine DNA, and the second target biomolecule may be a biomolecule selected from biomolecule group 1.

[0024] The first and second biomolecules can be selected from biomolecule group 1.

[0025] The present invention provides a method for analyzing a sample containing circulating microparticles or a sample derived from circulating microparticles, wherein the circulating microparticles contain at least three target molecules, at least two of which are genomic DNA fragments and at least one of which is an RNA fragment, and the method comprises measuring signals corresponding to the presence, absence, and / or level of each of the target molecules to generate at least two (informationally) linked signals for the circulating microparticles, where at least one of the linked signals corresponds to the presence, absence, and / or level of a genomic DNA fragment in the sample and at least one of the linked signals corresponds to the presence, absence, and / or level of an RNA fragment in the sample.

[0026] The present invention provides a method for analyzing a sample containing circulating microparticles or a sample derived from circulating microparticles, wherein the circulating microparticles contain at least three target molecules, at least two of which are genomic DNA fragments and at least one of which is an RNA fragment, and the method comprises measuring signals corresponding to the presence, absence, and / or level of each of the target molecules to generate a single signal for the circulating microparticles, the single signal corresponding to the presence, absence, and / or level of genomic DNA fragments and RNA fragments in the sample.

[0027] The present invention provides a method for analyzing a sample containing circulating microparticles or a sample derived from circulating microparticles, wherein the circulating microparticles contain at least three target molecules, at least two of the target molecules being fragments of target nucleic acids (e.g., genomic DNA), and at least one of the target molecules being a target biomolecule (e.g., a target polypeptide), and the method comprises measuring signals corresponding to the presence, absence, and / or level of each of the target molecules to generate a set of at least three (informationally) linked signals for the circulating microparticles, where at least two of the linked signals correspond to the presence, absence, and / or level of one of the fragments of target nucleic acids (e.g., genomic DNA) in the sample, and at least one of the linked signals corresponds to the presence, absence, and / or level of a target biomolecule (e.g., a target polypeptide) in the sample.

[0028] The present invention provides a method for analyzing a sample containing circulating microparticles or a sample derived from circulating microparticles, wherein the circulating microparticles contain at least three target molecules, at least two of the target molecules being fragments of a target nucleic acid (e.g., genomic DNA), and at least one of the target molecules being a target biomolecule (e.g., a target polypeptide), and the method comprises measuring signals corresponding to the presence, absence, and / or level of each of the target molecules to generate a set of at least two (informationally) linked signals for the circulating microparticles, where at least one of the linked signals corresponds to the presence, absence, and / or level of fragments of the target nucleic acid (e.g., genomic DNA) in the sample, and at least one of the linked signals corresponds to the presence, absence, and / or level of the target biomolecule (e.g., a target polypeptide) in the sample.

[0029] The present invention provides a method for analyzing a sample containing circulating microparticles or a sample derived from circulating microparticles, wherein the circulating microparticles contain at least three target molecules, at least two of which are fragments of a target nucleic acid (e.g., genomic DNA), and at least one of which is a target biomolecule (e.g., a target polypeptide), and the method comprises measuring signals corresponding to the presence, absence, and / or level of each of the target molecules to generate a single signal for the circulating microparticles, the single signal corresponding to the presence, absence, and / or level of the target nucleic acid (e.g., genomic DNA) fragments and target biomolecule (e.g., target polypeptide) in the sample.

[0030] A fragment of the target nucleic acid (e.g., genomic DNA) may contain a specific sequence of nucleotides and / or a fragment of the target nucleic acid (e.g., genomic DNA) may contain at least one modified nucleotide or nucleic acid base. A fragment of the target nucleic acid may not contain a specific sequence of nucleotides. A fragment of the target nucleic acid may contain non-target and / or unknown and / or randomly selected and / or randomly sampled nucleotide sequences. For example, the modified nucleotide or nucleic acid base may be 5-methylcytosine or 5-hydroxy-methylcytosine. A fragment of the target nucleic acid (e.g., genomic DNA) may contain one or a microsatellite sequence and / or a microsatellite genomic region (i.e., a short tandem repeat).

[0031] The target polypeptide may contain a specific amino acid sequence and / or post-translational modifications. For example, the target polypeptide may contain acetylated amino acid residues and / or methylated amino acid residues (e.g., specific acetylated amino acid residues and / or specific methylated amino acid residues on / within a specific polypeptide).

[0032] This method may involve measuring signals corresponding to the presence, absence, and / or level of each target molecule in the circulating microparticle to generate a set of at least three (informationally) linked signals for the circulating microparticle, where one of the linked signals corresponds to the presence, absence, and / or level of a first fragment of the target nucleic acid (e.g., genomic DNA) in the circulating microparticle, one of the linked signals corresponds to the presence, absence, and / or level of a second fragment of the target nucleic acid (e.g., genomic DNA) in the circulating microparticle, and one of the linked signals corresponds to the presence, absence, and / or level of a target biomolecule (e.g., target polypeptide) in the circulating microparticle.

[0033] The step of measuring signals corresponding to the presence, absence, and / or level of a target nucleic acid (e.g., genomic DNA) fragment may include analyzing the sequences of each of at least two of the at least two fragments of the target nucleic acid (e.g., genomic DNA), and optionally, the step of measuring signals corresponding to the presence, absence, and / or level of a target nucleic acid (e.g., genomic DNA) fragment may include sequencing at least a portion of each of the at least two of the at least two fragments of the target nucleic acid (e.g., genomic DNA) to generate at least two (informationally) concatenated sequence reads.

[0034] Steps to measure signals corresponding to the presence, absence, and / or level of a target nucleic acid (e.g., genomic DNA) fragment may include (a) ligating at least two of at least two fragments of the target nucleic acid (e.g., genomic DNA) to produce a set of at least two ligated fragments of the target nucleic acid (e.g., genomic DNA), and optionally (b) analyzing the sequences of each of at least two of the ligated fragments in the set. Step (b) may include sequencing at least a portion of each of the at least two of the ligated fragments in the set to produce at least two (informationally) ligated sequence reads.

[0035] Steps to measure signals corresponding to the presence, absence, and / or levels of a target nucleic acid (e.g., genomic DNA) fragment may include (a) attaching each of at least two of the at least two fragments of the target nucleic acid (e.g., genomic DNA) of the circulating microparticle to a barcode sequence to generate a set of linked fragments of the target nucleic acid (e.g., genomic DNA), and optionally (b) analyzing the sequences of each of at least two of the linked fragments in the set. Step (b) may include sequencing at least a portion of each of the at least two of the linked fragments in the set to generate at least two (informationally) linked sequence reads, the at least two linked sequence reads being linked by a barcode sequence. Optionally, each of at least two of the at least two fragments of the target nucleic acid may contain the same barcode sequence.

[0036] Steps to measure signals corresponding to the presence, absence, and / or level of a fragment of a target nucleic acid (e.g., genomic DNA) may include (a) attaching each of at least two of at least two fragments of the target nucleic acid (e.g., genomic DNA) from a circulating microparticle to different barcode sequences of a set of barcode sequences to generate a set of ligated fragments of the target nucleic acid (e.g., genomic DNA), and optionally, (b) analyzing the sequences of each of at least two of the ligated fragments in the set. Step (b) may include sequencing at least a portion of each of at least two of the ligated fragments in the set to generate at least two (informationally) ligated sequence reads. The at least two ligated sequence reads may be ligated by a set of barcode sequences (i.e., the barcode sequence attached to the first fragment of the target nucleic acid and the barcode sequence attached to the second fragment of the target nucleic acid ligate the two sequence reads together by being in the same set of barcode sequences).

[0037] Step (b) may include (a) adding a first barcode sequence to a first fragment of the target nucleic acid (e.g., genomic DNA) to generate a first barcoded target nucleic acid molecule, and adding a second barcode sequence to a second fragment of the target nucleic acid (e.g., genomic DNA) to generate a second barcoded target nucleic acid molecule, wherein the first and second barcode sequences each contain the same barcode sequence or each contains a different set of barcode sequences, and optionally (b) analyzing the sequences of each of the first and second barcoded target nucleic acid molecules. Step (b) may include sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules to generate at least two (informationally) concatenated sequence reads. The at least two concatenated sequence reads may be concatenated by the same barcode sequence or set of barcode sequences. Step (b) may include sequencing all or at least a portion of the first and second barcode sequences attached to the first and second fragments of the target nucleic acid.

[0038] Step (b) may include (a) adding a first barcode oligonucleotide to a first fragment of the target nucleic acid (e.g., genomic DNA) to generate a first barcode target nucleic acid molecule, and adding a second barcode oligonucleotide to a second fragment of the target nucleic acid (e.g., genomic DNA) to generate a second barcode target nucleic acid molecule, wherein the first and second barcode oligonucleotides each contain the same barcode sequence or each contains a different barcode sequence from a set of barcode sequences, and optionally (b) analyzing the sequences of each of the first and second barcode target nucleic acid molecules. Step (b) may include sequencing at least a portion of each of the first and second barcode target nucleic acid molecules to generate at least two (informationally) linked sequence reads. The at least two linked sequence reads may be linked by the same barcode sequence or set of barcode sequences. Step (b) may include sequencing all or at least some of the first and second barcoded oligonucleotides attached to the first and second fragments of the target nucleic acid.

[0039] The step of measuring a signal corresponding to the presence, absence, and / or level of a fragment of a target nucleic acid (e.g., genomic DNA) may include (a) contacting a sample with a multimerized barcoding reagent, the multimerized barcoding reagent comprising a first barcode region and a second barcode region linked together, each barcode region comprising a nucleic acid sequence; and (b) attaching the barcode sequences to each of the first and second fragments of the target nucleic acid of the microparticle to generate first and second barcoded target nucleic acid molecules for the microparticle, the first barcoded target nucleic acid molecule comprising the nucleic acid sequence of the first barcode region, and the second barcoded target nucleic acid molecule comprising the nucleic acid sequence of the second barcode region. The first and second barcode regions may each contain the same barcode sequence, or the first and second barcode regions may contain different barcode sequences of a set of barcode sequences. The method may further include (c) analyzing the sequences of each of the first and second barcoded target nucleic acid molecules. Step (c) may include sequencing at least a portion of each of the first and second barcode target nucleic acid molecules to generate at least two (informationally) concatenated sequence reads. The at least two concatenated sequence reads may be concatenated by the same barcode sequence or set of barcode sequences.

[0040] The step of measuring a signal corresponding to the presence, absence, and / or level of a fragment of a target nucleic acid (e.g., genomic DNA) may include (a) contacting a sample with a multimerized barcoding reagent, the multimerized barcoding reagent comprising a first barcoded oligonucleotide and a second barcoded oligonucleotide linked together, each containing a barcode region, and (b) attaching (e.g., annealing or ligating) the first and second barcoded oligonucleotides to first and second fragments of the target nucleic acid in particulate matter to generate first and second barcoded target nucleic acid molecules. The barcode regions of the first and second barcoded oligonucleotides may each contain the same barcode sequence, or the barcode regions of the first and second barcoded oligonucleotides may each contain different barcode sequences from a set of barcode sequences. The method may further include (c) analyzing the sequences of each of the first and second barcoded target nucleic acid molecules. Step (c) may include sequencing at least a portion of each of the first and second barcode target nucleic acid molecules to generate at least two (informationally) concatenated sequence reads. The at least two concatenated sequence reads may be concatenated by the same barcode sequence or set of barcode sequences.

[0041] A fragment of a target nucleic acid (e.g., genomic DNA) may contain at least one epigenetic modification (e.g., a modified nucleotide or nucleic acid base), and the step of measuring the signal corresponding to the presence, absence, and / or level of the fragment of the target nucleic acid (e.g., genomic DNA) may include measuring the signal corresponding to the presence, absence, and / or level of the epigenetic modification (e.g., a modified nucleotide or nucleic acid base) of the fragment of the target nucleic acid (e.g., genomic DNA). For example, the modified nucleotide or nucleic acid base may include 5-methylcytosine or 5-hydroxymethylcytosine.

[0042] The present invention provides a method for analyzing a sample containing circulating microparticles or a sample derived from circulating microparticles, wherein the circulating microparticles contain at least two target molecules, at least one of the target molecules being a fragment of a target nucleic acid (e.g., genomic DNA) containing epigenetic modifications, and at least one of the target molecules being a target biomolecule (e.g., a target polypeptide), and the method comprises measuring signals corresponding to the presence, absence, and / or level of each of the target molecules to generate a set of at least two (informationally) linked signals for the circulating microparticles, at least one of the linked signals corresponding to the presence, absence, and / or level of epigenetic modifications in the sample, and at least one of the linked signals corresponding to the presence, absence, and / or level of a target biomolecule (e.g., a target polypeptide) in the sample.

[0043] The present invention provides a method for analyzing a sample containing circulating microparticles or a sample derived from circulating microparticles, wherein the circulating microparticles contain at least two target molecules, at least one of which is a fragment of a target nucleic acid (e.g., genomic DNA) containing epigenetic modifications, and at least one of which is a target biomolecule (e.g., a target polypeptide), and the method comprises measuring signals corresponding to the presence, absence, and / or level of each of the target molecules to generate a single signal for the circulating microparticles, the single signal corresponding to the presence, absence, and / or level of the epigenetic modification fragment and the target biomolecule (e.g., a target polypeptide) in the sample.

[0044] This method may include the step of analyzing the sequence of a target nucleic acid (e.g., genomic DNA) that includes epigenetic modifications. Alternatively, this method may not include the step of analyzing the sequence of a target nucleic acid (e.g., genomic DNA) that includes epigenetic modifications.

[0045] Epigenetic modifications may include modified nucleotides, such as modified gDNA nucleotides or modified RNA nucleotides. Modified nucleotides may include modified bases. Modified bases may be methylated bases, such as 5-methylcytosine or 5-hydroxymethylcytosine. A fragment of target nucleic acid (e.g., genomic DNA) containing epigenetic modifications may contain 5-methylcytosine DNA or 5-hydroxymethylcytosine DNA.

[0046] Signals corresponding to the presence, absence, and / or level of epigenetic modifications (e.g., modified DNA or RNA nucleotides) can be measured using barcoded affinity probes. A barcoded affinity probe may comprise at least one affinity moiety linked to a barcoded oligonucleotide, where the barcoded oligonucleotide comprises at least one nucleotide (i.e., the barcoded oligonucleotide comprises a nucleotide sequence of at least one nucleotide length), and the affinity moiety is capable of binding to a target biomolecule (i.e., to an epigenetic modification). The signal can be measured by determining the presence, absence, and / or level of the barcoded oligonucleotide in the barcoded affinity probe (e.g., by sequencing or PCR).

[0047] Signals corresponding to the presence, absence, and / or level of epigenetic modifications (e.g., modified DNA or RNA nucleotides) can be measured by flow cytometry and / or fluorescence-activated cell sorting using optically labeled affinity probes and / or fluorescently labeled affinity probes. Optically labeled affinity probes and / or fluorescently labeled affinity probes can be measured and / or detected using optical microscopy and / or fluorescence microscopy visualization. For example, using a fluorescence microscope and / or using fluorescence laser-based detection and / or using fluorescence-activated cell sorting (FACS) instruments. Optically labeled affinity probes and / or fluorescently labeled affinity probes can be measured and / or detected using sorting processes, for example, using fluorescence-activated cell sorting (FACS).

[0048] Signals corresponding to the presence, absence, and / or level of epigenetic modifications (e.g., modified DNA or RNA nucleotides) can be measured using a method that includes a molecular transformation step. In the case of modified nucleotides (i.e., nucleotides containing modified bases such as 5-methylcytosine or 5-hydroxymethylcytosine), a molecular transformation step can be performed to convert the modified base(s) to a different modified or unmodified nucleotide. This is then detected (e.g., using PCR or sequencing) and provides a signal corresponding to the presence, absence, and / or level of epigenetic modifications. This transformation step may include a bisulfite transformation step, an oxidative bisulfite transformation step, or any other molecular transformation step. This method can be used to measure 5-methylcytosine in genomic DNA fragments of circulating microparticles.

[0049] The method may further include one or more steps of compartmentalizing a sample containing one or more circulating microparticles (or a sample derived from one or more circulating microparticles). In addition, or otherwise, the method may further include one or more steps of adding any one or more barcode sequences and / or compartmentalizing the barcode sequences and / or barcoded oligonucleotides into one or more fragments of a target nucleic acid. One or more barcode sequences and / or barcoded oligonucleotides may be provided by and / or contained therein in one or more multimerized barcoding reagents as described herein.

[0050] Signals corresponding to the presence, absence, and / or level of non-nucleic acid biomolecules (e.g., target polypeptides) can be measured using barcoded affinity probes. A barcoded affinity probe may comprise at least one affinity moiety linked to a barcoded oligonucleotide, where the barcoded oligonucleotide comprises at least one nucleotide (i.e., the barcoded oligonucleotide contains a nucleotide sequence of at least one nucleotide length), and the affinity moiety can bind to a target biomolecule (i.e., a target non-nucleic acid biomolecule (e.g., target polypeptide)). The signal can be measured by determining the presence, absence, and / or level of the barcoded oligonucleotide of the barcoded affinity probe (e.g., by sequencing or PCR).

[0051] Signals corresponding to the presence, absence, and / or levels of non-nucleic acid biomolecules (e.g., target polypeptides) can be measured by flow cytometry and / or fluorescence-activated cell sorting using optically labeled affinity probes and / or fluorescently labeled affinity probes. Optically labeled affinity probes and / or fluorescently labeled affinity probes can be measured and / or detected using optical microscopy and / or fluorescence microscopy visualization. For example, using a fluorescence microscope and / or using fluorescence laser-based detection and / or using fluorescence-activated cell sorting (FACS) instruments. Optically labeled affinity probes and / or fluorescently labeled affinity probes can be measured and / or detected using sorting processes, for example, using fluorescence-activated cell sorting (FACS).

[0052] Signals corresponding to the presence, absence, and / or level of non-nucleic acid biomolecules (e.g., target polypeptides) can be measured by a support labeled with an affinity probe. The support labeled with an affinity probe may include, for example, affinity probe-labeled beads (such as magnetic beads) labeled with an antibody specific to the target polypeptide. The presence, absence, and / or level of non-nucleic acid biomolecules (e.g., target polypeptides in circulating microparticles) can be measured by incubating and / or binding the non-nucleic acid biomolecules to the affinity probe(s) on the support. Optionally, support-bound fractions (i.e., microparticles(s) containing and / or containing high levels of the non-nucleic acid biomolecules) are further isolated and / or processed (e.g., by compartmentalization and / or barcoding and / or nucleic acid sequencing). Optionally, non-support-bound fractions (i.e., microparticles(s) containing and / or containing low levels of the non-nucleic acid biomolecules) are further isolated and / or processed (e.g., by compartmentalization and / or barcoding and / or nucleic acid sequencing).

[0053] Signals corresponding to the presence, absence, and / or level of non-nucleic acid biomolecules (e.g., target polypeptides) can be measured separately from signals corresponding to the presence, absence, and / or level of nucleic acid biomolecules. For example, signals corresponding to the presence, absence, and / or level of non-nucleic acid biomolecules (e.g., target polypeptides) can be measured by FACS, and signals corresponding to the presence, absence, and / or level of nucleic acid biomolecules can be measured by sequencing.

[0054] This method allows for the measurement of a set of linked signals corresponding to the presence, absence, and / or levels of (or each of) circulating microparticles, epigenetic modifications (e.g., modified nucleotides such as modified nucleotides containing 5-methylcytosine and / or 5-hydroxymethylcytosine), and target non-nucleic acid biomolecules (e.g., target polypeptides).

[0055] For example, in this method, the target molecules of the circulating microparticles may include at least two (different) fragments of a target nucleic acid (e.g., genomic DNA), at least one fragment of the target nucleic acid (e.g., genomic DNA) containing epigenetic modifications, and at least one target non-nucleic acid biomolecule (e.g., target polypeptide). This method may include measuring signals corresponding to the presence, absence, and / or level of each of the target molecules to generate a set of linked signals for the circulating microparticles. The method may provide (different) linked signals for each of the target molecules. In this method, at least two of the linked signals may correspond to the presence, absence, and / or level of one of the fragments of the target nucleic acid (e.g., genomic DNA). At least one of the linked signals may correspond to the presence, absence, and / or level of an epigenetic modification (e.g., a modified nucleotide such as a modified nucleotide containing 5-methylcytosine and / or 5-hydroxymethylcytosine). And at least one of the linked signals may correspond to the presence, absence, and / or level of a target non-nucleic acid biomolecule (e.g., target polypeptide).

[0056] The circulating microparticles may contain at least 3, at least 4, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10,000, at least 100,000, or at least 1,000,000 (different) target molecules, and optionally, the method includes generating a set of at least 3, at least 4, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10,000, at least 100,000, or at least 1,000,000 (different) linked signals for the circulating microparticles (i.e., each (different) linked signal for each target molecule of the circulating microparticles).

[0057] The target molecules of the circulating microparticles may include at least 2, at least 3, at least 4, at least 9, at least 49, at least 99, at least 49, at least 499, at least 999, at least 4999, at least 9,999, at least 99,999, or at least 999,999 (different) fragments of a target nucleic acid (e.g., genomic DNA), and at least one target non-nucleic acid biomolecule (e.g., a target polypeptide). Optionally, the method includes generating a set of at least 3, at least 4, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10,000, at least 100,000, or at least 1,000,000 (different) linked signals (i.e., each (different) linked signal of the target molecule of the circulating microparticles) for the circulating microparticles.

[0058] The target molecules of the circulating microparticles may include at least 2, at least 3, at least 4, at least 9, at least 49, at least 99, at least 49, at least 499, at least 4999, at least 9,999, at least 99,999, or at least 999,999 (different) target polypeptides and at least one fragment of a target nucleic acid (e.g., genomic DNA). Optionally, the method includes generating a set of at least 3, at least 4, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10,000, at least 100,000, or at least 1,000,000 (different) linked signals (i.e., each (different) linked signal of the target molecules of the circulating microparticles) for the circulating microparticles.

[0059] The sample may comprise first and second circulating particles, each circulating particle comprising a target molecule (e.g., at least two or at least three target molecules), and the method comprises: performing a measurement step (as described herein) to generate a set of coupled signals for the first circulating particle; and performing a measurement step (as described herein) to generate a set of coupled signals for the second circulating particle.

[0060] For example, the step of measuring a signal corresponding to the presence, absence, and / or level of a fragment of a target nucleic acid (e.g., genomic DNA) is to (a) contact the sample with a library containing at least two multimer barcoding reagents, each multimer barcoding reagent containing a first barcode region and a second barcode region linked together, each barcode region containing a nucleic acid sequence, and the first and second barcode regions of the first multimer barcoding reagent being different from the first and second barcode regions of the second multimer barcoding reagent in the library; and (b) attaching the barcode sequences to each of the first and second fragments of the target nucleic acid of the first microparticle to the first and second microparticles. The method includes generating barcode target nucleic acid molecules, wherein the first barcode target nucleic acid molecule includes the nucleic acid sequence of the first barcode region of the first multimer barcoding reagent, and the second barcode target nucleic acid molecule includes the nucleic acid sequence of the second barcode region of the first multimer barcoding reagent; and generating first and second barcode target nucleic acid molecules for the second microparticles by adding the barcode sequences to each of the first and second fragments of the target nucleic acid of the second microparticle, wherein the first barcode target nucleic acid molecule includes the nucleic acid sequence of the first barcode region of the second multimer barcoding reagent, and the second barcode target nucleic acid molecule includes the nucleic acid sequence of the second barcode region of the second multimer barcoding reagent.

[0061] For example, the step of measuring a signal corresponding to the presence, absence, and / or level of a fragment of a target nucleic acid (e.g., genomic DNA) is to (a) contact a sample with a library containing at least two multimer barcoding reagents, each multimer barcoding reagent containing first and second barcoding oligonucleotides linked together, each barcoding oligonucleotide containing a barcode region, and the barcode regions of the first and second barcoding oligonucleotides of the first multimer barcoding reagent in the library containing the first and second barcode regions of the second multimer barcoding reagent in the library. (b) bringing into contact with a region different from the barcode region of the barcoded oligonucleotide, and (b) adding (e.g., annealing or ligating) the first and second barcoded oligonucleotides of the first multimer barcoding reagent to the first and second fragments of the target nucleic acid of the first microparticle to generate the first and second barcoded target nucleic acid molecules, and adding (e.g., annealing or ligating) the first and second barcoded oligonucleotides of the second multimer barcoding reagent to the first and second fragments of the target nucleic acid of the second microparticle to generate the first and second barcoded target nucleic acid molecules.

[0062] The sample may comprise n circulating microparticles, where each circulating microparticle comprises a target molecule (e.g., at least two or at least three target molecules), and the method comprises performing the step of measuring (as described herein) for each circulating microparticle to generate a set of coupled signals for each circulating microparticle. Optionally, n is at least 3, at least 5, at least 10, at least 50, at least 100, at least 1000, at least 10,000, at least 100,000, at least 1,000,000, at least 10,000,000, or at least 100,000,000 circulating microparticles.

[0063] This method may further include a step of determining the identity of the cell and / or tissue of origin of the target biomolecule from which the linked set of signals originates. The step of determining the identity of the cell and / or tissue of origin may include identifying one or more signature signals in the linked set of signals. Signature signals may be signals corresponding to the presence, absence, and / or level of a signature target biomolecule, where the signature target biomolecule is a target biomolecule characteristic of a particular cell and / or tissue.

[0064] The signature signal may be a combined signature signal corresponding to the presence, absence, and / or level of any two or more signature target biomolecules, where the signature target biomolecules are characteristic of a particular cell and / or tissue (for example, the target biomolecules together are characteristic of a particular cell and / or tissue). For example, the combined signature signal may correspond to the presence, absence, and / or level of any two or more biomolecules from biomolecule group 1. Optionally, the combined signature signal may correspond to the presence, absence, and / or level of any two or more biomolecules from biomolecule group 1, as well as any one or more reference sequences, and any one or more epigenetic signals (e.g., one or more signals corresponding to 5-methylcytosine, and / or one or more signals corresponding to 5-hydroxymethylcytosine). The signature signal may be a combination signature signal corresponding to the presence, absence, and / or levels of any number of signature target biomolecules, such as at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, or at least 50 signature target biomolecules (and / or such list or group as a list or group as a list or group as a reference sequence, and / or a list or group as a signal corresponding to 5-methylcytosine and / or 5-hydroxymethylcytosine).

[0065] The cells of origin may be derived from a specific subject (e.g., fetal cells, maternal cells, or paternal cells). The cells of origin may be lung cells, liver cells, ovarian cells, kidney cells, pancreatic cells, uterine cells, skin cells, epithelial cells, endothelial cells, brain cells, bladder cells, blood cells, lymphocytes, prostate cells, mammary cells, colorectal cells, heart cells, vascular cells (arterial or venous cells, etc.), and / or any other type of cell.

[0066] The cells of origin may be cancerous or malignant cells. The cells of origin may be lung cancer cells, breast cancer cells, ovarian cancer cells, prostate cancer cells, kidney cancer cells, liver cancer cells, hematological cancer cells, leukemia cells, lymphoma cells, colorectal cancer cells, pancreatic cancer cells, brain cancer cells, uterine cancer cells, bile duct cancer cells, skin cancer cells, melanoma cells, bladder cancer cells, esophageal cancer cells, oral cancer cells, pharyngeal cancer cells, and / or any other type of cancer cell.

[0067] The tissue of origin may be derived from a specific subject (e.g., fetal tissue, maternal tissue, or paternal tissue). The tissue of origin may be lung tissue, liver tissue, ovarian tissue, cardiac tissue, vascular tissue, intravascular tissue, intravascular plaque tissue, stable intravascular plaque tissue, unstable and / or fragile intravascular plaque tissue, atherosclerotic tissue, thrombotic tissue, embolic tissue, cerebrovascular tissue, endocarditis tissue, myocarditis tissue, peripheral artery tissue, brain tissue, cardiomyopathy tissue, and / or any other tissue.

[0068] The tissue of origin may be cancerous or malignant tissue. The tissue of origin may be cancerous lung tissue, cancerous liver tissue, cancerous ovarian tissue, cancerous breast tissue, cancerous prostate tissue, cancerous blood tissue, cancerous leukemia tissue, cancerous lymphoma tissue, cancerous colorectal tissue, cancerous pancreatic tissue, cancerous brain tissue, cancerous skin tissue, cancerous melanoma tissue, cancerous bladder tissue, cancerous esophageal tissue, and / or any other cancerous tissue.

[0069] The signature signal may include signals corresponding to the presence, absence, and / or level of a first signature biomolecule and signals corresponding to the presence, absence, and / or level of a second signature biomolecule. The first and second signature biomolecules can take any of the forms described herein for the target biomolecule. For example, the signature signal may include signals corresponding to the presence, absence, and / or level of any one or more biomolecules listed in biomolecule group 1.

