Method for cyclic microparticle analysis

Abstract

The invention relates to a method for cyclic microparticle analysis. The present invention provides reagents and methods for analyzing free biomolecules (e.g., free nucleic acid molecules and free polypeptides) of circulating microparticles (i.e., blood-derived microparticles). The method comprises analyzing a sample comprising circulating microparticles or a sample derived from circulating microparticles. The methods include methods of measuring at least two linked signals, each signal corresponding to the presence, absence and / or level of a biomolecule of a circulating microparticle. The methods also include methods of determining the presence, absence, and / or level of biomolecules of circulating microparticles using barcoded affinity probes. In certain methods, nucleic acid biomolecules and non-nucleic acid biomolecules circulating microparticles are analyzed together. Reagents for use in the methods are also provided.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of December 21, 2018, an application number of 201880096851.7, and an invention title of "Method for Circulating Microparticle Analysis". Technical Field

[0002] The present invention relates to the analysis of free biomolecules (such as free nucleic acid molecules and free polypeptides). Specifically, it relates to the analysis of free biomolecules contained within or derived from circulating microparticles. Reagents and methods for analyzing the biomolecules of circulating microparticles are provided, including reagents and methods for analyzing the biomolecules of individual circulating microparticles. Background Art

[0003] Circulating free DNA (cfDNA) is typically fragmented (usually in the range of 100 - 200 base pairs in length), and thus methods for cfDNA analysis have traditionally focused on the biological signals that can be detected with these short DNA fragments. For example, detecting single nucleotide variants within individual molecules, or'molecular counting' of large numbers of sequencing fragments to indirectly infer the presence of large-scale chromosomal abnormalities, such as testing for fetal chromosomal trisomy to assess fetal DNA within maternal circulation (a so-called 'non-invasive prenatal test', or NIPT).

[0004] A variety of methods for analyzing circulating free DNA have been previously described. Depending on the specific application area, these analyses may use different terms for a broadly similar set of sample types and technical methods, such as circulating tumor DNA (ctDNA), free fetal DNA (cffDNA), and / or liquid biopsy, or non-invasive prenatal testing. Generally, these methods involve laboratory protocols for preparing circulating free DNA samples for sequencing, the sequencing reaction itself, and then an informatics framework to analyze the resulting sequences to detect relevant biological signals. The methods include steps for DNA purification and isolation prior to sequencing, which means that subsequent analysis must rely solely on the information contained within the DNA itself. After sequencing, such methods typically employ one or more informatics or statistical frameworks to analyze various aspects of the sequence data, such as detecting specific mutations therein and / or detecting selective enrichment or depletion of specific chromosomal or sub-chromosomal regions (e.g., which may indicate chromosomal aneuploidy in a developing fetus).

[0005] Many of these methods can be used for NIPT (e.g., U.S. Pat. Nos. 6,258,540 B1, 8,296,076 B2, 8,318,430 B2, 8,195,415 B2, 9,447,453 B2, and 8,442,774 B2). The most commonly used methods for performing non-invasive prenatal testing to detect fetal chromosomal abnormalities (e.g., trisomies and / or sub-chromosomal abnormalities (e.g., microdeletions)) include sequencing a large number of cfDNA molecules and mapping the resulting sequences to the genome (i.e., determining which chromosome and / or which part of a given chromosome the sequence is derived from), and then for one or more such chromosomal or sub-chromosomal regions, determining the number of sequences corresponding thereto (e.g., as an absolute read count or relative read count), and then comparing it to one or more normal or abnormal thresholds or cut-off values, and / or performing statistical tests to determine whether the region is likely to be over-represented in terms of sequence number (e.g., may correspond to chromosomal trisomy) and / or whether the region is likely to be under-represented in terms of sequence number (e.g., may correspond to a microdeletion).

[0006] Also, a variety of other or improved methods for analyzing cell-free DNA using data from unlinked single molecules have been described (e.g., WO2016094853 A1, US2015344970 A1, and US20150105267 A1).

[0007] Despite such a wide range of methods, there is still a need for new methods of analyzing cfDNA that will allow reliable detection of remote genetic information (e.g., phasing), and there is also a need for more sensitive methods. For example, in the case of NIPT, fetal cfDNA represents only a small fraction of the total cfDNA of a pregnant individual (most of the circulating DNA is normal maternal DNA). Thus, a major technical challenge for NIPT is to distinguish fetal cfDNA from maternal DNA. Similarly, in cancer patients, cfDNA represents only a small fraction of the total circulating DNA. Thus, there are similar technical challenges regarding the use of cfDNA analysis for cancer diagnosis or monitoring.

[0008] In addition, methods for isolating cell type-specific apoptotic bodies by fluorescence-activated cell sorting (FACS) (Atkin-Smith et al., 2017. Scientific Reports 7, 39846) and methods for allowing multiplex analysis of protein markers in single extracellular vesicles (Lee et al., 2018. ACS Nano. 23, 12(1), 494 - 503) have also been described. SUMMARY OF THE INVENTION

[0009] The present invention provides methods for analyzing a sample comprising circulating micron-sized particles (or a sample derived from circulating micron-sized particles), such as apoptotic bodies. The present invention is based on the multi-parametric measurement of different types of biomolecules contained within or derived from a single circulating micron-sized particle. In particular, the present invention allows the measurement of ligation signals corresponding to the presence, absence, and / or level of two or more types of target biomolecules in the same circulating micron-sized particle. As Figure 30 shown, a signal corresponding to the fragment level of genomic DNA can be generated (e.g., by partitioning, barcoding, and sequencing), and a signal corresponding to the level of a target polypeptide can be generated (e.g., using barcoded affinity probes). Additionally, a signal corresponding to the level of a modified nucleotide (e.g., a nucleotide containing 5-methylcytosine) can be generated (e.g., by an affinity-based enrichment method, such as a method using an enrichment probe specific for or preferentially binding to 5-methylcytosine in fragments of genomic DNA). These measurements and related techniques thus generate a series of ligation signals corresponding to the physical and biological state of the circulating micron-sized particles.

[0010] The multi-parametric methods provided herein add an additional layer of information to the earlier inventions provided by the inventors in PCT / GB2017 / 053820, PCT / GB2017 / 053812, and PCT / GB2017 / 053816.

[0011] In PCT / GB2017 / 053820, the inventors previously provided methods for analyzing nucleic acid fragments in circulating micron-sized particles (or micron-sized particles derived from blood). The invention was based on a ligation fragment method, in which nucleic acid fragments from a single micron-sized particle were ligated together. This ligation enabled the generation of a set of ligation sequence reads corresponding to the sequences of the fragments from a single micron-sized particle (i.e., a set of ligation signals).

[0012] The method of linked fragments provides highly sensitive cfDNA analysis and can also detect remote genetic information. The method is based on a combination of insights. First, the method exploits the insight that a single circulating micron particle (e.g., a single circulating apoptotic body) will contain many fragments of genomic DNA that are generated from the same single cell (somewhere in the body) that has undergone apoptosis. Second, a portion of such fragments of genomic DNA within an individual micron particle will preferentially contain sequences from one or more specific chromosomal regions. Cumulatively, circulating micron particles thus act as data-rich and multi-featured'molecular stethoscopes' to observe very complex genetic events that may occur in a limited somatic tissue space somewhere in the body; importantly, since most of such micron particles enter the circulation before being cleared or metabolized, they can be detected non-invasively. The present invention describes experimental and informational methods using these'stethoscopes', i.e., a set of linked fragments and linked sequence reads (in the form of individual micron particles, or in many embodiments, in the form of a complex sample containing a large number of individual circulating micron particles) to perform analytical and diagnostic tasks.

[0013] The present invention advances the concept of the'molecular stethoscope' by leveraging data provided by the co-localization of non-nucleic acid molecules (e.g., target polypeptides) and nucleic acid molecules (e.g., fragments of genomic DNA) in, for example, single circulating micron particles. This advance is based on the discovery that many biomolecules contained in the circulation are biophysically retained in circulating micron particles rather than being single and freely diffusible in the blood. The present invention exploits this rich source of information by measuring signals corresponding to the presence, absence, and / or levels of multiple target biomolecules of circulating micron particles to generate a set of (informational) linked signals for the circulating micron particles. Additionally, by including in this set one or more signals corresponding to one or more target biomolecules unique to a particular cell or tissue type, the cell origin of a particular set of linked signals derived from a single circulating micron particle can be determined. Compared to currently available methods, this provides a much richer source of information for a set of linked signals with a 'cellular context'. By doing so, the present invention provides analytical methods with high accuracy, sensitivity, and precision. Such methods have clear applications in a wide range of diagnostic and monitoring applications including cancer diagnosis and monitoring as well as NIPT.

[0014] The inventors have previously provided reagents and methods related to barcodes. In WO2016 / 207639, the inventors provided various reagents, kits, and methods for molecular barcoding, including multi-plex barcoding reagents. In PCT / GB2017 / 053812, the inventors provided other methods and reagents for molecular barcoding. In PCT / GB2017 / 053816, the inventors provided reagents and methods for molecular barcoding of nucleic acids of single cells.

[0015] WO2016 / 207639, PCT / GB2017 / 053812, PCT / GB2017 / 053816, and PCT / GB2017 / 053820 are hereby incorporated by reference in their entirety.

[0016] The present invention provides a method for analyzing a sample comprising circulating micron particles or a sample derived from circulating micron particles, wherein the circulating micron particles comprise at least two target molecules, wherein the at least two target molecules are biomolecules, and wherein the method comprises measuring signals corresponding to the presence, absence, and / or level of each target molecule to generate a set of at least two (information) linkage signals for the circulating micron particles, wherein at least one linkage signal corresponds to the presence, absence, and / or level of a first biomolecule in the sample, and at least one linkage signal corresponds to the presence, absence, and / or level of a second biomolecule in the sample.

[0017] The present invention provides a method for analyzing a sample comprising circulating micron particles or a sample derived from circulating micron particles, wherein the circulating micron particles comprise at least two target molecules, wherein the at least two target molecules are biomolecules, and wherein the method comprises measuring signals corresponding to the presence, absence, and / or level of each target molecule to generate a single signal for the circulating micron particles, wherein the single signal corresponds to the presence, absence, and / or level of a biomolecule in the sample.

[0018] The first biomolecule can be a fragment of a target nucleic acid (e.g., a fragment of genomic DNA), and the second biomolecule can be a target (or predefined) non-nucleic acid biomolecule (e.g., a target polypeptide). Optionally, the fragment of the target nucleic acid may comprise at least one modified nucleotide or nucleobase.

[0019] The target molecule may comprise at least one or preferably at least two fragments of a target nucleic acid (e.g., genomic DNA).

[0020] The first biomolecule can be a polypeptide, and the second target biomolecule can be a fragment of a target nucleic acid (e.g., genomic DNA) comprising an epigenetic modification (e.g., 5-hydroxymethylcytosine DNA or 5-methylcytosine DNA).

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

[0022] The first biomolecule can be 5-methylcytosine DNA, and the second target biomolecule can be a fragment of RNA.

[0023] The first biomolecule can be 5-hydroxymethylcytosine DNA, and the second target biomolecule can be a biomolecule selected from Biomolecule Group 1.

[0024] The first biomolecule can be 5-methylcytosine DNA, and the second target biomolecule can be a biomolecule selected from Biomolecule Group 1.

[0025] The first and second biomolecules can be selected from Biomolecule Group 1.

[0026] The present invention provides a method for analyzing a sample comprising circulating micron-sized particles or a sample derived from circulating micron-sized particles, wherein the circulating micron-sized particles comprise at least three target molecules, wherein at least two of the target molecules are fragments of genomic DNA and at least one of the target molecules is an RNA fragment, and wherein the method comprises measuring signals corresponding to the presence, absence, and / or level of each target molecule to generate at least two sets of (information) linkage signals for the circulating micron-sized particles, wherein at least one linkage signal corresponds to the presence, absence, and / or level of a fragment of genomic DNA in the sample, and at least one linkage signal corresponds to the presence, absence, and / or level of an RNA fragment in the sample.

[0027] The present invention provides a method for analyzing a sample comprising circulating micron-sized particles or a sample derived from circulating micron-sized particles, wherein the circulating micron-sized particles comprise at least three target molecules, wherein at least two of the target molecules are fragments of genomic DNA and at least one of the target molecules is an RNA fragment, and wherein the method comprises measuring signals corresponding to the presence, absence, and / or level of each target molecule to generate a single signal for the circulating micron-sized particles, wherein the single signal corresponds to the presence, absence, and / or level of fragments of genomic DNA and RNA in the sample.

[0028] The present invention provides a method for analyzing a sample comprising circulating micron-sized particles or a sample derived from circulating micron-sized particles, wherein the circulating micron-sized particles comprise at least three target molecules, wherein at least two of the target molecules are fragments of target nucleic acid (e.g., genomic DNA) and at least one of the target molecules is a target biomolecule (e.g., target polypeptide), and wherein the method comprises measuring signals corresponding to the presence, absence, and / or level of each target molecule to generate at least three sets of (information) linkage signals for the circulating micron-sized particles, wherein each of at least two of the linkage signals corresponds to the presence, absence, and / or level of a fragment of target nucleic acid (e.g., genomic DNA) in the sample, and at least one linkage signal corresponds to the presence, absence, and / or level of a target biomolecule (e.g., target polypeptide) in the sample.

[0029] The present invention provides a method for analyzing a sample comprising circulating micron particles or a sample derived from circulating micron particles, wherein the circulating micron particles comprise at least three target molecules, wherein at least two of the target molecules are fragments of a target nucleic acid (e.g., genomic DNA) and at least one of the target molecules is a target biomolecule (e.g., a target polypeptide), and wherein the method comprises measuring signals corresponding to the presence, absence, and / or level of each target molecule to generate a set of at least two (information) linkage signals for the circulating micron particles, wherein at least one of the linkage signals corresponds to the presence, absence, and / or level of a fragment of a target nucleic acid (e.g., genomic DNA) in the sample, and at least one of the linkage signals corresponds to the presence, absence, and / or level of a target biomolecule (e.g., a target polypeptide) in the sample.

[0030] The present invention provides a method for analyzing a sample comprising circulating micron particles or a sample derived from circulating micron particles, wherein the circulating micron particles comprise at least three target molecules, wherein at least two of the target molecules are fragments of a target nucleic acid (e.g., genomic DNA) and at least one of the target molecules is a target biomolecule (e.g., a target polypeptide), and wherein the method comprises measuring signals corresponding to the presence, absence, and / or level of each target molecule to generate a single signal for the circulating micron particles, wherein the single signal corresponds to the presence, absence, and / or level of a fragment of a target nucleic acid (e.g., genomic DNA) and a target biomolecule (e.g., a target polypeptide) in the sample.

[0031] The fragment of the target nucleic acid (e.g., genomic DNA) may comprise a specific sequence of nucleotides and / or the fragment of the target nucleic acid (e.g., genomic DNA) may comprise at least one modified nucleotide or nucleobase. The fragment of the target nucleic acid may not comprise a specific sequence of nucleotides. The fragment of the target nucleic acid may comprise an untargeted and / or unknown and / or randomly selected and / or randomly sampled nucleotide sequence. For example, the modified nucleotide or nucleobase may be 5-methylcytosine or 5-hydroxymethylcytosine. The fragment of the target nucleic acid (e.g., genomic DNA) may comprise one or more microsatellite sequences and / or microsatellite genomic regions (i.e., short tandem repeats).

[0032] The target polypeptide may comprise a specific amino acid sequence and / or the target polypeptide may comprise a post-translational modification. For example, the target polypeptide may comprise acetylated amino acid residues and / or methylated amino acid residues (e.g., specific acetylated amino acid residues on / within a specific polypeptide and / or specific methylated amino acid residues on / within a specific polypeptide).

[0033] The method includes measuring signals corresponding to the presence, absence, and / or level of each target molecule of the circulating micron particles to generate a set of at least three (information) linkage signals for the circulating micron particles, wherein one linkage signal corresponds to the presence, absence, and / or level of a first target nucleic acid (e.g., genomic DNA) fragment of the circulating micron particles, one linkage signal corresponds to the presence, absence, and / or level of a second target nucleic acid (e.g., genomic DNA) fragment of the circulating micron particles, and one linkage signal corresponds to the presence, absence, and / or level of a target biomolecule (e.g., target polypeptide) of the circulating micron particles.

[0034] The step of measuring signals corresponding to the presence, absence, and / or level of a fragment of a target nucleic acid (e.g., genomic DNA) may include analyzing the sequence of each of at least two of at least two fragments of the target nucleic acid (e.g., genomic DNA). Optionally, the step of measuring signals corresponding to the presence, absence, and / or level of a fragment of a target nucleic acid (e.g., genomic DNA) includes sequencing at least a portion of each of at least two of at least two fragments of the target nucleic acid (e.g., genomic DNA) to generate at least two (information) linkage sequence reads.