[0070] Signature biomolecules may be polypeptides expressed only in specific cell types or tissue types (e.g., cancer cells or fetal cells). Signature biomolecules may be polypeptides preferentially expressed in specific cell types or tissue types (e.g., cancer cells or fetal cells). Signature biomolecules may be nucleic acids (mRNA molecules or microRNA molecules, etc.) expressed only (or preferentially expressed) in specific cell types or tissue types (e.g., cancer cells or fetal cells, or intravascular tissues such as intravascular plaques). For example, signature biomolecules may include any one or more biomolecules listed in biomolecule group 1.

[0071] Signature biomolecules can be genomic DNA fragments containing epigenetic modifications, such as 5-hydroxymethylcytosine. Genomic DNA fragments containing 5-hydroxymethylcytosine may provide signature signals to cancerous and / or malignant cells or tissues.

[0072] The signature biomolecule may be a polypeptide or the RNA encoding that polypeptide, for example, TTF-1 (also known as NK2 homeobox 1) or TTF-1 RNA. TTF-1 (or TTF-1 RNA) may provide a signature signal to lung cells and / or tissues.

[0073] Signature signals for lung cancer may be provided by measuring signals corresponding to the presence, absence, and / or level of genomic DNA fragments containing 5-hydroxymethylcytosine (the first signature biomolecule) and signals corresponding to the presence, absence, and / or level of TTF-1 or TTF-1 RNA (as the second signature biomolecule).

[0074] The present invention provides a method for analyzing a sample containing circulating microparticles or a sample derived from circulating microparticles, the method comprising: (a) contacting the sample with a barcoded affinity probe, wherein the barcoded affinity probe comprises at least one affinity moiety linked to a barcoded oligonucleotide, the barcoded oligonucleotide comprises at least one nucleotide (i.e., the barcoded oligonucleotide comprises a nucleotide sequence of at least one nucleotide length), and the affinity moiety is capable of binding to a target biomolecule; (b) forming a reaction mixture, the step of forming the reaction mixture comprising binding to the target molecule, if an affinity moiety is present, to form a barcoded biomolecular complex comprising the barcoded affinity probe and the target biomolecule; and (c) determining the presence, absence, and / or level of a target biomolecule in the sample by measuring the presence, absence, and / or level of a barcoded oligonucleotide in the reaction mixture.

[0075] The present invention provides a method for analyzing a sample containing circulating microparticles or a sample derived from circulating microparticles, wherein the circulating microparticles contain a target biomolecule, and the method comprises: (a) contacting the sample with a barcoded affinity probe, wherein the barcoded affinity probe contains at least one affinity moiety linked to a barcoded oligonucleotide, the barcoded oligonucleotide contains at least one nucleotide (i.e., the barcoded oligonucleotide contains a nucleotide sequence of at least one nucleotide length), and the affinity moiety is capable of binding to the target biomolecule; (b) forming a reaction mixture, wherein the step of forming the reaction mixture includes binding the affinity moiety to the target biomolecule to form a barcoded biomolecular complex containing the barcoded affinity probe and the target biomolecule; and (c) determining the level of the target biomolecule in the sample by measuring the level of the barcoded oligonucleotide in the reaction mixture.

[0076] The present invention provides a method for analyzing a sample containing circulating microparticles or a sample derived from circulating microparticles, the method comprising: (a) contacting the sample with at least one affinity moiety such that the affinity moiety can bind to a target biomolecule; (b) forming a reaction mixture, the step of forming the reaction mixture comprising: (i) binding the affinity moiety, if present, to the target biomolecule; (ii) contacting the sample with a barcoded oligonucleotide and linking the barcoded oligonucleotide to the affinity moiety to form a barcoded biomolecular complex comprising a barcoded affinity probe and a target biomolecule, wherein the barcoded affinity probe comprises at least one affinity moiety linked to the barcoded oligonucleotide, and the barcoded oligonucleotide comprises at least one nucleotide (i.e., the barcoded oligonucleotide comprises a nucleotide sequence of at least one nucleotide length); and (c) determining the presence, absence, and / or level of the target biomolecule in the sample by measuring the presence, absence, and / or level of the barcoded oligonucleotide in the reaction mixture.

[0077] The present invention provides a method for analyzing a sample containing circulating microparticles or a sample derived from circulating microparticles, wherein the circulating microparticles contain a target biomolecule, and the method comprises (a) contacting the sample with at least one affinity moiety such that the affinity moiety can bind to the target biomolecule; and (b) forming a reaction mixture, wherein the step of forming the reaction mixture comprises (i) binding the affinity moiety to the target biomolecule; and (ii) contacting the sample with a barcoded oligonucleotide, ligating the barcoded oligonucleotide to the affinity moiety to form a barcoded biomolecular complex containing a barcoded affinity probe and a target biomolecule, wherein the barcoded affinity probe contains at least one affinity moiety ligated to the barcoded oligonucleotide, and the barcoded oligonucleotide contains at least one nucleotide (i.e., the barcoded oligonucleotide contains a nucleotide sequence of at least one nucleotide length); and (c) determining the level of the target biomolecule in the sample by measuring the level of the barcoded oligonucleotide in the reaction mixture.

[0078] The step of forming a reaction mixture may include incubating the reagent under conditions suitable for binding the affinity moiety to the target biomolecule.

[0079] Prior to the step of measuring the presence, absence, and / or level of barcoded oligonucleotides in the sample, this method may include removing or depleting barcoded affinity probes and / or barcoded oligonucleotides that are not part of the barcoded biomolecular complex.

[0080] Measuring the level of barcoded oligonucleotides in a reaction mixture may involve quantifying the level of barcoded oligonucleotides in the reaction mixture.

[0081] Barcoded oligonucleotides can be linked directly or indirectly (e.g., via one or more linker molecules) to their affinity moieties. A barcoded oligonucleotide may be linked to its affinity moiety via a linker molecule, which is added and / or linked and / or bonded (covalently or non-covalently) to both at least one affinity moiety and at least one barcoded oligonucleotide. A barcoded oligonucleotide can be linked to any affinity moiety by one or more covalent linkages (or bonds) (e.g., covalent bonds such as those created by Innova Biosciences' LighteningLink® antibody labeling kit), one or more non-covalent linkages (or bonds) (e.g., protein-protein interactions or streptavidin-biotin linkages; for example, the affinity moiety may contain a streptavidin domain, and the barcoded oligonucleotide may contain a biotin moiety), or nucleic acid hybridization linkages. Any one or more linker molecules may be biopolymers (e.g., nucleic acid molecules) or synthetic polymers. Any one or more linker molecules may contain one or more ethylene glycol and / or poly(ethylene) glycol (e.g., hexa-ethylene glycol or penta-ethylene glycol) units. Any one or more linker molecules may contain one or more ethyl groups, such as one or more C3 (three-carbon) spacers, C6 spacers, C12 spacers, or C18 spacers.

[0082] The sample can be brought into contact with a library of at least 2, at least 3, at least 5, at least 10, at least 20, or at least 30 different barcode affinity probes.

[0083] The barcoded affinity probe may optionally include an aptamer in which the barcoded affinity probe is an aptamer. The aptamer may provide both the affinity moiety and the barcoded oligonucleotide of the barcoded affinity probe.

[0084] An aptamer may comprise at least one affinity moiety linked to a barcoded oligonucleotide, the barcoded oligonucleotide comprising at least one nucleotide, and the affinity moiety capable of binding to a target biomolecule. An aptamer may comprise a barcode sequence. Any or all of the nucleic acid sequences of the aptamer may be relevant to and / or help identify the affinity moiety of the aptamer, and / or can identify the target biomolecule to which the affinity moiety of the aptamer can bind.

[0085] The affinity moiety may be able to bind to the target biomolecule. The affinity moiety may be able to bind specifically to the target biomolecule. The affinity moiety may be able to bind to the target biomolecule. The affinity moiety may bind to the target biomolecule and bind specifically to it. The affinity moiety may have high affinity for the target biomolecule.

[0086] The affinity moiety may include one or more of the following: antibodies, antibody fragments, light chain antibody fragments, single-strand variable fragments (scFv), peptides, cell-permeable peptides, aptamers, DNA aptamers, and / or RNA aptamers.

[0087] The affinity portion may contain an antibody or a fragment thereof, and the target molecule may be a polypeptide.

[0088] The affinity portion may include an antibody or a fragment thereof, and the target molecule may be a fragment of nucleic acid.

[0089] The affinity moiety may include an antibody or a fragment thereof, and the target molecule may be a nucleic acid fragment containing epigenetic modifications, such as 5-methylcytosine or 5-hydroxymethylcytosine.

[0090] The affinity moiety may contain an aptamer, and the target molecule may be a polypeptide.

[0091] The affinity moiety may contain an aptamer, and the target molecule may be a nucleic acid fragment.

[0092] The affinity moiety may contain an aptamer, and the target molecule may be a nucleic acid fragment containing epigenetic modifications, such as 5-methylcytosine or 5-hydroxymethylcytosine.

[0093] A barcoded affinity probe may contain an aptamer, where the aptamer is contained within an aptamer sequence within an affinity oligonucleotide. The barcoded affinity probe may contain an aptamer, where the aptamer is contained within an aptamer sequence within an affinity oligonucleotide, and the affinity oligonucleotide contains a barcode sequence. The barcoded affinity probe may contain an aptamer, where the aptamer is contained within an aptamer sequence within an affinity oligonucleotide, and the affinity oligonucleotide contains a barcode sequence, and all or part of the barcode sequence is composed of the aptamer sequence in part or completely. An aptamer and / or aptamer sequence and / or affinity oligonucleotide and / or barcode sequence may contain one or more DNA nucleotides. Optionally, any such aptamer and / or aptamer sequence and / or affinity oligonucleotide and / or barcode sequence may contain one or more RNA nucleotides.

[0094] A barcoded affinity probe may comprise at least two affinity moieties. The barcoded affinity probe may comprise at least a first and a second affinity moiety, wherein the first affinity moiety can bind to a first target biomolecule, and the second affinity moiety can bind to a second target biomolecule, wherein the first target biomolecule and the second target biomolecule are different.

[0095] A barcoded affinity probe may contain at least three, at least four, at least five, or at least ten different affinity moieties. Optionally, each affinity moiety can bind to a different target biomolecule.

[0096] A barcoded affinity probe may include at least two affinity moieties that are directly or indirectly linked. The at least two affinity moieties of the barcoded affinity probe may be linked to a support (e.g., a solid support), a molecular support, or a polymer support.

[0097] A barcoded affinity probe may contain at least two affinity moieties. Each affinity moiety may contain an aptamer. At least two affinity moieties of a barcoded affinity probe may be contained within a single aptamer. At least two affinity moieties of a barcoded affinity probe may be contained within a single adjacent nucleic acid sequence (e.g., a DNA sequence and / or an RNA sequence).

[0098] A barcode affinity probe may contain at least two different barcoded oligonucleotides.

[0099] A barcoded oligonucleotide contains at least one nucleotide. A barcoded oligonucleotide may contain a barcode sequence. A barcoded oligonucleotide contains a barcode sequence of at least 2, at least 3, at least 5, at least 10, at least 20, or at least 30 nucleotides.

[0100] A barcoded oligonucleotide may contain a barcode sequence that is related to and / or identifies the affinity moiety to which it is ligated. Each barcoded oligonucleotide ligated to the same affinity moiety (e.g., the same antibody specific to the same protein target) may contain the same sequence (e.g., the same barcode sequence). Each barcoded oligonucleotide ligated to the same affinity moiety may contain a different sequence (e.g., two or more different barcode sequences). Optionally, each barcoded oligonucleotide ligated to a different affinity moiety may contain a different sequence (e.g., two or more different barcode sequences).

[0101] A barcoded oligonucleotide may include an adapter and / or coupling sequence, wherein the sequence is at least 1, at least 2, at least 3, at least 5, at least 10, at least 20, or at least 30 nucleotides long. The adapter and / or coupling sequence of a barcoded oligonucleotide may include a sequence complementary to the target region of the barcoded oligonucleotide contained in any multimerized barcoded reagent and / or its library. The adapter and / or coupling sequence of a barcoded oligonucleotide may include a poly(A) sequence of 2 nucleotides or longer. The adapter and / or coupling sequence in a barcoded oligonucleotide may be contained within the 3' end and / or 5' end of the barcoded oligonucleotide.

[0102] A barcoding affinity probe may comprise one or more secondary barcoding oligonucleotides, wherein the secondary barcoding oligonucleotide comprises a sequence at least partially complementary to all or part of one or more (non-secondary) barcoding oligonucleotides. The secondary barcoding oligonucleotide may be fully or partially annealed (i.e., hybridized) with any one or more (non-secondary) barcoding oligonucleotides. The secondary barcoding oligonucleotide may be fully or partially annealed (i.e., hybridized) with any one or more (non-secondary) barcoding oligonucleotides in the secondary barcoding oligonucleotide annealing reaction. The secondary barcoding oligonucleotide annealing reaction may occur before and / or after and / or in between any of steps (a), (b), or (c). The secondary barcoding oligonucleotide may comprise one or more nucleotides of a barcode sequence, wherein the barcode sequence relates to and / or identifies the affinity moiety linked within the barcoding affinity probe.

[0103] A barcoded affinity probe may comprise one or more affinity moieties, one or more primary barcoded oligonucleotides, and one or more secondary barcoded oligonucleotides.

[0104] The sample may contain one or more circulating particles, and / or the sample may originate from one or more circulating particles.

[0105] Biomolecules can be polypeptides (e.g., proteins), carbohydrates, lipids, or nucleic acids. Biomolecules can also be metabolites.

[0106] The sample may comprise a first circulating microparticle and a second circulating microparticle, or the sample may be derived from the first circulating microparticle and the second circulating microparticle, and step (b) comprises forming at least one barcoded biomolecular complex comprising a barcoded affinity probe and a target biomolecule of the first circulating microparticle, and forming at least one barcoded biomolecular complex comprising a barcoded affinity probe and a target biomolecule of the second circulating microparticle. The sample may further comprise a fragment of the target nucleic acid of the first circulating microparticle and a fragment of the target nucleic acid of the second circulating microparticle.

[0107] In steps (a), (b), and / or (c), the barcoding affinity probe may be at any concentration, e.g., at least 100 nanomoles, at least 10 nanomoles, at least 1 nanomoles, at least 100 picomoles, at least 10 picomoles, at least 1 picomole, at least 100 femtomoles, at least 10 femtomoles, or at least 1 femtomol. The concentration may be 1 picomole to 100 nanomoles, 10 picomoles to 10 nanomoles, or 100 picomoles to 1 nanomoles.

[0108] Optionally, in any one or more steps of any method (any step of adding a coupling sequence and / or coupling molecule, any step of adding and / or linking and / or connecting a barcode sequence of a barcode oligonucleotide, etc. (any step of adding / linking / connecting a barcode sequence contained within a barcode oligonucleotide, etc.), the step(s) and / or method(s) may be carried out in a high viscosity solution. Optionally, such a high viscosity solution may consist of one or more poly(ethylene glycol) (PEG) solutions, such as PEG400, PEG1000, PEG2000, PEG4000, PEG5000, PEG8000, PEG10000, and / or PEG20,000. Optionally, such a solution may contain, by weight or volume, at least 5% poly(ethylene) glycol, at least 10% poly(ethylene) glycol, at least 20% poly(ethylene) glycol, at least 25% poly(ethylene) glycol, at least 30% poly(ethylene) glycol, at least 40% poly(ethylene) glycol, or at least 50% poly(ethylene) glycol. Optionally, such a solution may contain any two or more PEG molecules, where each of these two or more PEG molecules is present at one of these concentrations by weight or volume. Optionally, such a high-viscosity solution may include a solution used in any step of annealing a barcoded oligonucleotide to a target nucleic acid. Optionally, such high-viscosity solutions may have a dynamic viscosity of at least 1.0 cmpoise, at least 1.1 cmpoise, at least 1.2 cmpoise, at least 1.5 cmpoise, at least 2.0 cmpoise, at least 5.0 cmpoise, at least 10.0 cmpoise, at least 20.0 cmpoise, at least 50.0 cmpoise, at least 100.0 cmpoise, or at least 200.0 cmpoise (e.g., at 25°C at standard sea level pressure). Preferably, such high-viscosity solutions will have a dynamic viscosity of at least 1.5 cmpoise.The use of high-viscosity solutions can slow down the diffusion of reagents (such as barcoded oligonucleotides and / or polymer barcoded reagents), preventing or delaying their diffusion from target molecules such as target nucleic acids.

[0109] In any one or more steps of any method (such as any step of adding a coupling sequence and / or coupling molecule, any step of adding and / or linking and / or connecting a barcode sequence, etc. (such as any step of adding / linking / connecting a barcode sequence contained within a barcode sequence), etc.), the step(s) and / or method(s) may be carried out in a solution containing one or more molecular packing reagents. That is, the molecular packing reagent(s) may have the effect of increasing the effective concentration of the target molecule and / or barcode sequence and / or polymer barcode reagent and / or other components in the step(s). Optionally, any one or more molecular clustering reagents may include beads and / or other rigid supports of any size, e.g., micron-scale beads (such as beads with a diameter of at least 1.0, at least 2.0, at least 3.0, at least 5.0, at least 10, at least 20, at least 50, or at least 100 micrometers) and / or nanometer-scale beads (such as beads with a diameter of at least 1.0, at least 2.0, at least 3.0, at least 5.0, at least 10, at least 20, at least 50, or at least 100 nanometers).

[0110] One or more steps to remove and / or deplete unbound barcoded affinity probes can be performed during and / or after any step of binding one or more barcoded affinity probes to one or more biomolecules from one or more circulating microparticles.

[0111] Optionally, any method for measuring biomolecules from circulating microparticles may include measurements using a single barcode affinity probe, wherein the single barcode affinity probe comprises an oligonucleotide of at least one nucleotide length.

[0112] Optionally, any nucleotide and / or oligonucleotide sequence of at least single nucleotide length may be considered a barcode and / or barcode sequence (and / or barcode oligonucleotide) within a barcoding affinity probe. Such nucleotide and / or oligonucleotide sequence of at least single nucleotide length does not need to be different from any other nucleotide and / or oligonucleotide sequences within the barcoding affinity probe, and / or any other nucleotide and / or oligonucleotide sequences within any other barcoding affinity probe.

[0113] Step (c) of this method may include determining the presence, absence, and / or level of barcoded oligonucleotides by analyzing the nucleotide sequence of the barcoded oligonucleotides, optionally, the sequence being analyzed by sequencing (at least a portion of the barcoded oligonucleotides are sequenced) or PCR (at least a portion of the barcoded oligonucleotides are amplified).

[0114] Step (c) may include measuring the presence, absence, and / or level of barcoded oligonucleotides by primer extension and / or PCR reaction and / or quantitative or semi-quantitative PCR reaction (such as a real-time PCR reaction).

[0115] Step (c) may include measuring the presence, absence, and / or level of a barcode oligonucleotide by primer extension and / or PCR reaction and / or quantitative or semi-quantitative PCR reaction (such as a real-time PCR reaction), wherein at least one primer in the reaction is specific to and / or at least partially complementary (and / or at least partially identical) to at least a portion of the barcode oligonucleotide.

[0116] In this method, step (b) or step (c) may include linking together at least two barcoded biomolecular complexes of a first circulating microparticle and linking together at least two barcoded biomolecular complexes of a second circulating microparticle.

[0117] A sample containing one or more circulating particles can be chemically crosslinked (e.g., using formaldehyde). The circulating particles can be chemically crosslinked before steps (a), (b), and / or (c).

[0118] Samples containing one or more circulating particles may be permeated (e.g., using a chemical surfactant). Circulating particles may be permeated before steps (a) and / or (b).

[0119] A sample containing one or more circulating particles may be chemically crosslinked (e.g., with formaldehyde) and then permeated (e.g., with a chemical surfactant) prior to step (a) and / or (b).

[0120] This method may (optionally, as part of step (c)) include (i) contacting a reaction mixture with a multimerized barcoding reagent, wherein the multimerized barcoding reagent comprises first and second barcode regions linked together, each barcode region comprising a nucleic acid sequence; (ii) attaching the barcode sequences of the barcode regions of the multimerized barcoding reagent to the barcoded oligonucleotide of at least one barcoded biomolecular complex of circulating microparticles; and (iii) measuring the presence, absence, and / or level of the barcoded oligonucleotide in the reaction mixture by analyzing the attached barcode sequences of the barcode regions of the multimerized barcoding reagent.

[0121] The reaction mixture may further contain a fragment of a target nucleic acid of a circulating microparticle, wherein the method comprises (i) contacting the reaction mixture with a multimerized barcoding reagent, wherein the multimerized barcoding reagent comprises first and second barcode regions linked together, each barcode region containing a nucleic acid sequence; (ii) adding the barcode sequence of the first barcode region of the multimerized barcoding reagent to a barcoded oligonucleotide of at least one barcoded biomolecular complex of the circulating microparticle (i.e., a first fragment of the target nucleic acid) to generate a first barcoded target nucleic acid molecule, and adding the barcode sequence of the second barcode region of the multimerized barcoding reagent to a fragment of the target nucleic acid (i.e., a second fragment of the target nucleic acid) to generate a second barcoded target nucleic acid molecule; and (iii) analyzing the sequences of the first and second barcoded target nucleic acid molecules, respectively.

[0122] The step of analyzing the sequences of each of the first and second barcoded target nucleic acid molecules can be performed by sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules.

[0123] The method may further include sequencing at least a portion of each of the first and second barcode target nucleic acid molecules of the first circulating microparticle. The method may include generating a sequence read for the first barcode target nucleic acid molecule, wherein the sequence read comprises at least a portion of the sequence of the first barcode region of the multimer barcoding reagent and at least a portion of the sequence of the first fragment of the target nucleic acid of the circulating microparticle. The method may also include generating a sequence read for the second barcode target nucleic acid molecule, wherein the sequence read comprises at least a portion of the sequence of the second barcode region of the multimer barcoding reagent and at least a portion of the sequence of the second fragment of the target nucleic acid of the circulating microparticle.

[0124] This method may (optionally, as part of step (c)) partition the reaction mixture into at least first and second compartments and analyze the nucleotide sequences of the barcoded oligonucleotides of the barcoded biomolecular complex in each of the first and second compartments.

[0125] This method may include partitioning the reaction mixture into at least 3, at least 4, at least 5, at least 10, at least 100, at least 1000, at least 10,000, at least 100,000, at least 1,000,000, at least 10,000,000, or at least 1,000,000,000 compartments. Preferably, this method includes partitioning the reaction mixture into at least 1000 compartments.

[0126] The target nucleic acid molecules may include barcoded oligonucleotides of the barcoded biomolecular complex of the circulating microparticles. The barcoded oligonucleotides of the barcoded biomolecular complex of the circulating microparticles may be present within the barcoded biomolecular complex or may originate from the barcoded biomolecular complex.

[0127] Two or more target nucleic acid molecules may include both a fragment of the target nucleic acid of the microparticle and a barcoded oligonucleotide of the barcoded biomolecular complex of the circulating microparticle.

[0128] Two or more target nucleic acid molecules may include both a fragment of the target nucleic acid (e.g., genomic DNA) of the microparticle and a barcoded oligonucleotide of the barcoded biomolecular complex of the circulating microparticle.

[0129] Two or more target nucleic acid molecules may include both a fragment of the target nucleic acid (e.g., RNA) of the microparticle and a barcoded oligonucleotide of the barcoded biomolecular complex of the circulating microparticle.

[0130] The step of analyzing the nucleotide sequence of a barcode oligonucleotide in a barcoded biomolecular complex may include: adding a first compartmental barcode sequence to at least one barcoded oligonucleotide compartmentalized in the first compartment (the first fragment of the target nucleic acid in the first compartment) (generating a first barcoded target nucleic acid molecule in the first compartment), wherein the at least one barcoded oligonucleotide compartmentalized in the first compartment is contained in or derived from the barcoded biomolecular complex; and adding a second compartmental barcode sequence to at least one barcoded oligonucleotide compartmentalized in the second compartment (the first fragment of the target nucleic acid in the second compartment) (generating a first barcoded target nucleic acid molecule in the second compartment), wherein the at least one barcoded oligonucleotide compartmentalized in the first compartment is contained in or derived from the barcoded biomolecular complex. Preferably, the first and second compartments each contain a barcoded oligonucleotide contained in or derived from the barcoded biomolecular complex.

[0131] The first partition barcode sequence and the second partition barcode sequence may be different. The first partition barcode sequence may be contained within a first set of partition barcode sequences, and the second partition barcode sequence may be contained within a second set of partition barcode sequences, where the first and second sets of partition barcode sequences are different. The first partition barcode sequence may be the nucleic acid sequence of the barcode region of the first multimer barcoding reagent, and the second partition barcode sequence may be the nucleic acid sequence of the second multimer barcoding reagent, where the first and second multimer barcoding reagents each contain two or more barcode regions that are linked together.

[0132] The step of analyzing the nucleotide sequence of the barcoded oligonucleotide of the barcoded biomolecular complex may further include analyzing the compartmental barcode sequence added from each of the first and second compartments.

[0133] A fragment of target nucleic acid (e.g., gDNA or RNA) from a circulating microparticle (a second fragment of the target nucleic acid in the first compartment) may also be added to the first compartment barcode sequence of the first compartment (to generate a second barcoded target nucleic acid molecule in the first compartment), and / or a fragment of target nucleic acid (e.g., gDNA or RNA) from a different circulating microparticle (a second fragment of the target nucleic acid in the second compartment) may also be added to the second compartment barcode sequence of the second compartment (to generate a second barcoded target nucleic acid molecule in the second compartment).

[0134] The step of analyzing the sequences of each of the first and second barcoded target nucleic acid molecules can be performed by sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules.

[0135] This method may further include analyzing the sequences of the first and second barcode target nucleic acid molecules in the first compartment, and analyzing the sequences of the first and second barcode target nucleic acid molecules in the second compartment. Optionally, the step of analyzing sequences is performed by sequencing at least a portion of each of the first and second barcode target nucleic acid molecules.

[0136] The method may further include sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules of the first compartment. The method may include generating a sequence read for the first barcoded target nucleic acid molecule, wherein the sequence read comprises at least a portion of the sequence of the first compartment barcode and at least a portion of the sequence of the first fragment of the target nucleic acid of the first compartment. The method may also include generating a sequence read for the second barcoded target nucleic acid molecule, wherein the sequence read comprises at least a portion of the sequence of the first compartment barcode and at least a portion of the sequence of the second fragment of the target nucleic acid of the first compartment.

[0137] The method may further include sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules of the second compartment. The method may include generating a sequence read for the first barcoded target nucleic acid molecule, wherein the sequence read comprises at least a portion of the sequence of the second compartment barcode and at least a portion of the sequence of the first fragment of the target nucleic acid of the second compartment. The method may also include generating a sequence read for the second barcoded target nucleic acid molecule, wherein the sequence read comprises at least a portion of the sequence of the second compartment barcode and at least a portion of the sequence of the second fragment of the target nucleic acid of the second compartment. A sequence read may contain at least 5, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 5000, or at least 10,000 nucleotides derived from the target nucleic acid (genomic DNA). Preferably, each sequence read contains at least 5 nucleotides derived from the target nucleic acid. As used herein, “at least a portion of a sequence” means at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, or at least 5000 nucleotides of the relevant sequence. Preferably, as used herein, “at least a portion of a sequence” means at least 2 nucleotides of the relevant sequence.

[0138] This method may include a step of amplifying a signal from one or more barcoding affinity probes (i.e., a signal amplification step or process). The signal amplification process may include one or more chain substitution amplification reactions and / or one or more polysubstitution amplification reactions. The signal amplification process may include an in vitro transcription reaction. The signal amplification process may include a step of adding and / or binding and / or annealing (i.e., hybridizing) one or more secondary barcoding oligonucleotides to the barcoding affinity probe, such as (non-secondary) barcoding oligonucleotides in the barcoding affinity probe. The signal amplification process may include a step of adding and / or binding one or more secondary affinity moieties to the barcoding affinity probe (e.g., binding a secondary antibody to a (non-secondary) antibody in the barcoding affinity probe). Optionally, any number of at least two, at least three, at least five, or at least ten secondary barcoding oligonucleotides and / or secondary affinity moieties can be added and / or bound and / or annealed to any barcoding affinity probe. A method for adding and / or annealing and / or binding two or more secondary barcoded oligonucleotides and / or secondary affinity moieties to a barcoded affinity probe may be carried out in separate sequential steps, each of which is added and / or annealed and / or bound, or in a single parallel step.

[0139] Barcoded oligonucleotides and / or secondary barcoded oligonucleotides may contain templates for in vitro transcription reactions. Barcoded oligonucleotides and / or secondary barcoded oligonucleotides may contain promoter regions for in vitro transcription reactions, such as the promoter of T7 RNA polymerase.