[0035] The step of measuring signals corresponding to the presence, absence, and / or level of a fragment of a target nucleic acid (e.g., genomic DNA) may include: (a) ligating at least two of at least two fragments of the target nucleic acid (e.g., genomic DNA) to generate a set of at least two ligated fragments of the target nucleic acid (e.g., genomic DNA); and optionally, (b) analyzing the sequence 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 ligated sequence reads.

[0036] 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: (a) attaching each of at least two of at least two fragments of the target nucleic acid (e.g., genomic DNA) of the circulating micron particles to a barcode sequence to generate a set of ligated fragments of the target nucleic acid (e.g., genomic DNA); and optionally, (b) analyzing the sequence 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 (information) linkage sequence reads, wherein the at least two linkage sequence reads are linked by the barcode sequence. Optionally, each of at least two of at least two fragments of the target nucleic acid may include the same barcode sequence.

[0037] 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) can include: (a) attaching each of at least two of at least two fragments of the target nucleic acid (e.g., genomic DNA) of the circulating micron particles to a different barcode sequence in a set of barcode sequences to produce a set of ligated fragments of the target nucleic acid (e.g., genomic DNA); and optionally, (b) analyzing the sequence of each of at least two of the ligated fragments in the set. Step (b) can include sequencing at least a portion of each of at least two of the ligated fragments in the set to produce at least two ligated sequence reads. The at least two ligated sequence reads can be joined (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 join the two sequence reads to each other by being present within the same set of barcode sequences).

[0038] 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) can include: (a) attaching a first barcode sequence to a first fragment of the target nucleic acid (e.g., genomic DNA) to produce a first barcoded target nucleic acid molecule, and attaching a second barcode sequence to a second fragment of the target nucleic acid (e.g., genomic DNA) to produce a second barcoded target nucleic acid molecule, wherein the first and second barcode sequences each comprise the same barcode sequence, or each comprise different barcode sequences in a set of barcode sequences; and optionally, (b) analyzing the sequence of each of the first and second barcoded target nucleic acid molecules. Step (b) can include sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules to produce at least two (informative) ligated sequence reads. The at least two ligated sequence reads can be joined by the same barcode sequence or a set of barcode sequences. Step (b) can include sequencing all or at least a portion of each of the first and second barcode sequences 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) can include: (a) attaching (e.g., ligating or joining) a first barcoded oligonucleotide to a first fragment of the target nucleic acid (e.g., genomic DNA) to produce a first barcoded target nucleic acid molecule, and attaching (e.g., ligating or joining) a second barcoded oligonucleotide to a second fragment of the target nucleic acid (e.g., genomic DNA) to produce a second barcoded target nucleic acid molecule, wherein the first and second barcoded oligonucleotides each contain the same barcode sequence, or each contain different barcode sequences from a set of barcode sequences; and optionally, (b) analyzing the sequence of each of the first and second barcoded target nucleic acid molecules. Step (b) can include sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules to produce at least two (informative) linked sequence reads. The at least two linked sequence reads can be linked by the same barcode sequence or a set of barcode sequences. Step (b) can include sequencing all or at least a portion of each of the first and second barcoded oligonucleotides attached to the first and second fragments of the target nucleic acid.

[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) can include: (a) contacting a sample with a polybarcode reagent, wherein the polybarcode reagent comprises a first and a second barcode region linked together, wherein each barcode region comprises a nucleic acid sequence; and (b) attaching a barcode sequence to each of the first and second fragments of the target nucleic acid of a micron particle to produce a first and a second barcoded target nucleic acid molecule for the micron particle, wherein the first barcoded target nucleic acid molecule comprises the nucleic acid sequence of the first barcode region and the second barcoded target nucleic acid molecule comprises the nucleic acid sequence of the second barcode region. The first and second barcode regions can each contain the same barcode sequence, or the first and second barcode regions can contain different barcode sequences from a set of barcode sequences. The method can further include (c) analyzing the sequence of each of the first and second barcoded target nucleic acid molecules. Step (c) can include sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules to produce at least two (informative) linked sequence reads. The at least two linked sequence reads can be linked by the same barcode sequence or by a set of barcode sequences.

[0041] 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) can include: (a) contacting a sample with a polymeric barcoding reagent, wherein the polymeric barcoding reagent comprises first and second barcoded oligonucleotides linked together, and wherein the barcoded oligonucleotides each comprise a barcode region; and (b) attaching (e.g., ligating or joining) the first and second barcoded oligonucleotides to first and second fragments of the target nucleic acid of a micron particle to produce first and second barcoded target nucleic acid molecules. The barcode regions of the first and second barcoded oligonucleotides can each comprise the same barcode sequence, or the barcode regions of the first and second barcoded oligonucleotides can each comprise different barcode sequences from a set of barcode sequences. The method can further include (c) analyzing the sequence of each of the first and second barcoded target nucleic acid molecules. Step (c) can include sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules to produce at least two (informative) linked sequence reads. The at least two linked sequence reads can be linked by the same barcode sequence or a set of barcode sequences.

[0042] A fragment of a target nucleic acid (e.g., genomic DNA) can comprise at least one epigenetic modification (e.g., a modified nucleotide or nucleobase), and 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) can include measuring a signal corresponding to the presence, absence, and / or level of an epigenetic modification (e.g., a modified nucleotide or nucleobase) of the fragment of the target nucleic acid (e.g., genomic DNA). For example, the modified nucleotide or nucleobase can comprise 5-methylcytosine or 5-hydroxymethylcytosine.

[0043] The present invention provides a method of analyzing a sample comprising circulating micron particles or a sample derived from circulating micron particles, wherein the circulating micron particles comprise at least two target molecules, wherein at least one target molecule is a fragment of a target nucleic acid (e.g., genomic DNA) comprising an epigenetic modification and at least one target molecule is a target biomolecule (e.g., a target polypeptide), and wherein the method comprises measuring a signal corresponding to the presence, absence, and / or level of each target molecule to produce a set of at least two (informative) linked signals of the circulating micron particles, wherein at least one linked signal corresponds to the presence, absence, and / or level of an epigenetic modification in the sample, and at least one linked signal corresponds to the presence, absence, and / or level of a target biomolecule (e.g., a target polypeptide) in the sample.

[0044] The present invention provides a method for analyzing a sample comprising circulating micron-sized particles or a sample derived from circulating micron-sized particles, wherein the circulating micron-sized particles comprise at least two target molecules, wherein at least one target molecule is a fragment of a target nucleic acid (e.g., genomic DNA) comprising an epigenetic modification and at least one target molecule is a target biomolecule (e.g., a target polypeptide), and wherein the method comprises measuring a signal corresponding to the presence, absence, and / or level of each target molecule to generate a single signal for the circulating micron-sized particles, wherein the single signal corresponds to the presence, absence, and / or level of the fragment of the epigenetic modification and the target biomolecule (e.g., a target polypeptide) in the sample.

[0045] The method may comprise the step of analyzing the sequence of a target nucleic acid (e.g., genomic DNA) comprising an epigenetic modification. Alternatively, the method may not comprise the step of analyzing the sequence of a target nucleic acid (e.g., genomic DNA) comprising an epigenetic modification.

[0046] The epigenetic modification may comprise a modified nucleotide, such as a modified gDNA nucleotide or a modified RNA nucleotide. The modified nucleotide may comprise a modified base. The modified base may be a methylated base, such as 5-methylcytosine or 5-hydroxymethylcytosine. The fragment of the target nucleic acid (e.g., genomic DNA) comprising an epigenetic modification may comprise 5-methylcytosine DNA or 5-hydroxymethylcytosine DNA.

[0047] A barcoded affinity probe may be used to measure a signal corresponding to the presence, absence, and / or level of an epigenetic modification (e.g., a modified DNA or RNA nucleotide). The barcoded affinity probe may comprise at least one affinity moiety linked to a barcoded oligonucleotide, wherein the barcoded oligonucleotide comprises at least one nucleotide (i.e., wherein the barcoded oligonucleotide comprises a nucleotide sequence having a length of at least one nucleotide), and wherein the affinity moiety is capable of binding to the target biomolecule (i.e., capable of binding to the epigenetic modification). The signal may 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).

[0048] Signals corresponding to the presence, absence, and / or level of an epigenetic modification (e.g., a modified DNA or RNA nucleotide) can be measured by flow cytometry and / or fluorescence-activated cell sorting, using optically labeled affinity probes and / or fluorescently labeled affinity probes. The optically labeled affinity probes and / or fluorescently labeled affinity probes can be visualized using an optical microscope and / or a fluorescence microscope for measurement and / or detection. For example, using a fluorescence microscope, and / or using fluorescence laser-based detection, and / or using a fluorescence-activated cell sorting (FACS) instrument. The optically labeled affinity probes and / or fluorescently labeled affinity probes can be measured and / or detected using sorting methods, such as using fluorescence-activated cell sorting (FACS).

[0049] Methods involving a molecular conversion step can be used to measure signals corresponding to the presence, absence, and / or level of an epigenetic modification (e.g., a modified DNA or RNA nucleotide). In the case of a modified nucleotide (i.e., a nucleotide containing a modified base, such as 5-methylcytosine or 5-hydroxymethylcytosine), a molecular conversion step can be performed to convert the modified base into a different modified or unmodified nucleotide that can be detected (e.g., using PCR or sequencing) to provide a signal corresponding to the presence, absence, and / or level of the epigenetic modification. This conversion step can include a bisulfite conversion step, an oxidative bisulfite conversion step, or any other molecular conversion step. The method can be used to measure 5-methylcytosine in fragments of genomic DNA of circulating microparticles.

[0050] The method can further include one or more steps of partitioning a sample containing one or more circulating microparticles (or a sample derived from one or more circulating microparticles). Additionally or alternatively, the method can further include one or more steps of attaching any one or more barcode sequences and / or partition barcode sequences and / or barcoded oligonucleotides to one or more fragments of a target nucleic acid. The one or more barcode sequences and / or barcoded oligonucleotides can be provided by and / or included in one or more of the polybarcode reagents as described herein.

[0051] Barcoded affinity probes can be used to measure signals corresponding to the presence, absence, and / or level of a non-nucleic acid biomolecule (e.g., a target polypeptide). The barcoded affinity probe can include at least one affinity moiety linked to a barcoded oligonucleotide, where the barcoded oligonucleotide includes at least one nucleotide (i.e., where the barcoded oligonucleotide includes a nucleotide sequence of at least one nucleotide in length), and where the affinity moiety is capable of binding to the target biomolecule (i.e., the target non-nucleic acid biomolecule (e.g., the 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).

[0052] Signals corresponding to the presence, absence, and / or level of a non-nucleic acid biomolecule (e.g., a target polypeptide) can be measured by flow cytometry and / or fluorescence-activated cell sorting using optically labeled affinity probes and / or fluorescently labeled affinity probes. The optically labeled affinity probes and / or fluorescently labeled affinity probes can be visualized and measured and / or detected using an optical microscope and / or a fluorescence microscope. For example, using a fluorescence microscope, and / or using fluorescence laser-based detection, and / or using a fluorescence-activated cell sorting (FACS) instrument. The optically labeled affinity probes and / or fluorescently labeled affinity probes can be measured and / or detected using sorting methods, such as using fluorescence-activated cell sorting (FACS).

[0053] Signals corresponding to the presence, absence, and / or level of a non-nucleic acid biomolecule (e.g., a target polypeptide) can be measured by a carrier labeled with an affinity probe. The carrier labeled with an affinity probe can comprise beads (e.g., magnetic beads) labeled with an affinity probe, e.g., an antibody specific for the target polypeptide. The presence, absence, and / or level of a non-nucleic acid biomolecule (e.g., a target polypeptide in circulating micron-sized particles) can be measured by incubating and / or binding the non-nucleic acid biomolecule with the affinity probe on the carrier. Optionally, wherein the portion bound to the carrier (i.e., the micron-sized particles containing the non-nucleic acid biomolecule and / or containing a high level of the non-nucleic acid biomolecule) is further separated and / or processed (e.g., partitioned and / or barcoded and / or analyzed by nucleic acid sequencing), and optionally, wherein the portion not bound to the carrier (i.e., the micron-sized particles not containing and / or containing a low level of the non-nucleic acid biomolecule) is further separated and / or processed (e.g., partitioned and / or barcoded and / or analyzed by nucleic acid sequencing).

[0054] Signals corresponding to the presence, absence, and / or level of a non-nucleic acid biomolecule (e.g., a target polypeptide) and signals corresponding to the presence, absence, and / or level of a nucleic acid biomolecule can be measured separately. For example, signals corresponding to the presence, absence, and / or level of a non-nucleic acid biomolecule (e.g., a target polypeptide) can be measured by FACS, and signals corresponding to the presence, absence, and / or level of a nucleic acid biomolecule can be measured by sequencing.

[0055] In the method, a set of linked signals can be measured for (or for each) circulating micron-sized particle, the set of linked signals corresponding to the presence, absence, and / or level of a target nucleic acid (e.g., genomic DNA) fragment, an epigenetic modification (e.g., a modified nucleotide, e.g., a modified nucleotide containing 5-methylcytosine and / or 5-hydroxymethylcytosine), and a target non-nucleic acid biomolecule (e.g., a target polypeptide).

[0056] For example, in the method, the target molecules of the cyclic micron particles may include at least 2 (different) fragments of a target nucleic acid (such as genomic DNA), at least one fragment of a target nucleic acid (such as genomic DNA) containing epigenetic modifications, and at least one target non-nucleic acid biomolecule (such as a target polypeptide). The method may include measuring signals corresponding to the presence, absence, and / or level of each target molecule to generate a set of ligation signals with the cyclic micron particles. The method may provide (different) ligation signals for each target molecule. In the method, each of at least two ligation signals may correspond to the presence, absence, and / or level of a fragment of a target nucleic acid (such as genomic DNA); at least one ligation signal may correspond to the presence, absence, and / or level of an epigenetic modification (such as a modified nucleotide, such as a modified nucleotide containing 5-methylcytosine and / or 5-hydroxymethylcytosine); and at least one ligation signal may correspond to the presence, absence, and / or level of a target non-nucleic acid biomolecule (such as a target polypeptide).

[0057] The cyclic micron particles may include 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, wherein the method includes generating 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) ligation signals for the cyclic micron particles (i.e., (different) ligation signals for each target molecule of the cyclic micron particles).

[0058] The target molecules of the cyclic micron particles may include at least 2, at least 3, at least 4, at least 9, at least 49, at least 99, at least 499, at least 999, at least 4999, at least 9999, at least 99,999, or at least 999,999 (different) fragments of a target nucleic acid (such as genomic DNA) and at least one target non-nucleic acid biomolecule (such as a target polypeptide), optionally, wherein the method includes generating 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) ligation signals for the cyclic micron particles (i.e., (different) ligation signals for each target molecule of the cyclic micron particles).

[0059] The target molecules of the cyclic micron particles can comprise at least 2, at least 3, at least 4, at least 9, at least 49, at least 99, at least 499, at least 999, at least 4999, at least 9999, at least 99,999 or at least 999,999 (different) target polypeptides and at least one fragment of target nucleic acids (such as genomic DNA). Optionally, wherein the method comprises generating at least 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) ligation signals for the cyclic micron particles (i.e., (different) ligation signals for each target molecule of the cyclic micron particles).

[0060] The sample can comprise first and second cyclic micron particles, wherein each cyclic micron particle comprises target molecules (such as at least 2 or at least 3 target molecules), and wherein the method comprises performing a measurement step (as described herein) to generate a set of ligation signals for the first cyclic micron particle, and performing a measurement step as described herein to generate a set of ligation signals for the second cyclic micron particle.

[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 (such as genomic DNA) can comprise: (a) contacting the sample with a library comprising at least two polybarcode reagents, wherein each polybarcode reagent comprises a first and a second barcode region linked together, wherein each barcode region comprises a nucleic acid sequence, and wherein the first and second barcode regions of the first polybarcode reagent in the library are different from the first and second barcode regions of the second polybarcode reagent; and (b) attaching barcode sequences to each of the first and second fragments of the target nucleic acid of the first micron particle to generate first and second barcoded target nucleic acid molecules for the first micron particle, wherein the first barcoded target nucleic acid molecule comprises the nucleic acid sequence of the first barcode region of the first polybarcode reagent, and the second barcoded target nucleic acid molecule comprises the nucleic acid sequence of the second barcode region of the first polybarcode reagent, and attaching barcode sequences to each of the first and second fragments of the target nucleic acid of the second micron particle to generate first and second barcoded target nucleic acid molecules for the second micron particle, wherein the first barcoded target nucleic acid molecule comprises the nucleic acid sequence of the first barcode region of the second polybarcode reagent, and the second barcoded target nucleic acid molecule comprises the nucleic acid sequence of the second barcode region of the second polybarcode reagent.

[0062] 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) can include: (a) contacting a sample with a library comprising at least two polymeric barcoding reagents, wherein each polymeric barcoding reagent comprises a first and a second barcoded oligonucleotide linked together, wherein the barcoded oligonucleotides each comprise a barcode region, and wherein the barcode regions of the first and second barcoded oligonucleotides of the first polymeric barcoding reagent of the library are different from the barcode regions of the first and second barcoded oligonucleotides of the second polymeric barcoding reagent of the library; and (b) attaching (e.g., bonding or ligating) the first and second barcoded oligonucleotides of the first polymeric barcoding reagent to the first and second fragments of the target nucleic acid of a first micron particle to produce first and second barcoded target nucleic acid molecules, and attaching (e.g., bonding or ligating) the first and second barcoded oligonucleotides of the second polymeric barcoding reagent to the first and second fragments of the target nucleic acid of a second micron particle to produce first and second barcoded target nucleic acid molecules.

[0063] The sample can comprise n cyclic micron particles, wherein each cyclic micron particle comprises a target molecule (e.g., at least 2 or at least 3 target molecules), and wherein the method comprises performing the measurement step (as described herein) to generate a set of ligation signals for each cyclic micron particle, wherein 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 cyclic micron particles.

[0064] The method can further comprise the step of determining the identity of the origin cell and / or origin tissue of the target biomolecule from which the set of ligation signals is obtained. The step of determining the identity of the origin cell and / or origin tissue can include identifying one or more signature signals in the set of ligation signals. A signature signal can be a signal corresponding to the presence, absence, and / or level of a signature target biomolecule, wherein the signature target biomolecule is a target biomolecule that is characteristic of a particular cell and / or tissue.

[0065] Signature signals can be combinatorial signature signals corresponding to the presence, absence, and / or levels of any two or more signature target biomolecules, where the signature target biomolecules are target biomolecules characteristic of a particular cell and / or tissue (e.g., where the target biomolecules together are characteristic of a particular cell and / or tissue). For example, a combinatorial signature signal can correspond to the presence, absence, and / or levels of any two or more biomolecules from Biomolecule Group 1; optionally, a combinatorial signature signal can correspond to the presence, absence, and / or levels of any two or more biomolecules from Biomolecule Group 1, 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). Signature signals can be combinatorial signature signals corresponding to 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 a list or group thereof, such as a list or group of reference sequences, and / or a list or group corresponding to 5-methylcytosine and / or 5-hydroxymethylcytosine) in terms of their presence, absence, and / or levels.

[0066] The origin cells can be from a particular subject (e.g., fetal cells, maternal cells, or paternal cells). The origin cells can 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, breast cells, colorectal cells, brain cells, uterine cells, heart cells, vascular cells (e.g., artery cells or vein cells), and / or any other type of cell.

[0067] The origin cells can be cancer cells or malignant cells. The origin cells can be lung cancer cells, breast cancer cells, ovarian cancer cells, prostate cancer cells, kidney cancer cells, liver cancer cells, blood cancer cells, leukemia cells, lymphoma cells, colorectal cancer cells, pancreatic cancer cells, brain cancer cells, uterine cancer cells, cholangiocarcinoma 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 cells.

[0068] The origin tissue can be from a particular subject (e.g., fetal tissue, maternal tissue, or paternal tissue). The origin tissue can be lung tissue, liver tissue, ovarian tissue, heart 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.

[0069] The originating tissue can be cancerous or malignant tissue. The originating tissue can 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,

[0070] cancerous esophageal tissue and / or any other cancerous tissue.

[0071] The signature signal can include a signal corresponding to the presence, absence, and / or level of a first signature biomolecule and a signal corresponding to the presence, absence, and / or level of a second signature biomolecule. The first and second signature biomolecules can take any form of the target biomolecules described herein. For example, the signature signal can include a signal corresponding to the presence, absence, and / or level of any one or more of the biomolecules listed in Biomolecule Group 1.

[0072] The signature biomolecule can be a polypeptide that is expressed only in a specific cell type or tissue type (e.g., cancer cells or fetal cells). The signature biomolecule can be a polypeptide that is preferentially expressed in a specific cell type or tissue type (e.g., cancer cells or fetal cells). The signature biomolecule can be a nucleic acid (e.g., an mRNA molecule or a microRNA molecule) that is expressed (or preferentially expressed) only in a specific cell type or tissue type (e.g., cancer cells or fetal cells, or intravascular tissue such as intravascular plaque). For example, the signature biomolecule can include any one or more of the biomolecules listed in Biomolecule Group 1.

[0073] The signature biomolecule can be an epigenetic modification, such as a genomic DNA fragment containing 5-hydroxymethylcytosine. The genomic DNA fragment containing 5-hydroxymethylcytosine can provide a signature signal for cancerous and / or malignant cells or tissues.

[0074] The signature biomolecule can be a polypeptide or an RNA encoding the polypeptide, such as TTF-1 (also known as NK2 homeobox 1) or TTF-1 RNA. TTF-1 (or TTF-1 RNA) can provide a signature signal for lung cells and / or tissues.

[0075] A signature signal for lung cancer can be provided by measuring a signal corresponding to the presence, absence, and / or level of a genomic DNA fragment containing 5-hydroxymethylcytosine (the first signature biomolecule) and a signal corresponding to the presence, absence, and / or level of TTF-1 or TTF-1 RNA (as the second signature biomolecule).

[0076] The present invention provides a method for analyzing a sample comprising circulating micron particles or a sample derived from circulating micron particles, and wherein the method comprises: (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, wherein the barcoded oligonucleotide comprises at least one nucleotide (i.e., wherein the barcoded oligonucleotide comprises a nucleotide sequence having a length of at least one nucleotide), and wherein the affinity moiety is capable of binding to a target biomolecule; (b) forming a reaction mixture, wherein the step of forming the reaction mixture comprises binding the affinity moiety to the target molecule, if 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 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 comprising circulating micron particles or a sample derived from circulating micron particles, wherein the circulating micron particles comprise a target biomolecule, and wherein the method comprises: (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, wherein the barcoded oligonucleotide comprises at least one nucleotide (i.e., wherein the barcoded oligonucleotide comprises a nucleotide sequence having a length of at least one nucleotide), and wherein the affinity moiety is capable of binding to a target biomolecule; (b) forming a reaction mixture, wherein the step of forming the reaction mixture comprises binding the affinity moiety to the target molecule, if present, to form a barcoded biomolecular complex comprising 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.

[0078] The present invention provides a method for analyzing a sample comprising circulating micron particles or a sample derived from circulating micron particles, and wherein the method comprises: (a) contacting the sample with at least one affinity moiety, and wherein the affinity moiety is capable of binding to a target biomolecule; (b) forming a reaction mixture, wherein the step of forming the reaction mixture comprises (i) binding the affinity moiety to the target biomolecule, if present, and (ii) contacting the sample with a barcoded oligonucleotide and ligating the barcoded oligonucleotide to the affinity moiety to form a barcoded biomolecular complex comprising the barcoded affinity probe and the target biomolecule, wherein the barcoded affinity probe comprises at least one affinity moiety linked to the barcoded oligonucleotide, and wherein the barcoded oligonucleotide comprises at least one nucleotide (i.e., wherein the barcoded oligonucleotide comprises a nucleotide sequence of at least one nucleotide in 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.

[0079] The present invention provides a method for analyzing a sample comprising circulating micron particles or a sample derived from circulating micron particles, wherein the circulating micron particles comprise a target biomolecule, and wherein the method comprises: (a) contacting the sample with at least one affinity moiety, and wherein the affinity moiety is capable of binding to the target biomolecule; (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 and ligating the barcoded oligonucleotide to the affinity moiety to form a barcoded biomolecular complex comprising the barcoded affinity probe and the target biomolecule, wherein the barcoded affinity probe comprises at least one affinity moiety linked to the barcoded oligonucleotide, and wherein the barcoded oligonucleotide comprises at least one nucleotide (i.e., wherein the barcoded oligonucleotide comprises a nucleotide sequence of at least one nucleotide in 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.

[0080] The step of forming the reaction mixture may comprise incubating the reagents under conditions suitable for binding of the affinity moiety to the target biomolecule.

[0081] Prior to the step of measuring the presence, absence, and / or level of the barcoded oligonucleotide in the sample, the method may comprise removing or depleting the barcoded affinity probe and / or the barcoded oligonucleotide that is not part of the barcoded biomolecular complex.

[0082] Measuring the level of the barcoded oligonucleotide in the reaction mixture may comprise quantifying the level of the barcoded oligonucleotide in the reaction mixture.

[0083] The oligonucleotides of the barcode can be directly or indirectly (e.g., via one or more linker molecules) linked to an affinity moiety. The barcode-bearing oligonucleotides can be linked to an affinity moiety via a linker molecule, where the linker molecule is attached and / or linked and / or bound (covalently or non-covalently) to at least one affinity moiety and at least one barcode-bearing oligonucleotide. The barcode-bearing oligonucleotides can be linked to any affinity moiety via one or more covalent bonds (or linkages) (e.g., via covalent bonds generated by the antibody labeling kit from Innova Biosciences, e.g., linkages), one or more non-covalent bonds (or linkages) (e.g., protein-protein interactions or streptavidin-biotin bonds, e.g., the affinity moiety can comprise a streptavidin domain and the barcode-bearing oligonucleotide can comprise a biotin moiety) or nucleic acid hybridization bonds. Any one or more linker molecules can be a biopolymer (e.g., a nucleic acid molecule) or a synthetic polymer. Any one or more linker molecules can comprise one or more ethylene glycols and / or poly(ethylene glycol) (e.g., hexaethylene glycol or pentaethylene glycol) units. Any one or more linker molecules can comprise one or more ethyl groups, e.g., one or more C3 (three-carbon) spacers, C6 spacers, C12 spacers or C18 spacers.

[0084] The 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 barcode-bearing affinity probes.

[0085] The barcode-bearing affinity probe can comprise an aptamer, optionally, where the barcode-bearing affinity probe is an aptamer. The aptamer can provide the affinity moiety of the barcode-bearing affinity probe and the barcode-bearing oligonucleotide.

[0086] The aptamer can comprise at least one affinity moiety linked to the barcode-bearing oligonucleotide, where the barcode-bearing oligonucleotide comprises at least one nucleotide, and where the affinity moiety is capable of binding to a target biomolecule. The aptamer can comprise a barcode sequence. Any or all of the nucleic acid sequences of the aptamer can associate with the affinity moiety of the aptamer, and / or be used to identify the affinity moiety of the aptamer, and / or identify the target biomolecule to which the affinity moiety of the aptamer is capable of binding.

[0087] The affinity moiety may be capable of binding to a target biomolecule. The affinity moiety may be capable of specifically binding to a target biomolecule. The affinity moiety can bind to a target biomolecule. The affinity moiety can specifically bind to a target biomolecule. The affinity moiety can have a high affinity for the target biomolecule.

[0088] The affinity moiety can comprise one or more of the following: an antibody, an antibody fragment, a light chain antibody fragment, a single-chain variable fragment (scFv), a peptide, a cell-penetrating peptide, an aptamer, a DNA aptamer and / or an RNA aptamer.

[0089] The affinity moiety may comprise an antibody or a fragment thereof, and the target molecule may be a polypeptide.

[0090] The affinity moiety may comprise an antibody or a fragment thereof, and the target molecule may be a nucleic acid fragment.

[0091] The affinity moiety may comprise an antibody or a fragment thereof, and the target molecule may be a nucleic acid fragment comprising an epigenetic modification such as 5-methylcytosine or 5-hydroxymethylcytosine.

[0092] The affinity moiety may comprise an aptamer, and the target molecule may be a polypeptide.

[0093] The affinity moiety may comprise an aptamer, and the target molecule may be a nucleic acid fragment.

[0094] The affinity moiety may comprise an aptamer, and the target molecule may be a nucleic acid fragment comprising an epigenetic modification such as 5-methylcytosine or 5-hydroxymethylcytosine.

[0095] The barcoded affinity probe may comprise an aptamer, wherein the aptamer is within an aptamer sequence within an affinity oligonucleotide. The barcoded affinity probe may comprise an aptamer, wherein the aptamer is within an aptamer sequence within an affinity oligonucleotide, wherein the affinity oligonucleotide comprises a barcode sequence. The barcoded affinity probe may comprise an aptamer, wherein the aptamer is within an aptamer sequence within an affinity oligonucleotide, wherein the affinity oligonucleotide comprises a barcode sequence, wherein all or part of the barcode sequence is partially or completely constituted by the aptamer sequence. The aptamer and / or the aptamer sequence and / or the affinity oligonucleotide and / or the barcode sequence may comprise one or more DNA nucleotides. Optionally, any of the aptamer and / or the aptamer sequence and / or the affinity oligonucleotide and / or the barcode sequence may comprise one or more RNA nucleotides.

[0096] The barcoded affinity probe may comprise at least two affinity moieties. The barcoded affinity probe may comprise at least first and second affinity moieties, wherein the first affinity moiety is capable of binding to a first target biomolecule, and wherein the second affinity moiety is capable of binding to a second target biomolecule, wherein the first and second probe target biomolecules are different.

[0097] The barcoded affinity probe may comprise at least 3, at least 4, at least 5 or at least 10 different affinity moieties. Optionally, each affinity moiety is capable of binding to a different target biomolecule.

[0098] The barcoded affinity probe may comprise at least two directly or indirectly linked affinity moieties. At least two affinity moieties of the barcoded affinity probe may be linked to a carrier (such as a solid support), a molecular carrier or a macromolecular carrier.

[0099] A barcoded affinity probe can comprise at least two affinity moieties. Each affinity moiety can comprise an aptamer. At least two affinity moieties of the barcoded affinity probe can be comprised within a single aptamer. At least two affinity moieties of the barcoded affinity probe can be comprised within a single continuous nucleic acid sequence (e.g., a DNA sequence and / or an RNA sequence).

[0100] A barcoded affinity probe can comprise at least two different barcoded oligonucleotides.

[0101] A barcoded oligonucleotide comprises at least one nucleotide. A barcoded oligonucleotide can comprise a barcode sequence. The barcoded oligonucleotide comprises a barcode sequence of at least 2, at least 3, at least 5, at least 10, at least 20, or at least 30 nucleotides.

[0102] A barcoded oligonucleotide can comprise a barcode sequence that associates with and / or recognizes the affinity moiety to which it is linked. Each barcoded oligonucleotide linked to the same affinity moiety (e.g., the same antibody specific for the same protein target) can comprise the same sequence (e.g., the same barcode sequence). Each barcoded oligonucleotide linked to the same affinity moiety comprises a different sequence (e.g., two or more different barcode sequences). Optionally, each barcoded oligonucleotide linked to a different affinity moiety can comprise a different sequence (e.g., two or more different barcode sequences).

[0103] A barcoded oligonucleotide can comprise a linker and / or a coupling sequence, where the length of 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. The linker and / or coupling sequence of the barcoded oligonucleotide can comprise a sequence complementary to a target region of any polybarcoded reagent and / or of the barcoded oligonucleotides comprised in its library. The linker and / or coupling sequence of the barcoded oligonucleotide can comprise a poly(A) sequence of 2 or more nucleotides. The linker and / or coupling sequence within the barcoded oligonucleotide can be comprised within the 3' end and / or the 5' end of the barcoded oligonucleotide.

[0104] A barcoded affinity probe can comprise one or more secondary barcoded oligonucleotides, wherein the secondary barcoded oligonucleotides comprise sequences that are at least partially complementary to all or part of one or more (non-secondary) barcoded oligonucleotides. The secondary barcoded oligonucleotides can bind (i.e., hybridize) fully or partially to any one or more (non-secondary) barcoded oligonucleotides. The secondary barcoded oligonucleotides can bind (i.e., hybridize) fully or partially to any one or more (non-secondary) barcoded oligonucleotides in a secondary barcoded oligonucleotide binding reaction. The secondary barcoded oligonucleotide binding reaction can be performed before, and / or after, and / or during any one of steps (a), (b), or (c). The secondary barcoded oligonucleotides can comprise one or more nucleotides of a barcode sequence, wherein the barcode sequence associates with and / or identifies the affinity moiety to which it is attached within the barcoded affinity probe.

[0105] A barcoded affinity probe can comprise one or more affinity moieties, and one or more primary barcoded oligonucleotides, and one or more secondary barcoded oligonucleotides.

[0106] The sample can comprise one or more circulating microparticles and / or the sample can be derived from one or more circulating microparticles.

[0107] The biomolecule can be a polypeptide (e.g., a protein), a carbohydrate, a lipid, or a nucleic acid. The biomolecule can be a metabolite.

[0108] The sample can comprise a first circulating microparticle and a second circulating microparticle, or wherein the sample is derived from a first circulating microparticle and a second circulating microparticle, wherein step (b) comprises forming at least one barcoded biomolecular complex of a barcoded affinity probe and a target biomolecule that comprises the first circulating microparticle, and forming at least one barcoded biomolecular complex of a barcoded affinity probe and a target biomolecule that comprises the second circulating microparticle. The sample can further comprise fragments of the target nucleic acid of the first circulating microparticle and fragments of the target nucleic acid of the second circulating microparticle.