[0140] Barcoded oligonucleotides and / or secondary barcoded oligonucleotides may include cyclic (e.g., cyclic) oligonucleotides (such as cyclic DNA oligonucleotides or cyclic RNA oligonucleotides). A cyclic barcoded oligonucleotide may include one or more complementary primer oligonucleotides of at least one nucleotide length, where the complementary primer oligonucleotide is annealed to one or more sequences in the cyclic barcoded oligonucleotide. A cyclic barcoded oligonucleotide can be used as a template for one or more strand substitution amplification reactions and / or one or more polysubstituted amplification reactions, for example, reactions using a strand substitution polymerase such as phi29 DNA polymerase (optionally, one or more complementary primer oligonucleotides may be used as primers for such amplification reactions). The strand substitution amplification reactions and / or polysubstituted amplification reactions may occur before and / or after and / or during any step of binding any one or more barcoded affinity probes to any target biomolecule from a sample. The products of any one or more such strand substitution amplification reactions and / or one or more such polysubstituted amplification reactions may include target nucleic acid molecules for any method described herein. Any one or more of the chain substitution amplification reactions and / or the products of one or more of the polysubstituted amplification reactions may be added to any barcode sequence (such as any partition barcode sequence, any barcoded oligonucleotide, any barcode sequence and / or barcoded oligonucleotide contained in any multimerized barcode reagent).

[0141] This method (optionally as part of step (c)) involves (i) contacting a reaction mixture with a library containing at least two multimer barcoding reagents, each multimer barcoding reagent containing a jointly linked first barcode region and a second barcode region, each barcode region containing a nucleic acid sequence, and the first and second barcode regions of the first multimer barcoding reagent being different from the first and second barcode regions of the second multimer barcoding reagent in the library, and (ii) attaching the barcode sequences to the first and second barcode target nucleic acid molecules of the first microparticles. The method comprises generating a first barcode target nucleic acid molecule comprising the nucleic acid sequence of the first barcode region of the first multimer barcoding reagent and a second barcode target nucleic acid molecule comprising the nucleic acid sequence of the second barcode region of the first multimer barcoding reagent, and adding the barcode sequences to the first fragment and the second fragment of the target nucleic acid of the second microparticle to generate first and second barcode target nucleic acid molecules for the second microparticle, wherein the first barcode target nucleic acid molecule comprising the nucleic acid sequence of the first barcode region of the second multimer barcoding reagent and a second barcode target nucleic acid molecule comprising the nucleic acid sequence of the second barcode region of the second multimer barcoding reagent.

[0142] The first fragment of the target nucleic acid of the first microparticle may be a barcoded oligonucleotide of at least one barcoded biomolecular complex of the first circulating microparticle, and the first fragment of the target nucleic acid of the second microparticle may be a barcoded oligonucleotide of at least one barcoded biomolecular complex of the second circulating microparticle.

[0143] The reaction mixture may further contain a fragment of the target nucleic acid of the first circulating microparticle, the second fragment of the target nucleic acid of the first circulating microparticle being a fragment of the target nucleic acid of the first circulating microparticle.

[0144] The reaction mixture may further contain a fragment of the target nucleic acid of the second circulating microparticle, the second fragment of the target nucleic acid of the second circulating microparticle being a fragment of the target nucleic acid of the second circulating microparticle.

[0145] The step of contacting the reaction mixture with the library of polymer barcoding reagents can be carried out in a single adjacent aqueous volume. Step (c) may be carried out in a single adjacent aqueous volume, optionally steps (b) and (c) may be carried out in a single adjacent aqueous volume, optionally steps (a), (b), and (c) may be carried out in a single adjacent aqueous volume.

[0146] This method may further include analyzing the sequences of the first and second barcoded target nucleic acid molecules of the first circulating microparticles, and analyzing the sequences of the first and second barcoded target nucleic acid molecules of the second circulating microparticles. Optionally, the sequence analysis step is performed by sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules.

[0147] The method may further include sequencing at least a portion of each of the first and second barcode target nucleic acid molecules of the first circulating microparticle. The method may include generating a sequence read for the first barcode target nucleic acid molecule, wherein the sequence read comprises at least a portion of the sequence of the first barcode region of the first multimer barcoding reagent and at least a portion of the sequence of the first fragment of the target nucleic acid of the first circulating microparticle. The method may also include generating a sequence read for the second barcode target nucleic acid molecule, wherein the sequence read comprises at least a portion of the sequence of the second barcode region of the first multimer barcoding reagent and at least a portion of the sequence of the second fragment of the target nucleic acid of the first circulating microparticle.

[0148] The method may further include sequencing at least a portion of each of the first and second barcode target nucleic acid molecules of the second circulating microparticle. The method may include generating a sequence read for the first barcode target nucleic acid molecule, wherein the sequence read comprises at least a portion of the sequence of the first barcode region of the second multimer barcoding reagent and at least a portion of the sequence of the first fragment of the target nucleic acid of the second circulating microparticle. The method may also include generating a sequence read for the second barcode target nucleic acid molecule, wherein the sequence read comprises at least a portion of the sequence of the second barcode region of the second multimer barcoding reagent and at least a portion of the sequence of the second fragment of the target nucleic acid of the second circulating microparticle.

[0149] A sequence read may contain at least 5, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 5000, or at least 10,000 nucleotides derived from the target nucleic acid (genomic DNA). Preferably, each sequence read contains at least 5 nucleotides derived from the target nucleic acid. As used herein, “at least a portion of a sequence” means at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, or at least 5000 nucleotides of the relevant sequence. Preferably, as used herein, “at least a portion of a sequence” means at least 2 nucleotides of the relevant sequence.

[0150] The method may further comprise partitioning a sample or reaction mixture into at least first and second compartments, and analyzing the nucleotide sequences of barcoded oligonucleotides within each of the first and second compartments, wherein the first compartment comprises at least one barcoded oligonucleotide contained in or derived from at least one barcoded biomolecular complex of a first circulating microparticle, and the second compartment comprises at least one barcoded oligonucleotide contained in or derived from at least one barcoded biomolecular complex of a second circulating microparticle. The partitioning step may be performed before step (a), before step (b), and / or before step (c).

[0151] This method may include partitioning the sample into at least 3, at least 4, at least 5, at least 10, at least 100, at least 1000, at least 10,000, at least 100,000, at least 1,000,000, at least 10,000,000, or at least 1,000,000,000 compartments. Preferably, the method includes partitioning the sample into at least 1000 compartments.

[0152] The step of analyzing the nucleotide sequence of the barcoded oligonucleotide of the barcoded biomolecular complex may include (i) adding the barcode sequence of the first compartment to at least one barcoded oligonucleotide of the first compartment, and (ii) adding the barcode sequence of the second compartment to at least one barcoded oligonucleotide of the second compartment.

[0153] The barcode arrangement for the first section and the barcode arrangement for the second section may be different.

[0154] The first partition barcode sequence may originate from a first set of partition barcode sequences, the second partition barcode sequence may originate from a second set of partition barcode sequences, and the first and second sets of partition barcode sequences are different.

[0155] The first partition barcode sequence may be the nucleic acid sequence of the barcode region of the first multimer barcoding reagent, and the second partition barcode sequence may be the nucleic acid sequence of the barcode region of the second multimer barcoding reagent, with the first and second multimer barcoding reagents each containing two or more barcode regions linked together.

[0156] The first compartment may further contain a fragment of the target nucleic acid of the first circulating microparticle, and the second compartment may further contain a fragment of the target nucleic acid of the second circulating microparticle.

[0157] The step of analyzing the nucleotide sequence of the barcoded oligonucleotide of the barcoded biomolecular complex may include (i) attaching a first compartmental barcode sequence to at least one barcoded oligonucleotide in the first compartment and attaching the first compartmental barcode sequence to at least one fragment of the target nucleic acid of the first circulating microparticle, and (ii) attaching a second compartmental barcode sequence to at least one barcoded oligonucleotide in the second compartment and attaching the second compartmental barcode sequence to at least one fragment of the target nucleic acid of the second circulating microparticle, wherein the first compartmental barcode sequence and the second compartmental barcode sequence are different.

[0158] The step of analyzing the nucleotide sequence of the barcoded oligonucleotide of a barcoded biomolecular complex may include (i) attaching the first compartment barcode sequence of the first compartment barcode sequence set to at least one barcoded oligonucleotide in the first compartment and attaching the second compartment barcode sequence of the first compartment barcode sequence set to at least one fragment of the target nucleic acid of the first circulating microparticle, and (ii) attaching the first compartment barcode sequence of the second compartment barcode sequence set to at least one barcoded oligonucleotide in the second compartment and attaching the second compartment barcode sequence of the second compartment barcode sequence set to at least one fragment of the target nucleic acid of the second circulating microparticle, wherein the first compartment barcode sequence set and the second compartment barcode sequence set are different.

[0159] The first and second partition barcode sequences of the first partition barcode sequence set may be the nucleic acid sequences of the first and second barcode regions of the first multimer barcoding reagent, and the first and second partition barcode sequences of the second partition barcode sequence set may be the nucleic acid sequences of the first and second barcode regions of the second multimer barcoding reagent, with the first multimer barcoding reagent and the second multimer barcoding reagent each containing two or more barcode regions linked together.

[0160] The first compartment may further contain a fragment of a target nucleic acid, and the second compartment may further contain a fragment of a target nucleic acid, and the step of analyzing the nucleotide sequence of the barcoded oligonucleotide of the barcoded biomolecular complex includes (i) attaching the first compartment barcode sequence to at least one barcoded oligonucleotide of the first compartment and attaching the first compartment barcode sequence to at least one fragment of the target nucleic acid of the first compartment, and (ii) attaching the second compartment barcode sequence to at least one barcoded oligonucleotide of the second compartment and attaching the second compartment barcode sequence to at least one fragment of the target nucleic acid of the second compartment, wherein the first compartment barcode sequence and the second compartment barcode sequence are different. Alternatively, the step of analyzing the nucleotide sequence of a barcoded oligonucleotide of a barcoded biomolecular complex includes (i) attaching the first partition barcode sequence of the first partition barcode sequence set to at least one barcoded oligonucleotide of the first partition, and attaching the second partition barcode sequence of the first partition barcode sequence set to at least one fragment of the target nucleic acid of the first partition, and (ii) attaching the first partition barcode sequence of the second partition barcode sequence set to at least one barcoded oligonucleotide of the second partition, and attaching the second partition barcode sequence of the second partition barcode sequence set to at least one fragment of the target nucleic acid of the second partition, wherein the first partition barcode sequence set and the second partition barcode sequence set are different.

[0161] The first and second partition barcode sequences of the first partition barcode sequence set may be the nucleic acid sequences of the first and second barcode regions of the first multimer barcoding reagent, and the first and second partition barcode sequences of the second partition barcode sequence set may be the nucleic acid sequences of the first and second barcode regions of the second multimer barcoding reagent, with the first multimer barcoding reagent and the second multimer barcoding reagent each containing two or more barcode regions linked together.

[0162] The present invention provides the use of a barcoded affinity probe for determining the presence, absence, and / or level of a target biomolecule in circulating microparticles or a sample derived therefrom, wherein the barcoded affinity probe comprises at least one affinity moiety linked to a barcoded oligonucleotide, the barcoded oligonucleotide comprising at least one nucleotide, and the affinity moiety capable of binding to the target biomolecule.

[0163] The present invention provides a barcoded affinity probe for determining the presence, absence, and / or level of a target biomolecule, wherein the barcoded affinity probe comprises at least one affinity moiety linked to a barcoded oligonucleotide, the barcoded oligonucleotide comprises at least one nucleotide, and the affinity moiety is capable of binding to the target biomolecule.

[0164] The barcoded affinity probe, target biomolecule, affinity moiety, and barcoded oligonucleotide may take any of the forms described herein. In particular, they may take any of the forms described herein with respect to this method.

[0165] The present invention provides a library of barcoded affinity probes for determining the presence, absence, and / or level of at least two target biomolecules, wherein the library comprises: (i) a first barcoded affinity probe comprising at least one affinity moiety linked to a barcoded oligonucleotide, wherein the barcoded oligonucleotide comprises at least one nucleotide and the affinity moiety can bind to a first target biomolecule; and (ii) a second barcoded affinity probe comprising at least one affinity moiety linked to a barcoded oligonucleotide, wherein the barcoded oligonucleotide comprises at least one nucleotide and the affinity moiety can bind to a second target biomolecule, wherein the first target biomolecule and the second target biomolecule are different.

[0166] The barcoded affinity probes, the library of barcoded affinity probes, target biomolecules, affinity moieties, and barcoded oligonucleotides may take any of the forms described herein. In particular, they may take any of the forms described herein with respect to this method.

[0167] The first target biomolecule may be a polypeptide, and the second target biomolecule may be a barcoded oligonucleotide or a fragment of a target nucleic acid (genomic DNA).

[0168] The first target biomolecule may be a polypeptide, and the second target biomolecule may be a fragment of target nucleic acid (e.g., genomic DNA) containing epigenetic modifications (e.g., 5-hydroxymethylcytosine DNA or 5-methylcytosine DNA).

[0169] The first target biomolecule may be 5-hydroxymethylcytosine DNA, and the second target biomolecule may be a biomolecule selected from biomolecule group 1.

[0170] The first target biomolecule may be 5-methylcytosine DNA, and the second target biomolecule may be a biomolecule selected from biomolecule group 1.

[0171] The first and second target biomolecules may be selected from biomolecule group 1.

[0172] Optionally, any library of two or more barcode affinity probes may comprise a single mixed solution containing the two or more barcode affinity probes. Optionally, any library of two or more barcode affinity probes may comprise two or more separate solutions, each containing a solution of one of the two or more barcode affinity probes. Optionally, any library of two or more barcode affinity probes may be provided in the form of a kit, the kit comprising two or more separate solutions, each containing a solution of one of the two or more barcode affinity probes.

[0173] A sample can be contacted with a library of at least 2, at least 3, at least 5, at least 10, at least 20, or at least 30 different barcoded affinity probes. Preferably, the library contains at least two different barcoded affinity probes. Each barcoded affinity probe may contain at least one affinity moiety linked to a barcoded oligonucleotide, the barcoded oligonucleotide containing at least one nucleotide, and the affinity moiety can bind to a target biomolecule. Each affinity moiety of different barcoded affinity probes in the library may be able to bind to a different target biomolecule. The library of barcoded affinity probes may be able to bind to at least 2, at least 3, at least 5, at least 10, at least 20, or at least 30 different target biomolecules. Preferably, the library of barcoded affinity probes can bind to at least two different target biomolecules.

[0174] Optionally, in any library of two or more barcode affinity probes, barcode affinity probes containing the same affinity moiety (and / or affinity moiety capable of binding to the same target biomolecule) may contain the same barcode oligonucleotide. Optionally, in any library of barcode affinity probes, barcode affinity probes containing the same affinity moiety (and / or affinity moiety having affinity for the same target biomolecule) may contain different barcode oligonucleotides or different barcode sequences from two or more sets of different barcode sequences, and / or from sets of at least 10 different barcode sequences, and / or from sets of at least 100 different barcode sequences, and / or from sets of at least 1000 different barcode sequences, and / or from sets of at least 10,000 different barcode sequences, and / or from sets of at least 1,000,000 different barcode sequences.

[0175] Optionally, in any library of two or more different barcoded affinity probes, each barcoded affinity probe may contain two or more sets of different affinity moieties (for example, each barcoded affinity probe may contain two or more different affinity moieties, each capable of binding to a different target biomolecule). Optionally, in any library of barcoded affinity probes, barcoded affinity probes containing the same set of two or more different affinity moieties (and / or the same set of affinity moieties capable of binding to the same target biomolecule(s)) may contain the same barcoded oligonucleotide. Optionally, any library of barcoded affinity probes containing the same set of two or more different affinity moieties (and / or a set of affinity moieties capable of binding to the same target biomolecule(s)) may contain different barcode sequences from different barcode sequences or sets of two or more different barcode sequences, and / or from sets of at least 10 different barcode sequences, and / or from sets of at least 100 different barcode sequences, and / or from sets of at least 1000 different barcode sequences, and / or from sets of at least 10,000 different barcode sequences, and / or from sets of at least 1,000,000 different barcode sequences.

[0176] A library of two or more different barcode affinity probes may comprise a barcode affinity probe, each containing one or more affinity moieties, one or more primary barcode oligonucleotides, and one or more secondary barcode oligonucleotides, wherein each primary barcode oligonucleotide in the library contains the same sequence, and each secondary barcode oligonucleotide in the library contains a different sequence.

[0177] An optically labeled affinity probe and / or a fluorescently labeled affinity probe is provided, wherein the optically labeled affinity probe and / or the fluorescently labeled affinity probe comprises at least one affinity moiety having affinity and / or specificity for any one or more biomolecules (or target biomolecules) selected from biomolecule group 1. An optically labeled affinity probe and / or a fluorescently labeled affinity probe is provided, wherein the optically labeled affinity probe and / or the fluorescently labeled affinity probe comprises at least one affinity moiety having affinity and / or specificity for any one or more biomolecules (or target biomolecules) selected from biomolecule group 1, and comprises at least one optical and / or fluorescent label.

[0178] A library of two or more optically labeled affinity probes and / or fluorescently labeled affinity probes is provided, comprising at least first and second affinity probes for at least first and second biomolecules (or target biomolecules) selected from biomolecule group 1. Each optically labeled affinity probe and / or fluorescently labeled affinity probe comprises at least one optical label and / or fluorescent label. A library of two or more optically labeled affinity probes and / or fluorescently labeled affinity probes is provided, comprising a first optically labeled affinity probe and / or fluorescently labeled affinity probe having affinity and / or specificity for 5-methylcytosine DNA or 5-hydroxy-methylcytosine DNA, and at least second optically labeled affinity probes and / or fluorescently labeled affinity probes having affinity and / or specificity for any one or more biomolecules (or target biomolecules) selected from biomolecule group 1.

[0179] One or more oligonucleotides are provided, each containing a sequence identical and / or complementary to any DNA and / or RNA sequence of any biomolecule in biomolecule group 1. One or more primers are provided, each containing a sequence identical and / or complementary to any DNA and / or RNA sequence of any biomolecule in biomolecule group 1. One or more oligonucleotide probes for an in-situ hybridization (ISH) process are provided, each containing a sequence identical and / or complementary to any DNA and / or RNA sequence of any biomolecule in biomolecule group 1. One or more oligonucleotide probes for a fluorescent in-situ hybridization (FISH) process are provided, each containing a sequence identical and / or complementary to any DNA and / or RNA sequence of any biomolecule in biomolecule group 1. Optionally, any of these oligonucleotides, and / or primers, and / or oligonucleotide probes may include optical and / or fluorescent labeling. Optionally, any of these oligonucleotides, and / or primers, and / or oligonucleotide probes may include adapter sequences and / or coupling sequences. Selectively, any of the oligonucleotides, and / or primers, and / or oligonucleotide probes can be used in the reverse transcription process, and / or primer extension process, and / or PCR process, and / or in situ hybridization (ISH) process, and / or fluorescent in situ hybridization (FISH) process. A library of two or more oligonucleotides is provided, each oligonucleotide containing a sequence identical and / or complementary to any DNA and / or RNA sequence of any biomolecule of biomolecule group 1.

[0180] In this method, the circulating microparticles may include at least two fragments of a target nucleic acid, wherein the method includes (a) preparing a sample for sequencing, which involves ligating at least two of the at least two fragments of the target nucleic acid to produce a set of at least two ligated fragments of the target nucleic acid, and (b) sequencing at least two of the ligated fragments in the set to produce at least two (informationally) ligated sequence reads.

[0181] In this method, the circulating microparticles may include at least two fragments of a target nucleic acid, wherein the method includes (a) preparing a sample for sequencing, which involves ligating at least two of the at least two fragments of the target nucleic acid to produce a set of at least two ligated fragments of the target nucleic acid, and (b) sequencing at least two of the ligated fragments in the set to produce at least two (informationally) ligated sequence reads.

[0182] In this method, the circulating microparticles comprise at least two fragments of genomic DNA, wherein the method comprises (a) preparing a sample for sequencing, which includes (a) ligating at least two of the at least two fragments of genomic DNA to produce a set of at least two ligated fragments of genomic DNA, and (b) sequencing at least two of the ligated fragments in the set to produce at least two ligated sequence reads.

[0183] In this method, the circulating microparticles may contain at least two fragments of genomic DNA, wherein the method includes (a) preparing a sample for sequencing, which involves ligating at least two of the at least two fragments of genomic DNA to produce a set of at least two ligated fragments of genomic DNA, and (b) sequencing at least two of the ligated fragments in the set to produce at least two ligated sequence reads.

[0184] In this method, at least 3, at least 4, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10,000, at least 100,000, or at least 1,000,000 fragments of the target nucleic acid of the microparticles may be ligated together as a set, then sequenced to produce at least 3, at least 4, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10,000, at least 100,000, or at least 1,000,000 ligated sequence reads. Preferably, at least 5 fragments of the target nucleic acid of the microparticles may be ligated together as a set, then sequenced to produce at least 5 ligated sequence reads.

[0185] In this method, each of the ligated sequence reads may provide a sequence of at least 1 nucleotide, at least 5 nucleotides, at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 500 nucleotides, at least 1000 nucleotides, or at least 10,000 nucleotides from the ligated fragment. Preferably, each of the ligated sequence reads may provide a sequence of at least 20 nucleotides from the ligated fragment.

[0186] This method may generate a total of at least 2, at least 10, at least 100, at least 1,000, at least 10,000, at least 100,000, at least 1,000,000, at least 10,000,000, at least 100,000,000, at least 1,000,000,000, at least 10,000,000,000, at least 100,000,000,000, or at least 1,000,000,000,000 sequence reads. Preferably, a total of at least 500,000 sequence reads are generated.

[0187] A sequence read may contain at least 5, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 5000, or at least 10,000 nucleotides derived from the target nucleic acid (genomic DNA). Preferably, each sequence read contains at least 5 nucleotides derived from the target nucleic acid.

[0188] A sequence read may include a portion of the raw sequence reads generated from a sequencing instrument, for example, a raw sequence read of a 50-nucleotide sequence generated from an Illumina sequencing instrument. A sequence read may include a merged sequence from both reads of a paired-end sequencing run, for example, a merged sequence from both the first and second reads of a paired-end sequencing run on an Illumina sequencing instrument. A sequence read may include a portion of the raw sequence reads generated from the sequencing instrument within a 150-nucleotide raw sequence read generated by an Illumina sequencing instrument, for example, 20 adjacent nucleotides. A single raw sequence read may include at least two concatenated sequence reads generated by the method of the present invention.

[0189] Sequence reads can be generated by any method known in the art, for example, by chain termination or Sanger sequencing. Preferably, sequencing is performed by next-generation sequencing methods such as synthetic sequencing, synthetic sequencing with reversible terminators (e.g., Illumina sequencing), pyrosequencing (e.g., 454 sequencing), ligation sequencing (e.g., SOLiD sequencing), single-molecule sequencing (e.g., single-molecule real-time (SMRT) sequencing (Pacific Biosciences)), or nanopore sequencing (e.g., on Minion or Promethion platforms (Oxford Nanopore Technologies)). Most preferably, sequence reads are generated by synthetic sequencing with reversible terminators (e.g., Illumina sequencing).

[0190] This method may include a further step of mapping each of the ligated sequence reads to a reference genome sequence. The ligated sequence reads may include sequences mapped to the same chromosome in the reference genome sequence, or sequences mapped to two or more different chromosomes in the reference genome sequence.

[0191] The fine particles may have a diameter of at least 100 nm, at least 110 nm, at least 125 nm, at least 150 nm, at least 175 nm, at least 200 nm, at least 250 nm, or at least 500 nm. Preferably, the fine particles have a diameter of at least 200 nm. The diameter of the fine particles may be between 100 and 5000 nm. The diameter of the fine particles may be between 10 and 10,000 nm (e.g., 100 to 10,000 nm, 110 to 10,000 nm), 50 to 5000 nm, 75 to 5,000 nm, or 100 to 3,000 nm. The diameter of the fine particles may be 10-90 nm, 50-100 nm, 90-200 nm, 100-200 nm, 100-500 nm, 100-1000 nm, 1000-2000 nm, 90-5000 nm, or 2000-10,000 nm. Preferably, the diameter of the fine particles is 100-5000 nm. Most preferably, the fine particles have a diameter of 200-5000 nm. The sample may contain fine particles of at least two different sizes, or at least three different sizes, or a range of different sizes.

[0192] A linked fragment of genomic DNA can originate from a single genomic DNA molecule.

[0193] This method may further include the step of estimating or determining the genomic sequence length of the ligated fragment of genomic DNA. Optionally, this step may be performed by sequencing substantially the entire sequence of the ligated fragment (i.e., from its approximately 5' end to its approximately 3' end) and counting the number of nucleotides sequenced within it. Optionally, this may be performed by sequencing a sufficient number of nucleotides at the 5' end of the ligated fragment's sequence to map the 5' end to a locus in a reference genomic sequence (e.g., the human genomic sequence), similarly sequencing a sufficient number of nucleotides at the 3' end of the ligated fragment to map the 3' end to a locus in the reference genomic sequence, and then determining the genomic sequence length of the ligated fragment using the reference genomic sequence (i.e., the number of nucleotides sequenced at the 3' end of the ligated fragment + the number of nucleotides sequenced at the 5' end of the ligated fragment + the number of nucleotides between these sequences in the reference genomic sequence (i.e., the unsequenced portion)).

[0194] In this method, the sample may comprise first and second circulating microparticles, each microparticle comprising at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method comprises: (a) performing step (a) to generate a first set of ligated fragments of the target nucleic acid for the first microparticle and a second set of ligated fragments of the target nucleic acid for the second microparticle; and (b) performing step (b) to generate a first set of ligated sequence reads (i.e., a set of ligated signals) for the first microparticle and a second set of ligated sequence reads (i.e., a set of ligated signals) for the second microparticle.

[0195] In this method, the set of concatenated sequence reads (i.e., the set of concatenated signals) generated for the first microparticle may be distinguishable from the set of concatenated sequence reads (i.e., the set of concatenated signals) generated for the second microparticle.

[0196] In this method, the sample may contain n microparticles derived from blood, each microparticle containing at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method comprises: (a) generating n linked target nucleic acid fragment sets (one set per n microparticles); and (b) generating n linked sequence read sets (i.e., linked signal sets) (one set per n microparticles).

[0197] In this method, n may be at least 3, at least 5, at least 10, at least 50, at least 100, at least 1000, at least 10,000, at least 100,000, at least 1,000,000, at least 10,000,000, at least 100,000,000, at least 1,000,000,000, or at least 100,000,000,000. Preferably, n is at least 100,000 particles.

[0198] In this method, the sample consists of at least 3, at least 5, at least 10, at least 50, at least 100, at least 1000, at least 10,000, at least 100,000, at least 1,000,000, at least 10,000,000, at least 100,000,000, at least 10,000,000,000, or at least 100,000,000,000 particles (and / Alternatively, a sample derived from at least 3, at least 5, at least 10, at least 50, at least 100, at least 1000, at least 10,000, at least 100,000, at least 1,000,000, at least 10,000,000, at least 100,000,000, at least 1,000,000,000, or at least 100,000,000,000 particles can be evaluated. Here, the fine particles (and / or samples derived therefrom) are contained in a single adjacent aqueous volume during any step of the method, such as any step of contacting the sample with a library of polymeric barcoding reagents, and / or any step of adding and / or linking and / or ligating a barcode sequence (e.g., barcoded oligonucleotide) to a target nucleic acid, and / or any step of adding a coupling sequence to a target nucleic acid, and / or any step of adding and / or linking and / or ligating a coupling molecule to a target nucleic acid or other target biomolecule, and / or any step of crosslinking or permeabilization.

[0199] The set of concatenated sequence reads (i.e., the set of concatenated signals) generated for each microparticle may be distinguishable from the set of concatenated sequence reads generated for other microparticles.

[0200] This method may further include the step of partitioning the sample into at least two different reaction volumes prior to step (a).

[0201] In the present invention, two sequences or sequence reads (determined, for example, by a sequencing reaction) can be informationally linked by any means that enable such sequences to relate to or interact with one another in any way within a computer system, an algorithm, or a dataset. Such linkages can consist of and / or be established and / or represented by individual specific linkages, or shared properties, or any indirect methods that link, interact with, or correlate two or more such sequences.

[0202] A concatenation may consist of, and / or be established and / or represented by, a sequence within the sequencing reaction itself (e.g., the form of a barcode sequence determined by the sequencing reaction, or the form of two different parts or segments of a single determined sequence that together contain first and second concatenated sequences), or may be established, constitute, or represented independently of such a sequence (e.g., established by the merit of being contained within the same flow cell or the same lane of a flow cell, or within the same compartment or region of a sequencing instrument, or within the same sequencing run of a sequencing instrument, or with some spatial proximity within a biological sample, and / or with some spatial proximity within a sequencing instrument or sequencing flow cell). A concatenation may consist of, and / or be established and / or represented by, measurements or parameters corresponding to physical locations or compartments within a sequencing instrument, such as pixels or pixel positions in an image and / or in a multi-pixel camera or multi-pixel charge-coupled device, and / or the positions of nanopores in a nanopore or nanopore sequencing instrument or nanopore film.