[0109] In step (a), (b), and / or (c), the barcoded affinity probe can be at any concentration, such as a concentration of at least 100 nanomolar, at least 10 nanomolar, at least 1 nanomolar, at least 100 picomolar, at least 10 picomolar, at least 1 picomolar, at least 100 femtomolar, at least 10 femtomolar, or at least 1 femtomolar. The concentration can be from 1 picomolar to 100 nanomolar, from 10 picomolar to 10 nanomolar, or from 100 picomolar to 1 nanomolar.

[0110] Optionally, in any one or more steps of any method (e.g., any step of attaching a coupling sequence and / or a coupling molecule, any step of attaching a barcode sequence, e.g., any step of attaching and / or ligating and / or conjugating a barcoded oligonucleotide (e.g., any step of attaching / ligating / conjugating the barcode sequence contained in the barcoded oligonucleotide)), the step and / or method can be performed in a high-viscosity solution. Optionally, such a high-viscosity solution can consist of a poly(ethylene glycol) (PEG) solution, such as one or more of the following: PEG 400, PEG 1000, PEG 2000, PEG 4000, PEG 5000, PEG 8000, PEG 10000, and / or PEG 20,000. Optionally, such a solution can contain 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) by weight or volume; optionally, such a solution can contain any two or more PEG molecules, where each of such two or more PEG molecules is present at one of these stated concentrations by weight or volume. Optionally, such a high-viscosity solution can contain the solution employed in any step of adhering a barcoded oligonucleotide to a target nucleic acid. Optionally, the dynamic viscosity of such a high-viscosity solution can be at least 1.0 centipoise, at least 1.1 centipoise, at least 1.2 centipoise, at least 1.5 centipoise, at least 2.0 centipoise, at least 5.0 centipoise, at least 10.0 centipoise, at least 20.0 centipoise, at least 50.0 centipoise, at least 100.0 centipoise, or at least 200.0 centipoise (e.g., at 25 degrees Celsius at standard sea-level pressure). Preferably, such a high-viscosity solution will have a dynamic viscosity of at least 1.5 centipoise. The use of a high-viscosity solution can slow the diffusion of reagents (e.g., barcoded oligonucleotides and / or polybarcode reagents) to prevent or delay diffusion away from their target molecules, e.g., target nucleic acids.

[0111] Optionally, in any one or more steps of any method (e.g., any step of attaching a coupling sequence and / or a coupling molecule, any step of attaching a barcode sequence, e.g., any step of attaching and / or ligating and / or conjugating a barcoded oligonucleotide (e.g., any step of attaching / ligating / conjugating the barcode sequence contained in the barcoded oligonucleotide)), the step and / or method can be performed in a solution containing one or more molecular crowding reagents, i.e., where the molecular crowding reagent has the effect of increasing the effective concentration of the target molecule and / or the barcoded oligonucleotide and / or the polybarcode reagent and / or other components in the step. Optionally, any one or more molecular crowding reagents can include beads and / or other solid carriers of any size, such as micron-sized beads (e.g., 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 microns), and / or nano-sized beads (e.g., 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).

[0112] One or more steps of removing and / or depleting 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 any one or more cyclic microparticles.

[0113] Optionally, any method of measuring a biomolecule from cyclic microparticles can include measurement with a single barcoded affinity probe. Optionally, any method of measuring a biomolecule from cyclic microparticles can include measurement with a single barcoded affinity probe, where the single barcoded affinity probe includes an oligonucleotide having a length of at least a single nucleotide.

[0114] Optionally, any nucleotide and / or oligonucleotide sequence having a length of at least a single nucleotide can be considered a barcode and / or barcode sequence (and / or barcoded oligonucleotide) within the barcoded affinity probe. The nucleotide and / or oligonucleotide sequence having a length of at least a single nucleotide need not be different from any other nucleotide and / or oligonucleotide sequence within the barcoded affinity probe and / or from any other nucleotide and / or oligonucleotide sequence within any other barcoded affinity probe.

[0115] Step (c) of the method can include measuring the presence, absence, and / or level of the barcoded oligonucleotide by analyzing the nucleotide sequence of the barcoded oligonucleotide, optionally where the sequence is analyzed by sequencing (where at least a portion of the barcoded oligonucleotide is sequenced) or PCR (where at least a portion of the barcoded oligonucleotide is amplified).

[0116] Step (c) may include measuring the presence, absence, and / or level of the barcoded oligonucleotide by primer extension and / or PCR reaction and / or quantitative or semi-quantitative PCR reaction (such as real-time PCR reaction).

[0117] Step (c) may include measuring the presence, absence, and / or level of the barcoded oligonucleotide by primer extension and / or PCR reaction and / or quantitative or semi-quantitative PCR reaction (such as real-time PCR reaction), wherein at least one primer in the reaction is specific to at least a portion of the barcoded oligonucleotide and / or is at least partially complementary (and / or at least partially identical) thereto.

[0118] In the method, step (b) or step (c) may include linking together at least two barcoded biomolecular complexes of the first cycle of micron particles, and linking together at least two barcoded biomolecular complexes of the second cycle of micron particles.

[0119] A sample containing one or more cycles of micron particles can be chemically crosslinked (e.g., with formaldehyde). The cycle of micron particles can be chemically crosslinked before step (a), (b), and / or (c).

[0120] A sample containing one or more cycles of micron particles can be permeabilized (e.g., with a chemical surfactant). The cycle of micron particles can be permeabilized before step (a) and / or (b).

[0121] Before step (a) and / or (b), a sample containing one or more cycles of micron particles can be chemically crosslinked (e.g., with formaldehyde) and then permeabilized (e.g., with a chemical surfactant).

[0122] The method may (optionally as part of step (c)) include: (i) contacting the reaction mixture with a polybarcode reagent, wherein the polybarcode reagent includes a first and a second barcode region linked together, and each barcode region includes a nucleic acid sequence; (ii) attaching the barcode sequences of the barcode regions of the polybarcode reagent to the barcoded oligonucleotide of at least one barcoded biomolecular complex of the cycle of micron particles; 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 polybarcode reagent.

[0123] The reaction mixture may further comprise fragments of the target nucleic acid of the circulating micron particles, and wherein the method: (i) contacting the reaction mixture with a polymeric barcoding reagent, wherein the polymeric barcoding reagent comprises first and second barcoding regions linked together, wherein each barcoding region comprises a nucleic acid sequence; (ii) attaching the barcoding sequence of the first barcoding region of the polymeric barcoding reagent to the barcoded oligonucleotide of at least one barcoded biomolecular complex of the circulating micron particles (i.e., the first fragment of the target nucleic acid) to produce a first barcoded target nucleic acid molecule, and attaching the barcoding sequence of the second barcoding region of the polymeric barcoding reagent to the fragment of the target nucleic acid (i.e., the second fragment of the target nucleic acid) to produce a second barcoded target nucleic acid molecule; and (iii) analyzing the sequence of each of the first and second barcoded target nucleic acid molecules.

[0124] The step of analyzing the sequence of each of the first and second barcoded target nucleic acid molecules may be performed by sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules.

[0125] The method may further comprise sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules of the first circulating micron particles. The method may comprise generating sequence reads for the first barcoded target nucleic acid molecule, wherein the sequence reads comprise at least a portion of the sequence of the first barcoding region of the polymeric barcoding reagent and at least a portion of the sequence of the first fragment of the target nucleic acid of the circulating micron particles. The method may comprise generating sequence reads for the second barcoded target nucleic acid molecule, wherein the sequence reads comprise at least a portion of the sequence of the second barcoding region of the polymeric barcoding reagent and at least a portion of the sequence of the second fragment of the target nucleic acid of the circulating micron particles.

[0126] The method may (optionally as part of step (c)) comprise dividing the reaction mixture into at least first and second partitions, and analyzing the nucleotide sequence of the barcoded oligonucleotide of the barcoded biomolecular complex in each of the first and second partitions.

[0127] The method may comprise dividing 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, at least 100,000,000 or at least 1,000,000,000 partitions. Preferably, the method comprises dividing the reaction mixture into at least 1000 partitions.

[0128] The target nucleic acid molecule may comprise a barcoded oligonucleotide of a barcoded biomolecular complex of circulating micron particles. The barcoded oligonucleotide of the barcoded biomolecular complex of circulating micron particles may be present in or derived from the barcoded biomolecular complex.

[0129] Two or more target nucleic acid molecules may comprise a fragment of the target nucleic acid of the micron particle and a barcoded oligonucleotide of a barcoded biomolecular complex of circulating micron particles.

[0130] Two or more target nucleic acid molecules may comprise a fragment of the target nucleic acid (e.g., genomic DNA) of the micron particle and a barcoded oligonucleotide of a barcoded biomolecular complex of circulating micron particles.

[0131] Two or more target nucleic acid molecules may comprise a fragment of the target nucleic acid (e.g., RNA) of the micron particle and a barcoded oligonucleotide of a barcoded biomolecular complex of circulating micron particles.

[0132] The step of analyzing the nucleotide sequence of the barcoded oligonucleotide of the barcoded biomolecular complex may comprise attaching a first partition barcode sequence to at least one barcoded oligonucleotide (a first fragment of the target nucleic acid of the first partition), the oligonucleotide being divided into the first partition (to generate a first barcoded target nucleic acid molecule of the first partition), wherein at least one barcoded oligonucleotide divided into the first partition is included in or derived from the barcoded biomolecular complex, and attaching a second partition barcode sequence to at least one barcoded oligonucleotide (a first fragment of the target nucleic acid of the second partition), the oligonucleotide being divided into the second partition (to generate a first barcoded target nucleic acid molecule of the second partition), wherein at least one barcoded oligonucleotide divided into the first partition is included in or derived from the barcoded biomolecular complex. Preferably, each of the first and second partitions comprises a barcoded oligonucleotide that is included in or derived from the barcoded biomolecular complex.

[0133] The first and second partition barcode sequences may be different. The first partition barcode sequence may be included in a first set of partition barcode sequences, and the second partition barcode sequence may be included in a second set of partition barcode sequences, wherein 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 a first multipolymer barcode reagent, and the second partition barcode sequence may be the nucleic acid sequence of a second multipolymer barcode reagent, wherein each of the first and second multipolymer barcode reagents comprises two or more barcode regions linked together;

[0134] The step of analyzing the nucleotide sequence of the barcoded oligonucleotide of the biomolecular complex with a barcode may further comprise analyzing the attached partition barcode sequences from each of the first and second partitions.

[0135] Fragments of the target nucleic acid (e.g., gDNA or RNA) from the cyclic microparticles (the second fragment of the target nucleic acid of the first partition) may also be attached to the first partition barcode sequence of the first partition (to generate the second barcoded target nucleic acid molecule of the first partition), and / or fragments of the target nucleic acid (e.g., gDNA or RNA) from different cyclic microparticles (the second fragment of the target nucleic acid of the second partition) may also be attached to the second partition barcode sequence of the second partition (to generate the second barcoded target nucleic acid molecule of the second partition).

[0136] The step of analyzing the sequences of each of the first and second barcoded target nucleic acid molecules may be performed by sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules.

[0137] The method may further comprise analyzing the sequences of each of the first and second barcoded target nucleic acid molecules of the first partition, and analyzing the sequences of each of the first and second barcoded target nucleic acid molecules of the second partition. Optionally, the step of analyzing the sequences is performed by sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules.

[0138] The method may further comprise sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules of the first partition. The method may comprise generating sequence reads for the first barcoded target nucleic acid molecule, wherein the sequence reads comprise at least a portion of the sequence of the first partition barcode and at least a portion of the sequence of the first fragment of the target nucleic acid of the first partition. The method may comprise generating sequence reads for the second barcoded target nucleic acid molecule, wherein the sequence reads comprise at least a portion of the sequence of the first partition barcode and at least a portion of the sequence of the second fragment of the target nucleic acid of the first partition.

[0139] The method may further comprise sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules of the second partition. The method may comprise generating sequence reads for the first barcoded target nucleic acid molecule, wherein the sequence reads comprise at least a portion of the sequence of the second partition barcode and at least a portion of the sequence of the first fragment of the target nucleic acid of the second partition. The method may comprise generating sequence reads for the second barcoded target nucleic acid molecule, wherein the sequence reads comprise at least a portion of the sequence of the second partition barcode and at least a portion of the sequence of the second fragment of the target nucleic acid of the second partition.

[0140] Sequence reads can 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 from a target nucleic acid (e.g., genomic DNA). Preferably, each sequence read contains at least 5 nucleotides from the target nucleic acid. "At least a portion of a sequence" herein 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, "at least a portion of a sequence" herein means at least 2 nucleotides of the relevant sequence.

[0141] The method can include the step of amplifying the signal from one or more barcoded affinity probes (i.e., the signal amplification step or process). The signal amplification process can include one or more strand displacement amplification reactions and / or one or more multiple displacement amplification reactions. The signal amplification process can include an in vitro transcription reaction. The signal amplification process can include the steps of attaching and / or binding and / or ligating (i.e., hybridizing) one or more secondary barcoded oligonucleotides to the barcoded affinity probe, e.g., the (non-secondary) barcoded oligonucleotide within the barcoded affinity probe. The signal amplification process can include the step of attaching and / or binding one or more secondary affinity moieties to the barcoded affinity probe (e.g., binding a secondary antibody to the (non-secondary) antibody within the barcoded affinity probe). Optionally, any number of at least 2, at least 3, at least 5, or at least 10 secondary barcoded oligonucleotides and / or secondary affinity moieties can be attached and / or bound and / or ligated to any barcoded affinity probe. The method of attaching and / or ligating and / or binding two or more secondary barcoded oligonucleotides and / or secondary affinity moieties to a barcoded affinity probe can be performed in separate sequential steps of attaching and / or ligating and / or binding them individually, or can be performed in a single parallel step.

[0142] The barcoded oligonucleotide and / or the secondary barcoded oligonucleotide can contain a template for an in vitro transcription reaction. The barcoded oligonucleotide and / or the secondary barcoded oligonucleotide can contain a promoter region for an in vitro transcription reaction, e.g., a promoter for T7 RNA polymerase.

[0143] Barcoded oligonucleotides and / or secondarily barcoded oligonucleotides can comprise circularized (e.g., circular) oligonucleotides (e.g., circular DNA oligonucleotides or circular RNA oligonucleotides). The circular barcoded oligonucleotides can comprise one or more complementary primer oligonucleotides of at least one nucleotide in length, wherein the complementary primer oligonucleotides are ligated to one (or more) sequences within the circular barcoded oligonucleotide. The circular barcoded oligonucleotides can be used as templates for one or more strand displacement amplification reactions and / or one or more multiple displacement amplification reactions, such as reactions using a strand displacement polymerase, such as phi29 DNA polymerase (optionally, wherein one or more complementary primer oligonucleotides are used as primers for such amplification reactions). The strand displacement amplification reaction and / or multiple displacement amplification reaction can be performed before and / or after and / or during any step of binding any one or more barcoded affinity probes to any target biomolecule in a sample. The product of any one or more of the strand displacement amplification reactions and / or one or more of the multiple displacement amplification reactions can comprise target nucleic acid molecules for any method described herein. The product of any one or more of the strand displacement amplification reactions and / or one or more of the multiple displacement amplification reactions can be attached to any barcode sequence (e.g., any partition barcode sequence, any barcoded oligonucleotide, any barcode sequence contained in any polybarcode reagent, and / or barcoded oligonucleotide).

[0144] The method can (optionally as part of step (c)) comprise: (i) contacting a reaction mixture with a library comprising at least two polybarcode reagents, wherein each polybarcode reagent comprises first and second barcode regions linked together, wherein each barcode region comprises a nucleic acid sequence, and wherein the first and second barcode regions of the first polybarcode reagent of the library are different from the first and second barcode regions of the second polybarcode reagent; and (ii) attaching barcode sequences to each of a first fragment of a target nucleic acid and a second fragment of the target nucleic acid of a first micron particle to generate first and second barcoded target nucleic acid molecules for the first micron particle, wherein the first barcoded target nucleic acid molecule comprises the nucleic acid sequence of the first barcode region of the first polybarcode reagent and the second barcoded target nucleic acid molecule comprises the nucleic acid sequence of the second barcode region of the first polybarcode reagent, and attaching barcode sequences to each of a first fragment of a target nucleic acid and a second fragment of the target nucleic acid of a second micron particle to generate first and second barcoded target nucleic acid molecules for the second micron particle, wherein the first barcoded target nucleic acid molecule comprises the nucleic acid sequence of the first barcode region of the second polybarcode reagent and the second barcoded target nucleic acid molecule comprises the nucleic acid sequence of the second barcode region of the second polybarcode reagent.

[0145] The first fragment of the target nucleic acid of the first micron particle can be a barcoded oligonucleotide of at least one barcoded biomolecular complex of the first cycle micron particle, and wherein the first fragment of the target nucleic acid of the second micron particle can be a barcoded oligonucleotide of at least one barcoded biomolecular complex of the second cycle micron particle.

[0146] The reaction mixture can further comprise a fragment of the target nucleic acid of the first cycle micron particle, and wherein the second fragment of the target nucleic acid of the first cycle micron particle is a fragment of the target nucleic acid of the first cycle micron particle.

[0147] The reaction mixture can further comprise a fragment of the target nucleic acid of the second cycle micron particle, and wherein the second fragment of the target nucleic acid of the second cycle micron particle is a fragment of the target nucleic acid of the second cycle micron particle.

[0148] The step of contacting the reaction mixture with a library of polybarcode reagents can be performed in a single continuous aqueous volume. Step (c) can be performed in a single continuous aqueous volume, optionally, wherein steps (b) and (c) are performed in a single continuous aqueous volume, optionally, wherein steps (a), (b), and (c) are performed in a single continuous aqueous volume.

[0149] The method can further comprise analyzing the sequence of each of the first and second barcoded target nucleic acid molecules of the first cycle micron particle, and analyzing the sequence of each of the first and second barcoded target nucleic acid molecules of the second cycle micron particle. Optionally, the step of analyzing the sequence is performed by sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules.

[0150] The method can further comprise sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules of the first cycle micron particle. The method can comprise generating sequence reads for the first barcoded target nucleic acid molecule, wherein the sequence reads comprise at least a portion of the sequence of the first barcode region of the first polybarcode reagent and at least a portion of the sequence of the first fragment of the target nucleic acid of the first cycle micron particle. The method can comprise generating sequence reads for the second barcoded target nucleic acid molecule, wherein the sequence reads comprise at least a portion of the sequence of the second barcode region of the first polybarcode reagent and at least a portion of the sequence of the second fragment of the target nucleic acid of the first cycle micron particle.

[0151] 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 round of micron particles. The method may include generating sequence reads for the first barcoded target nucleic acid molecule, wherein the sequence reads include at least a portion of the sequence of the first barcode region of the second polybarcode reagent and at least a portion of the sequence of the first fragment of the target nucleic acid of the second round of micron particles. The method may include generating sequence reads for the second barcoded target nucleic acid molecule, wherein the sequence reads include at least a portion of the sequence of the second barcode region of the second polybarcode reagent and at least a portion of the sequence of the second fragment of the target nucleic acid of the second round of micron particles.

[0152] The sequence reads may include 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 from the target nucleic acid (e.g., genomic DNA). Preferably, each sequence read includes at least 5 nucleotides 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, "at least a portion of a sequence" as used herein means at least 2 nucleotides of the relevant sequence.

[0153] The method further includes partitioning the sample or reaction mixture into at least first and second partitions and analyzing the nucleotide sequences of the barcoded oligonucleotides in each of the first and second partitions, wherein the first partition contains at least one barcoded oligonucleotide contained in or derived from at least one barcoded biomolecular complex of the first round of micron particles, and wherein the second partition contains at least one barcoded oligonucleotide contained in or derived from at least one barcoded biomolecular complex of the second round of micron particles. The partitioning step may be performed before step (a), before step (b), and / or before step (c).

[0154] The 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, at least 100,000,000, or at least 1,000,000,000 partitions. Preferably, the method includes partitioning the sample into at least 1000 partitions.

[0155] The steps of analyzing the nucleotide sequence of a barcoded oligonucleotide of a barcoded biomolecular complex may include: (i) attaching a first partition barcode sequence to at least one barcoded oligonucleotide of a first partition; and (ii) attaching a second partition barcode sequence to at least one barcoded oligonucleotide of a second partition.

[0156] The first and second partition barcode sequences may be different.

[0157] The first partition barcode sequence may be from a first set of partition barcode sequences, and the second partition barcode sequence may be from a second set of partition barcode sequences, and wherein the first and second sets of partition barcode sequences are different.

[0158] The first partition barcode sequence may be the nucleic acid sequence of the barcode region of a first polymeric barcode reagent, and the second partition barcode sequence may be the nucleic acid sequence of the barcode region of a second polymeric barcode reagent, and wherein the first and second polymeric barcode reagents each comprise two or more barcode regions linked together.

[0159] The first partition may further comprise a fragment of the target nucleic acid of a first set of circulating microparticles, and wherein the second partition may further comprise a fragment of the target nucleic acid of a second set of circulating microparticles.

[0160] The steps of analyzing the nucleotide sequence of a barcoded oligonucleotide of a barcoded biomolecular complex may include: (i) attaching a first partition barcode sequence of a first set of partition barcode sequences to at least one barcoded oligonucleotide of a first partition and attaching a second partition barcode sequence of the first set of partition barcode sequences to at least one fragment of the target nucleic acid of a first set of circulating microparticles; (ii) attaching a first partition barcode sequence of a second set of partition barcode sequences to at least one barcoded oligonucleotide of a second partition and attaching a second partition barcode sequence of the second set of partition barcode sequences to at least one fragment of the target nucleic acid of a second set of circulating microparticles; and wherein the first and second partition barcode sequences are different.

[0161] The steps of analyzing the nucleotide sequence of a barcoded oligonucleotide of a barcoded biomolecular complex may include: (i) attaching a first partition barcode sequence of a first set of partition barcode sequences to at least one barcoded oligonucleotide of a first partition and attaching a second partition barcode sequence of the first set of partition barcode sequences to at least one fragment of the target nucleic acid of a first set of circulating microparticles; and (ii) attaching a first partition barcode sequence of a second set of partition barcode sequences to at least one barcoded oligonucleotide of a second partition and attaching a second partition barcode sequence of the second set of partition barcode sequences to at least one fragment of the target nucleic acid of a second set of circulating microparticles; and wherein the first and second sets of partition barcode sequences are different.

[0162] The first and second partition barcode sequences of the first set of partition barcode sequences can be nucleic acid sequences of the first and second barcode regions of a first multiplex barcode reagent, and wherein the first and second partition barcode sequences of the second set of partition barcode sequences can be nucleic acid sequences of the first and second barcode regions of a second multiplex barcode reagent, and wherein each of the first and second multiplex barcode reagents comprises two or more barcode regions linked together.

[0163] The first partition may further comprise a fragment of a target nucleic acid, and wherein the second partition may further comprise a fragment of a target nucleic acid, and wherein the step of analyzing the nucleotide sequence of a barcoded oligonucleotide of a barcoded biomolecular complex comprises: (i) attaching the first partition barcode sequence to at least one barcoded oligonucleotide of the first partition, and attaching the first partition barcode sequence to at least one fragment of the target nucleic acid of the first partition; (ii) attaching the second partition barcode sequence to at least one barcoded oligonucleotide of the second partition, and attaching the second partition barcode sequence to at least one fragment of the target nucleic acid of the second partition; wherein the first and second partition barcode sequences are different. Alternatively, the step of analyzing the nucleotide sequence of a barcoded oligonucleotide of a barcoded biomolecular complex comprises: (i) attaching the first partition barcode sequence of the first set of partition barcode sequences to at least one barcoded oligonucleotide of the first partition, and attaching the second partition barcode sequence of the first set of partition barcode sequences 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 set of partition barcode sequences to at least one barcoded oligonucleotide of the second partition, and attaching the second partition barcode sequence of the second set of partition barcode sequences to at least one fragment of the target nucleic acid of the second partition; wherein the first and second sets of partition barcode sequences are different.

[0164] The first and second partition barcode sequences of the first set of partition barcode sequences can be nucleic acid sequences of the first and second barcode regions of a first multiplex barcode reagent, and wherein the first and second partition barcode sequences of the second set of partition barcode sequences can be nucleic acid sequences of the first and second barcode regions of a second multiplex barcode reagent, and wherein each of the first and second multiplex barcode reagents comprises two or more barcode regions linked together.

[0165] The present invention provides the use of a barcoded affinity probe for determining the presence, absence, and / or level of a target biomolecule in a circulating micron particle or a sample derived therefrom, wherein the barcoded affinity probe comprises at least one affinity moiety linked to a barcoded oligonucleotide, wherein the barcoded oligonucleotide comprises at least one nucleotide, and wherein the affinity moiety is capable of binding to the target biomolecule.

[0166] 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, wherein the barcoded oligonucleotide comprises at least one nucleotide, and wherein the affinity moiety is capable of binding to the target biomolecule.

[0167] The barcoded affinity probe, the target biomolecule, the affinity moiety, and the barcoded oligonucleotide can take any form described herein. Specifically, they can take any form described herein that is relevant to the method.

[0168] 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 wherein the affinity moiety is capable of binding 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 wherein the affinity moiety is capable of binding to a second target biomolecule; and wherein the first target biomolecule and the second target biomolecule are different.

[0169] The library of barcoded affinity probes, the barcoded affinity probe, the target biomolecule, the affinity moiety, and the barcoded oligonucleotide can take any form described herein. Specifically, they can take any form described herein that is relevant to the method.

[0170] The first target biomolecule can be a polypeptide, and the second target biomolecule can be a fragment of a barcoded oligonucleotide or a target nucleic acid (e.g., genomic DNA).

[0171] The first target biomolecule can be a polypeptide, and the second target biomolecule can be a fragment of a target nucleic acid (e.g., genomic DNA) comprising an epigenetic modification (e.g., 5-hydroxymethylcytosine DNA or 5-methylcytosine DNA).

[0172] The first target biomolecule can be 5-hydroxymethylcytosine DNA, and the second target biomolecule can be a biomolecule selected from Biomolecule Group 1.

[0173] The first target biomolecule can be 5-methylcytosine DNA, and the second target biomolecule can be a biomolecule selected from Biomolecule Group 1.

[0174] The first and second target biomolecules can be selected from Biomolecule Group 1.

[0175] Optionally, any library of two or more barcoded affinity probes may contain a single mixed solution comprising the two or more barcoded affinity probes. Optionally, any library of two or more barcoded affinity probes may comprise two or more separate solutions, where each solution comprises one of the two or more barcoded affinity probes. Optionally, any library of two or more barcoded affinity probes may be provided in the form of a kit, where the kit consists of two or more separate solutions, where each solution comprises one of the two or more barcoded affinity probes.

[0176] The sample may 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 comprises at least 2 different barcoded affinity probes. Each barcoded affinity probe may comprise at least one affinity moiety linked to a barcoded oligonucleotide, where the barcoded oligonucleotide comprises at least one nucleotide, and where the affinity moiety is capable of binding to a target biomolecule. The affinity moieties of each different barcoded affinity probe in the library may be capable of binding to different target biomolecules. The library of barcoded affinity probes may be capable of binding 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 is capable of binding to at least 2 different target biomolecules.

[0177] Optionally, in any library of two or more barcoded affinity probes, the barcoded affinity probes comprising the same affinity moiety (and / or comprising affinity moieties capable of binding to the same target biomolecule) may comprise the same barcoded oligonucleotide. Optionally, in any library of barcoded affinity probes, the barcoded affinity probes comprising the same affinity moiety (and / or comprising affinity moieties having an affinity for the same target biomolecule) may comprise different barcoded oligonucleotides or different barcode sequences, the barcode sequences being from a set of two or more different barcode sequences, and / or from a set of at least 10 different barcode sequences, and / or from a set of at least 100 different barcode sequences, and / or from a set of at least 1000 different barcode sequences, and / or from a set of at least 10,000 different barcode sequences, and / or from a set of at least 1,000,000 different barcode sequences.

[0178] Optionally, in any library of two or more different barcoded affinity probes, each barcoded affinity probe may comprise a collection of two or more different affinity moieties (e.g., each barcoded affinity probe may comprise two or more different affinity moieties, each capable of binding a different target biomolecule). Optionally, in any library of barcoded affinity probes, barcoded affinity probes comprising the same collection of two or more different affinity moieties (and / or comprising a collection of affinity moieties capable of binding to the same target biomolecule) may comprise the same barcoded oligonucleotide. Optionally, in any library of barcoded affinity probes, barcoded affinity probes comprising the same collection of two or more different affinity moieties (and / or comprising a collection of affinity moieties capable of binding to the same target biomolecule) may comprise different barcode sequences, or a collection of two or more different barcode sequences, and / or a collection of at least 10 different barcode sequences, and / or a collection of at least 100 different barcode sequences, and / or a collection of at least 1000 different barcode sequences, and / or a collection of at least 10,000 different barcode sequences, and / or different barcode sequences from a collection of at least 1,000,000 different barcode sequences.

[0179] A library of two or more different barcoded affinity probes may comprise barcoded affinity probes each comprising one or more affinity moieties, and one or more first barcoded oligonucleotides and one or more second barcoded oligonucleotides, wherein each first barcoded oligonucleotide in the library comprises the same sequence, and wherein each second barcoded oligonucleotide in the library comprises a different sequence.

[0180] There is provided an optically labeled and / or fluorescently labeled affinity probe, wherein the optically labeled and / or 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. There is provided an optically labeled and / or fluorescently labeled affinity probe, wherein the optically labeled and / or 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.

[0181] A library of two or more affinity probes with optical and / or fluorescent labels is provided, which contains at least first and second affinity probes for at least first and second biomolecules (or target biomolecules) selected from Biomolecule Group 1, wherein each affinity probe with an optical and / or fluorescent label contains at least one optical and / or fluorescent label. A library of two or more affinity probes with optical and / or fluorescent labels is provided, which contains a first affinity probe with an optical and / or fluorescent label having affinity and / or specificity for 5-methylcytosine DNA or 5-hydroxymethylcytosine DNA, and at least a second affinity probe with an optical and / or fluorescent label having affinity and / or specificity for any one or more biomolecules (or target biomolecules) selected from Biomolecule Group 1.

[0182] One or more oligonucleotides are provided, wherein the oligonucleotides contain sequences identical and / or complementary to any DNA and / or RNA sequences of any biomolecules in Biomolecule Group 1. One or more primers are provided, wherein the primers contain sequences identical and / or complementary to any DNA and / or RNA sequences of any biomolecules in Biomolecule Group 1. One or more oligonucleotide probes for in situ hybridization (ISH) methods are provided, wherein the oligonucleotide probes contain sequences identical and / or complementary to any DNA and / or RNA sequences of any biomolecules in Biomolecule Group 1. One or more oligonucleotide probes for fluorescence in situ hybridization (FISH) methods are provided, wherein the oligonucleotide probes contain sequences identical and / or complementary to any DNA and / or RNA sequences of any biomolecules in Biomolecule Group 1. Optionally, any of the oligonucleotides and / or primers and / or oligonucleotide probes may contain optical and / or fluorescent labels. Optionally, any of the oligonucleotides and / or primers and / or oligonucleotide probes may contain linker sequences and / or coupling sequences. Optionally, any of the oligonucleotides and / or primers and / or oligonucleotide probes may be used in reverse transcription methods, and / or primer extension methods; and / or PCR methods, and / or in situ hybridization (ISH) methods, and / or fluorescence in situ hybridization (FISH) methods. A library of two or more oligonucleotides is provided, wherein each of the oligonucleotides contains sequences identical and / or complementary to any DNA and / or RNA sequences of any biomolecules in Biomolecule Group 1.

[0183] In the method, the cyclic microparticles may contain at least two fragments of the target nucleic acid, and wherein the method comprises: (a) preparing a sample for sequencing, which includes 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 (informative) ligated sequence reads.

[0184] In the method, the circular micron particles may contain at least two fragments of the target nucleic acid, and wherein the method comprises: (a) preparing a sample for sequencing, which comprises 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 (informative) ligated sequence reads.

[0185] In the method, the circular micron particles contain at least two fragments of genomic DNA, and wherein the method comprises: (a) preparing a sample for sequencing, which comprises 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.

[0186] In the method, the circular micron particles may contain at least two fragments of genomic DNA, and wherein the method comprises: (a) preparing a sample for sequencing, which comprises 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.

[0187] In the 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 micron particles may be ligated into a set, and 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 micron particles may be ligated into a set, and then sequenced to produce at least 5 ligated sequence reads.

[0188] In the method, each ligated sequence read may provide the 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 200 nucleotides, at least 500 nucleotides, at least 1000 nucleotides or at least 10,000 nucleotides of the ligated fragment. Preferably, each ligated sequence read may provide the sequence of at least 20 nucleotides of the ligated fragment.

[0189] In the method, at least 2, 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, 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 can be generated in total. Preferably, at least 500,000 sequence reads are generated in total.

[0190] The sequence reads can 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 from a target nucleic acid (such as genomic DNA). Preferably, each sequence read contains at least 5 nucleotides from the target nucleic acid.