[0203] Linking can be absolute (i.e., two sequences are either linked or not linked, and there is no other quantitative, semi-quantitative, or qualitative / categorical relationship). Linking can also be relative, probabilistic, established, constitute, or represent, for example, the degree, probability, or range of linking, with respect to one or more parameters that can hold one of the quantitative, semi-quantitative, or qualitative / categorical values ​​of a series. For example, two (or more) sequences may be informationally linked by a quantitative, semi-quantitative, or qualitative / categorical parameter that represents, includes, estimates, or embodies the proximity of those two (or more) sequences in a sequencing instrument or in a biological sample.

[0204] For any analysis involving two or more sequences that are informationally linked in any such manner, the presence (or absence) of linkage can be used as a parameter of the analysis or evaluation step or the algorithm for performing it. For any analysis involving two or more sequences that are informationally linked in any such manner, the degree, probability, or range of linkage can be used as a parameter of the analysis or evaluation step or the algorithm for performing it.

[0205] In one version of such concatenation, a given set of two or more concatenated sequences may be associated with a specific identifier, such as an alphanumeric identifier, a barcode, or a barcode sequence. In one further version, a given set of two or more concatenated sequences may be associated with a barcode or barcode sequence, where the barcode or barcode sequence is contained within a sequence determined by a sequencing reaction. For example, each sequence determined by a sequencing reaction may contain both a barcode sequence and a sequence corresponding to a genomic DNA sequence. Optionally, a particular sequence or concatenated sequence may be represented or associated with two or more barcodes or identifiers.

[0206] In another version of concatenation, two or more concatenated arrays may be held in separate partitions within a computer, or in a computer network, on a hard drive, or on any kind of storage medium, or any other means of storing array data. Optionally, a particular array or concatenated array may be held in two or more partitions within such computer or data medium.

[0207] An informationally linked array may contain one or more sets of informationally linked arrays. All arrays within a set of linked arrays may share the same linking function or representation. For example, all arrays within a linked set may be associated with the same barcode or the same identifier, or may be contained within the same partition in a computer or storage medium. All arrays may share any other form of linking, interrelationship, and / or correlation. One or more arrays within a linked set may be exclusive members of that set and therefore may not be members of any other set. Alternatively, one or more arrays within a linked set may be non-exclusive members of that set and therefore may be represented and / or associated with two or more different sets of linked arrays.

[0208] The present invention provides a method for analyzing a sample containing at least two circulating microparticles or a sample derived from at least two circulating microparticles, comprising: (i) partitioning the sample into at least two compartments, each compartment containing, on average, fewer than n circulating microparticles; and (ii) determining the presence, absence, and / or levels of at least two target biomolecules in each of the at least two of the at least two compartments. Optionally, n is 1000, 500, 200, 100, 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, 0.001, 0.0005, or 0.0001. Preferably, where n is 0.5. Optionally, step (i) includes partitioning the sample into at least 3 compartments, at least 5 compartments, at least 10 compartments, at least 100 compartments, at least 1000 compartments, at least 10,000 compartments, at least 100,000 compartments, at least 1,000,000 compartments, at least 100,000,000 compartments, or at least 1,000,000,000 compartments. Preferably, step (i) includes partitioning the sample into at least 1000 compartments.

[0209] (ii) The step of determining the presence, absence, and / or level of at least two target biomolecules may be carried out by a method of analyzing a sample containing circulating microparticles (i.e., the sample in the compartment) or a sample derived from circulating microparticles for each of at least two of the at least two compartments, wherein the circulating microparticles contain at least two target molecules, and at least two target molecules are biomolecules, and the method comprises measuring signals corresponding to the presence, absence, and / or level of each of the target molecules to generate at least two (informationally) coupled sets of signals for the circulating microparticles (i.e., at least two (informationally) coupled sets of signals for the compartment), wherein at least one of the coupled signals corresponds to the presence, absence, and / or level of a first biomolecule in the sample (i.e., the sample in the compartment), and at least one of the coupled signals corresponds to the presence, absence, and / or level of a second biomolecule in the sample (i.e., the sample in the compartment). The method may be carried out by any of the methods provided herein, which comprises generating at least two coupled sets of signals for the microparticles. The method can generate at least two coupled sets of signals for each of at least two of the at least two compartments.

[0210] The present invention provides a method for analyzing a sample comprising at least two circulating microparticles or a sample derived from at least two circulating microparticles, comprising: (i) partitioning the sample into at least two compartments, wherein the first compartment comprises at least first and second target biomolecules of the first circulating microparticle, the second compartment comprises at least first and second target biomolecules of the second circulating microparticle, and each of the at least two compartments comprises, on average, a total DNA mass less than [X]; and (ii) determining the presence, absence, and / or level of at least two target biomolecules in each of the at least two compartments, optionally, where [X] is 1.0 attgram of DNA, 10 attgrams of DNA, 100 attgrams of DNA, 1.0 femtgram of DNA, 10 femtgrams of DNA, 100 femtgrams of DNA, 1.0 picogram of DNA, 10 picograms of DNA, 100 picograms of DNA, or 1.0 nanogram of DNA. Preferably, here, [X] is 100 femtograms of DNA.

[0211] (ii) The step of determining the presence, absence, and / or level of at least two target biomolecules may be carried out by a method of analyzing a sample containing circulating microparticles (i.e., the sample in the compartment) or a sample derived from circulating microparticles for each of at least two of the at least two compartments, wherein the circulating microparticles contain at least two target molecules, and at least two target molecules are biomolecules, and the method comprises measuring signals corresponding to the presence, absence, and / or level of each of the target molecules to generate at least two (informationally) coupled sets of signals for the circulating microparticles (i.e., at least two (informationally) coupled sets of signals for the compartment), wherein at least one of the coupled signals corresponds to the presence, absence, and / or level of a first biomolecule in the sample (i.e., the sample in the compartment), and at least one of the coupled signals corresponds to the presence, absence, and / or level of a second biomolecule in the sample (i.e., the sample in the compartment). The method may be carried out by any of the methods provided herein, which comprises generating at least two coupled sets of signals for the microparticles. The method can generate at least two coupled sets of signals for each of at least two of the at least two compartments.

[0212] The present invention provides a method for analyzing a sample containing at least two circulating microparticles or a sample derived from at least two circulating microparticles, comprising: (i) partitioning the sample into at least two compartments, wherein the first compartment contains at least first and second target biomolecules of the first circulating microparticle, the second compartment contains at least first and second target biomolecules of the second circulating microparticle, and each of the at least two compartments contains, on average, a total polypeptide mass less than [Y]; and (ii) determining the presence, absence, and / or level of at least two target biomolecules in each of the at least two compartments, optionally, where [Y] is 1.0 attgram of polypeptide, 10 attgrams of polypeptide, 100 attgrams of polypeptide, 1.0 femtgram of polypeptide, 10 femtgrams of polypeptide, 100 femtgrams of polypeptide, 1.0 picogram of polypeptide, 10 picograms of polypeptide, 100 picograms of polypeptide, or 1.0 nanogram of polypeptide. Preferably, here, [Y] is 100 femtograms of polypeptide.

[0213] (ii) The step of determining the presence, absence, and / or level of at least two target biomolecules may be carried out by a method of analyzing a sample containing circulating microparticles (i.e., the sample in the compartment) or a sample derived from circulating microparticles for each of at least two of the at least two compartments, wherein the circulating microparticles contain at least two target molecules, and at least two target molecules are biomolecules, and the method comprises measuring signals corresponding to the presence, absence, and / or level of each of the target molecules to generate at least two (informationally) coupled sets of signals for the circulating microparticles (i.e., at least two (informationally) coupled sets of signals for the compartment), wherein at least one of the coupled signals corresponds to the presence, absence, and / or level of a first biomolecule in the sample (i.e., the sample in the compartment), and at least one of the coupled signals corresponds to the presence, absence, and / or level of a second biomolecule in the sample (i.e., the sample in the compartment). The method may be carried out by any of the methods provided herein, which comprises generating at least two coupled sets of signals for the microparticles. The method can generate at least two coupled sets of signals for each of at least two of the at least two compartments.

[0214] This method may further include analyzing the sequences of at least two target nucleic acid molecules partitioned within each of the first and second compartments.

[0215] This method may include partitioning the sample into at least 3, at least 4, at least 5, at least 10, at least 100, at least 1000, at least 10,000, at least 100,000, at least 1,000,000, at least 10,000,000, or at least 1,000,000,000 compartments. Preferably, this method includes partitioning the sample into at least 1000 compartments.

[0216] The first target biomolecule may be a polypeptide, and the second target biomolecule may be a barcoded oligonucleotide or a fragment of a target nucleic acid (genomic DNA).

[0217] The first target biomolecule may be a polypeptide, and the second target biomolecule may be a fragment of target nucleic acid (e.g., genomic DNA) containing epigenetic modifications (e.g., 5-hydroxymethylcytosine DNA or 5-methylcytosine DNA).

[0218] The first target biomolecule may be 5-hydroxymethylcytosine DNA, and the second target biomolecule may be a biomolecule selected from biomolecule group 1.

[0219] The first target biomolecule may be 5-methylcytosine DNA, and the second target biomolecule may be a biomolecule selected from biomolecule group 1.

[0220] The first and second target biomolecules may be selected from biomolecule group 1.

[0221] Any one or more steps of determining (or measuring) the presence, absence, and / or level of a target biomolecule (or measuring a signal corresponding to the presence, absence, and / or level of a target biomolecule) may be performed using one or more barcoding affinity probes (provided herein), for example, by binding the barcoding affinity probes to the target biomolecule. Any one or more steps of determining (or measuring) the presence, absence, and / or level of a target biomolecule (or measuring a signal corresponding to the presence, absence, and / or level of a target biomolecule) may be performed according to any method comprising contacting a sample with a barcoding affinity probe (provided herein). Optionally, this method comprises binding at least one barcoding affinity probe to the target biomolecule, where the barcode sequence from a multimeric barcoding reagent is added to the barcoding oligonucleotide of the barcoding affinity probe. Optionally, the measurement is performed by analyzing the barcode sequence of the multimeric barcoding reagent and / or by analyzing the barcode sequence from the barcoding oligonucleotide of the barcoding affinity probe.

[0222] Any one or more steps of determining (or measuring) the presence, absence, and / or level of a target biomolecule (or measuring a signal corresponding to the presence, absence, and / or level of a target biomolecule) may be performed using one or more optical and / or fluorescent / fluorescent measurement processes, for example, using one or more optically labeled and / or fluorescently labeled affinity probes. For example, the measurement step may be performed using one or more optically labeled and / or fluorescently labeled affinity probes, where at least one optically labeled and / or fluorescently labeled affinity probe is bound to the target biomolecule, and the measurement is performed using at least one optical measurement step or at least one fluorescence detection step (for example, the measurement is performed by measuring the optical and / or fluorescent signals from the optically labeled and / or fluorescently labeled affinity probes).

[0223] Optionally, any one or more optical and / or fluorescence measurement processes may include optical and / or fluorescence measurements of a sample containing one or more circulating microparticles and / or biomolecules from one or more circulating microparticles, the sample contained in an aqueous volume and / or aqueous droplet (such as a droplet analyzed by a fluorescence-activated cell sorting (FACS) instrument). Optionally, any such optical and / or fluorescence measurement process may further include sorting and / or selection processes, for example, any one or more optical and / or fluorescence measurements of circulating microparticles used to sort and / or select any group and / or subset of any given circulating microparticles and / or two or more circulating microparticles (e.g., to sort a sample containing circulating microparticles into a first subset of circulating microparticles exhibiting high levels of a particular target biomolecule and a second subset of circulating microparticles exhibiting high levels of the same particular target biomolecule).

[0224] Optionally, any one or more optical and / or fluorescence measurement processes may include optical and / or fluorescence measurements of a sample containing one or more circulating microparticles and / or biomolecules from one or more circulating microparticles, the sample being contained on a plane (such as a flat glass surface like a microscope slide, or any other plane). Optionally, any one or more optical and / or fluorescence measurement processes may include optical and / or fluorescence measurements of a sample containing one or more circulating microparticles and / or biomolecules from one or more circulating microparticles, the sample being visualized with an optical microscope and / or fluorescence microscope.

[0225] Optionally, any one or more fluorescently labeled affinity probes may comprise a fluorophore having a specific absorption spectrum and / or emission spectrum. Optionally, any one or more fluorescently labeled affinity probes included in a pool and / or library and / or set of two or more fluorescently labeled affinity probes may comprise a fluorophore having an absorption spectrum and / or emission spectrum different from at least one and / or at least two of the other fluorescently labeled affinity probes in the pool and / or library and / or set.

[0226] Optionally, all fluorescently labeled affinity probes and / or sets of two or more fluorescently labeled affinity probes containing the same target biomolecule within a pool and / or library may contain fluorophores having the same absorption and / or emission spectra. Optionally, all fluorescently labeled affinity probes and / or sets of two or more fluorescently labeled affinity probes containing the same target biomolecule within a pool and / or library may contain the same fluorophores. Optionally, all fluorescently labeled affinity probes and / or sets of two or more fluorescently labeled affinity probes containing the same target biomolecule within a pool and / or library may contain two or more different fluorophores (e.g., two or more different fluorophores containing two or more different absorption and / or emission spectra). Optionally, each fluorescently labeled affinity probe and / or set of two or more fluorescently labeled affinity probes in a pool and / or library may contain fluorophores from a set of two or more different fluorophores (e.g., two or more different fluorophores containing two or more different absorption and / or emission spectra). Here, all such fluorescently labeled affinity probes having affinity for the same target biomolecule share the same fluorophore, and optionally, each fluorophore identifies and / or associates with the target biomolecule of the fluorescently labeled affinity probe. Optionally, in any pool and / or library and / or set of two or more fluorescently labeled affinity probes, a number of different fluorophores (e.g., any number of different fluorophores including different absorption and / or emission spectra) such as at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 50 may be used.

[0227] Any method for analyzing a sample containing at least one circulating microparticle, optionally, includes any sample, and / or solution, and / or reactants or reaction mixtures, and / or aqueous volume, and / or mixtures containing any number or concentration of circulating microparticles, and / or any number or concentration of biomolecules from one or more circulating microparticles, and / or any number or concentration of (identical or different) barcodes, and / or any number or concentration of (identical or different) barcode molecules, and / or any number or concentration of (identical or different) barcode sequences, and / or any number or concentration of (identical or different) barcoded oligonucleotides, and / or any number or concentration of (identical or different) multimer barcoded reagents, and / or any number or concentration of (identical or different) affinity moieties, and / or any number or concentration of (identical or different) barcoded affinity probes, and / or any number or concentration of (identical or different) adapter oligonucleotides, and / or any number or concentration of (identical or different) ) Coupling sequences, and / or any number or concentrations of (identical or different) concentrated probes, and / or any number or concentrations of (identical or different) primers, and / or any number or concentrations of (identical or different) hybridization probes, and / or any number or concentrations of (identical or different) fluorescent in-situ hybridization probes are contained within a single compartment or within at least the first and second compartments during and / or before and / or after any one or more steps of the method. It may be rare (for example, partitioned or divided into first and second partitions), or it may be contained within any number of partitions, such as at least three partitions, at least four partitions, at least five partitions, at least ten partitions, at least 100 partitions, at least 1,000 partitions, at least 10,000 partitions, at least 100,000 partitions, at least 1,000,000 partitions, at least 10,000,000 partitions, or at least 1,000,000,000 partitions (for example, partitioned or divided).

[0228] Optionally, in any method, any one or more target biomolecules may be measured and / or analyzed by optical measurement and / or optical quantification processes. Optionally, in any method, any one or more target biomolecules may be measured and / or analyzed with optically labeled and / or fluorescently labeled affinity probes, wherein the affinity probes have affinity and / or specificity for the target biomolecules.

[0229] Optionally, any method for measuring and / or analyzing biomolecules may include one or more steps of direct detection. Optionally, any method for measuring and / or analyzing biomolecules may include one or more steps of indirect detection.

[0230] To avoid misunderstanding, in the present invention and in any manner herein, any term referring to any one or more biomolecules that are “in a circulating microparticle” and / or “inside a circulating microparticle” and / or “of a circulating microparticle” and / or “from a circulating microparticle” and / or “contained in a circulating microparticle” and / or “contained inside a circulating microparticle” means broadly referring to such biomolecules that are found (and / or potentially found) entirely or partially within any form or location of the circulating microparticle (including being fully or partially encapsulated within a membrane, and / or being fully or partially embedded on the outer and / or inner surfaces of a membrane, and / or fully or partially embedded within a membrane).

[0231] Optionally, any step of analyzing the sequence of one or more target nucleic acid molecules in any manner may be carried out by a primer extension reaction. Optionally, any step of analyzing the sequence of one or more target nucleic acid molecules in any manner may be carried out by polymerase chain reaction (PCR) using a primer set that provides amplification (and therefore measurement and detection) of a specific target sequence (such as a specific DNA, RNA, or cDNA target sequence). Optionally, any step of analyzing the sequence of one or more target nucleic acid molecules in any manner may be carried out by a reverse transcription reaction, optionally accompanied by one or more subsequent primer extension or PCR steps.

[0232] Any step of analyzing the sequence of one or more target nucleic acid molecules in any way and at any discretion may be performed by an in situ hybridization (ISH) process, such as a fluorescence in situ hybridization (FISH) process.

[0233] The method of the present invention can be deterministic (e.g., a single barcode sequence can be used to identify sequence reads from a single microparticle) or probabilistic (e.g., a barcode sequence can be used to identify sequence reads that are likely to be from a single microparticle). As a further example, in this method, the compartmentalization step may aim to achieve an average of one circulating microparticle per compartment. However, this is an inherently statistical process and cannot guarantee that each compartment contains only biomolecules from a single microparticle. Therefore, it cannot be guaranteed that a set of concatenated signals corresponding to biomolecules from a particular compartment corresponds to biomolecules from a single microparticle. For example, if a particular compartment contains two different microparticles, the set of concatenated signals may correspond to two microparticles.

[0234] The present invention further includes a system and apparatus for analyzing a sample containing one or more circulating particles (or two or more such samples, e.g., each containing one or more circulating particles). Optionally, such a system includes at least one algorithm or part of an algorithm and / or computer program (e.g., an algorithm or part of an algorithm and / or computer program contained within a computer system and / or within a web-based or internet-based computer storage system) for analyzing one or more sets of concatenated signals derived from the measurement of at least one circulating particle (e.g., one or more sets of concatenated sequences derived from the measurement of at least one circulating particle) (any one or more algorithms and / or computer programs configured to calculate any one of any parameter values, such as any parameter values ​​described herein). 、 / or at least one reference sequence and / or set of reference sequences (such as one or more reference sequences contained in a computer data storage system such as a computer system and / or a server and / or a hard disk), and / or at least one set of barcoded oligonucleotides, and / or at least one multimer barcoded reagent and / or a library thereof, and / or at least one physical device including one or more compartments (such as one or more tubes, each containing a compartment, and / or one or more plates, each containing a well, and each well containing a compartment, and / or one or more devices, each containing two or more compartments, wherein each such compartment contains a droplet such as a microfluidic device (such as the Chromium system provided by 10X Genomics) that contains or can generate microfluidic droplets, or a plane on which one or more droplets are contained), and / or at least one enzyme or enzyme solution (such as any ligase enzyme, polymerase enzyme, and / or transposase enzyme),The system may also include at least one algorithm and / or computer program configured to report the results of any one or more analyses described herein (such as the results of any one or more methods of diagnosis and / or diagnostic testing based on the analysis of two or more linked signals from a sample containing one or more circulating microparticles) to a physician and / or other healthcare professional and / or patient. For example, a system for analyzing a sample containing one or more circulating microparticles may include at least one algorithm or part thereof for analyzing one or more sets of linked signals derived from the measurement of at least one circulating microparticle, and at least one physical device including at least one set of barcoded oligonucleotides (configured to be together with the circulating microparticles or to be attached to target biomolecules derived from the circulating microparticles), and one or more compartments. Alternatively, such a system may include at least one algorithm or part thereof for analyzing one or more sets of linked signals derived from the measurement of at least one circulating microparticle, and at least one library of multimer barcoded reagents. Alternatively, such a system may include at least one algorithm or part thereof for analyzing one or more sets of concatenated signals derived from the measurement of at least one circulating microparticle, and at least one physical device comprising at least one library of multimer barcoding reagents and one or more compartments. Alternatively, such a system may include at least one algorithm or part thereof for analyzing one or more sets of concatenated signals derived from the measurement of at least one circulating microparticle, and at least one set of barcoded oligonucleotides (configured to be attached to target biomolecules derived from the circulating microparticles or to be combined with the circulating microparticles), and at least one algorithm and / or computer program configured to report the results of any one or more analyses described herein.

[0235] 1. Sample of circulating particulate matter A sample for use in the method of the present invention may contain at least one circulating microparticle (i.e., a microparticle derived from blood (e.g., human blood)) and / or may be derived from at least one circulating microparticle. The microparticle(s) may be derived from maternal blood. The microparticle(s) may be derived from the blood of a patient with a disease (e.g., cancer). The sample may be, for example, a blood sample, a plasma sample, or a serum sample. The sample may be a mammalian sample. Preferably, the sample is a human sample.

[0236] Circulating microparticles may be one or more of the various cell-free microparticles found in the blood, plasma, and / or serum of humans and / or other animals (Orozco et al, Cytometry Part A (2010).77A:502 514, 2010). "Cell-free" refers to the fact that such microparticles are not cells. Instead, the microparticles originate, for example, from secretory or post-apoptotic cells. These microparticles are diverse in the tissues and cells from which they originate, as well as the biophysical processes underlying their formation, and their respective sizes, molecular structures, and compositions. Microparticles may contain one or more components from the cell membrane (e.g., including phospholipid components) and one or more intracellular and / or nuclear components. Microparticles may be selected from one or more exosomes, apoptotic bodies (also known as apoptotic vesicles), and / or extracellular microvesicles.

[0237] Microparticles can be defined as membrane vesicles containing at least two fragments of a target nucleic acid (e.g., genomic DNA). Microparticles may have a diameter of 100 to 5000 nm. Preferably, microparticles have a diameter of 100 to 3000 nanometers.

[0238] Exosomes are one of the smallest circulating microparticles, typically ranging in diameter from 50 to 100 nanometers, and are thought to originate from the cell membrane of viable, intact cells. They contain both protein and RNA components (including both mRNA molecules and / or degraded mRNA molecules, as well as small regulatory RNA molecules such as microRNA molecules) within an outer phospholipid component. Exosomes are thought to be formed by exocytosis of the cytoplasmic multivesicle (Gyorgy et al, Cell. Mol. Life Sci. (2011) 68:2667-2688). Exosomes are thought to play various roles in intercellular signaling and extracellular functions (Kanada et al, PNAS (2015) 1418401112). Techniques for quantifying or sequencing microRNA and / or mRNA molecules found in exosomes have been previously described (e.g., U.S. Patent Application No. 13 / 456,121, European Patent Application No. EP2626433A1).

[0239] Microparticles include apoptotic bodies (also known as apoptotic vesicles) and extracellular microvesicles, which together range in diameter from 1 micron to 2-5 microns, and are generally thought to be larger than 100 nanometers in diameter (Lichtenstein et al, Ann NY Acad Sci. (2001); 945:239-49). All classes of circulating microparticles are thought to be generated by a large number of diverse cells in the body (Thierry et al, Cancer Metastasis Rev35(3), 347-376.9(2016) / s10555-016-9629-x).

[0240] Preferably, the microparticles are not exosomes, but rather any microparticles having a larger diameter than exosomes.

[0241] Samples for use in this method may include samples containing at least one circulating microparticle, and samples derived from at least one circulating microparticle. For example, the step of measuring the signal or measuring a reagent (e.g., a barcoded oligonucleotide) may be performed on a sample containing at least one intact circulating microparticle (e.g., the sample or reaction mixture contains intact circulating microparticles at the time of signal measurement or reagent measurement). Alternatively, the step of measuring the signal or measuring a reagent (e.g., a barcoded oligonucleotide) may be performed on a sample containing biomolecules derived from circulating microparticles (e.g., biomolecules purified and / or processed and / or fractionated and / or isolated from circulating microparticles). The sample may not contain intact circulating microparticles at the time of signal measurement or reagent measurement.

[0242] The sample may contain at least 2, at least 3, at least 4, at least 5, at least 7, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 500, at least 1000, at least 5000, at least 10,000, at least 20,000, at least 50,000, at least 100,000, at least 1,000,000, at least 10,000,000, at least 1,000,000,000, or at least 100,000,000,000 different target biomolecules and / or target epitopes. Preferably, the sample contains at least 100 target biomolecules and / or target epitopes.

[0243] In the sample, nucleic acid fragments (e.g., genomic DNA) may be present at concentrations of less than 1.0 picogram of DNA per microliter, less than 10 picograms of DNA per microliter, less than 100 picograms of DNA per microliter, less than 1.0 nanogram of DNA per microliter, less than 10 nanograms of DNA per microliter, less than 100 nanograms of DNA per microliter, or less than 1000 nanograms of DNA per microliter.

[0244] The sample may contain (or be derived from) at least 2, at least 3, at least 4, at least 5, at least 7, at least 10, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10,000, at least 50,000, at least 100,000, at least 1,000,000, at least 10,000,000, or at least 100,000,000 circulating particles. Preferably, the sample contains (or is derived from) at least 100 circulating particles.

[0245] In the sample, the fine particles may be present in concentrations of less than 0.001, less than 0.01, less than 0.1, less than 1.0, less than 10, less than 100, less than 1000, less than 1000, less than 10,000, less than 100,000, less than 100,000, less than 1,000,000, less than 10,000,000, or less than 100,000,000 per microliter.

[0246] The circulating microparticles may contain at least 2, at least 3, at least 4, at least 5, at least 7, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 500, at least 1000, at least 5000, at least 10,000, at least 20,000, at least 100,000, at least 500,000, at least 1,000,000, or at least 10,000,000 different target biomolecules and / or target epitopes. Preferably, the circulating microparticles contain at least 10 target biomolecules and / or target epitopes.

[0247] The method of the present invention allows for the measurement and / or analysis of any number of one or more different target biomolecules and / or target epitopes. Optionally, in any method, a group of at least 2, at least 3, at least 4, at least 5, at least 7, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 500, at least 1000, at least 5000, at least 10,000, or at least 20,000 different target biomolecules and / or target epitopes can be measured and / or analyzed. Preferably, a group of at least three different target biomolecules and / or target epitopes is measured and / or analyzed.

[0248] This method allows for the measurement and / or analysis of the same target biomolecule (and / or target epitope), and / or the same group of two or more target biomolecules (and / or target epitopes), for all circulating microparticles (or compartments) in the sample. Optionally, in any method, a specific target biomolecule (and / or target epitope), and / or a specific group of two or more target biomolecules (and / or target epitopes), can be measured and / or analyzed for a subset of circulating microparticles in the sample. Optionally, in any method, a sample of circulating microparticles can be divided into any number of two or more subsamples, and different specific target biomolecules (and / or target epitopes), and / or different specific groups of two or more target biomolecules (and / or target epitopes), can be measured and / or analyzed for each subsample.

[0249] Selectively and by any method, two or more different target epitopes of the same biomolecule can be measured and / or analyzed. For example, a target biomolecule (such as a target protein) can be measured or analyzed using two or more different affinity probes (such as two or more different antibodies) that have affinity or specificity for two or more different epitopes within the target biomolecule.

[0250] Biomolecules (also referred to herein as target biomolecules) may be chemical or molecular species present in or derived from circulating microparticles. Biomolecules may be macromolecules. Biomolecules may be polypeptides (e.g., proteins), carbohydrate molecules, lipid molecules, or nucleic acid molecules. Biomolecules may be metabolites. Preferably, the biomolecules are human biomolecules.

[0251] The target biomolecule may have a predetermined (or pre-defined) sequence. For example, the target polypeptide may have a predetermined (or pre-defined) amino acid sequence or epitope. Similarly, a fragment of the target nucleic acid may have a predetermined (or pre-defined) nucleotide sequence. This method may include measuring a signal corresponding to the presence, absence, and / or level of a predetermined (or pre-defined) sequence or epitope using a target-specific reagent, such as a barcoded affinity probe or an affinity probe.

[0252] The biomolecule can be a nucleic acid biomolecule or a non-nucleic acid biomolecule.

[0253] As used herein, the term "polypeptide" includes a chain of at least two amino acid monomers linked by peptide bonds, peptides, and proteins, such as post-translationally modified proteins like glycoproteins. One or more biomolecules can be one or more protein isoforms.

[0254] The biomolecule can include an epitope of an antigen present in or derived from circulating microparticles. For example, the epitope can be an epitope of a polypeptide or protein. The biomolecule can include a specific epitope, such as a specific protein epitope and / or a specific epitope generated by post-translational modification of a protein (e.g., lysine methylation modification). The biomolecule can include a specific nucleic acid epitope, such as a specific nucleic acid modification (e.g., 5-methylcytosine DNA epitope and / or 5-hydroxy-methylcytosine DNA epitope). The biomolecule can include a specific epitope recognized by one or more affinity probes (e.g., barcoded affinity probes), such as a specific epitope recognized by an antibody.