[0191] The sequence reads can contain the original sequence reads of a part generated by a sequencing instrument, such as a 50-nucleotide sequence original sequence read generated by an Illumina sequencing instrument. The sequence reads can contain the combined sequences of two reads from a paired-end sequencing run, such as the concatenated or combined sequences of the first and second reads from paired-end sequencing run on an Illumina sequencing instrument. The sequence reads can contain a part of the original sequence reads generated by a sequencing instrument, such as 20 consecutive nucleotides in a 150-nucleotide original sequence read generated by an Illumina sequencing instrument. A single original sequence read can contain at least two ligated sequence reads generated by the method of the present invention.

[0192] The sequence reads can be generated by any method known in the art. For example, by chain termination or Sanger sequencing. Preferably, sequencing is performed as follows: by next-generation sequencing methods, such as sequencing by synthesis, synthesis sequencing using reversible terminators (such as Illumina sequencing), pyrosequencing (such as 454 sequencing), ligation sequencing (such as SOLiD sequencing), single-molecule sequencing (such as single-molecule, real-time (SMRT) sequencing, Pacific Biosciences) or by nanopore sequencing (such as on the Minion or Promethion platforms, Oxford Nanopore Technologies). Most preferably, the sequence reads are generated by synthesis sequencing using reversible terminators (such as Illumina sequencing).

[0193] The method may further include another step of mapping each ligated sequence read to a reference genomic sequence. The ligated sequence reads may include sequences mapped to the same chromosome of the reference genomic sequence or sequences mapped to two or more different chromosomes of the reference genomic sequence.

[0194] The diameter of the microparticles can be 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 diameter of the microparticles is at least 200 nm. The diameter of the microparticles can be 100 - 5000 nm. The diameter of the microparticles can be 10 - 10,000 nm (e.g., 100 - 10,000 nm, 110 - 10,000 nm), 50 - 5000 nm, 75 - 5,000 nm, 100 - 3,000 nm. The diameter of the microparticles can 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 microparticles is 100 to 5000 nm. Most preferably, the diameter of the microparticles is 200 to 5000 nm. The sample can include at least two different sizes, or at least three different sizes, or a series of different sizes of microparticles.

[0195] The ligated fragments of genomic DNA can be derived from a single genomic DNA molecule.

[0196] The method may further include the step of estimating or determining the genomic sequence length of the ligated fragments of genomic DNA. Optionally, this step can be performed by sequencing substantially the entire sequence of the ligated fragment (i.e., from its approximate 5' end to its approximate 3' end) and counting the number of nucleotides sequenced therein. Optionally, this can be performed as follows: by sequencing a sufficient number of nucleotides at the 5' end of the sequence of the ligated fragment to map the 5' end to a locus within a reference genomic sequence (e.g., a human genomic sequence), and also sequencing a sufficient number of nucleotides at the 3' end of the ligated fragment to map the 3' end to a locus within the reference genomic sequence, and then using the reference genomic sequence to determine the genomic sequence length of the ligated fragment (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 genome (i.e., the unsequenced portion)).

[0197] In the method, the sample can comprise first and second circular micron particles, where each micron particle contains at least two fragments of a target nucleic acid (e.g., genomic DNA), and where the method comprises performing step (a) to generate a first set of ligated fragments of the target nucleic acid for the first micron particle and a second set of ligated fragments of the target nucleic acid for the second micron particle, and performing step (b) to generate a first set of ligated sequence reads (i.e., a set of ligation signals) for the first micron particle and a second set of ligated sequence reads (i.e., a set of ligation signals) for the second micron particle.

[0198] In the method, the set of ligated sequence reads (i.e., the set of ligation signals) generated for the first micron particle can be distinguished from the set of ligated sequence reads (i.e., the set of ligation signals) generated for the second micron particle.

[0199] In the method, the sample can comprise n blood-derived micron particles, where each micron particle contains at least two fragments of a target nucleic acid (e.g., genomic DNA), and where the method comprises performing step (a) to generate n sets of ligated fragments of the target nucleic acid, one set being generated for each of the n micron particles, and performing step (b) to generate n sets of ligated sequence reads (i.e., sets of ligation signals), one set being generated for each of the n micron particles.

[0200] In the method, n can 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, at least 10,000,000,000 or at least 100,000,000,000. Preferably, n is at least 100,000 micron particles.

[0201] In the method, the sample can comprise 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, at least 10,000,000,000 or at least 100,000,000,000 micron particles (and / or 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, at least 10,000,000,000 or at least 100,000,000,000 micron particles), wherein the micron particles (and / or the sample derived therefrom) are contained within a single continuous aqueous volume at any step of the method (e.g., any step of contacting the sample with a library of polybarcode reagents, and / or any step of attaching and / or ligating and / or coupling a barcode sequence (e.g., a barcoded oligonucleotide) to a target nucleic acid, and / or any step of attaching a coupling sequence to a target nucleic acid, and / or any step of attaching and / or ligating and / or coupling a coupling molecule to a target nucleic acid or other target biomolecule, and / or any step of crosslinking or permeabilizing).

[0202] The set of ligation sequence reads generated for each micron particle (i.e., the set of ligation signals) can be distinguished from the set of ligation sequence reads generated for other micron particles.

[0203] The method can further comprise the step of partitioning the sample into at least two different reaction volumes prior to step (a).

[0204] In the present invention, two sequences or sequence reads (e.g., sequences determined by a sequencing reaction) can be informationally linked in any manner that allows such sequences to be associated or correlated with each other in any way within a computer system, within an algorithm, or within a data set. Such a linkage can be constituted by, and / or established by, and / or represented by a discrete identifying linkage, or by a shared attribute, or by any indirect method of linking, correlating, or associating two or more such sequences.

[0205] The linkage can be constituted by, and / or established by, and / or represented by sequences within the sequencing reaction itself (e.g., in the form of a barcode sequence determined by the sequencing reaction, or in the form of two different portions or segments of a single determined sequence that jointly contain a first and a second linkage sequence), or can be established, contained, or represented independently of such sequences (e.g., established by virtue of being contained within the same flow cell, or within the same channel of a flow cell, or within the same compartment or region of a sequencing instrument, or contained within the same sequencing run of a sequencing instrument, or contained with a certain degree of spatial proximity within a biological sample, and / or with a certain degree of spatial proximity within a sequencing instrument or a sequencing flow cell. The linkage can be constituted by, and / or established by, and / or represented by a measure or parameter corresponding to a physical location or partition within a sequencing instrument, and / or, for example, an image and / or a pixel or pixel location within a multi-pixel camera or a multi-pixel charge-coupled device, and / or, for example, the location of a nanopore or nanopores within a nanopore sequencing instrument or a nanopore membrane).

[0206] The linkage can be absolute (i.e., two sequences are linked or unlinked, with no other quantitative, semi-quantitative, or qualitative / categorical relationship). The linkage can also be relative, probabilistic, or established, contained, or represented with respect to the degree, probability, or extent of the linkage, e.g., with respect to one or more parameters (or represented thereby) that can accommodate one of a range of quantitative, semi-quantitative, or qualitative / categorical values. For example, two (or more) sequences can be linked by quantitative, semi-quantitative, or qualitative / categorical parameter information that represents, contains, estimates, or embodies the proximity of the two (or more) sequences within a sequencing instrument, or the proximity of the two (or more) sequences within a biological sample).

[0207] For any analysis of two or more sequences that are informationally linked in any such manner, the presence (or absence) of the linkage can be used as a parameter in any analysis or evaluation step or any algorithm that performs such a step. For any analysis of two or more sequences that are informationally linked in any such manner, the degree, probability, or extent of the linkage can be used as a parameter in any analysis or evaluation step or any algorithm that performs such a step.

[0208] In one form of such a linkage, a given set of two or more linked sequences can be associated with a specific identifier, such as an alphanumeric identifier, or a barcode, or a barcode sequence. In another form, a given set of two or more linked sequences can be associated with a barcode or a barcode sequence that is contained within a sequence determined by a sequencing reaction. For example, each sequence determined in a sequencing reaction can contain a barcode sequence and a sequence corresponding to a genomic DNA sequence. Optionally, certain sequences or linked sequences can be represented by or associated with two or more barcodes or identifiers.

[0209] In another form of connection, two or more connection sequences may be stored within a computer or computer network, within discrete partitions within a hard disk drive or any kind of storage medium or any other device for storing sequence data. Optionally, certain sequences or connection sequences may be stored in two or more partitions within such a computer or data medium.

[0210] The sequence of information connections may include one or more sets of information connection sequences. The sequences within a set of connection sequences may all share the same connection function or its representation; for example, all sequences within a connection set may be associated with the same barcode or the same identifier, or may be contained within the same partition of a computer or storage medium; all sequences may share any other form of connection, interrelationship, and / or correlation. One or more sequences within a connection set may be exclusive members of that set and thus not members of any other set. Alternatively, one or more sequences within a connection set may be non-exclusive members of that set and thus the sequences may be represented and / or associated with two or more different sets of connection sequences.

[0211] 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, wherein the method comprises: (i) dividing the sample into at least two partitions, wherein each partition on average contains less than n circulating microparticles; and (ii) determining the presence, absence, and / or level of at least two target biomolecules in each of at least two of the at least two partitions. Optionally, wherein 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, wherein n is 0.5. Optionally, wherein step (i) comprises dividing the sample into at least 3 partitions, at least 5 partitions, at least 10 partitions, at least 100 partitions, at least 1000 partitions, at least 10,000 partitions, at least 100,000 partitions, at least 1,000,000 partitions, at least 10,000,000 partitions, at least 100,000,000 partitions, or at least 1,000,000,000 partitions. Preferably, wherein step (i) comprises dividing the sample into at least 1000 partitions.

[0212] (ii) The step of determining the presence, absence, and / or level of at least two target biomolecules can be performed for each of at least two of at least two partitions by analyzing a sample comprising circulating microparticles (i.e., the sample in the partition) or a sample derived from circulating microparticles, wherein the circulating microparticles comprise at least two target molecules, wherein the at least two target molecules are biomolecules, and wherein the method comprises measuring signals corresponding to the presence, absence, and / or level of each target molecule to generate at least two sets of (information) linking signals for the circulating microparticles (i.e., at least two sets of (information) linking signals for the at least two partitions), wherein at least one linking signal corresponds to the presence, absence, and / or level of a first biomolecule in the sample (i.e., the sample in the partition), and at least one linking signal corresponds to the presence, absence, and / or level of a second biomolecule in the sample (i.e., the sample in the partition). The method can be performed by any method provided herein, the method comprising generating at least two sets of linking signals for the microparticles. The method can generate at least two sets of linking signals for each of at least two of at least two partitions.

[0213] The present invention provides a method of analyzing a sample comprising at least two circulating microparticles or a sample derived from at least two circulating microparticles, wherein the method comprises: (i) dividing the sample into at least two partitions, wherein a first partition comprises at least a first and a second target biomolecule of a first circulating microparticle, and a second partition comprises at least a first and a second target biomolecule of a second circulating microparticle, and wherein each of at least two of at least two partitions comprises on average less than [X] of the total mass of DNA; and (ii) determining the presence, absence, and / or level of at least two target biomolecules in each of at least two of at least two partitions. Optionally, wherein [x] is 1.0 attogram of DNA, 10 attograms of DNA, 100 attograms of DNA, 1.0 femtogram of DNA, 10 femtograms of DNA, 100 femtograms of DNA, 1.0 picogram of DNA, 10 picograms of DNA, 100 picograms of DNA, or 1.0 nanogram of DNA. Preferably, wherein [X] is 100 femtograms of DNA.

[0214] (ii) The step of determining the presence, absence, and / or level of at least two target biomolecules can be performed for each of at least two of at least two partitions by a method of analyzing a sample comprising circulating microparticles (i.e., the sample in the partition) or a sample derived from circulating microparticles, wherein the circulating microparticles comprise at least two target molecules, wherein the at least two target molecules are biomolecules, and wherein the method comprises measuring signals corresponding to the presence, absence, and / or level of each target molecule to generate at least two sets of (information) connection signals for the circulating microparticles (i.e., at least two sets of (information) connection signals for the partition), wherein at least one connection signal corresponds to the presence, absence, and / or level of a first biomolecule in the sample (i.e., the sample in the partition), and at least one connection signal corresponds to the presence, absence, and / or level of a second biomolecule in the sample (i.e., the sample in the partition). The method can be performed by any method provided herein, the method comprising generating at least two sets of connection signals for the microparticles. The method can generate at least two sets of connection signals for each of at least two of at least two partitions.

[0215] The present invention provides a method of analyzing a sample comprising at least two circulating microparticles or a sample derived from at least two circulating microparticles, wherein the method comprises: (i) dividing the sample into at least two partitions, wherein the first partition comprises at least a first and a second target biomolecule of a first circulating microparticle, and the second partition comprises at least a first and a second target biomolecule of a second circulating microparticle, and wherein each of at least two of at least two partitions comprises on average less than [Y] of the total mass of the polypeptide; and (ii) determining the presence, absence, and / or level of at least two target biomolecules in each of at least two of at least two partitions. Optionally, wherein [Y] is 1.0 attogram of polypeptide, 10 attograms of polypeptide, 100 attograms of polypeptide, 1.0 femtogram of polypeptide, 10 femtograms of polypeptide, 100 femtograms of polypeptide, 1.0 picogram of polypeptide, 10 picograms of polypeptide, 100 picograms of polypeptide, or 1.0 nanogram of polypeptide. Preferably, wherein [Y] is 100 femtograms of polypeptide.

[0216] (ii) The step of determining the presence, absence, and / or level of at least two target biomolecules can be performed for each of at least two of at least two partitions by analyzing a sample containing circulating microparticles (i.e., the sample in the partition) or a sample derived from circulating microparticles, wherein the circulating microparticles contain at least two target molecules, wherein the at least two target molecules are biomolecules, and wherein the method comprises measuring signals corresponding to the presence, absence, and / or level of each target molecule to generate at least two sets of (information) connection signals for the circulating microparticles (i.e., at least two sets of (information) connection signals for the partition), wherein at least one connection signal corresponds to the presence, absence, and / or level of a first biomolecule in the sample (i.e., the sample in the partition), and at least one connection signal corresponds to the presence, absence, and / or level of a second biomolecule in the sample (i.e., the sample in the partition). The method can be performed by any method provided herein, the method comprising generating at least two sets of connection signals for the microparticles. The method can generate at least two sets of connection signals for each of at least two of at least two partitions.

[0217] The method can further comprise analyzing the sequences of at least two target nucleic acid molecules that have been separated into each of the first and second partitions.

[0218] The method can comprise dividing 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, at least 100,000,000 partitions or at least 1,000,000,000 partitions. Preferably, the method comprises dividing the sample into at least 1000 partitions.

[0219] The first target biomolecule can be a polypeptide, and the second target biomolecule can be a barcoded oligonucleotide or a fragment of a target nucleic acid (e.g., genomic DNA).

[0220] The first target biomolecule can be a polypeptide, and the second target biomolecule can be a fragment of a target nucleic acid (e.g., genomic DNA) containing an epigenetic modification (e.g., 5-hydroxymethylcytosine DNA or 5-methylcytosine DNA).

[0221] The first target biomolecule can be 5-hydroxymethylcytosine DNA, and the second target biomolecule can be a biomolecule selected from Biomolecule Group 1.

[0222] The first target biomolecule can be 5-methylcytosine DNA, and the second target biomolecule can be a biomolecule selected from Biomolecule Group 1.

[0223] The first and second target biomolecules can be selected from Biomolecule Group 1.

[0224] 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) can be performed using one or more barcoded affinity probes (such as those provided herein), for example, by binding the barcoded affinity probe 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) can be performed according to any method that includes contacting a sample with a barcoded affinity probe (such as those provided herein). Optionally, the method includes binding at least one barcoded affinity probe to the target biomolecule, wherein a barcode sequence from a polymeric barcode reagent is attached to the barcoded oligonucleotide of the barcoded affinity probe. Optionally, the measurement is performed by analyzing the barcode sequence of the polymeric barcode reagent and / or by analyzing the barcode sequence of the barcoded oligonucleotide from the barcoded affinity probe.

[0225] 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) can be performed using one or more optical and / or fluorescent / fluorescence measurement methods, for example, by using one or more optically labeled and / or fluorescently labeled affinity probes. For example, the measurement step can be performed using one or more optically labeled and / or fluorescently labeled affinity probes, wherein at least one optically labeled and / or fluorescently labeled affinity probe binds to the target biomolecule, and wherein the measurement is performed using at least one optical measurement step or at least one fluorescence detection step (e.g., wherein the measurement is performed by measuring the optical and / or fluorescence signal from the optically labeled and / or fluorescently labeled affinity probe).

[0226] Optionally, any one or more optical and / or fluorescence / fluorescent measurement processes may include performing an optical and / or fluorescent measurement on a sample that includes one or more circulating micron-sized particles and / or includes biomolecules from one or more circulating micron-sized particles, where the sample is contained within an aqueous volume and / or an aqueous droplet (e.g., a droplet analyzed using a fluorescence-activated cell sorting (FACS) instrument). Optionally, any such optical and / or fluorescence measurement process may further include a sorting and / or selection process, e.g., where any one or more optical and / or fluorescent measurements of the circulating micron-sized particles are used to sort and / or select any given circulating micron-sized particle and / or any group and / or subset of two or more circulating micron-sized particles (e.g., sorting a sample containing circulating micron-sized particles into a first subset of circulating micron-sized particles that exhibit a high level of a particular target biomolecule, and a second subset of circulating micron-sized particles that exhibit a high level of the particular target biomolecule).

[0227] Optionally, any one or more optical and / or fluorescence / fluorescent measurement processes may include performing an optical and / or fluorescent measurement on a sample that includes one or more circulating micron-sized particles and / or includes biomolecules from one or more circulating micron-sized particles, where the sample is contained on a flat surface (e.g., a flat glass surface such as a microscope slide, or any other flat surface). Optionally, any one or more optical and / or fluorescence / fluorescent measurement processes may include performing an optical and / or fluorescent measurement on a sample that includes one or more circulating micron-sized particles and / or includes biomolecules from one or more circulating micron-sized particles, where the sample is visualized using an optical microscope and / or a fluorescence microscope.

[0228] Optionally, any one or more fluorescently labeled affinity probes may include a fluorophore having a specific absorption spectrum and / or emission spectrum. Optionally, any one or more fluorescently labeled affinity probes contained within a pool and / or library and / or collection of two or more fluorescently labeled affinity probes may include a fluorophore having an absorption spectrum and / or emission spectrum that is different from that of at least one and / or at least two other fluorescently labeled affinity probes within the pool and / or library and / or collection.

[0229] Optionally, all fluorescently labeled affinity probes that have affinity for the same target biomolecule contained within a pool and / or library and / or collection of two or more fluorescently labeled affinity probes may comprise fluorophores having the same absorption spectrum and / or emission spectrum. Optionally, all fluorescently labeled affinity probes that have affinity for the same target biomolecule contained within a pool and / or library and / or collection of two or more fluorescently labeled affinity probes may comprise the same fluorophore. Optionally, fluorescently labeled affinity probes that have affinity for the same target biomolecule contained within a pool and / or library and / or collection of two or more fluorescently labeled affinity probes may comprise two or more different fluorophores (e.g., two or more different fluorophores comprising two or more different absorption spectra and / or emission spectra). Optionally, fluorescently labeled affinity probes within a pool and / or library and / or collection of two or more fluorescently labeled affinity probes may each comprise fluorophores from a collection of two or more different fluorophores (e.g., two or more different fluorophores comprising two or more different absorption spectra and / or emission spectra), wherein all of the fluorescently labeled affinity probes having affinity for the same target biomolecule share the same fluorophore, optionally, wherein each fluorophore identifies and / or associates with the target biomolecule of the fluorescently labeled affinity probe. Optionally, within any pool and / or library and / or collection of two or more fluorescently labeled affinity probes, multiple different fluorophores may be used (e.g., any number of different fluorophores comprising different absorption spectra 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.

[0230] Optionally, in any method for analyzing a sample comprising at least one circulating micron particle, during and / or before and / or after any one or more steps of the method, any sample, and / or solution, and / or reactant or reaction mixture, and / or aqueous volume, and / or mixture comprising any number or concentration of circulating micron particles, and / or any number or concentration of biomolecules from one or more circulating micron particles, and / or any number or concentration of (same or different) barcodes, and / or any number or concentration of (same or different) barcode molecules, and / or any number or concentration of (same or different) barcode sequences, and / or any number or concentration of (same or different) barcoded oligonucleotides, and / or any number or concentration of (same or different) polybarcode reagents, and / or any number or concentration of (same or different) affinity moieties, and / or any number or concentration of (same or different) barcoded affinity probes, and / or any number or concentration of (same or different) adapter oligonucleotides, and / or any number or concentration of (same or different) coupling sequences, and / or any number or concentration of (same or different) enrichment probes, and / or any number or concentration of (same or different) primers, and / or any number or concentration of (same or different) hybridization probes, and / or any number or concentration of (same or different) fluorescence in situ hybridization probes may be contained in a single partition, or at least first and second partitions (e.g., divided or split into first and second partitions), or contained in (e.g., divided or split into) any number of partitions, such as at least 3 partitions, at least 4 partitions, at least 5 partitions, at least 10 partitions, at least 100 partitions, at least 1000 partitions, at least 10,000 partitions, at least 100,000 partitions, at least 1,000,000 partitions, at least 10,000,000 partitions, at least 100,000,000 partitions or at least 1,000,000,000 partitions.

[0231] Optionally, in any method, any one or more target biomolecules may be measured and / or analyzed by optical measurement and / or optical quantification methods. Optionally, in any method, any one or more target biomolecules may be measured and / or analyzed using optically labeled and / or fluorescently labeled affinity probes that have affinity and / or specificity for the target biomolecules.

[0232] Optionally, any method for measuring and / or analyzing biomolecules may comprise one or more direct detection steps. Optionally, any method for measuring and / or analyzing biomolecules may comprise one or more indirect detection steps.

[0233] For the avoidance of doubt, in the present invention and in any method herein, any reference to any one or more biomolecules 'in' a circulating micron particle and / or 'within' a circulating micron particle, and / or 'belonging to' a micron particle, and / or 'from' a circulating micron particle, and / or 'contained in' a circulating micron particle, and / or 'contained within' a circulating micron particle is to be construed broadly to mean that the biomolecule(s) is / are found (and / or potentially found) in whole or in part within any form or location of the circulating micron particle (including wholly or partly enclosed within a membrane, and / or wholly or partly on the outer and / or inner surface of a membrane, and / or wholly or partly embedded within a membrane).

[0234] Optionally, in any method, any step of analyzing the sequence of one or more target nucleic acid molecules can be carried out by primer extension reaction. Optionally, in any method, any step of analyzing the sequence of one or more target nucleic acid molecules can be carried out by polymerase chain reaction (PCR), optionally using a primer set that provides amplification (and thus measurement and detection) of a specific target sequence (such as a specific DNA, RNA or cDNA target sequence). Optionally, in any method, any step of analyzing the sequence of one or more target nucleic acid molecules can be carried out by reverse transcription reaction, optionally with one or more subsequent primer extension or PCR steps.

[0235] Optionally, in any method, any step of analyzing the sequence of one or more target nucleic acid molecules can be carried out by in situ hybridization (ISH) methods, such as fluorescence in situ hybridization (FISH) methods.

[0236] The methods of the present invention can be deterministic (e.g., a barcode sequence can be used to identify sequence reads from a single micron particle) or probabilistic (e.g., a barcode sequence can be used to identify sequence reads that may be from a single micron particle). As another example, in the method, the partitioning step can be designed to achieve an average of only 1 circulating micron particle per partition. However, this is an inherently statistical process that cannot guarantee that each partition will contain only biomolecules from a single micron particle; thus, it cannot guarantee that the set of ligation signals corresponding to the biomolecules from a particular partition will correspond to the biomolecules from a single micron particle. For example, if a particular partition contains two different micron particles, the set of ligation signals can correspond to the two micron particles.

[0237] The present invention further includes systems and devices for analyzing a sample (or two or more such samples, e.g., each containing one or more circulating micron-sized particles) containing one or more circulating micron-sized particles. Optionally, such a system may include at least one algorithm or a portion of an algorithm and / or computer program (e.g., an algorithm or a portion of an algorithm and / or computer program included within a computer system and / or included within a network- or Internet-based computer storage system) for analyzing one or more sets of ligation signals (e.g., one or more sets of ligation sequences) derived from measurements of at least one circulating micron-sized particle (e.g., any one or more algorithms and / or computer programs configured to calculate any one or more parameter values, such as any of the parameter values described herein), and / or at least one reference sequence and / or set of reference sequences (e.g., one or more reference sequences included within a computer system and / or computer data storage system, such as a server and / or hard drive), and / or at least one set of barcoded oligonucleotides, and / or at least one multiplex barcoding reagent and / or library thereof, and / or at least one physical device containing one or more partitions (e.g., one or more tubes, each containing a partition; and / or one or more plates containing wells, where each well contains a partition; and / or one or more devices containing two or more partitions, where each such partition contains droplets, e.g., a microfluidic device containing or capable of generating microfluidic droplets (e.g., the Chromium system provided by 10X Genomics), or a flat surface containing one or more droplets thereon), and / or at least one enzyme or enzyme solution capable of attaching a barcode sequence to a target nucleic acid (e.g., any ligase, polymerase, and / or transposase), and / or at least one algorithm and / or computer program configured to report the results of any one or more of the analyses herein to a physician and / or other healthcare provider and / or patient (e.g., the results of any one or more diagnostic methods and / or diagnostic tests based on the analysis of two or more ligation signals from a sample containing one or more circulating micron-sized particles).For example, a system for analyzing a sample comprising one or more circulating micron-sized particles may include at least one algorithm or a portion thereof for analyzing one or more sets of ligation signals derived from measurements of at least one circulating micron-sized particle, and at least one set of barcoded oligonucleotides (configured to attach to a target biomolecule contained in or derived from a circulating micron-sized particle), and at least one physical device comprising one or more partitions; alternatively, such a system may include at least one algorithm or a portion thereof for analyzing one or more sets of ligation signals derived from measurements of at least one circulating micron-sized particle, and at least one library of polymeric barcode reagents; alternatively, such a system may include at least one algorithm or a portion thereof for analyzing one or more sets of ligation signals derived from measurements of at least one circulating micron-sized particle, and at least one library of polymeric barcode reagents, and at least one physical device comprising one or more partitions; alternatively, such a system may include at least one algorithm or a portion thereof for analyzing one or more sets of ligation signals derived from measurements of at least one circulating micron-sized particle, and at least one set of barcoded oligonucleotides (configured to attach to a target biomolecule contained in or derived from a circulating micron-sized particle), and at least one algorithm and / or computer program configured to report the results of any one or more of the analyses herein.

[0238] 1. Sample of circulating micron-sized particles

[0239] A sample for use in the methods of the present invention may comprise at least one circulating micron-sized particle (i.e., a micron-sized particle derived from blood (e.g., human blood)) and / or a sample for use in the methods of the present invention may be derived from at least one circulating micron-sized particle. The micron-sized particle may be derived from maternal blood. The micron-sized particle may be derived from the blood of a patient having 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.

[0240] Circulating micron-sized particles may be one or more of the various free micron-sized particles that have been 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). "Free" refers to the fact that such micron-sized particles are not cells. Instead, the micron-sized particles are derived from cells, for example, by secretion or after apoptosis. These micron-sized particles differ in the tissues and cells from which they originate, as well as in the biophysical methods underlying their formation, and in their respective sizes and molecular structures and compositions. The micron-sized particle may comprise one or more components from the cell membrane (e.g., incorporated phospholipid components) and one or more intracellular and / or nuclear components. The micron-sized particle may be selected from one or more of exosomes, apoptotic bodies (also referred to as apoptotic vesicles), and / or extracellular microvesicles.

[0241] Microparticles can be defined as membranous vesicles containing at least two fragments of target nucleic acids (such as genomic DNA). The diameter of the microparticles can be 100 - 5000 nm. Preferably, the diameter of the microparticles is 100 - 3000 nanometers.

[0242] Exosomes are the smallest circulating microparticles, typically in the diameter range of 50 to 100 nanometers, and are thought to originate from the cell membranes of intact living cells and contain protein and RNA components (including mRNA molecules and / or degraded mRNA molecules, as well as small regulatory RNA molecules such as microRNA molecules) contained in the outer phospholipid component. Exosomes are thought to be formed by the exocytosis of multivesicular bodies (Gyorgy et al., Cell. Mol. Life Sci. (2011) 68:2667 - 2688). Exosomes are thought to play diverse roles in cell - cell signaling and extracellular functions (Kanada et al., Proc. Natl. Acad. Sci. USA (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 13 / 456,121, European Application EP2626433 A1).

[0243] Microparticles also include apoptotic bodies (also known as apoptotic vesicles) and extracellular microvesicles, with a total diameter range up to 1 micrometer and even 2 to 5 micrometers, and are generally considered to have a diameter greater than 100 nanometers (Lichtenstein et al., Ann N Y Acad Sci. (2001); 945:239 - 49). All classes of circulating microparticles are thought to be produced by a large number and variety of cells in the body (Thierry et al., Cancer Metastasis Rev 35(3), 347 - 376.9 (2016) / s10555 - 016 - 9629 - x).

[0244] Preferably, the microparticles are not exosomes, for example, the microparticles are any microparticles with a diameter greater than that of exosomes.

[0245] Samples for the 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 a signal or a measuring reagent (such as a barcoded oligonucleotide) may be performed on a sample containing at least one intact circulating microparticle (e.g., where the sample or reaction mixture contains intact circulating microparticles when measuring the signal or the measuring reagent). Alternatively, the step of measuring a signal or a measuring reagent (such as 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 separated from circulating microparticles). When measuring the signal or the measuring reagent, the sample may not contain intact circulating microparticles.

[0246] 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 100,000,000, 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.

[0247] In the sample, the concentration of nucleic acid (e.g., genomic DNA) fragments may be less than 1.0 picogram DNA / μL, less than 10 picogram DNA / μL, less than 100 picogram DNA / μL, less than 1.0 nanogram DNA / μL, less than 10 nanogram DNA / μL, less than 100 nanogram DNA / μL, or less than 1000 nanogram DNA / μL.

[0248] 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 microparticles. Preferably, the sample contains (or is derived from) at least 100 circulating microparticles.

[0249] In a sample, the concentration of micron particles can be less than 0.001 micron particles per microliter, less than 0.01 micron particles per microliter, less than 0.1 micron particles per microliter, less than 1.0 micron particles per microliter, less than 10 micron particles per microliter, less than 100 micron particles per microliter, less than 1000 micron particles per microliter, less than 10,000 micron particles per microliter, less than 100,000 micron particles per microliter, less than 1,000,000 micron particles per microliter, less than 10,000,000 micron particles per microliter, or less than 100,000,000 micron particles per microliter.

[0250] Circulating micron particles can comprise 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 micron particles comprise at least 10 target biomolecules and / or target epitopes.

[0251] In the methods of the present invention, any number of one or more different target biomolecules and / or target epitopes can be measured and / or analyzed. Optionally, in any method, a set 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 set of at least 3 different target biomolecules and / or target epitopes is measured and / or analyzed.

[0252] In the methods, the same target biomolecule (and / or target epitope) and / or two or more target biomolecules (and / or target epitopes) of the same set can be measured and / or analyzed for all circulating micron particles (or partitions) within a sample. Optionally, in any method, a specific target biomolecule (and / or target epitope) and / or two or more target biomolecules (and / or target epitopes) of a specific set can be measured and / or analyzed for a subset of the circulating micron particles within a sample. Optionally, in any method, a sample of circulating micron particles can be divided into any number of two or more sub-samples, wherein different specific target biomolecules (and / or target epitopes), and / or two or more target biomolecules (and / or target epitopes) of different specific sets, can be measured and / or analyzed for each said sub-sample.

[0253] Optionally, in any method, two or more different target epitopes of the same biomolecule can be measured and / or analyzed. For example, two or more different affinity probes (e.g., two or more different antibodies) that have affinity or specificity for two or more different epitopes within a target biomolecule (e.g., a target protein) can be used to measure or analyze the target biomolecule.

[0254] A biomolecule (also referred to herein as a target biomolecule) can be a chemical or molecular substance present in or derived from circulating microparticles. A biomolecule can be a macromolecule. A biomolecule can be a polypeptide (e.g., a protein), a carbohydrate molecule, a lipid molecule, or a nucleic acid molecule. A biomolecule can be a metabolite. Preferably, the biomolecule is a human biomolecule.

[0255] The target biomolecule can have a predetermined (or predefined) sequence. For example, a target polypeptide can have a predetermined (or predefined) amino acid sequence or epitope. Similarly, a fragment of a target nucleic acid can have a predetermined (or predefined) nucleotide sequence. The method can include using a target-specific reagent, such as a barcoded affinity probe or an affinity probe, to measure a signal corresponding to the presence, absence, and / or level of a predetermined (or predefined) sequence or epitope.

[0256] The biomolecule can be a nucleic acid biomolecule or a non-nucleic acid biomolecule.

[0257] 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, such as glycoproteins. One or more biomolecules can be one or more protein isoforms.

[0258] The biomolecule can contain an epitope of an antigen present in or derived from circulating microparticles. For example, the epitope can be an epitope of a polypeptide or a protein. The biomolecule can contain 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 contain a specific nucleic acid epitope, such as a specific nucleic acid modification (e.g., 5-methylcytosine DNA epitope and / or 5-hydroxymethylcytosine DNA epitope). The biomolecule can contain a specific epitope recognized by one or more affinity probes (e.g., a barcoded affinity probe), such as an epitope recognized by an antibody.