[0255] Biomolecules can contain epitopes that are not nucleic acid epitopes. The biomolecule may not be a 5-methylcytosine DNA molecule (i.e., the biomolecule may be an epitope that is not a 5-methylcytosine DNA epitope), and / or the biomolecule may not be a 5-hydroxy-methylcytosine DNA molecule (i.e., the biomolecule may be an epitope that is not a 5-hydroxy-methylcytosine DNA epitope).

[0256] The biomolecule can be a DNA-binding protein. Optionally, the biomolecule is not a DNA-binding protein.

[0257] The biomolecule can be a histone protein (e.g., histone H1, histone H2A, histone H2B, histone H3, and / or histone H4, and / or any histone variant). The histone protein can be a post-translationally modified histone protein (e.g., histone H3 lysine 4 trimethylation, histone H3 lysine 27 trimethylation, and / or any histone acetylation modification). Optionally, the biomolecule is not a histone protein.

[0258] The biomolecule can be a chromatin protein. Optionally, the biomolecule is not a chromatin protein.

[0259] The biomolecule can be a membrane protein or polypeptide. Optionally, the biomolecule is not a membrane protein or polypeptide. The biomolecule can be a polypeptide or protein that immunoprecipitates with DNA. Optionally, the biomolecule is not a polypeptide or protein that immunoprecipitates with DNA.

[0260] A biomolecule may be a biomolecule that binds to DNA. Optionally, a biomolecule may not be a biomolecule that binds to DNA. A biomolecule may be a membrane biomolecule or a membrane-related biomolecule. Optionally, a biomolecule may not be a membrane biomolecule or a membrane-related biomolecule. A biomolecule may be a biomolecule that immunoprecipitates with DNA. Optionally, a biomolecule may not be a biomolecule that immunoprecipitates with DNA.

[0261] Biomolecules may be entirely or partially contained within the inner and / or outer surface of the membrane of the circulating microparticle (e.g., the lipid bilayer membrane of the circulating microparticle). Biomolecules may be entirely or partially encapsulated within the membrane of the circulating microparticle (e.g., encapsulated within the lipid bilayer of the circulating microparticle). Biomolecules may be contained within and / or across the membrane of the circulating microparticle, and / or any combination thereof. Biomolecules may be contained entirely or partially embedded within the membrane of the circulating microparticle (e.g., entirely or partially embedded within the lipid bilayer membrane of the circulating microparticle).

[0262] Biomolecules may originate from the inner and / or outer surfaces of the circulating microparticles, and / or from within the circulating microparticles (for example, from within the membrane of the circulating microparticles), and / or from within and / or across the membrane of the circulating microparticles, and / or from any combination thereof.

[0263] Biomolecules can be DNA (e.g., double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA)), RNA (e.g., double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA)), or fragments thereof. Biomolecules can also be genomic DNA or RNA (e.g., mRNA), or fragments thereof.

[0264] One or more biomolecules (or target biomolecules) may be DNA fragments, RNA fragments, and / or polypeptides selected from (or encoding) biomolecule group 1, which includes the following: Plasma-based protein markers of cancer and / or cancer aggressiveness, including prostate-specific antigen (PSA) and CA-125. Cell surface and immunocytotype markers including CD3, CD4, CD8, CD19, CD20, CD41, CD45, CD61, CD62, CD146, CD235a, and CD326. • Genes and proteins involved in oncogenesis and malignant transformation, as well as antigens such as KI-67 (Ki-67), NK2 homeobox 1 (TTF-1), B-cell lymphoma 2 (BCL2), BRAF, C-kit / CD117, c-Myc, c-Raf, Ras, Survivin, vascular endothelial growth factor receptor (VEGFR), tumor-associated glycoprotein 72 (TAG-72), epidermal growth factor receptor (EGFR), estrogen receptor, programmed death ligand 1 (PD-L1), cyclin B1, epidermal cell adhesion molecule (EpCAM), HER2 / Neu, progesterone receptor, K-ras, Genes used as immunocytochemical markers to assess cancer cell types and subtypes include NRAS, beta-2 microglobulin (B2M), calcitonin, CA19-9, CA15-3 / CA27.29, chromogranin A (CgA), neuron-specific enolase, lactate dehydrogenase, thyroglobulin, claudin-1 (CLDN1), HE4, platelet-derived growth factor receptor (PDGF-R), nuclear matrix protein 22, cytokeratin 8 (CK-8), cytokeratin 18 (CK-18), cytokeratin fragment 21-1, and OVX1. • Markers associated with pregnancy (i.e., plasma protein markers) or complications of pregnancy, including alpha-fetoprotein (AFP), beta-human chorionic gonadotropin (beta-nCG), and Toll-like receptor 4 (TLR4). Proteins associated with circulating lipoprotein particles and / or intravascular plaques, including annexin V, apolipoprotein A1 (Apo A-1), plasminogen activator inhibitor (PAI-1), CD31, CD144, and urokinase plasminogen activator (uPA). MicroRNA molecules (miRNAs) associated with (and / or differentially expressed within) intravascular plaque, including miR-1, miR-19b, miR-21, miR-22, miR-29b, miR-92a, miR-99a, miR-100, miR-126, miR-127, miR-133a, miR-133b, miR-143, miR-145, miR-199a, miR-210, and let-7f. • Markers of lymphocytes and / or other immune cells, including LY6G6D and immunoglobulins. Other target biomolecules such as transthyretin, C-reactive protein (CRP), and troponin.

[0265] The biomolecules (or target biomolecules) provided above are collectively referred to as "Biomolecule Group 1" in this specification.

[0266] Such DNA fragments may comprise all or part of the DNA sequence of one or more protein-coding genes (e.g., genome sequence, exon region sequence, intron region sequence, promoter region sequence, and / or terminator region sequence). Such RNA fragments may comprise all or part of the RNA sequence of one or more protein-coding genes (e.g., exon RNA sequence, intron RNA sequence, 5' untranslated region sequence, and / or 3' untranslated region sequence). Such polypeptides may comprise all or part of one or more proteins. Such polypeptides may comprise one or more post-translational modifications of the polypeptide (e.g., the polypeptide is acetylated or methylated at any one or more amino acid residues). Preferably, the biomolecule is a human biomolecule (e.g., human Ki-67).

[0267] Biomolecules may contain epigenetic modifications. Epigenetic modifications may include modified nucleotides, e.g., modified gDNA nucleotides or modified RNA nucleotides. Modified nucleotides may include modified bases. Modified bases may be methylated bases, e.g., 5-methylcytosine or 5-hydroxymethylcytosine. Biomolecules (such as fragments of target nucleic acids (e.g., genomic DNA)) may contain 5-methylcytosine (i.e., 5-methylcytosine DNA and / or 5-methylcytosine DNA nucleotides). Biomolecules (such as fragments of target nucleic acids (e.g., genomic DNA)) may contain 5-hydroxymethylcytosine (i.e., 5-hydroxymethylcytosine DNA and / or 5-hydroxymethylcytosine DNA nucleotides). Epigenetic modifications may include post-translational modifications of proteins. Post-translational modifications may include methylation, phosphorylation, acetylation, ubiquitination, and / or SUMOylation. Post-translationally modified polypeptides may be histone proteins. For example, post-translational modified histone proteins (e.g., histone H3 lysine 4 trimethylation, histone H3 lysine 27 trimethylation, and / or any histone acetylation modification).

[0268] Biomolecules may include exogenously administered molecules such as exogenously administered polypeptides (exogenously administered antibodies, etc.) and / or exogenously administered nucleic acids (exogenously administered oligonucleotides, etc., for example, exogenously administered barcode sequences such as barcoded oligonucleotides, etc.).

[0269] The biomolecule may include the barcoded oligonucleotide (or its barcoded sequence) of the barcoded affinity probe.

[0270] At least two of the biomolecules of the circulating microparticles may be fragments of a target nucleic acid (e.g., molecules of fragmented genomic DNA). These molecules of fragmented genomic DNA, and / or sequences contained within these molecules of fragmented genomic DNA, may be concatenated by any method described herein.

[0271] The fragment of the target nucleic acid can be a DNA fragment (e.g., a molecule of fragmented genomic DNA) or an RNA fragment (e.g., a fragment of mRNA). Preferably, the fragment of the target nucleic acid is a fragment of genomic DNA.

[0272] The DNA fragment can be a mitochondrial DNA fragment. The DNA fragment can be a mitochondrial DNA fragment from maternal cells or tissues. The DNA fragment can be a mitochondrial DNA fragment from a fetus or placental tissue. The DNA fragment can be a mitochondrial DNA fragment from diseased tissue and / or cancerous tissue.

[0273] The microparticles can contain platelets. The microparticles can contain tumorized platelets. The target nucleic acid can contain platelet RNA (e.g., a fragment of platelet RNA, and / or a fragment of tumorized platelet RNA). A sample containing one or more platelets can contain platelet-rich plasma (e.g., platelet-rich plasma containing tumorized platelets).

[0274] The fragment of the target nucleic acid can contain double-stranded or single-stranded nucleic acid. The fragment of genomic DNA can contain double-stranded DNA or single-stranded DNA. The fragment of the target nucleic acid can contain partially double-stranded nucleic acid. The fragment of genomic DNA can contain partially double-stranded DNA.

[0275] The fragment of the target nucleic acid can be a fragment derived from a single nucleic acid molecule or a fragment derived from two or more nucleic acid molecules. For example, the fragment of genomic DNA can be derived from a single genomic DNA molecule.

[0276] As those skilled in the art will understand, as used herein, the term "fragment of target nucleic acid" refers to the original fragment present in the microparticle, and its copies or amplicons. For example, the term "fragment of gDNA" refers to the original gDNA fragment present in the microparticle, and, for example, refers to a DNA molecule that can be prepared from the original genomic DNA fragment by a primer extension reaction. As a further example, the term "fragment of mRNA" refers to the original mRNA fragment present in the microparticle, and, for example, refers to a cDNA molecule that can be prepared from the original mRNA fragment by reverse transcription.

[0277] A fragment of the target nucleic acid (e.g., genomic DNA) may be at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, or at least 50 nucleotides. A fragment of the target nucleic acid (e.g., genomic DNA) may be 15 to 100,000 nucleotides, 20 to 50,000 nucleotides, 25 to 25,000 nucleotides, 30 to 10,000 nucleotides, 35 to 5,000 nucleotides, 40 to 1,000 nucleotides, or 50 to 500 nucleotides. A fragment of the target nucleic acid (e.g., genomic DNA) may be 20 to 200 nucleotides long, 100 to 200 nucleotides long, 200 to 1,000 nucleotides long, 50 to 250 nucleotides long, 1,000 to 10,000 nucleotides long, 10,000 to 100,000 nucleotides long, or 50 to 100,000 nucleotides long. Preferably, the fragmented genomic DNA molecules are 50 to 500 nucleotides long.

[0278] Optionally, any method for analyzing a sample containing one or more circulating microparticles (and / or a sample derived from one or more circulating microparticles) may include combinational measurements (e.g., measurements of presence, absence, and / or levels) including the measurement of any combination of any two or more different biomolecules (e.g., any two or more different target biomolecules). For example, any such method may include the measurement of a linked fragment of genomic DNA (e.g., by barcoding and / or sequencing), and optionally, the measurement of a linked fragment of genomic DNA may further include the measurement and / or estimation of the genomic or nucleotide sequence length of the fragment of genomic DNA, and optionally, the measurement of a linked fragment of genomic DNA may further include the measurement and / or estimation of the genomic coordinates (or genomic location) of the 3' end (or multiple) and / or 5' end of the linked fragment of genomic DNA, and the measurement of one or more modified nucleotides or nucleic acid bases (e.g., measurement of 5-methylcytosine, measurement of 5-hydroxymethylcytosine), and the measurement of one or more polypeptide biomolecules (e.g., measurement of one or more biomolecules from biomolecule 1). Optionally, any such combination of measurements may (further) include the measurement of one or more plasma-based protein markers of cancer and / or cancer attack, as well as one or more cell surface or immune cell type markers, as well as one or more proteins involved in oncogenesis and malignant transformation or immunocytochemical markers for assessing cancer cell types and cell types, as well as one or more markers associated with pregnancy or pregnancy complications, as well as one or more proteins associated with circulating lipoprotein particles and / or intravascular plaques, and one or more microRNA molecules (such as markers provided in the list included in Biomolecule Group 1).For example, a combination measurement may include the measurement of a linked fragment of genomic DNA, and optionally, the measurement of a linked fragment of genomic DNA may further include the measurement and / or estimation of the genomic or nucleotide sequence length of the said fragment of genomic DNA, and optionally, the measurement of a linked fragment of genomic DNA may further include the measurement and / or estimation of the genomic coordinates (or genomic position) of the 3' end (or multiple) and / or 5' end of the linked fragment of genomic DNA, and the measurement of one or more modified nucleotides or nucleic acid bases (e.g., measurement of 5-methylcytosine, measurement of 5-hydroxymethylcytosine), as well as the measurement of PSA, CA-125, CD4, CD8, Ki-67, BCL2, and EGFR. Optionally, such a combination measurement may further include the measurement of TTF-1 and / or Ras and / or c-Myc and / or PD-L1 and / or estrogen receptor and / or cyclin B1.

[0279] Optionally, any combination measurement may include separate such combination measurements of two or more samples from a single individual (e.g., a single patient), the two or more samples being taken / prepared from the same individual but separated by one or more time periods (at least one month, at least three months, at least six months, at least twelve months, at least eighteen months, at least two years, at least three years, at least four years, at least five years, and / or at least ten years, and / or any other time period). For example, a particular combination measurement (of any kind described herein) may be performed on a first sample taken from an individual and separately on a second sample taken from the same individual at a later time period. Any number of such consecutive (time-separated) samples from an individual can be analyzed, such as at least three, at least four, at least five, at least six, at least eight, at least ten, at least fifteen, at least twenty, at least twenty-five, or at least thirty consecutive samples, or more or similar numbers.

[0280] 2. Isolation of circulating microparticle samples Numerous methods for isolating circulating microparticles (and / or specific subsets, categories, or fractions of circulating microparticles) have been previously described. European Patent No. ES2540255(B1) and U.S. Patent No. 9005888B2 describe a method for isolating specific circulating microparticles, such as apoptotic bodies, based on a centrifugation procedure. Numerous methods for isolating different types of cell-free microparticles by centrifugation, ultracentrifugation, and other techniques have been previously well described and developed (Gyorgy et al, Cell. Mol. Life Sci. (2011) 68:2667-2688).

[0281] This method may further include isolating a sample containing one or more circulating microparticles from blood, plasma, or serum. The microparticles(s) can be isolated from blood, plasma, or serum. This method may further include the step of isolating microparticles from blood, plasma, or serum.

[0282] The particles(s) can be isolated by centrifugation, size exclusion chromatography, and / or filtering.

[0283] The isolation step may include centrifugation. The particle(s) may be isolated by pelletizing in a centrifugation step and / or ultracentrifugation step, or by a series of two or more centrifugation steps and / or ultracentrifugation steps at two or more different rates, and the pellet and / or supernatant from one centrifugation / ultracentrifugation step may be further processed in a second centrifugation / ultracentrifugation step and / or fractionation centrifugation process.

[0284] The centrifugal separation or ultracentrifugation step(s) may be performed at speeds of 100–500,000G, 100–1,000G, 1,000–10,000G, 10,000–100,000G, 500–100,000G, or 100,000–500,000G. The centrifugal separation or ultracentrifugation step may be performed for at least 5 seconds, at least 10 seconds, at least 30 seconds, at least 60 seconds, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 60 minutes, or at least 3 hours.

[0285] The isolation step may include a column-based size exclusion chromatography process, such as size exclusion chromatography, which may involve a column containing a Sepharose-based matrix or a Cefacryl-based matrix.

[0286] Size exclusion chromatography may involve using a matrix or filter containing pore sizes of at least 50 nanometers, at least 100 nanometers, at least 200 nanometers, at least 500 nanometers, at least 1.0 micrometer, at least 2.0 micrometers, or at least 5.0 micrometers in size or diameter.

[0287] The isolation step may include filtering the sample. The filtrate may provide the particulate matter(s) analyzed in this manner. Optionally, a filter may be used to isolate particulate matter below a certain size, the filter preferentially or completely removing particles larger than 100 nanometers, 200 nanometers, 300 nanometers, 500 nanometers, 1.0 micrometer, 2.0 micrometers, 3.0 micrometers, 5.0 micrometers, or 10.0 micrometers. Optionally, two or more such filtering steps may be performed using filters having the same or different size filtering parameters. Optionally, the filtrate from one or more filtering steps may contain particulate matter from which concatenated sequence reads are generated.

[0288] 3. Preparation of circulating particulate samples for analysis This method allows for the measurement and / or analysis of any one or more target biomolecules while the circulating microparticles are intact. Optionally, any one or more target biomolecules can be measured and / or analyzed while the circulating microparticles are not intact (i.e., after one or more biomolecules have been released from the circulating microparticles).

[0289] A sample containing one or more circulating microparticles may be chemically crosslinked (e.g., using formaldehyde). A sample containing one or more circulating microparticles may be permeated (e.g., using a chemical surfactant). A sample containing one or more circulating microparticles may be chemically crosslinked (e.g., using formaldehyde). The chemical crosslinking and / or permeation step(s) may be performed before measuring and / or analyzing the target biomolecule(s) of the one or more circulating microparticles.

[0290] The crosslinking step may be carried out using a chemical crosslinking agent, such as formaldehyde, paraformaldehyde, glutaraldehyde, disuccinimidyl glutarate, ethylene glycol bis(succinimidyl succinate), a homobifunctional crosslinking agent, or a heterobifunctional crosslinking agent. Any such crosslinking step may be further terminated by a quenching step, for example by quenching the formaldehyde crosslinking step by mixing with a glycine solution. Any such crosslinking may be removed before certain subsequent steps of the protocol, for example, before primer extension, PCR, or nucleic acid purification steps. The crosslinking step with a chemical crosslinking agent serves the purpose of keeping biomolecules (genomic DNA and / or polypeptide fragments) within each microparticle in physical proximity to one another, thereby allowing the sample to be manipulated and processed while preserving the fundamental structural properties of the microparticles (i.e., while preserving the physical proximity of genomic DNA fragments and / or polypeptides derived from the same microparticle).

[0291] The particulate matter(s) may be permeated in an incubation step. The incubation step involves a chemical surfactant (e.g., Triton X-100(C)). 14 H 22 O(C2H4O) n The incubation step can be carried out in the presence of (n=9~10), NP-40, Tween20, Tween80, saponin, digitonin, or sodium dodecyl sulfate. The incubation step can be carried out at a temperature of at least 20 degrees Celsius, at least 30 degrees Celsius, at least 37 degrees Celsius, at least 45 degrees Celsius, at least 50 degrees Celsius, at least 60 degrees Celsius, at least 65 degrees Celsius, at least 70 degrees Celsius, or at least 80 degrees Celsius. The incubation step can be at least 1 second, at least 5 seconds, at least 10 seconds, at least 30 seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 60 minutes, or at least 3 hours.

[0292] After transferring one or more of the reagents described herein (e.g., barcoded oligonucleotides, multimer barcoded reagents, affinity probes, barcoded affinity probes, etc.) to one or more microparticles, one or more target biomolecules can be measured and / or analyzed. This method may include the step of transferring one or more of the reagents described herein (e.g., barcoded oligonucleotides, multimer barcoded reagents, affinity probes, barcoded affinity probes, etc.) to one or more circulating microparticles.

[0293] In this method, one or more of the reagents described herein can be transferred to one or more circulating microparticles by complex formation with a transfection reagent or lipid carrier (e.g., liposomes or micelles). The transfection reagent may be a lipid transfection reagent, such as a cationic lipid transfection reagent. Optionally, the cationic lipid transfection reagent includes at least two alkyl chains. Optionally, the cationic lipid transfection reagent may be a commercially available cationic lipid transfection reagent such as lipofectamine.

[0294] In this method, reagents for analyzing the first circulating microparticles may be contained within the first lipid carrier, and reagents for analyzing the second circulating microparticles may be contained within the second lipid carrier. The lipid carrier may be liposomes or micelles.

[0295] Prior to the transfer step, this method may include a step of crosslinking biomolecules (e.g., fragments of genomic DNA and / or target polypeptides) in the microparticles. Prior to the transfer step, and optionally after the crosslinking step, this method may further include a step of permeabilizing the microparticles.

[0296] Following the step of releasing target biomolecules from one or more circulating microparticles, any one or more target biomolecules can be measured and / or analyzed. One or more target biomolecules may be released from the circulating microparticles(or microparticles) by steps of dissolving, permeating, and / or scattering the circulating microparticles(or microparticles). The method of the present invention may include releasing target biomolecules from one or more circulating microparticles (for example, by dissolving, permeating, and / or scattering the circulating microparticles). This release step may be carried out in conjunction with a high-temperature incubation step and / or incubation with a molecular solvent or chemical surfactant.

[0297] Any one or more target biomolecules can be measured and / or analyzed after a step of purifying and / or isolating and / or processing any one or more target biomolecules from one or more circulating microparticles. The method of the present invention may include one or more steps of processing, purifying, fractionating and / or isolating any or all target biomolecules and / or other components of the circulating microparticles before, and / or during, and / or after any step of analyzing the sample. This method may include a step of purifying and / or isolating nucleic acids (such as DNA molecules and / or RNA molecules). This method may include a step of purifying and / or isolating polypeptides (such as proteins and / or post-translationally modified proteins).

[0298] Any one or more target biomolecules can be measured and / or analyzed after the step of binding and / or adding any one or more such target biomolecules and / or target nucleic acid molecules to a support such as a solid support and / or semi-solid support and / or gel support.

[0299] This method may include the step of attaching one or more molecules (such as one or more nucleic acid molecules, including DNA molecules and / or RNA molecules, and / or any polypeptide molecules, including proteins or post-translation modified proteins) to a support. Any number or fraction of such molecules from a sample containing one or more circulating microparticles can be attached to one or more supports. Optionally, at least 0.01%, at least 0.1%, at least 1%, at least 10%, at least 50%, or 100% of such molecules can be attached to one or more supports.

[0300] Any one or more such molecules can be linked to any form of support (e.g., polymers, solid or semi-solid supports, or dendrimers). Any support may be beads (e.g., gel beads, agarose beads, silica beads, styrofoam beads, gel beads (such as those available from 10x Genomics®), antibody conjugate beads, oligo-dT conjugate beads, streptavidin beads, or magnetic beads (e.g., superparamagnetic beads)). Any beads may be of any size and / or molecular structure (e.g., 10 nanometers to 100 microns in diameter, 100 nanometers to 10 microns in diameter, or 1 micron to 5 microns in diameter). Molecules can be linked to a support directly or indirectly (e.g., via linker molecules). Molecules can be linked by binding to a support and / or by binding to or annealing to a linker molecule bound to a support. Molecules can be attached to a support (or linker molecule) by covalent linkage, non-covalent linkage (e.g., protein-protein interactions or streptavidin-biotin linkages), or nucleic acid hybridization. The linker molecule may be a biopolymer (e.g., a nucleic acid molecule) or a synthetic polymer. The linker molecule may contain one or more ethylene glycol and / or poly(ethylene) glycol (e.g., hexa-ethylene glycol or penta-ethylene glycol) units. The linker molecule may contain one or more ethyl groups, such as a C3 (3-carbon) spacer, a C6 spacer, a C12 spacer, or a C18 spacer. Any support can be functionalized to allow the attachment of two or more molecules. This functionalization can be achieved by adding a chemical moiety (e.g., a carboxylated group, an alkyne, an azide, an acrylate group, an amino group, a sulfate group, or a succinimide group) and / or a protein-based moiety (e.g., streptavidin, avidin, or protein G) to the support.

[0301] Molecules can be linked by polymers by binding to and / or annealing to them. The polymers may be nucleic acids, each containing two or more nucleotides that can bind to a barcode molecule. In addition, the nucleic acids may each contain two or more regions that can hybridize to a barcode molecule. The polymers may be synthetic polymers (e.g., dendrimers) or biopolymers, such as nucleic acids (e.g., single-stranded nucleic acids such as single-stranded DNA), peptides, polypeptides, or proteins (e.g., multimeric proteins). The dendrimers may comprise at least two, at least three, at least five, or at least ten generations.

[0302] This method may include attaching one or more circulating microparticles to a support by a method comprising: (a) attaching a coupling molecule containing one or more biotin moieties to a target molecule (such as a target nucleic acid molecule or a target polypeptide molecule) by any method, and / or attaching a biotin conjugate affinity probe to the target molecule to create a biotin conjugate target molecule; and (b) attaching the biotin conjugate target molecule to one or more streptavidin conjugate supports (such as one or more streptavidin conjugate beads). Optionally, before and / or during step (b), the biotin conjugate target molecule is compartmentalized into two or more compartments.

[0303] Following the step of compartmentalizing the sample into two or more compartments, any one or more target biomolecules can be measured and / or analyzed. This method may include compartmentalizing the sample into two or more compartments. Optionally, each compartment may contain one or more supports, and molecules from the microparticles (or more) compartmentalized in each compartment are each attached to the supports contained within the same compartment. Optionally, samples containing any number of microparticles (e.g., at least 1,000, at least 1,000,000, or at least 100,000,000) can be attached by this process. Optionally, any number and / or average number of microparticles can be compartmentalized into each compartment (for example, microparticles with an average of less than 100, less than 10, less than 1, less than 0.5, less than 0.2, less than 0.1, less than 0.05, less than 0.01, less than 0.001, less than 0.00001, or less than 0.000001 can be compartmentalized into each compartment). Each compartment may contain, or on average contain, any number of supports, such as an average of 0.1 supports, an average of 0.5 supports, an average of 1 supports, an average of 2 supports, an average of 5 supports, an average of 10 supports, or an average of 100 supports. Optionally, following any process of adding molecules from a sample containing two or more circulating microparticles to supports within a compartment, all or any fractions of the solutions contained in any fraction and / or all compartments can be merged together to form a single, uncompartmentalized support-added reaction mixture. Here, the uncompartmentalized support addition reaction mixture contains a support to which molecules from the sample have been thus added. Optionally, the uncompartmentalized support addition reaction mixture can then be used in any process to analyze a sample containing two or more circulating microparticles, such as any method for measuring fragments of genomic DNA, any method for measuring modified nucleotides or nucleic acid bases, and / or any method for measuring one or more target polypeptides.Optionally, two or more target molecules attached to a support in an uncompartmentalized support addition reaction mixture (e.g., two or more molecules from the same circulating microparticle, such as two or more fragments of genomic DNA, and / or two or more polypeptides bound to a barcoding affinity probe) can be linked to the same barcode sequence or to different barcode sequences from a set of barcode sequences. Optionally, any such process for adding a barcode sequence may involve adding two or more barcode oligonucleotides from a multimerized barcoding reagent to two or more target molecules attached to the same support in an uncompartmentalized support addition reaction mixture. Optionally, any process for adding a barcode sequence may comprise contacting an uncompartmentalized support addition reaction mixture with a library of at least 2, at least 100, at least 1,000, at least 10,000, at least 1,000,000, at least 10,000,000, or at least 1,000,000,000 multimerized barcoding reagents, and adding the barcoded oligonucleotides contained within the multimerized barcoding reagents to a target molecule attached to a support in the uncompartmentalized support addition reaction mixture. Any one or more uncompartmentalized support addition reaction mixtures may comprise a sample derived from one or more circulating microparticles for use in any one or more of the methods described herein. Optionally, in any such method, any number of compartments (at least 10, at least 1,000, at least 1,000,000, or at least 1,000,000,000 compartments, etc.), any type of compartment (reaction tubes, droplets, or droplets in emulsion, etc.), and / or any volume of a compartment (less than or greater than 100 femtoliters, less than or greater than 1.0, 10.0, or 100.0 picoliters, less than or greater than 1.0, 10.0, or 100.0 nanoliters, or less than or greater than 1.0, 10.0, or 100.0 microliters) may be used.For example, the number, type, or volume of parcels described herein and / or PCT / GB2017 / 053820 are arbitrary and their contents are incorporated herein by reference.

[0304] 4. Linking by barcode The present invention provides a method for preparing a sample for sequencing, wherein the sample comprises circulating microparticles (or microparticles derived from blood), the microparticles comprising at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method comprises attaching at least two fragments of the target nucleic acid from the microparticles to different barcode sequences of a barcode sequence, or a set of barcode sequences, to generate a set of linked fragments of the target nucleic acid.

[0305] The present invention provides a method for preparing a sample for sequencing, wherein the sample comprises circulating microparticles, each circulating microparticle comprising at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method comprises attaching at least two fragments of the target nucleic acid from the circulating microparticles to a barcode sequence, or different barcode sequences of a set of barcode sequences, to generate a set of linked fragments of the target nucleic acid.

[0306] Prior to the step of attaching at least two fragments of the target nucleic acid of the microparticle to a barcode sequence, or different barcode sequences of a set of barcode sequences, the method may include attaching a coupling sequence to each of the fragments of the target nucleic acid of the microparticle (e.g., genomic DNA), and then the coupling sequence is attached to different barcode sequences of the barcode sequence, or a set of barcode sequences, to generate a set of linked fragments of the target nucleic acid.

[0307] In this method, the sample may comprise first and second microparticles derived from blood, each microparticle comprising at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method may comprise: adding at least two fragments of the target nucleic acid from the first microparticle to a first barcode sequence or different barcode sequences of a first set of barcode sequences to produce a concatenated fragment of the first set of target nucleic acid; and adding at least two fragments of the target nucleic acid from the second microparticle to a second barcode sequence or different barcode sequences of a second set of barcode sequences to produce a concatenated fragment of the second set of target nucleic acid.

[0308] The first barcode sequence may differ from the second barcode sequence. The first set of barcode sequences may differ from the second set of barcode sequences.

[0309] In this method, the sample may consist of n microparticles derived from blood, each microparticle containing at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method comprises performing step (a) to generate n linked sets of target nucleic acid fragments (one set for each n microparticles).

[0310] In this method, n may be at least 3, at least 5, at least 10, at least 50, at least 100, at least 1000, at least 10,000, at least 100,000, at least 1,000,000, at least 10,000,000, at least 100,000,000, at least 1,000,000,000, or at least 100,000,000,000. Preferably, n is at least 100,000 particles.

[0311] Preferably, each set of concatenated array reads (i.e., a set of concatenated signals) is concatenated by a different barcode array or a different set of barcode arrays. Each barcode array in a barcode array set may be different from the barcode arrays of at least 1, at least 4, at least 9, at least 49, at least 999, at least 999, at least 9,999, at least 999,999, at least 999,999, at least 999,999,999, at least 999,999,999, at least 999,999,999, at least 99,999,999,999, or at least 999,999,999,999 other barcode array sets in the library. Each barcode array in a barcode array set may be different from the barcode arrays of all other sets of barcode arrays in the library. Preferably, each barcode array in a barcode array set is different from the barcode arrays of at least 9 other sets of barcode arrays in the library.

[0312] The present invention provides a method for analyzing a sample comprising blood-derived microparticles, wherein the microparticles comprise at least two fragments of a target nucleic acid, and the method comprises (a) preparing a sample for sequencing, which includes (a) attaching at least two fragments of the target nucleic acid (e.g., genomic DNA) of the microparticles to a barcode sequence to generate a set of linked fragments of the target nucleic acid, and (b) sequencing each of the linked fragments in the set to generate at least two linked sequence reads, wherein the at least two linked sequence reads are linked by a barcode sequence.

[0313] A barcode sequence may contain unique sequences. Each barcode sequence may contain at least 5, at least 10, at least 15, at least 20, at least 25, at least 50, or at least 100 nucleotides. Preferably, each barcode sequence contains at least 5 nucleotides. Preferably, each barcode sequence contains a deoxyribonucleotide, and optionally, all nucleotides in the barcode sequence are deoxyribonucleotides. One or more of the deoxyribonucleotides may be modified deoxyribonucleotides (e.g., deoxyribonucleotides modified with a biotin moiety or deoxyuracil nucleotide). A barcode sequence may contain one or more degenerate nucleotides or sequences. A barcode sequence may not contain any degenerate nucleotides or sequences.

[0314] In this method, prior to the step of attaching at least two fragments of the target nucleic acid of the microparticle to the barcode sequence, the method may include attaching a coupling sequence to each of the nucleic acid fragments of the microparticle, and then the coupling sequence is attached to the barcode sequence to generate a set of concatenated fragments.

[0315] In this method, the sample may comprise first and second microparticles derived from blood, each microparticle comprising at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method comprises: (a) performing step (a) to generate a first set of ligated fragments of the target nucleic acid for the first microparticle and a second set of ligated fragments of the target nucleic acid for the second microparticle; and (b) performing step (b) to generate a first set of ligated sequence reads (i.e., a set of ligated signals) for the first microparticle and a second set of ligated sequence reads (i.e., a set of ligated signals) for the second microparticle, wherein at least two ligated sequence reads of the first microparticle are ligated to at least two ligated sequence reads of the second microparticle by different barcode sequences.

[0316] A first set of concatenated fragments can be concatenated to a second set of concatenated fragments by a different barcode sequence.

[0317] In this method, the sample may contain n microparticles derived from blood, each microparticle containing at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method comprises: (a) generating n linked target nucleic acid fragment sets (one set per n microparticles); and (b) generating n linked sequence read sets (i.e., linked signal sets) (one set per n microparticles).

[0318] In this method, n may be at least 3, at least 5, at least 10, at least 50, at least 100, at least 1000, at least 10,000, at least 100,000, at least 1,000,000, at least 10,000,000, at least 100,000,000, at least 1,000,000,000, or at least 100,000,000,000. Preferably, n is at least 100,000 particles.

[0319] Preferably, each set of concatenated array reads (i.e., a set of concatenated signals) is concatenated by a different barcode array.

[0320] In this method, different barcode sequences can be provided as a library of barcode sequences. The library used in this method may contain at least 2, at least 5, at least 10, at least 50, at least 100, at least 1000, at least 10,000, at least 100,000, at least 1,000,000, at least 10,000,000, at least 100,000,000, at least 1,000,000,000, at least 10,000,000,000, or at least 1,000,000,000,000 different barcode sequences. Preferably, the library used in this method contains at least 1,000,000 different barcode sequences.

[0321] In this method, each barcode sequence in the library can only be attached to a fragment from a single microparticle.

[0322] This method can be deterministic (i.e., one barcode sequence can be used to identify sequence reads from a single microparticle) or probabilistic (i.e., one barcode sequence can be used to identify a likely sequence read from a single microparticle). In certain embodiments, one barcode sequence may be appended to fragments of genomic DNA from two or more microparticles.

[0323] The method may include (a) preparing a sample for sequencing, which involves (a) adding each of at least two fragments of a target nucleic acid (e.g., genomic DNA) of microparticles to different barcode sequences of a set of barcode sequences to generate a ligated fragment of the target nucleic acid, and (b) sequencing each of the ligated fragments in the set to generate at least two ligated sequence reads, wherein the at least two ligated sequence reads are ligated by the set of barcode sequences.

[0324] In this method, prior to the step of attaching each of the at least two fragments of the target nucleic acid of the microparticle to a different barcode sequence, the method may include attaching a coupling sequence to each of the fragments of the target nucleic acid of the microparticle, where each of the at least two fragments of the target nucleic acid of the microparticle is attached by its coupling sequence to a different barcode sequence of the set of barcode sequences.

[0325] In this method, the sample may comprise first and second microparticles derived from blood, each microparticle comprising at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method may comprise step (a) of generating a first set of ligated fragments of the target nucleic acid for the first microparticle and a second set of ligated fragments of the target nucleic acid for the second microparticle, and step (b) of generating a first set of ligated sequence reads (i.e., a set of ligated signals) for the first microparticle and a second set of ligated sequence reads (i.e., a set of ligated signals) for the second microparticle, where the first set of ligated sequence reads is ligated to the second set of ligated sequence reads by a different set of barcode sequences.

[0326] In this method, the sample may consist of n microparticles derived from blood, each microparticle containing at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method may consist of step (a) generating n linked target nucleic acid fragment sets (one set per n microparticles) and step (b) generating n linked sequence read sets (i.e., linked signal sets) (one set per n microparticles).

[0327] In this method, n may be at least 3, at least 5, at least 10, at least 50, at least 100, at least 1000, at least 10,000, at least 100,000, at least 1,000,000, at least 10,000,000, at least 100,000,000, at least 1,000,000,000, or at least 100,000,000,000. Preferably, n is at least 100,000 particles.

[0328] Preferably, each set of concatenated array reads (i.e., the set of concatenated signals) is concatenated by a different set of barcode arrays.

[0329] In this method, different sets of barcode sequences can be provided as a library of barcode sequence sets. The library used in this method may contain at least 2, at least 5, at least 10, at least 50, at least 100, at least 1000, at least 10,000, at least 100,000, at least 1,000,000, at least 10,000,000, at least 100,000,000, at least 1,000,000,000, at least 10,000,000,000, or at least 1,000,000,000,000 sets of different barcode sequences. Preferably, the library used in this method contains at least 1,000,000 sets of different barcode sequences.

[0330] Each barcode sequence in a barcode sequence set may be different from barcode sequences in at least 1, at least 4, at least 9, at least 49, at least 99, at least 999, at least 999, at least 9,999, at least 999,999, at least 999,999, at least 999,999,999, at least 999,999,999, at least 999,999,999, at least 99,999,999,999, or at least 999,999,999,999 other barcode sequence sets in the library. Each barcode sequence in a set of barcode sequences may be different from barcode sequences in all other sets of barcode sequences in the library. Preferably, each barcode sequence in a set of barcode sequences is different from barcode sequences in at least 9 other sets of barcode sequences in the library.

[0331] In this method, barcode sequences from a set of barcode sequences in a library can only be attached to fragments from single microparticles.

[0332] This method can be deterministic (i.e., a single set of barcode sequences can be used to identify sequence reads from a single microparticle) or probabilistic (i.e., a single set of barcode sequences can be used to identify the most likely sequence reads from a single microparticle).

[0333] The method may include preparing first and second samples for sequencing, each sample comprising at least one microparticle derived from blood, each microparticle comprising at least two fragments of a target nucleic acid (e.g., genomic DNA), and each barcode sequence comprising a sample identifier region, the method comprising (i) performing step (a) for each sample, wherein the barcode sequence(optional) attached to the target nucleic acid fragment from the first sample has a different sample identifier region than the barcode sequence(optional) attached to the target nucleic acid fragment from the second sample; (ii) performing step (b) for each sample, wherein each concatenated sequence read comprises a sequence of the sample identifier region; and (iii) determining the sample from which each concatenated sequence read originates its sample identifier region.

[0334] In this method, before, during, and / or after the step of adding barcode sequences and / or coupling sequences, the method may include a step of crosslinking genomic DNA fragments in microparticles.

[0335] In this method, before, during, and / or after the step of adding barcode sequences and / or coupling sequences, and / or optionally after the step of crosslinking genomic DNA fragments in the microparticles, the method may include a step of permeabilizing the microparticles. Before the transfer step, and optionally after the crosslinking step, the method includes permeabilizing the microparticles.

[0336] The barcode sequence may be contained within the barcoded oligonucleotide in a solution. Such a barcoded oligonucleotide may be single-stranded, double-stranded, or single-stranded with one or more double-stranded regions. The barcoded oligonucleotide may be ligated to a target nucleic acid fragment during a single-stranded or double-stranded ligation reaction. The barcoded oligonucleotide may contain a single-stranded 5' or 3' region that can be ligated to a target nucleic acid fragment. Each barcoded oligonucleotide may be ligated to a target nucleic acid fragment during a single-stranded ligation reaction. Alternatively, the barcoded oligonucleotide may contain a smooth, recessed, or overhanging 5' or 3' region that can be ligated to a target nucleic acid fragment. Each barcoded oligonucleotide may be ligated to a target nucleic acid fragment during a double-stranded ligation reaction.

[0337] In a particular method, the ends of a target nucleic acid fragment may be converted to blunt double-stranded ends in a blunting reaction, and the barcoded oligonucleotide may contain blunt double-stranded ends. Each barcoded oligonucleotide can be ligated to a target nucleic acid fragment during a blunt-end ligation reaction. In a particular method, the ends of a target nucleic acid fragment may be converted to blunt double-stranded ends in a blunting reaction, and then converted to a form having a single 3' adenosine overhang, where the barcoded oligonucleotide contains a double-stranded end having a single 3' thymine overhang that can anneal to a single 3' adenosine overhang of the target nucleic acid fragment. Each barcoded oligonucleotide can be ligated to a target nucleic acid fragment during a double-stranded A / T ligation reaction.

[0338] In a particular manner, barcoded oligonucleotides include a target region at their 3' or 5' end that can be annealed to a target region of a target nucleic acid and / or coupling sequence, and the barcoded sequence can be attached to the target nucleic acid by annealing the barcoded oligonucleotide to the target nucleic acid and / or coupling sequence, and optionally by extending and / or ligating the barcoded oligonucleotide to the nucleic acid target and / or coupling sequence.

[0339] In certain methods, the coupling sequence can be added to a fragment of genomic DNA before the barcoded oligonucleotide is attached.

[0340] This method may include a step of compartmentalizing the nucleic acid sample into at least two different reaction volumes prior to the addition step.

[0341] 5. Linking by barcoding using a multimer barcoding reagent. The present invention provides a method for preparing a sample for sequencing, wherein the sample comprises circulating microparticles (i.e., microparticles derived from blood), the microparticles comprising at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method may comprise the following steps: (a) contacting the sample with a multimerized barcoding reagent, the multimerized barcoding reagent comprising a first barcode region and a second barcode region linked together, each barcode region comprising a nucleic acid sequence; and (b) attaching the barcode sequences to each of the first and second fragments of the target nucleic acid of the microparticles to generate first and second barcoded target nucleic acid molecules for the microparticles, the first barcoded target nucleic acid molecule comprising the nucleic acid sequence of the first barcode region, and the second barcoded target nucleic acid molecule comprising the nucleic acid sequence of the second barcode region.

[0342] The present invention provides a method for preparing a sample for sequencing, wherein the sample comprises microparticles derived from blood, the microparticles comprising at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method may comprise the following steps: (a) contacting the sample with a multimerized barcoding reagent, the multimerized barcoding reagent comprising first and second barcoded oligonucleotides linked together, each barcoded oligonucleotide comprising a barcode region; and (b) annealing or ligating the first and second barcoded oligonucleotides to the first and second fragments of the target nucleic acid of the microparticles to generate first and second barcoded target nucleic acid molecules.

[0343] The present invention provides a method for preparing a sample for sequencing, wherein the sample comprises first and second microparticles derived from blood, each microparticle comprising at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method comprises the following steps: (a) contacting the sample with a library comprising at least two multimer barcoding reagents, each multimer barcoding reagent comprising a first barcode region and a second barcode region linked together, each barcode region comprising a nucleic acid sequence, wherein the first and second barcode regions of the first multimer barcoding reagent are different from the first and second barcode regions of the second multimer barcoding reagent in the library; and (b) adding the barcode sequences to each of the first and second fragments of the target nucleic acid of the first microparticle. The method includes generating first and second barcode target nucleic acid molecules for a first microparticle, wherein the first barcode target nucleic acid molecule includes the nucleic acid sequence of the first barcode region of the first multimer barcoding reagent, and the second barcode target nucleic acid molecule includes the nucleic acid sequence of the second barcode region of the first multimer barcoding reagent; and generating first and second barcode target nucleic acid molecules for a second microparticle by adding the barcode sequences to each of the first and second fragments of the target nucleic acid of the second microparticle, wherein the first barcode target nucleic acid molecule includes the nucleic acid sequence of the first barcode region of the second multimer barcoding reagent, and the second barcode target nucleic acid molecule includes the nucleic acid sequence of the second barcode region of the second multimer barcoding reagent.

[0344] The present invention provides a method for preparing a sample for sequencing, wherein the sample comprises first and second microparticles derived from blood, each microparticle comprising at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method comprises the following steps: (a) contacting the sample with a library comprising at least two multimer barcoding reagents, each multimer barcoding reagent comprising first and second barcoding oligonucleotides linked together, each barcoding oligonucleotide comprising a barcode region, and the barcode regions of the first and second barcoding oligonucleotides of the first multimer barcoding reagent in the library are live (b) contacting the first and second barcoded oligonucleotides of the second multimerized barcoding reagent of Larry, which are different from the barcode regions of the first and second barcoded oligonucleotides of the first multimerized barcoding reagent, to generate first and second barcoded target nucleic acid molecules, and to generate first and second barcoded target nucleic acid molecules.

[0345] Barcoded oligonucleotides can be ligated to fragments of target nucleic acids during single-stranded or double-stranded ligation reactions.

[0346] In this method, the barcoded oligonucleotide may contain a single-stranded 5' or 3' region that can be ligated to a fragment of the target nucleic acid. Each barcoded oligonucleotide can be ligated to a fragment of the target nucleic acid during a single-stranded ligation reaction.

[0347] In this method, the barcoded oligonucleotide may contain a smooth, recessed, or overhanging 5' or 3' region that can be ligated to the target nucleic acid fragment. Each barcoded oligonucleotide can be ligated to the target nucleic acid fragment during the double-stranded ligation reaction.

[0348] In this method, the ends of the target nucleic acid fragment can be converted to blunt double-stranded ends in the blunt-end ligation reaction, and the barcoded oligonucleotide may contain blunt double-stranded ends. Each barcoded oligonucleotide can be ligated to the target nucleic acid fragment during the blunt-end ligation reaction.

[0349] In this method, the ends of the target nucleic acid fragment can be converted to blunt double-stranded ends in a blunting reaction, and then converted to a form having a single 3' adenosine overhang, where the barcoded oligonucleotide contains a double-stranded end having a single 3' thymine overhang that can anneal to the single 3' adenosine overhang of the target nucleic acid fragment. Each barcoded oligonucleotide can be ligated to the target nucleic acid fragment during a double-stranded A / T ligation reaction.

[0350] In this method, the ends of a fragment of the target nucleic acid can be brought into contact with a restriction enzyme, which digests each fragment at the restriction sites to create ligation junctions, and the barcoded oligonucleotide has ends that are compatible with these ligation junctions. Each barcoded oligonucleotide can be ligated to the fragment of the target nucleic acid at the ligation junction during the double-stranded ligation reaction. Optionally, the restriction enzyme may be EcoRI, HindIII, or BglII.

[0351] In this method, prior to the step of annealing or ligating the first and second barcoded oligonucleotides to the first and second fragments of the target nucleic acid, the method may include adding a coupling sequence to each fragment of the target nucleic acid, and then the first and second barcoded oligonucleotides are annealed or ligated to the coupling sequences of the first and second fragments of the target nucleic acid.

[0352] In this method, step (b) may include (i) annealing the first and second barcoding oligonucleotides of the first multimer barcoding reagent to the first and second fragments of the target nucleic acid of the first microparticle, and annealing the first and second barcoding oligonucleotides of the second multimer barcoding reagent to the first and second fragments of the target nucleic acid of the second microparticle, and (ii) extending the first and second barcoding oligonucleotides of the first multimer barcoding reagent to produce first and second different barcoding target nucleic acid molecules, and extending the first and second barcoding oligonucleotides of the second multimer barcoding reagent to produce first and second different barcoding target nucleic acid molecules, where each barcoding target nucleic acid molecule includes at least one nucleotide synthesized from a fragment of the target nucleic acid as a template.

[0353] This method involves (a) contacting a sample with a library containing at least two multimer barcoding reagents, each multimer barcoding reagent containing a first and second barcoding oligonucleotide linked together, each barcoding oligonucleotide containing a target region and a barcode region in the 5' to 3' direction, and the barcode regions of the first and second barcoding oligonucleotides of the first multimer barcoding reagent in the library being the barcode regions of the first and second barcoding oligonucleotides of the second multimer barcoding reagent in the library Unlike the coding region, the sample is further contacted with the first and second target primers of each multimer barcoding reagent, and (b) for each microparticle, the following steps are performed: (i) annealing the target region of the first barcoding oligonucleotide to the first subsequence of the first fragment of the target nucleic acid (e.g., genomic DNA) of the microparticle, and annealing the target region of the second barcoding oligonucleotide to the first subsequence of the second fragment of the target nucleic acid (e.g., genomic DNA) of the microparticle, and (ii) annealing the first target primer to the target nucleic acid of the microparticle (iii) Annealing the first fragment to a second subsequence, wherein the second subsequence is 3' of the first subsequence; and annealing the second target primer to a second subsequence of the second fragment of the target nucleic acid of the microparticle, wherein the second subsequence is 3' of the first subsequence; (iii) Using the first fragment of the target nucleic acid of the microparticle as a template, extending the first target primer to reach the first subsequence to produce a first extended target primer; and using the second fragment of the target nucleic acid of the microparticle, (iv) extending the second target primer to reach the first subsequence to generate a second extended target primer, and (iv) ligating the 3' end of the first extended target primer to the 5' end of the first barcoding oligonucleotide to generate a first barcoding target nucleic acid molecule, and ligating the 3' end of the second extended target primer to the 5' end of the second barcoding oligonucleotide to generate a second barcoding target nucleic acid molecule, wherein the first and second barcoding target nucleic acid molecules are different.Each contains at least one nucleotide synthesized from the target nucleic acid as a template.

[0354] Each multimer barcoding reagent may comprise (i) a first and second hybridization molecule linked together (each hybridization molecule containing a nucleic acid sequence including a hybridization region), and (ii) a first and second barcoding oligonucleotide (the first barcoding oligonucleotide being annealed to the hybridization region of the first hybridization molecule, and the second barcoding oligonucleotide being annealed to the hybridization region of the second hybridization molecule).

[0355] Each multimerized barcoding reagent may comprise (i) a first and second barcode molecule linked together (each barcode molecule comprising a nucleic acid sequence containing a barcode region), and (ii) a first and second barcoding oligonucleotide (the first barcoding oligonucleotide comprising a barcode region annealed to the barcode region of the first barcode molecule, and the second barcoding oligonucleotide comprising a barcode region annealed to the barcode region of the second barcode molecule).

[0356] The present invention provides a method for preparing a sample for sequencing, wherein the sample comprises at least two microparticles derived from blood, each microparticle comprising at least two fragments of a target nucleic acid, and the method comprises the following steps: (a) contacting the sample with a library comprising first and second multimer barcoding reagents, each multimer barcoding reagent comprising first and second barcode molecules linked together, each barcode molecule optionally comprising a nucleic acid sequence comprising a barcode region and an adapter region in the 5' to 3' direction; and (b) adding coupling sequences to the first and second fragments of the target nucleic acid (e.g., genomic DNA) of the first and second microparticles. (c) Annealing the coupling sequence of a first fragment to the adapter region of a first barcode molecule and the coupling sequence of a second fragment to the adapter region of a second barcode molecule for each of the multimer barcoding reagents, and (d) Adding a barcode sequence to each of at least two fragments of a microparticle target nucleic acid for each of the multimer barcoding reagents to produce first and second different barcode target nucleic acid molecules, wherein the first barcode target nucleic acid molecule includes the nucleic acid sequence of the barcode region of the first barcode molecule, and the second barcode target nucleic acid molecule includes the nucleic acid sequence of the barcode region of the second barcode molecule.

[0357] In this method, each barcode molecule may contain a nucleic acid sequence including a barcode region and an adapter region in the 5' to 3' direction, where step (d) includes, for each of the multimerized barcoding reagents, extending the coupling sequence of a first fragment using the barcode region of a first barcode molecule as a template to generate a first barcode target nucleic acid molecule, and extending the coupling sequence of a second fragment using the barcode region of a second barcode molecule as a template to generate a second barcode target nucleic acid molecule, where the first barcode target nucleic acid molecule contains a sequence complementary to the barcode region of the first barcode molecule, and the second barcode target nucleic acid molecule contains a sequence complementary to the barcode region of the second barcode molecule.

[0358] In this method, each barcode molecule may contain a nucleic acid sequence including an adapter region and a barcode region in the 5' to 3' direction, where step (d) is to (i) anneal and extend a first extension primer using the barcode region of the first barcode molecule as a template to produce a first barcode oligonucleotide, and to anneal and extend a second extension primer using the barcode region of the second barcode molecule as a template to produce a second barcode oligonucleotide, wherein the first barcode (ii) annealing and elongating a barcode-containing oligonucleotide comprising a sequence complementary to the barcode region of a first barcode molecule, and a second barcode-containing oligonucleotide comprising a sequence complementary to the barcode region of a second barcode molecule, and (ii) ligating the 3' end of the first barcode-containing oligonucleotide to the 5' end of the coupling sequence of a first fragment to generate a first barcode-target nucleic acid molecule, and ligating the 3' end of the second barcode-containing oligonucleotide to the 5' end of the coupling sequence of a second fragment to generate a second barcode-target nucleic acid molecule.

[0359] In this method, each barcode molecule may contain a nucleic acid sequence comprising an adapter region, a barcode region, and a priming region in the 5' to 3' direction, where step (d) is to (i) anneal a first extension primer to the priming region of the first barcode molecule, extend the first extension primer using the barcode region of the first barcode molecule as a template to generate a first barcode oligonucleotide, and anneal a second extension primer to the priming region of the second barcode molecule, extend the second extension primer using the barcode region of the second barcode molecule as a template (ii) Annealing to generate a second barcode oligonucleotide, wherein the first barcode oligonucleotide includes a sequence complementary to the barcode region of the first barcode molecule, and the second barcode oligonucleotide includes a sequence complementary to the barcode region of the second barcode molecule; and (ii) Ligate the 3' end of the first barcode oligonucleotide to the 5' end of the coupling sequence of the first fragment to generate a first barcode target nucleic acid molecule, and ligate the 3' end of the second barcode oligonucleotide to the 5' end of the coupling sequence of the second fragment to generate a second barcode target nucleic acid molecule.

[0360] This method comprises (a) contacting a sample with a library containing first and second multimer barcoding reagents, each multimer barcoding reagent containing first and second barcode molecules linked together, each barcode molecule containing a nucleic acid sequence including a barcode region and an adapter region in the 5' to 3' direction, and the sample being further contacted with first and second adapter oligonucleotides for each multimer barcoding reagent, each containing an adapter region; (b) ligating the first and second adapter oligonucleotides of the first multimer barcoding reagent to first and second fragments of the target nucleic acid of the first microparticle, and ligating the first and second adapter oligonucleotides of the second multimer barcoding reagent to first and second fragments of the target nucleic acid of the second microparticle; and (c) multimer barcoding (d) For each reagent, the adapter region of the first adapter oligonucleotide is annealed to the adapter region of the first barcode molecule, and the adapter region of the second adapter oligonucleotide is annealed to the adapter region of the second barcode molecule; (d) For each of the multimer barcoding reagents, the first adapter oligonucleotide is extended using the barcode region of the first barcode molecule as a template to generate a first barcoding target nucleic acid molecule, and the second adapter oligonucleotide is extended using the barcode region of the second barcode molecule as a template to generate a second barcoding target nucleic acid molecule, wherein the first barcoding target nucleic acid molecule contains a sequence complementary to the barcode region of the first barcode molecule, and the second barcoding target nucleic acid molecule contains a sequence complementary to the barcode region of the second barcode molecule.

[0361] This method involves the following steps: (a) contacting a sample with a library containing first and second multimer barcoding reagents, each multimer barcoding reagent comprising (i) first and second barcode molecules linked together (each barcode molecule optionally comprising a nucleic acid sequence including an adapter region and a barcode region in the 5' to 3' direction), and (ii) first and second barcoding oligonucleotides (the first barcoding oligonucleotide comprises a barcode region annealed to the first barcode region, and the second barcoding oligonucleotide comprises a barcode region annealed to the second barcode region, and the barcode regions of the first and second barcoding oligonucleotides of the first multimer barcoding reagent in the library differ from the barcode regions of the first and second barcoding oligonucleotides of the second multimer barcoding reagent in the library, and the sample is further contacted with the first and second adapter oligonucleotides for each of the multimer barcoding reagents, and the first and second (b) Contacting the adapter oligonucleotides (each including an adapter region), (c) Annealing or ligating the first and second adapter oligonucleotides of the first multimer barcoding reagent to the first and second fragments of the target nucleic acid (e.g., genomic DNA) of the first microparticle, and annealing or ligating the first and second adapter oligonucleotides of the second multimer barcoding reagent to the first and second fragments of the target nucleic acid (e.g., genomic DNA) of the second microparticle, (d) For each multimer barcoding reagent, annealing the adapter region of the first adapter oligonucleotide to the adapter region of the first barcode molecule, and annealing the adapter region of the second adapter oligonucleotide to the adapter region of the second barcode molecule, and (e.g., for each multimer barcoding reagent, ligating the 3' end of the first barcoding oligonucleotide to the 5' end of the first adapter oligonucleotide to generate the first barcoding target nucleic acid molecule.This may include ligating the 3' end of a second barcoded oligonucleotide to the 5' end of a second adapter oligonucleotide to generate a second barcoded target nucleic acid molecule.

[0362] In this method, step (b) comprises annealing the first and second adapter oligonucleotides of the first multimer barcoding reagent to the first and second fragments of the target nucleic acid (e.g., genomic DNA) of the first microparticle, and annealing the first and second adapter oligonucleotides of the second multimer barcoding reagent to the first and second fragments of the target nucleic acid (e.g., genomic DNA) of the second microparticle, wherein (i) for each of the multimer barcoding reagents, step (d) comprises ligating the 3' end of the first barcoding oligonucleotide to the 5' end of the first adapter oligonucleotide to produce the first barcoding adapter oligonucleotide, and the second barcoding oligonucleotide The method comprises ligating the 3' end of one to the 5' end of a second adapter oligonucleotide to generate a second barcoding adapter oligonucleotide, and extending the first and second barcoding adapter oligonucleotides to generate first and second different barcoding target nucleic acid molecules, each containing at least one nucleotide synthesized from a fragment of target nucleic acid as a template, or (ii) prior to step (d), for each of the multimerized barcoding reagents, the method comprises extending the first and second adapter oligonucleotides to generate first and second different target nucleic acid molecules, each containing at least one nucleotide synthesized from a fragment of target nucleic acid as a template.

[0363] In this method, prior to the step of annealing or ligating the first and second adapter oligonucleotides to the first and second fragments of the target nucleic acid, the method may include adding a coupling sequence to each of the fragments of the target nucleic acid, and then the first and second adapter oligonucleotides are annealed or ligated to the coupling sequences of the first and second fragments of the target nucleic acid.

[0364] In any method described herein, the method may include a step of crosslinking fragments of target nucleic acid (e.g., genomic DNA) in microparticles. This step may be carried out using a chemical crosslinking agent, such as formaldehyde, paraformaldehyde, glutaraldehyde, disuccinimidyl glutarate, ethylene glycol bis(succinimidyl succinate), a homobifunctional crosslinking agent, or a heterobifunctional crosslinking agent. This step may be carried out before any permeabilization step, after any permeabilization step, before any compartmentalization step, before any step of adding coupling sequences, after any step of adding coupling sequences, before any step of adding barcode sequences (e.g., before step (b)), after any step of adding barcode sequences (e.g., after step (d)), during the addition of barcode sequences, or in any combination thereof. For example, a sample containing microparticles can be crosslinked before contacting the sample containing microparticles with a library of two or more multimer barcoding reagents. Any crosslinking step may be further terminated by a quenching step, for example by quenching the formaldehyde crosslinking step by mixing with a glycine solution. Any such crosslinking may be removed before certain subsequent steps of the protocol, for example, before primer extension, PCR, or nucleic acid purification steps.

[0365] In this method, between steps (b), (c), and / or (d) (i.e., the step of adding the barcode sequence), the microparticles and / or fragments of the target nucleic acid may be contained within a gel or hydrogel such as an agarose gel or polyacrylamide gel, or any covalently crosslinked gel such as a covalently crosslinked poly(ethylene glycol) gel, or a covalently crosslinked gel containing a mixture of thiol-functionalized poly(ethylene glycol) and acrylate-functionalized poly(ethylene glycol).

[0366] In any method described herein, optionally, after any step of crosslinking, the method may include permeation of the microparticles. The microparticles may be permeated in an incubation step. The incubation step can be carried out in the presence of a chemical surfactant. Optionally, this permeation step can be carried out before the addition of the barcode sequence (e.g., before step (b)), after the addition of the barcode sequence (e.g., after step (d)), or both before and after the addition of the barcode sequence. The incubation step can be carried out at a temperature of at least 20 degrees Celsius, at least 30 degrees Celsius, at least 37 degrees Celsius, at least 45 degrees Celsius, at least 50 degrees Celsius, at least 60 degrees Celsius, at least 65 degrees Celsius, at least 70 degrees Celsius, or at least 80 degrees Celsius. The incubation step may be at least 1 second long, at least 5 seconds long, at least 10 seconds long, at least 30 seconds long, at least 1 minute long, at least 5 minutes long, at least 10 minutes long, at least 30 minutes long, at least 60 minutes long, or at least 3 hours long. This step may be performed after any crosslinking step, before any permeation step, after any permeation step, before any compartmentalization step, before any step of adding a coupling sequence, after any step of adding a coupling sequence, before any step of adding a barcode sequence (e.g., before step (b)), after any step of adding a barcode sequence (e.g., after step (d)), during the addition of a barcode sequence, or in any combination thereof. For example, a sample containing microparticles may be crosslinked and then permeated in the presence of a chemical surfactant before contacting the sample containing microparticles with a library of two or more polymer barcoding reagents.

[0367] In any of the methods described herein, a sample of particulate matter may be digested in a proteinase digestion step, such as digestion with the enzyme proteinase K. Optionally, this proteinase digestion step may be at least 10 seconds long, at least 30 seconds long, at least 60 seconds long, at least 5 minutes long, at least 10 minutes long, at least 30 minutes long, at least 60 minutes long, at least 3 hours long, at least 12 hours long, or at least 24 hours long. This step may be performed after any crosslinking step, before any permeation step, after any permeation step, before any compartmentalization step, before any step of adding a coupling sequence, after any step of adding a coupling sequence, before any step of adding a barcode sequence (e.g., before step (b)), after any step of adding a barcode sequence (e.g., after step (d)), during the addition of a barcode sequence, or in any combination thereof. For example, a sample containing microparticles can be crosslinked and then partially digested in a proteinase K digestion step before contacting it with a library of two or more polymer barcoding reagents.

[0368] In this method, steps (a) and (b), and optionally (c) and (d), may be performed on at least two particles in a single reaction volume.

[0369] This method may further include, prior to step (b), the step of compartmentalizing the nucleic acid sample into at least two different reaction volumes.

[0370] The present invention provides a method for analyzing a sample comprising blood-derived microparticles, wherein the microparticles comprise at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method comprises (a) preparing a sample for sequencing, which includes (i) contacting the sample with a multimerized barcoding reagent comprising first and second barcode regions linked together, each barcode region comprising a nucleic acid sequence, and (ii) adding the barcode sequences to each of at least two fragments of the target nucleic acid of the microparticles to generate first and second distinct barcoded target nucleic acid molecules, the first barcoded target nucleic acid molecule comprising the nucleic acid sequence of the first barcode, and the second barcoded target nucleic acid molecule comprising the nucleic acid sequence of the second barcode, and (b) sequencing each of the barcoded target nucleic acid molecules to generate at least two linked sequence reads.

[0371] In this method, prior to the step of attaching a barcoding sequence to each of at least two fragments of the microparticle's genomic DNA, the method may include attaching a coupling sequence to each of the fragments of the microparticle's genomic DNA, and then the sequence is attached to the coupling sequence of each of the at least two fragments of the microparticle's genomic DNA to generate first and second different barcoding target nucleic acid molecules.

[0372] During step (a), the target nucleic acid particles and / or fragments may be contained within a gel or hydrogel such as an agarose gel or polyacrylamide gel, or any covalently crosslinked gel such as a covalently crosslinked poly(ethylene glycol) gel, or a covalently crosslinked gel containing a mixture of thiol-functionalized poly(ethylene glycol) and acrylate-functionalized poly(ethylene glycol).

[0373] The particulate sample can be digested using a proteinase digestion step, such as digestion with the proteinase K enzyme. Optionally, this proteinase digestion step may be at least 10 seconds long, at least 30 seconds long, at least 60 seconds long, at least 5 minutes long, at least 10 minutes long, at least 30 minutes long, at least 60 minutes long, at least 3 hours long, at least 12 hours long, or at least 24 hours long. This step can be performed before permeabilization, after permeabilization, before barcode sequencing (e.g., before step (a)(ii)), after barcode sequencing (e.g., after step (a)(ii)), during barcode sequencing, or any combination thereof.

[0374] Step (a) of this method can be carried out by any method for preparing a sample (or nucleic acid sample) for sequencing as described herein.

[0375] The method may include preparing first and second samples for sequencing, each sample comprising at least one microparticle derived from blood, each microparticle comprising at least two fragments of a target nucleic acid (e.g., genomic DNA), and each barcode sequence comprising a sample identifier region, the method comprising (i) performing step (a) for each sample, wherein the barcode sequence attached to the nucleic acid fragment from the first sample has a different sample identifier region than the barcode sequence attached to the target nucleic acid fragment from the second sample; (ii) performing step (b) for each sample, wherein each sequence read comprises a sequence of the sample identifier region; and (iii) determining the sample from which each sequence read originates its sample identifier region.

[0376] The method may include analyzing a sample comprising at least two microparticles derived from blood, each microparticle comprising at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method comprises the following steps: (a) preparing a sample for sequencing, (i) for each of two or more microparticles, contacting the sample with a library of multimer barcoding reagents comprising a multimer barcoding reagent, wherein each multimer barcoding reagent is as defined herein; and (ii) adding a barcode sequence to each of the at least two fragments of the target nucleic acid of each microparticle, wherein at least two barcoded target nucleic acid molecules are generated from each of the at least two microparticles, and each of the at least two barcoded target nucleic acid molecules generated from a single microparticle comprises a nucleic acid sequence of a barcode region from the same multimer barcoding reagent; and (b) sequencing each of the barcoded target nucleic acid molecules to generate at least two concatenated sequence reads for each microparticle.

[0377] The barcode sequence can be added to the genomic DNA fragment of the microparticle in a single reaction volume. That is, step (a) of the method can be carried out in a single reaction volume.

[0378] Prior to the additional step (step (a)(ii)), this method may further include the step of partitioning the sample into at least two different reaction volumes.

[0379] In any method prior to the step of adding the barcode sequence, the multimerized barcoding reagent can be separated, fractionated, or dissolved into two or more components, for example, by releasing barcoded oligonucleotides.

[0380] In any method, the multimer barcoding reagent may be at concentrations of less than 1.0 femtomol, less than 10 femtomol, less than 100 femtomol, less than 1.0 picomole, less than 10 picomole, less than 100 picomole, less than 1 nanomol, less than 10 nanomol, less than 100 nanomol, or less than 1.0 micromol.

[0381] 6. Linking by joining fragments together The present invention provides a method for analyzing a sample comprising blood-derived microparticles, wherein the microparticles comprise at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method comprises (a) preparing a sample for sequencing, which includes (a) ligating together at least two fragments of the target nucleic acid of the microparticles to produce a single nucleic acid molecule comprising the sequences of at least two fragments of the target nucleic acid, and (b) sequencing each of the fragments within the single nucleic acid molecule to produce at least two ligated sequence reads.

[0382] At least two fragments of the target nucleic acid (e.g., genomic DNA) may be adjacent to each other within a single nucleic acid molecule.

[0383] At least two concatenated sequence reads may be provided within a single raw sequence read.

[0384] This method may include, prior to the linking step, adding a coupling sequence to at least one of the target nucleic acid fragments (e.g., genomic DNA), and then linking at least two of the target nucleic acid fragments together by the coupling sequence.

[0385] Fragments of target nucleic acids (e.g., genomic DNA) can be linked together by a solid support, where two or more fragments are linked to the same solid support (directly or indirectly, e.g., via a coupling sequence). Optionally, the solid support may be beads such as polystyrene beads, superparamagnetic beads, or agarose beads.

[0386] Fragments of target nucleic acid (e.g., genomic DNA) can be joined together by ligation reactions, such as double-strand ligation or single-strand ligation reactions.

[0387] The ends of a target nucleic acid fragment can be converted to blunt, ligable double-stranded ends in a blunt-end reaction, and this method may involve ligating two or more fragments to each other by a blunt-end ligation reaction.

[0388] The ends of a target nucleic acid fragment can be brought into contact with a restriction enzyme, which digests the fragment at restriction sites to create ligation junctions at these restriction sites, and this method may include ligating two or more fragments to each other by a ligation reaction at the ligation junctions. Any target nucleic acid can be brought into contact with a restriction enzyme, which digests the fragment at restriction sites to create ligation junctions at these restriction sites, and this method may include ligating two or more fragments to each other by a ligation reaction at the ligation junctions. Optionally, the restriction enzyme may be EcoRI, HindIII, or BglII.

[0389] Coupling sequences can be attached to two or more fragments of a target nucleic acid before the fragments are joined together. Two or more different coupling sequences are selectively attached to a population of fragments of the target nucleic acid.

[0390] The coupling sequence may include a ligation junction at at least one end, where the first coupling sequence is attached to a first fragment of the target nucleic acid, and the second coupling sequence is attached to a second fragment of the target nucleic acid. The two coupling sequences are then ligated together, thus linking the two fragments of the target nucleic acid together.

[0391] The coupling sequences may include an annealing region at at least one 3' end, the first coupling sequence is attached to a first fragment of the target nucleic acid, the second coupling sequence is attached to a second fragment of the target nucleic acid, the two coupling sequences are complementary to each other along a segment of at least one nucleotide length and are annealed to each other, and at least one of the 3' ends of the first coupling sequence is extended by at least one nucleotide to the sequence of the second fragment of the target nucleic acid using DNA polymerase, thereby linking the two fragments of the target nucleic acid (e.g., genomic DNA).

[0392] Before linking at least two fragments together, this method may further include the step of crosslinking the fine particles with a chemical crosslinking agent such as formaldehyde, paraformaldehyde, glutaraldehyde, disuccinimidyl glutarate, ethylene glycol bis(succinimidyl succinate), homobifunctional crosslinking agents, or heterobifunctional crosslinking agents.

[0393] Before linking at least two fragments together, this method may further involve partitioning the microparticles into two or more compartments.

[0394] This method may further include permeation treatment of the microparticles during the incubation step. This step may be performed before (if done), after (if done), before and / or after the fragments have been joined.

[0395] The incubation step involves a chemical surfactant (e.g., Triton X-100(C)). 14 H 22 O(C2H4O) n (n=9~10)) This can be done in the presence of NP-40, Tween20, Tween80, saponin, digitonin, or sodium dodecyl sulfate.

[0396] The incubation step is carried out at a temperature of at least 20 degrees Celsius, at least 30 degrees Celsius, at least 37 degrees Celsius, at least 45 degrees Celsius, at least 50 degrees Celsius, at least 60 degrees Celsius, at least 65 degrees Celsius, at least 70 degrees Celsius, at least 80 degrees Celsius, at least 90 degrees Celsius, or at least 95 degrees Celsius.

[0397] The incubation step may be at least 1 second long, at least 5 seconds long, at least 10 seconds long, at least 30 seconds long, at least 1 minute long, at least 5 minutes long, at least 10 minutes long, at least 30 minutes long, at least 60 minutes long, or at least 3 hours long.

[0398] This method may include digesting a particulate sample with a proteinase digestion step, such as digestion with the proteinase K enzyme. Optionally, this proteinase digestion step may be at least 10 seconds long, at least 30 seconds long, at least 60 seconds long, at least 5 minutes long, at least 10 minutes long, at least 30 minutes long, at least 60 minutes long, at least 3 hours long, at least 12 hours long, or at least 24 hours long. This step may be performed before compartmentalization (if performed), after compartmentalization (if performed), before and / or after ligation of the fragments.

[0399] This method may involve amplifying a (original) fragment of a target nucleic acid and then linking two or more of the resulting nucleic acid molecules together.

[0400] The step of linking the fragments together can create a linked nucleic acid molecule containing at least 3, at least 5, at least 10, at least 50, at least 100, at least 500, or at least 1000 nucleic acid molecules attached to one another from a single adjacent nucleic acid molecule.

[0401] This method may be used to generate a concatenated array read of at least 3 microparticles, at least 5 microparticles, at least 10 microparticles, at least 50 microparticles, at least 100 microparticles, at least 1000 microparticles, at least 10,000 microparticles, at least 100,000 microparticles, at least 1,000,000 microparticles, at least 10,000,000,000 microparticles, or at least 100,000,000,000 microparticles.

[0402] The sample may comprise at least two microparticles derived from blood, where each microparticle comprises at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method comprises: (a) generating a single nucleic acid molecule comprising the sequences of at least two fragments of the target nucleic acid from each microparticle; and (b) generating a concatenated sequence read from each microparticle.

[0403] Prior to, during, and / or after the step of linking together at least two fragments of a target nucleic acid (e.g., genomic DNA), the method may include a step of crosslinking the fragments of the target nucleic acid in microparticles. The crosslinking step may be carried out using a chemical crosslinking agent, such as formaldehyde, paraformaldehyde, glutaraldehyde, disuccinimidyl glutarate, ethylene glycol bis(succinimidyl succinate), a homobifunctional crosslinking agent, or a heterobifunctional crosslinking agent.

[0404] Prior to, during, and / or after, and / or optionally after, the step of crosslinking the target nucleic acid fragments in the microparticles, the method includes a step of permeabilizing the microparticles.

[0405] Prior to step (a), the method may further include the step of compartmentalizing the nucleic acid sample into at least two different reaction volumes.

[0406] In one embodiment of a method for ligating together at least two fragments of a target nucleic acid from circulating microparticles to produce a single nucleic acid molecule containing sequences of at least two fragments of the target nucleic acid, the sample is a sample containing at least one circulating microparticle (e.g., the sample is obtained and / or purified by any method disclosed herein), which is crosslinked in a solution of 1% formaldehyde at room temperature for 10 minutes, and the formaldehyde crosslinking step is then quenched with glycine. The microparticles are pelletized in a centrifugation step (e.g., 3000xG for 5 minutes), resuspended in 1X NEBuffer2 (New England Biolabs) containing 1.0% sodium dodecyl sulfate (SDS), and permeabilized by incubation at 45 degrees Celsius for 10 minutes. The SDS is quenched by adding Triton X-100, and the solution is incubated overnight with AluI (New England Biolabs) at 37 degrees Celsius to create smooth, ligateable ends. The enzyme is inactivated by adding SDS to a final concentration of 1.0% and incubating at 65°C for 15 minutes. SDS is quenched by adding Triton X-100, and the solution is diluted at least 10-fold with 1X buffer for T4 DNA ligase to a total DNA concentration of up to 1.0 nanogram per microliter. The diluted solution is incubated overnight with T4 DNA ligase at 16°C to ligate fragments from the circulating microparticles. Next, the crosslinks are reversed and the protein components are degraded by incubating overnight at 65°C in a solution of proteinase K. The ligated DNA is then purified (e.g., using a Qiagen spin column PCR purification kit and / or Ampure XP beads, etc.). Next, the Nextera in vitro transposition method (Illumina, according to the manufacturer's protocol) is added to the Illumina sequencing adapter sequence, and the ligated material is amplified by performing an appropriate number of PCR cycles. Next, the amplified and purified DNA, sized appropriately, is sequenced using an Illumina sequencer (e.g., Illumina NextSeq 500 or MiSeq) with paired-end reads of at least 50 bases each.Each end of a paired-end sequence is individually mapped to a reference human genome to elucidate the concatenated sequence read (for example, a read where the two ends contain sequences from different fragments of genomic DNA from a single circulating microparticle).

[0407] A method for ligating at least two fragments of a target nucleic acid from microparticles to produce a single nucleic acid molecule containing the sequences of at least two fragments of the target nucleic acid may have various inherent properties and characteristics that make it desirable as a method for ligating sequences from one or more circulating microparticles. In one embodiment, such a method enables the ligation of sequences from circulating microparticles without complex equipment (e.g., microfluidics for compartmentalization-based approaches). Furthermore, this approach can be carried out (broadly) in a single, separate reaction that may contain a large number of circulating microparticles (hundreds, thousands, or more), and thus can process a large number of circulating microparticles without requiring multiple reactions that may be required in, for example, combinatorial indexing approaches. Moreover, since this method does not necessarily require the use of barcode and / or multimer barcoding reagents, it is not limited by the size of the barcode library (and / or multimer barcoding reagent library) for achieving useful molecular measurements of ligated sequences from circulating microparticles.

[0408] 7. Connection through partitioning This method can be performed on nucleic acid samples containing at least two microparticles partitioned into at least two different reaction volumes (or compartments).

[0409] In any manner, a nucleic acid sample containing at least two microparticles can be partitioned into at least two different reaction volumes (or compartments). The different reaction volumes (or compartments) can be provided by different reaction vessels (or different physical reaction vessels). The different reaction volumes (or compartments) can be provided by different water droplets, e.g., different water droplets in an emulsion, or different water droplets on a solid support (e.g., a slide).

[0410] For example, a nucleic acid sample may be compartmentalized before a barcode sequence is added to the target nucleic acid fragments of the microparticles. Alternatively, a nucleic acid sample may be compartmentalized before at least two fragments of the target nucleic acid of the microparticles are joined together.

[0411] For any method including a compartmentalization step, any step in the method following the compartmentalization step may be any step of adding a barcode sequence or coupling sequence, or any step of ligation, annealing, primer extension, or PCR, in which reagents (oligonucleotides, enzymes, buffers, etc.) can be added directly to each compartment, independently of each compartment. In a method in which compartments contain water droplets in an emulsion, such addition steps may be performed via a process of merging water droplets in the emulsion using a microfluidic droplet merging conduit, etc., and optionally using a mechanical or thermal mixing step.

[0412] Each compartment contains different droplets of aqueous solution within the emulsion, which is a water-in-oil emulsion, and the droplets are generated by physical shaking or eddy flow steps, or by the merging of aqueous solution and oil solution within a microfluidic conduit or joint.

[0413] If the compartment contains water droplets within the emulsion, such water-in-oil emulsions can be generated by any method or tool known in the Art. Optionally, this may include commercially available microfluidic systems such as the Chromium system or other systems available from 10X Genomics Inc., digital droplet generators from Raindance Technologies or Bio-Rad, and component-based systems for microfluidic generation and manipulation such as Drop-Seq (Macosko et al., 2015, Cell 161, 1202-1214) and inDrop (Klein et al., 2015, Cell 161, 1187-1201).

[0414] The compartments may include different, physically non-overlapping spatial volumes within a covalently crosslinked gel, such as an agarose gel, a polyacrylamide gel, or any covalently crosslinked gel, such as a covalently crosslinked poly(ethylene glycol) gel, or a mixture of thiol-functionalized poly(ethylene glycol) molecules and acrylate-functionalized poly(ethylene glycol) molecules.

[0415] A sample of particulate matter can be separated into a total of at least 10, at least 100, at least 1,000, at least 10,000, at least 100,000, at least 1,000,000, at least 10,000,000, or at least 1,000,000,000 compartments. Preferably, a solution of particulate matter is separated into a total of at least 1,000 compartments.

[0416] The particulate sample can be separated into sections where each section contains an average of less than 0.0001 particles, less than 0.001 particles, less than 0.01 particles, less than 0.1 particles, less than 1.0 particles, less than 10 particles, less than 100 particles, less than 1,000 particles, less than 10,000 particles, less than 100,000 particles, less than 1,000,000 particles, or less than 100,000,000 particles. Preferably, each section contains an average of less than 1.0 particles.

[0417] The solution of fine particles can be compartmentalized so that each compartment contains an average of less than 1.0 atg of DNA, less than 10 atg of DNA, less than 100 atg of DNA, less than 1.0 femtogram of DNA, less than 10 femtograms of DNA, less than 100 femtograms of DNA, less than 1.0 picogram of DNA, less than 10 picograms of DNA, less than 100 picograms of DNA, or less than 1.0 nanogram of DNA. Preferably, each compartment contains less than 10 picograms of DNA.

[0418] The compartments may have volumes of less than 100 femtoliters, less than 1.0 picoliter, less than 10 picoliters, less than 100 picoliters, less than 1.0 nanoliter, less than 10 nanoliters, less than 100 nanoliters, less than 1.0 microliter, less than 10 microliters, less than 100 microliters, or less than 1.0 milliliter.

[0419] A barcode sequence may be provided in each section. For each of two or more sections containing a barcode sequence, the barcode sequence contained therein may contain multiple copies of the same barcode sequence or different barcode sequences from the same set of barcode sequences.

[0420] After the particulate matter is separated into two or more compartments, the particulate matter may be permeated in an incubation step according to any of the methods described herein.

[0421] The particulate sample can be digested using a proteinase digestion step, such as digestion with the proteinase K enzyme. Optionally, this proteinase digestion step may be at least 10 seconds long, at least 30 seconds long, at least 60 seconds long, at least 5 minutes long, at least 10 minutes long, at least 30 minutes long, at least 60 minutes long, at least 3 hours long, at least 12 hours long, or at least 24 hours long. This step can be performed before compartmentalization, after compartmentalization, before barcode sequencing, after barcode sequencing, and / or during barcode sequencing.

[0422] Addition of sequences by combinatorial barcoding process A method for adding barcode sequences may comprise at least two steps of a combinatorial barcoding process, where a first barcoding step is performed, in which a sample of microparticles is partitioned into two or more compartments, each compartment comprising a different barcode sequence or set of different barcode sequences, which are then added to sequences from fragments of target nucleic acid (e.g., genomic DNA) of the microparticles contained within that compartment; the barcoded nucleic acid molecules from at least two compartments are then merged into a second sample mixture, which is then partitioned into two or more new compartments, each new compartment comprising a different barcode sequence or set of different barcode sequences, which are then added to sequences from fragments of target nucleic acid (e.g., genomic DNA) of the microparticles contained within the two or more new compartments.

[0423] Optionally, the combinatorial barcoding process may include a first barcoding step, where A) a first sample mixture containing at least first and second circulating particles is partitioned into at least first and second original compartments (for example, at least first circulating particles from the sample are partitioned into the first original compartment, and at least second circulating particles from the sample are partitioned into the second original compartment), the first original compartment contains a barcode sequence (or set of barcode sequences) different from the barcode sequence (or set of barcode sequences) contained in the second original compartment, the barcode sequence (or barcode sequence from the set of barcode sequences) contained in the first original compartment is attached to at least first and second fragments of the target nucleic acid of the first circulating particle, and the barcode sequence (or barcode sequence from the set of barcode sequences) contained in the second original compartment is attached to at least first and second fragments of the target nucleic acid of the second circulating particle, and at least one circulating particle contained in the first original compartment A) The particles and at least one circulating microparticle contained in the second original compartment are merged to produce a second sample mixture and a second barcoding step, B) the microparticles contained in the second sample are compartmentalized into at least first and second new compartments (for example, at least first circulating microparticles from the second sample mixture are compartmentalized into the first new compartment, and at least second circulating microparticles from the second sample mixture are compartmentalized into the second new compartment), the first new compartment contains a barcode sequence (or set of barcode sequences) different from the barcode sequence (or set of barcode sequences) contained in the second new compartment, the barcode sequence (or barcode sequence from set of barcode sequences) contained in the first new compartment is attached to at least first and second fragments of the target nucleic acid of the first circulating microparticle, and the barcode sequence (or barcode sequence from set of barcode sequences) contained in the second new compartment is attached to at least first and second fragments of the target nucleic acid of the second circulating microparticle.

[0424] Alternative processes for combinatorial barcoding are described in PCT / GB2017 / 053820, which is incorporated herein by reference.

[0425] Optionally, in any combinatorial barcoding process, one or more steps of chemical crosslinking can be performed before and / or after any step of any combinatorial barcoding process.

[0426] Optionally, in any combinatorial barcoding process, crosslinked particles may be permeated in a step following the chemical crosslinking step. Further details are provided in PCT / GB2017 / 053820, which is incorporated herein by reference.

[0427] Optionally, in any combinatorial barcoding process, crosslinking may be partially or completely reversed in any one or more steps following the chemical crosslinking step. Further details are provided in PCT / GB2017 / 053820, which is incorporated herein by reference.

[0428] Optionally, in any combinatorial barcoding process, a barcode sequence may be added by any one or more of the methods described herein (single-strand ligation, double-strand ligation, blunt-end ligation, A-tail ligation, sticky-end mediated ligation, hybridization, hybridization and extension, hybridization and extension and ligation, and / or transposition, etc.).

[0429] Optionally, during any step of any combinatorial barcoding process, at least 2, at least 3, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1000, at least 2000, at least 500, at least 10,000, at least 50,000, at least 100,000, at least 500,000, or at least 1,000,000 circulating particles may be contained within a compartment (and / or within each of at least the first and second compartments, and / or within any more compartments). Preferably, at least 50 circulating particles may be contained within a compartment (and / or within each of at least the first and second compartments, and / or within any more compartments).

[0430] Optionally, at least 2, at least 3, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1000, at least 2000, at least 5000, at least 1000, at least 2000, at least 5000, at least 10,000, at least 50,000, at least 100,000, at least 500,000, at least 1,000,000, at least 10,000,000, or at least 100,000,000 compartments can be used during any step of any combinatorial barcoding process (for example, circulating particles can be compartmentalized into such number of compartments). Preferably, at least 24 compartments can be used during any step of any combinatorial barcoding process (for example, circulating particles can be compartmentalized into such number of compartments).

[0431] Optionally, during any step of any combinatorial barcoding process, a sample of particulate matter can be separated into compartments containing, on average, less than 0.0001, less than 0.001, less than 0.01, less than 0.1, less than 1.0, less than 10, less than 100, less than 1,000, less than 10,000, less than 100,000, less than 1,000,000, less than 10,000,000, or less than 100,000,000 particulate matter per compartment. Preferably, each compartment contains, on average, less than 1.0 particulate matter.

[0432] Optionally, during any step of any combinatorial barcoding process, a solution of particulate matter can be compartmentalized such that each compartment contains an average of less than 1.0 atg of DNA, less than 10 atg of DNA, less than 100 atg of DNA, less than 1.0 femtogram of DNA, less than 10 femtograms of DNA, less than 100 femtograms of DNA, less than 1.0 picogram of DNA, less than 10 picograms of DNA, less than 100 picograms of DNA, or less than 1.0 nanogram of DNA. Preferably, each compartment contains less than 10 picograms of DNA.

[0433] Optionally, during any step of any combinatorial barcoding process, a partition may have a volume of less than 100 femtoliters, less than 1.0 picoliters, less than 10 picoliters, less than 100 picoliters, less than 1.0 nanoliter, less than 10 nanoliters, less than 100 nanoliters, less than 1.0 microliter, less than 10 microliters, less than 100 microliters, or less than 1.0 milliliter.

[0434] Optionally, any combinatorial barcoding process may include at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 500, or at least 1000 different barcoding steps. Each of the barcoding steps may be as described herein for the first and second barcoding steps.

[0435] Optionally, in any combinatorial barcoding process, any one or more compartmentalization steps may include probabilistic features. For example, an estimated number of circulating particles (rather than the actual or exact number) can be compartmentalized into one or more compartments. That is, the number of circulating particles per compartment may be subject to statistical or probabilistic uncertainty (e.g., subject to Poisson loading and / or distributed statistics).

[0436] Optionally, in any combinatorial barcoding process, a set of barcodes attached to a specific sequence (e.g., a set attached to a sequence of genomic DNA fragments, e.g., a set including a first barcode attached to the sequence during a first barcoding step and a second barcode attached to the sequence during a second barcoding step) may be used to concatenate sequences from a single microparticle and / or from a set of two or more microparticles. Optionally, in any combinatorial barcoding process, the same set of two (or more) barcodes may be attached to a specific sequence from two or more circulating microparticles (e.g., attached to a sequence of genomic DNA fragments) (e.g., the two or more circulating microparticles are partitioned into the same set of first and second compartments during the first and second barcoding steps, respectively). Optionally, in any combinatorial barcoding process, two (or more) identical sets of barcodes may be appended to a specific sequence from only one circulating microparticle (e.g., appended to a sequence of genomic DNA fragments) (e.g., only one circulating microparticle is partitioned into a specific set of first and second compartments during the first and second barcoding steps, respectively).

[0437] Optionally, in any combinatorial barcoding process, the number of compartments used in any one or more barcoding steps, and the number of different barcoding steps, can be combinatorially combined such that, on average, each set of two (or more) barcodes is added to an array from only one circulating particulate. Further details are provided in PCT / GB2017 / 053820, incorporated herein by reference.

[0438] Combinatorial barcoding processes can offer advantages over alternative barcoding processes by reducing the requirements for advanced and / or complex instruments to achieve a large number of potential specific barcode sets for the purpose of adding barcodes to sequences from circulating microparticles (e.g., from fragments of genomic DNA). For example, a combinatorial barcoding process using 96 different compartments in two different barcoding steps (e.g., easily implemented in a standard 96-well plate widely used in molecular biology) can achieve a net of (96 x 96 =) 9216 different barcode sets. This significantly reduces the amount of compartments required to perform such indexing compared to alternative non-combinatorial approaches. Very high levels of combinatorial indexing resolution can be achieved by increasing the number of barcoding steps and / or the number of compartments used in one or more such barcoding steps. Furthermore, combinatorial barcoding processes can eliminate the need for complex instruments used in alternative barcoding processes (e.g., microfluidic instruments such as the 10X Genomics Chromium System).

[0439] 8. Linking by spatial sequencing, in situ sequencing, or in situ library construction. The present invention provides a method for preparing a sample for sequencing, wherein the sample comprises microparticles derived from blood, and the microparticles comprise at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method comprises (a) preparing a sample for sequencing, wherein at least two fragments of the target nucleic acid in the microparticles are ligated together by being in close proximity to each other on a sequencing instrument to produce a set of at least two ligated fragments of the target nucleic acid, and (b) sequencing each of the ligated fragments of the target nucleic acid using a sequencing instrument to produce at least two ligated sequence reads.

[0440] The nucleic acid sample may comprise at least two microparticles derived from blood, where each microparticle comprises at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method comprises: (a) performing a step to generate a set of ligated fragments of the target nucleic acid of each microparticle, wherein the fragments of the target nucleic acid of each microparticle are spatially distinct on a sequencing instrument; and (b) performing a step to generate ligated sequence reads of each microparticle.

[0441] At least two fragments from a microparticle can maintain physical proximity to each other inside or on the sequencing device itself, and this physical proximity can be known, determined, or observed by the sequencing device, or by its operation, or during its operation, and the measurement of this physical proximity helps in linking at least two sequences.

[0442] This method may involve sequencing using an in-situ library construction process. In this method, intact or partially intact microparticles from a sample can be placed on a sequencer, and two or more fragments of a target nucleic acid (e.g., genomic DNA) are processed into a sequenceable template within the sequencer, i.e., sequenced using an in-situ library construction process. In-situ library construction is described in Schwartz et al (2012) PNAS 109(46):18749-54.

[0443] This method may include in-situ sequencing. In this method, the sample may remain intact (e.g., mostly or partially intact), and fragments of the target nucleic acid (e.g., genomic DNA) within the microparticles are directly sequenced using a "FISSEQ" fluorescence in-situ sequencing technique, such as that described, for example, Lee et al. (2014) Science, 343, 6177, 1360-1363.

[0444] Optionally, a sample of microparticles may be crosslinked with a chemical crosslinking agent, then placed in or on a sequencing apparatus, and then held in physical proximity to one another. Optionally, two or more fragments of a target nucleic acid (e.g., genomic DNA) from microparticles placed in or on a sequencing apparatus may have all or part of their sequences determined by the sequencing process. Optionally, such fragments may be sequenced by fluorescence in situ sequencing, where the sequence of the fragment is determined by an optical sequencing process. Optionally, one or more coupling, adapter, or amplification sequences may be added to the fragment of the target nucleic acid. Optionally, the fragment may be amplified in an amplification process, where the amplified products remain in physical proximity to or in physical contact with the fragment from which they were amplified. Optionally, these amplified products are sequenced by an optical sequencing process. Optionally, the amplified products are added to a plane, such as a sequencing flow cell. Optionally, each of the amplified products generated from a single fragment constitutes a single cluster within the flow cell. Optionally, in any of the above methods, the distance between any two or more sequenced molecules may be known a priori by the configuration within the sequencing apparatus or may be determined or observed during the sequencing process. Optionally, each sequenced molecule may be mapped within a cluster field or within an array of pixels, and the distance between any two or more sequenced molecules may be determined by the distance between the cluster or pixels. Optionally, any two or more determined sequences may be concatenated using any measurement or estimation of distance or proximity.

[0445] Optionally, sequences determined by any of the above methods can be further evaluated, and distance or proximity measurements between two or more sequenced molecules can be compared to one or more cutoff values ​​or thresholds to determine that only molecules within a specific range, or above or below a specific threshold or cutoff value, are linked to the information. Optionally, two or more such cutoffs or thresholds or sets of their ranges can be used to determine different degrees of linkage between any two or more sequenced molecules and / or classes and / or categories.

[0446] 9. Concatenation by individual sequencing processes The present invention provides a method for preparing a sample for sequencing, wherein the sample comprises microparticles derived from blood, each microparticle comprising at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method comprises (a) preparing a sample for sequencing, wherein at least two fragments of the target nucleic acid (e.g., genomic DNA) in each microparticle are ligated by loading them into separate sequencing processes to produce a set of at least two ligated fragments of the target nucleic acid, and (b) sequencing each of the ligated fragments of the target nucleic acid using a sequencing instrument to produce a set of at least two ligated sequence reads (i.e., a set of at least two ligated signals).

[0447] The sample may comprise at least two microparticles derived from blood, wherein each microparticle comprises at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method may comprise step (a) generating a ligated fragment of the target nucleic acid of each microparticle, wherein at least two fragments of the target nucleic acid of each microparticle are ligated by being loaded into separate sequencing processes, and step (b) for each sequencing process generating a ligated sequence read of each microparticle.

[0448] In this method, a fragment of a first single microparticle (or group of microparticles) can be sequenced independently of fragments of other microparticles, and the resulting sequence reads are informationally concatenated. A fragment contained within a second single microparticle (or group of microparticles) is sequenced independently of the first microparticle or group of microparticles, and the resulting sequence reads are informationally concatenated.

[0449] Optionally, the first and second sequencing processes (of all sequencing processes) may be performed using different sequencing instruments and / or using the same sequencing instrument, but at two different times or within two different sequencing processes. Optionally, the first and second sequencing processes may be performed using the same sequencing instrument, but in two different regions, sections, compartments, conduits, flow cells, lanes, nanopores, microscaffolds, arrays of microscaffolds, or integrated circuits of the sequencing instrument. Optionally, three or more, ten or more, 1,000 or more, 1,000,000 or more, or 1,000,000,000 or more particles or groups of particles may be linked in the manner described above.

[0450] 10. Amplify the original fragments before concatenating them. As those skilled in the art will understand, the terms “fragment” (e.g., “fragment of genomic DNA,” or “fragment of target nucleic acid,” or “fragment of genomic DNA of / from a microparticle”) as used herein refer to the original fragment present in the microparticle, and parts, copies, or amplicons (e.g., the amplicon) containing only copies of a portion of the original fragment, as well as modified fragments or copies (e.g., fragments to which coupling sequences have been added). For example, the term “fragment of genomic DNA” refers to the original genomic DNA fragment present in the microparticle, and, for example, refers to DNA molecules that can be prepared from the original genomic DNA fragment by a primer extension reaction. As a further example, the term “mRNA fragment” refers to the original mRNA fragment present in the microparticle, and, for example, refers to cDNA molecules that can be prepared from the original mRNA fragment by reverse transcription. As used herein, “fragment of target nucleic acid” also refers to barcoded oligonucleotides (e.g., barcoded oligonucleotides of barcoded affinity probes) and other nucleic acid reagents described herein.

[0451] This method may further include a step of amplifying the original fragment of the target nucleic acid in microparticles by, for example, a primer extension step or a polymerase chain reaction step, prior to the step of attaching the barcode sequence. The barcode sequence can then be attached to the amplicon or copy of the original fragment of the target nucleic acid using any of the methods described herein.

[0452] The primer extension step or polymerase chain reaction step can be carried out using one or more primers containing one or more segments of degenerate bases.

[0453] The primer extension step or polymerase chain reaction step can be performed using one or more primers specific to a particular target nucleic acid sequence (e.g., a specific target genomic DNA sequence).

[0454] The amplification step can be carried out using a chain-substitution polymerase such as Phi29 DNA polymerase, Bst polymerase, or Bsm polymerase, or a modified derivative of Phi29, Bst, or Bsm polymerase. Amplification can be carried out by a polysubstitution amplification reaction and a set of primers containing one or more degenerate base regions. Optionally, random hexamers, random heptamers, random octamers, random nonamers, or random decamer primers can be used.

[0455] The amplification step may involve DNA polymerase extension of single-strand nicks in the original target nucleic acid fragment. Nicks can be generated by enzymes that exhibit single-strand DNA cleavage behavior, or by sequence-specific nick restriction endonucleases.

[0456] The amplification step may include incorporating at least one dUTP nucleotide into a synthesized DNA strand by replicating or amplifying at least a portion of one or more fragments of genomic DNA with a DNA polymerase, and the nicks are generated by a uracil excision enzyme such as uracil DNA glycosylase.

[0457] The amplification step may include the generation of a priming sequence on a nucleic acid containing a fragment of genomic DNA, the priming sequence being generated by a primase enzyme such as Thermus Thermophilus PrimPol polymerase or TthPrimPol polymerase, and a DNA polymerase using this priming sequence as a primer to copy at least one nucleotide of the sequence of the genomic DNA fragment.

[0458] The amplification step can be carried out by a linear amplification reaction, such as an RNA amplification process performed via an in vitro transcription process.

[0459] The amplification step can be carried out by a primer extension step or a polymerase chain reaction step, and the primers or multiple primers used therefor are universal primers corresponding to one or more universal priming sequences. Universal priming sequences can be added to fragments of genomic DNA by ligation reactions, primer extension or polymerase chain reactions, or in vitro transposition reactions.

[0460] 11. Add coupling sequences to the fragments before concatenation. In any manner, the barcode sequence may be directly or indirectly (e.g., by annealing or ligation) attached to a fragment of target nucleic acid (e.g., gDNA) of a microparticle. The barcode sequence may also be attached to a coupling sequence (e.g., a synthetic sequence) attached to the fragment.

[0461] A method comprising linking together at least two fragments of a target nucleic acid in microparticle form to produce a single nucleic acid molecule, wherein a coupling sequence may first be attached to each of the at least two fragments, and then the fragments may be linked together by the coupling sequence.

[0462] The coupling sequence can be attached to the original fragment of the target nucleic acid of the microparticle, or to a copy or amplicon thereof.

[0463] The coupling sequence can be added to the 5' or 3' ends of two or more fragments of a nucleic acid sample. In this method, the target region (of the barcoded oligonucleotide) may contain a sequence complementary to the coupling sequence.

[0464] The coupling sequence may be contained within a double-stranded or single-stranded coupling oligonucleotide. The coupling oligonucleotide can be added to a target nucleic acid by a double-stranded or single-stranded ligation reaction. The coupling oligonucleotide may contain a single-stranded 5' or 3' region that can be ligated to the target nucleic acid, and the coupling sequence can be added to the target nucleic acid by a single-stranded ligation reaction.

[0465] The coupling oligonucleotide may contain a smooth, recessed, or overhanging 5' or 3' region that can ligate to the target nucleic acid, and the coupling sequence may be added to the target nucleic acid in a double-stranded ligation reaction.

[0466] The target nucleic acid's terminal(s) may be converted to blunt double-stranded terminal(s) during the blunt-end ligation reaction, and the coupling oligonucleotide may contain blunt double-stranded terminals, where the coupling oligonucleotide may be ligated to the target nucleic acid during the blunt-end ligation reaction.

[0467] The terminal(s) of the target nucleic acid may be converted to blunt double-stranded terminals(s) during a blunt-end reaction, and then converted to (a) a form having a single 3'-adenosine overhang(s), where the coupling oligonucleotide may contain a double-stranded terminal having a single 3'-thymine overhang that can anneal to the single 3'-adenosine overhang of the target nucleic acid, and the coupling oligonucleotide is ligated to the target nucleic acid during a double-stranded A / T ligation reaction.

[0468] The target nucleic acid can be brought into contact with a restriction enzyme, which digests the target nucleic acid at the restriction site to create (a) ligation sites at the restriction site(s), and the coupling oligonucleotide has a compatible end to the ligation site, and the coupling oligonucleotide is then ligated to the target nucleic acid during a double-strand ligation reaction.

[0469] Coupling oligonucleotides can be added by primer extension or polymerase chain reaction steps.

[0470] The coupling oligonucleotide can be added by a primer extension or polymerase chain reaction step using one or more oligonucleotides that include a priming segment containing one or more degenerate bases.

[0471] The coupling oligonucleotide may be added by a primer extension or polymerase chain reaction step using one or more oligonucleotides further containing priming or hybridization segments specific to a particular target nucleic acid sequence.

[0472] The coupling sequence may be added by a polynucleotide tailing reaction. The coupling sequence may be added by a terminal transferase enzyme (e.g., a terminal deoxynucleotidyltransferase enzyme). The coupling sequence may be added by a polynucleotide tailing reaction carried out using a terminal deoxynucleotidyltransferase enzyme, where the coupling sequence includes at least two adjacent nucleotides of the homopolymer sequence.

[0473] The coupling sequence may include a homopolymer 3' tail (e.g., a poly(A) tail). Optionally, in this method, the target region (of the barcoded oligonucleotide) may include a complementary homopolymer 3' tail (e.g., a poly(T) tail).

[0474] The coupling sequence may be contained within the synthetic transposon and can be added via an in vitro rearrangement reaction.

[0475] The coupling sequence may be added to a target nucleic acid, where the barcode oligonucleotide is added to the target nucleic acid by at least one primer extension step or polymerase chain reaction step, and the barcode oligonucleotide includes a region of at least 1 nucleotide length that is complementary to the coupling sequence. Optionally, this complementary region is located at the 3' end of the barcode oligonucleotide. Optionally, this complementary region is at least 2 nucleotides long, at least 5 nucleotides long, at least 10 nucleotides long, at least 20 nucleotides long, or at least 50 nucleotides long.

[0476] 12. Methods for using coupling molecules for coupling molecule and particulate matter analysis This method may include (a) adding one or more coupling molecules to one or more target biomolecules of or from the circulating microparticles to create one or more add-coupling molecules, and (b) ligating one or more barcode sequences to the add-coupling molecules to create one or more barcoded add-coupling molecules. Optionally, any such step of ligating one or more barcode sequences to the add-coupling molecules may include adding one or more barcoded oligonucleotides to the add-coupling molecule(s), and optionally, the barcoded oligonucleotide(s) may be contained within one or more multimerized barcoding reagents (e.g., a library of two or more multimerized barcoding reagents).

[0477] This method may include (a) performing one or more steps to crosslink the sample; (b) performing one or more steps to create one or more add-coupling molecules by adding one or more coupling molecules to one or more target biomolecules of or from the circulating microparticles; and (c) creating one or more barcode sequences (e.g., barcoded oligonucleotides such as barcoded oligonucleotides contained in one or more multimerized barcoding reagents) to the add-coupling molecules to create one or more barcoded add-coupling molecules. Optionally, one or more steps of permeabilizing the sample and / or microparticles may be performed following any step of crosslinking. Optionally, one or more steps of partially or completely reversing the crosslinking may be performed following any step of crosslinking. Optionally, one or more steps of partially or completely digesting the sample with proteinase may be performed following any step of crosslinking.

[0478] Optionally, following any one or more steps to create one or more barcoded addition coupling molecules, the process may optionally further include one or more barcode ligation steps, where one or more barcode sequences are added to one or more target nucleic acid molecules. Optionally, any one or more such barcode ligation steps may include a process of annealing and / or ligating one or more barcode sequences in one or more barcoded addition coupling molecules to one or more target nucleic acid molecules in the barcoded addition coupling molecule(s). Optionally, any one or more barcode ligation steps may occur after one or more steps of crosslinking a sample of one or more microparticles and / or after one or more steps of partially or completely reversing the crosslinking and / or after one or more steps of partial or complete proteinase digestion.

[0479] This method comprises (a) one or more steps of crosslinking the sample, then (optionally) one or more steps of permeabilizing the sample; (b) one or more steps of adding one or more coupling molecules to one or more target biomolecules of or from the circulating microparticles to create one or more (single and / or double and / or multiple) addition coupling molecules, wherein one or more such target biomolecules include a target nucleic acid molecule; and (c) one or more steps of linking at least one barcode sequence to the addition coupling molecule to create one or more barcoded addition coupling molecules (for example, at least one barcoded oligonucleotide contained in one or more multimerized barcoding reagents). (d) performing one or more steps of linking coding oligonucleotides, and (d) performing one or more barcode ligation steps, wherein a barcode sequence in a barcode addition coupling molecule is added to a target nucleic acid molecule in the barcode addition coupling molecule, and optionally, one or more steps of reversing the crosslinking and / or one or more steps of proteinase digestion are performed before and / or during step (d) of performing one or more barcode ligation steps, and optionally, one or more of the barcode ligation steps include one or more steps of annealing and / or ligating one or more barcode sequences in the barcode addition coupling molecule to one or more target nucleic acid molecules in the barcode addition coupling molecule.

[0480] This method may include two or more steps of adding one or more coupling molecules to one or more target biomolecules of the circulating microparticles to create one or more add-coupling molecules. This method may include one or more steps of adding two or more coupling molecules to each of one or more target biomolecules of the circulating microparticles to create one or more multiple add-coupling molecules (i.e., one or more add-coupling molecules). This method may include a first step of adding a first coupling molecule to each of one or more target biomolecules of the circulating microparticle(s) to create one or more single add-coupling molecules, and then a second step of adding a second coupling molecule to each of the single add-coupling molecules(s) to create one or more double add-coupling molecules (i.e., one or more add-coupling molecules). One or more multiple addition coupling molecules (i.e., one or more addition coupling molecules) can be created by performing any number of (sequential or simultaneous) steps of adding coupling molecules to single and / or double and / or multiple addition coupling molecules, optionally followed by one or more steps of reversing the crosslinking, and / or optionally followed by any one or more barcode connection steps. Any step of adding a coupling molecule may include adding the coupling molecule directly or indirectly to the addition coupling molecule.

[0481] This method may comprise one or more steps of diluting a sample and / or derived sample and / or any solution and / or reaction mixture, wherein the concentration of nucleic acids (such as DNA and / or RNA) and / or polypeptides in the sample is reduced to or below a specific concentration, such as less than 1.0 picogram of DNA (and / or RNA and / or protein) per microliter, less than 10 picograms of DNA (and / or RNA and / or protein) per microliter, less than 100 picograms of DNA (and / or RNA and / or protein) per microliter, less than 1.0 nanogram of DNA (and / or RNA and / or protein) per microliter, less than 10 nanograms of DNA (and / or RNA and / or protein) per microliter, or less than 1000 nanograms of DNA (and / or RNA and / or protein) per microliter. Optionally, any such step of dilution may be performed before, during, and / or after, any one or more steps and / or processes in any method for analyzing a sample containing one or more circulating particles and / or a sample derived from one or more circulating particles. Optionally, any such step of dilution may be performed after any one or more steps that partially or completely reverse crosslinking and / or after any one or more steps of proteinase digestion and / or before any one or more barcode connection steps.

[0482] Any step of attaching one or more coupling molecules to one or more target biomolecules and / or one or more (single-addition and / or double-addition and / or multiple-addition) coupling molecules of or from the circulating microparticles may be performed on one, two, two or more, all, or any number and / or fractions and / or parts of the target biomolecules and / or t...

Claims

1. A method for analyzing a sample containing first circulating microparticles, wherein the first circulating microparticles are membrane vesicles, the first circulating microparticles contain at least three target molecules, at least two of the target molecules are genomic DNA fragments, and at least one of the target molecules is a target polypeptide, the method comprising the step of measuring signals corresponding to the presence, absence, and / or level of each of the target molecules to generate a set of signals for the first circulating microparticles, the set of signals comprising at least two informationally associated signals, at least one of the at least two informationally associated signals corresponding to the presence, absence, and / or level of the genomic DNA fragments in the sample, at least one of the at least two informationally associated signals corresponding to the presence, absence, and / or level of the target polypeptide in the sample, and the step of measuring signals corresponding to the presence, absence, and / or level of the genomic DNA fragments comprising ligating at least two of the at least two genomic DNA fragments to generate a set of genomic DNA fragments, the set of genomic DNA fragments comprising at least two ligated genomic DNA fragments.

2. The method according to claim 1, wherein at least two target molecules, which are genomic DNA fragments, contain a specific nucleotide sequence, and / or at least two target molecules, which are genomic DNA fragments, contain at least one modified nucleotide or modified nucleic acid base.

3. The method according to claim 2, wherein the modified nucleotide or modified nucleic acid base is 5-methylcytosine or 5-hydroxymethylcytosine.

4. The method according to any one of claims 1 to 3, wherein the target polypeptide comprises a specific amino acid sequence and / or the target polypeptide comprises post-translational modifications.

5. The method according to claim 4, wherein the target polypeptide comprises an acetylated amino acid residue and / or a methylated amino acid residue.

6. The method according to any one of claims 1 to 5, wherein the method comprises the step of measuring the signal corresponding to the presence, absence, and / or level of each of the target molecules of the first circulating microparticle to generate a set of signals for the circulating microparticle, the set of signals comprising at least three informationally associated signals, one of the at least three informationally associated signals corresponding to the presence, absence, and / or level of a first genomic DNA fragment of the first circulating microparticle, one of the at least three informationally associated signals corresponding to the presence, absence, and / or level of a second genomic DNA fragment of the first circulating microparticle, and one of the at least three informationally associated signals corresponding to the presence, absence, and / or level of the target polypeptide of the first circulating microparticle.

7. The method according to any one of claims 1 to 6, wherein the step of measuring a signal corresponding to the presence, absence, and / or level of the genomic DNA fragments includes analyzing the sequences of each of at least two of the at least two genomic DNA fragments.

8. The method according to claim 7, wherein the step of measuring a signal corresponding to the presence, absence, and / or level of the genomic DNA fragment comprises sequencing at least a portion of each of at least two of the at least two genomic DNA fragments.

9. The method according to any one of claims 1 to 8, wherein the step of measuring a signal corresponding to the presence, absence, and / or level of the genomic DNA fragments comprises sequencing at least a portion of each of at least two of the concatenated fragments in the set of genomic DNA fragments to generate at least two concatenated sequence reads.

10. The step of measuring a signal corresponding to the presence, absence, and / or level of the genomic DNA fragment, (a) The method according to any one of claims 1 to 9, comprising adding at least two of the at least two genomic DNA fragments of the first circulating microparticle to a barcode sequence to generate a set of linked genomic DNA fragments.

11. The step of measuring a signal corresponding to the presence, absence, and / or level of the genomic DNA fragment, (b) The method of claim 10, further comprising sequencing at least a portion of each of at least two of the linked fragments in the set of genomic DNA fragments to generate at least two linked sequence reads, wherein the at least two linked sequence reads are linked by the barcode sequence.

12. The step of measuring a signal corresponding to the presence, absence, and / or level of the genomic DNA fragment, (a) The method according to any one of claims 1 to 9, comprising adding at least two of the at least two genomic DNA fragments of the first circulating microparticle to different barcode sequences of a barcode sequence set to generate a set of linked genomic DNA fragments.

13. The step of measuring a signal corresponding to the presence, absence, and / or level of the genomic DNA fragment, (b) The method of claim 12, further comprising sequencing at least a portion of each of at least two of the linked fragments in the set of genomic DNA fragments to generate at least two linked sequence reads, wherein the at least two linked sequence reads are linked by the barcode sequence set.

14. The method according to any one of claims 1 to 13, wherein the at least two target molecules, which are genomic DNA fragments, comprise at least one modified nucleotide or modified nucleic acid base, and the step of measuring a signal corresponding to the presence, absence, and / or level of the genomic DNA fragments comprises the step of measuring a signal corresponding to the presence, absence, and / or level of the modified nucleotide or modified nucleic acid base of the genomic DNA fragments.

15. The method of claim 14, wherein the signal corresponding to the presence, absence, and / or level of the modified nucleotide or modified nucleic acid base is measured using (i) a barcoded affinity probe comprising at least one affinity moiety linked to a barcoded oligonucleotide, the barcoded oligonucleotide comprising at least one nucleotide, and the affinity moiety capable of binding to the modified nucleotide or modified nucleic acid base, and / or (ii) an optically labeled affinity probe and / or a fluorescently labeled affinity probe.

16. The method according to any one of claims 1 to 15, wherein the signal corresponding to the presence, absence, and / or level of the target polypeptide is measured using (i) a barcoded affinity probe comprising at least one affinity moiety linked to a barcoded oligonucleotide, the barcoded oligonucleotide comprising at least one nucleotide, and the affinity moiety capable of binding to the target polypeptide, and / or (ii) an optically labeled affinity probe and / or a fluorescently labeled affinity probe.

17. The method according to claim 16, wherein in (i), the signal is measured by determining the presence, absence, and / or level of the barcoded oligonucleotide by sequencing, and / or in (ii), the signal is measured by flow cytometry and / or fluorescence-activated cell sorting.

18. The method according to any one of claims 1 to 17, wherein the first circulating microparticle comprises at least three target molecules, and the method comprises generating a set of signals for the first circulating microparticle, wherein the set of signals comprises at least three informationally associated signals.

19. The method according to any one of claims 1 to 18, wherein the target molecule comprises at least three genomic DNA fragments, and the method comprises generating a set of signals for the first circulating microparticle, wherein the set of signals comprises at least three informationally associated signals.

20. The method according to any one of claims 1 to 19, wherein the target molecule comprises at least two target polypeptides, and the method comprises generating a set of signals for the first circulating microparticles, wherein the set of signals comprises at least three informationally associated signals.

21. The method according to any one of claims 1 to 20, wherein the sample comprises a first circulating microparticle and a second circulating microparticle, the first circulating microparticle and the second circulating microparticle are membrane vesicles, and each circulating microparticle comprises at least three target molecules as defined in any one of claims 1 to 20, and the method comprises: performing the measuring step according to any one of claims 1 to 20 to generate a set of signals for the first circulating microparticle, wherein the set of signals comprises at least two informationally associated signals; and performing the measuring step according to any one of claims 1 to 20 to generate a set of signals for the second circulating microparticle, wherein the set of signals comprises at least two informationally associated signals.

22. The method according to claim 21, wherein the sample comprises n circulating microparticles, each circulating microparticle being a membrane vesicle, each circulating microparticle comprising at least three target molecules as defined in any one of claims 1 to 20, and the method comprises performing the measuring step according to any one of claims 1 to 20 for each circulating microparticle to generate a set of signals for each circulating microparticle, the set of signals comprising at least two informationally associated signals, and n being at least 3.

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