[0259] The biomolecule can contain an epitope that is not a nucleic acid epitope. 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-hydroxymethylcytosine DNA molecule (i.e., the biomolecule may be an epitope that is not a 5-hydroxymethylcytosine DNA epitope).

[0260] The biomolecule can be a DNA-binding protein. Optionally, the biomolecule is not a DNA-binding protein.

[0261] The biomolecule can be a histone (such as histone H1, histone H2A, histone H2B, histone H3, and / or histone H4 and / or any histone variant). The histone can be a post-translationally modified histone (such as trimethylated histone H3 lysine 4, trimethylated histone H3 lysine 27, and / or any histone acetylation modification). Optionally, the biomolecule is not a histone.

[0262] The biomolecule can be a chromatin protein. Optionally, the biomolecule is not a chromatin protein.

[0263] 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 immunoprecipitated with DNA. Optionally, the biomolecule is not a polypeptide or protein immunoprecipitated with DNA.

[0264] The biomolecule can be a DNA-binding biomolecule. Optionally, the biomolecule is not a DNA-binding biomolecule. The biomolecule can be a membrane biomolecule or a membrane-associated biomolecule. Optionally, the biomolecule is not a membrane biomolecule or a membrane-associated biomolecule. The biomolecule can be a biomolecule immunoprecipitated with DNA. Optionally, the biomolecule is not a biomolecule immunoprecipitated with DNA.

[0265] The biomolecule can be completely or partially located on the inner surface and / or outer surface of the membrane of the circulating microparticle (such as the lipid bilayer membrane of the circulating microparticle). The biomolecule can be completely or partially enclosed within the membrane of the circulating microparticle (such as enclosed within the lipid bilayer of the circulating microparticle). The biomolecule can be contained within the membrane of the circulating microparticle and / or across the membrane and / or any combination thereof. The biomolecule can be completely or partially embedded within the membrane of the circulating microparticle (such as completely or partially embedded within the lipid bilayer membrane of the circulating microparticle).

[0266] The biomolecule can be derived from the inner surface and / or outer surface of the circulating microparticle, and / or derived from within the circulating microparticle (such as derived from within the membrane of the circulating microparticle), and / or derived from within and / or across the membrane of the circulating microparticle, and / or any combination thereof.

[0267] The biomolecule can be DNA (such as double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA)), RNA (such as double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA)), or a fragment thereof. The biomolecule can be genomic DNA or RNA (such as mRNA) or a fragment thereof.

[0268] One or more biomolecules (or target biomolecules) may be DNA fragments, RNA fragments, and / or polypeptides selected from (or encoding) Biomolecule Group 1, which include:

[0269] ● Plasma-based protein markers for cancer and / or cancer invasiveness, including prostate-specific antigen (PSA) and CA-125;

[0270] ● Cell surface and immune cell type markers, including CD3, CD4, CD8, CD19, CD20, CD20, CD41, CD45, CD61, CD62, CD146, CD235a, and CD326;

[0271] ● Genes and proteins involved in tumor formation and malignant transformation, and genes used as immunocytochemical markers for evaluating cancer cell types and subtypes, including antigen 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, epithelial cell adhesion molecule (EpCAM), HER2 / Neu, progesterone receptor, K-ras, 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;

[0272] ● Markers related to pregnancy (i.e., plasma protein markers) (e.g., fetal markers or placental markers) or markers related to pregnancy complications, including alpha-fetoprotein (AFP), beta-human chorionic gonadotropin (beta-nCG), and Toll-like receptor 4 (TLR4);

[0273] ● Proteins related to 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);

[0274] ● MicroRNA molecules (miRNAs) associated with (and / or differentially expressed in) intravascular plaques, 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;

[0275] ● Markers of lymphocytes and / or other immune cells, including LY6G6D and immunoglobulins; and

[0276] ● And other target biomolecules, including transthyretin, C-reactive protein (CRP), and troponin.

[0277] The biomolecules (or target biomolecules) provided above are collectively referred to herein as "biomolecule set 1".

[0278] Such DNA fragments may include all or part of the DNA sequences of one or more protein-coding genes (e.g., genomic sequences, exon region sequences, intron region sequences, promoter region sequences, and / or terminator region sequences). Such RNA fragments may include all or part of the RNA sequences of one or more protein-coding genes (e.g., exon RNA sequences, intron RNA sequences, 5' untranslated region sequences, and / or 3' untranslated region sequences). Such polypeptides may include all or part of one or more proteins. Such polypeptides may include one or more post-translational modified forms of the polypeptide (e.g., wherein 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).

[0279] Biomolecules can contain epigenetic modifications. Epigenetic modifications can include modified nucleotides, such as modified gDNA nucleotides or modified RNA nucleotides. Modified nucleotides can include modified bases. Modified bases can be methylated bases, such as 5-methylcytosine or 5-hydroxymethylcytosine. A biomolecule (e.g., a fragment of a target nucleic acid such as genomic DNA) can contain 5-methylcytosine (i.e., can contain 5-methylcytosine DNA and / or can contain 5-methylcytosine DNA nucleotides). A biomolecule (such as a fragment of a target nucleic acid (e.g., genomic DNA)) can contain 5-hydroxymethylcytosine (i.e., can contain 5-hydroxymethylcytosine DNA and / or can contain 5-hydroxymethylcytosine DNA nucleotides). Epigenetic modifications can include post-translational modifications of proteins. Post-translational modifications can be methylation, phosphorylation, acetylation, ubiquitination, and / or sumoylation. Post-translationally modified polypeptides can be histones. For example, post-translationally modified histones (e.g., histone H3 lysine 4 trimethylation, histone H3 lysine 27 trimethylation, and / or any histone acetylation modification).

[0280] Biomolecules can contain exogenously administered molecules, such as exogenously administered polypeptides (e.g., exogenously administered antibodies), and / or exogenously administered nucleic acids (e.g., exogenously administered oligonucleotides, such as exogenously administered barcode sequences, such as barcoded oligonucleotides).

[0281] Biomolecules can contain barcoded oligonucleotides (or their barcode sequences) of barcoded affinity probes.

[0282] At least two biomolecules of the circulating microparticles can be fragments of a target nucleic acid (e.g., molecules of fragmented genomic DNA). These fragmented genomic DNA molecules and / or the sequences contained within these fragmented genomic DNA molecules can be ligated by any method described herein.

[0283] Fragments of the target nucleic acid can be fragments of DNA (e.g., molecules of fragmented genomic DNA) or fragments of RNA (e.g., fragments of mRNA). Preferably, the fragments of the target nucleic acid are fragments of genomic DNA.

[0284] Fragments of DNA can be fragments of mitochondrial DNA. Fragments of DNA can be fragments of mitochondrial DNA from maternal cells or tissues. Fragments of DNA can be fragments of mitochondrial DNA from fetal or placental tissues. Fragments of DNA can be fragments of mitochondrial DNA from diseased and / or cancerous tissues.

[0285] The micron-sized particles may comprise platelets. The micron-sized particles may comprise tumor-conditioned platelets. The target nucleic acid may comprise platelet RNA (e.g., fragments of platelet RNA and / or fragments of tumor-conditioned platelet RNA). A sample comprising one or more platelets may comprise platelet-rich plasma (e.g., platelet-rich plasma comprising tumor-conditioned platelets).

[0286] Fragments of the target nucleic acid may comprise double-stranded or single-stranded nucleic acid. Fragments of genomic DNA may comprise double-stranded DNA or single-stranded DNA. Fragments of the target nucleic acid may comprise partially double-stranded nucleic acid. Fragments of genomic DNA may comprise partially double-stranded DNA.

[0287] Fragments of the target nucleic acid may be fragments derived from a single nucleic acid molecule, or fragments derived from two or more nucleic acid molecules. For example, fragments of genomic DNA may be derived from a single genomic DNA molecule.

[0288] As will be understood by those skilled in the art, as used herein, the term "fragment of the target nucleic acid" refers to the original fragment present in the micron-sized particles and its copies or amplicons. For example, the term "gDNA fragment" refers to the original gDNA fragment present in the micron-sized particles and, for example, to a DNA molecule that can be prepared from the original genomic DNA fragment by primer extension reaction. As another example, the term "mRNA fragment" refers to the original mRNA fragment present in the micron-sized particles and, for example, to a cDNA molecule that can be prepared from the original mRNA fragment by reverse transcription.

[0289] Fragments 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. Fragments 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 1000 nucleotides or 50 to 500 nucleotides. The length of fragments of the target nucleic acid (e.g., genomic DNA) may be 20 to 200 nucleotides, a length of 100 to 200 nucleotides, a length of 200 to 1000 nucleotides, a length of 50 to 250 nucleotides, a length of 1000 to 10,000 nucleotides, a length of 10,000 to 100,000 nucleotides, or a length of 50 to 100,000 nucleotides. Preferably, the length of the fragmented genomic DNA molecules is 50 to 500 nucleotides.

[0290] Optionally, any method of analyzing a sample that includes one or more circulating microparticles (and / or a sample derived from one or more circulating microparticles) can include combinatorial measurements (e.g., measuring presence, absence, and / or levels) that include measuring any combination of any two or more different biomolecules (e.g., any two or more different target biomolecules). For example, any such method can include measuring ligated fragments of genomic DNA (e.g., by barcoding and / or sequencing), optionally where the measurement of the ligated fragments of genomic DNA further includes measuring and / or estimating the genomic or nucleotide sequence length of the fragments of the genomic DNA, and optionally where the measurement of the ligated fragments of genomic DNA further includes measuring and / or estimating the genetic coordinates (or genomic positions) of the 3' and / or 5' ends of the ligated fragments of genomic DNA, as well as one or more modified nucleotides or nucleobases (e.g., measuring 5-methylcytosine, and measuring 5-hydroxymethylcytosine), and measuring one or more polypeptide biomolecules (e.g., measuring any one or more biomolecules from Biomolecule Group 1). Optionally, any such combinatorial measurement can (further) include measuring one or more plasma-based protein markers for cancer and / or cancer invasiveness, and one or more cell surface or immunocyte type markers, and one or more proteins involved in tumorigenesis and malignant transformation or immunocytochemical markers for assessing cancer cell types and cell types, and one or more markers related to pregnancy or pregnancy complications, and one or more proteins related to circulating lipoprotein particles and / or intravascular plaques, and one or more microRNA molecules (e.g., any such markers provided in the list contained within Biomolecule Group 1). For example, the combinatorial measurement can include measuring ligated fragments of genomic DNA, optionally where the measurement of the ligated fragments of genomic DNA further includes measuring and / or estimating the genomic or nucleotide sequence length of the fragments of the genomic DNA, and optionally where the measurement of the ligated fragments of genomic DNA further includes measuring and / or estimating the genetic coordinates (or genomic positions) of the 3' and / or 5' ends of the ligated fragments of genomic DNA, and measuring one or more modified nucleotides or nucleobases (e.g., measuring 5-methylcytosine, and measuring 5-hydroxymethylcytosine), and measuring PSA, and CA-125, and CD4, and CD8, and Ki-67, and BCL2, and EGFR; optionally, such combinatorial measurement can further include measuring TTF-1 and / or Ras and / or c-Myc and / or PD-L1 and / or estrogen receptor and / or cyclin B1.

[0291] Optionally, any combinatorial measurement may include separate such combinatorial measurements on two or more samples from a single individual (e.g., a single patient), where the two or more samples are obtained / made from the same individual but are separated by one or more durations (e.g., at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years, and / or at least 10 years, and / or any other duration). For example, a particular combinatorial measurement (any kind described herein) may be performed on a first sample obtained from an individual and separately on a second sample obtained from the individual at a later time period. Any number of such sequential (time-separated) samples from an individual may be analyzed in this way, e.g., at least 3, at least 4, at least 5, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, or at least 30, or any greater or similar number of sequential samples.

[0292] 2. Sample of isolated circulating micron-sized particles

[0293] Numerous methods for isolating circulating micron-sized particles (and / or specific subsets, classes, or portions of circulating micron-sized particles) have been previously described. European Patent ES2540255 (B1) and U.S. Patent 9005888 B2 describe methods for isolating specific circulating micron-sized particles (e.g., apoptotic bodies) based on centrifugation procedures. Numerous methods for separating different types of free micron-sized particles by centrifugation, ultracentrifugation, and other techniques have been previously well described and developed (Gyorgy et al., Cellular and Molecular Life Sciences (2011) 68:2667 - 2688).

[0294] The method may further comprise isolating a sample comprising one or more circulating micron-sized particles from blood, plasma, or serum. Micron-sized particles may be isolated from blood, plasma, or serum. The method may further comprise the step of isolating micron-sized particles from blood, plasma, or serum.

[0295] Micron-sized particles may be isolated by centrifugation, size exclusion chromatography, and / or filtration.

[0296] The isolation step may comprise centrifugation. Micron-sized particles may be isolated by precipitation by means of a centrifugation step and / or an ultracentrifugation step, or a series of two or more centrifugation steps and / or ultracentrifugation steps at two or more different speeds, where the pellet and / or supernatant from one centrifugation / ultracentrifugation step is further processed in a second centrifugation / ultracentrifugation step and / or differential centrifugation.

[0297] The centrifugation or ultracentrifugation step can be carried out at a speed of 100 - 500,000 G, 100 - 1000 G, 1000 - 10,000 G, 10,000 - 100,000 G, 500 - 100,000 G or 100,000 - 500,000 G. The centrifugation or ultracentrifugation step can be performed for a duration of 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.

[0298] The separation step can include size exclusion chromatography, such as column-based size exclusion chromatography, such as chromatography including columns containing an agarose-based matrix or a sephacryl-based matrix.

[0299] Size exclusion chromatography can include using a matrix or filter with a pore size 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.

[0300] The separation step can include filtering the sample. The filtrate can provide micron-sized particles for analysis in the method. Optionally, the filter is used to separate micron-sized particles below a certain size, and wherein the filter preferentially or completely removes particles with a size greater than 100 nanometers, a size greater than 200 nanometers, a size greater than 300 nanometers, a size greater than 500 nanometers, a size greater than 1.0 micrometer, a size greater than 2.0 micrometers, a size greater than 3.0 micrometers, a size greater than 5.0 micrometers or a size greater than 10.0 micrometers. Optionally, filters with the same size filtration parameters or with different size filtration parameters can be used to perform two or more such filtration steps. Optionally, the filtrate of one or more filtration steps contains micron-sized particles and thereby generates ligated sequence reads.

[0301] 3. Prepare a sample of circulating micron-sized particles for analysis

[0302] In the method, any one or more target biomolecules can be measured and / or analyzed when the circulating micron-sized particles are intact. Optionally, any one or more target biomolecules can be measured and / or analyzed when the circulating micron-sized particles are not intact (i.e., after one or more biomolecules have been released from the circulating micron-sized particles).

[0303] A sample containing one or more circulating micron-sized particles can be chemically cross-linked (e.g., with formaldehyde). A sample containing one or more circulating micron-sized particles can be permeabilized (e.g., with a chemical surfactant). A sample containing one or more circulating micron-sized particles can be chemically cross-linked (e.g., with formaldehyde). The chemical cross-linking and / or permeabilization step can be carried out before measuring and / or analyzing the target biomolecules of one or more circulating micron-sized particles.

[0304] The cross-linking step can be carried out using a chemical cross-linking agent, such as formaldehyde, paraformaldehyde, glutaraldehyde, succinimidyl glutarate, ethylene glycol bis(succinimidyl succinate), homobifunctional cross-linking agents or heterobifunctional cross-linking agents. Any such cross-linking step can be further terminated by a quenching step, such as quenching the formaldehyde cross-linking step by mixing with a glycine solution. Any such cross-linking can be removed prior to a specific subsequent step of the protocol (e.g., prior to primer extension, PCR or nucleic acid purification steps). The purpose of the cross-linking step with a chemical cross-linking agent is to keep the biomolecules (e.g., fragments of genomic DNA and / or polypeptides) within each micron particle physically close to each other so that the sample can be manipulated and processed while maintaining the basic structural properties of the micron particle (i.e., while keeping the genomic DNA fragments and / or polypeptides derived from the same micron particle physically close).

[0305] The micron particles can be permeabilized using an incubation step. The incubation step can be carried out in the presence of a chemical surfactant (such as Triton X-100 (C 14 H 22 O(C2H4O) n (n = 9 - 10)), NP-40, Tween 20, Tween 80, saponin, digitonin or sodium dodecyl sulfate). The incubation step can be carried out at a temperature of at least 20 °C, at least 30 °C, at least 37 °C, at least 45 °C, at least 50 °C, at least 60 °C, at least 65 °C, at least 70 °C or at least 80 °C. The incubation step can 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.

[0306] After the step of transferring any one or more of the reagents described herein (e.g., barcoded oligonucleotides, polybarcode reagents, affinity probes, barcoded affinity probes, etc.) to one or more micron particles, any one or more target biomolecules can be measured and / or analyzed. The method can include the step of transferring any one or more of the reagents described herein (e.g., barcoded oligonucleotides, polybarcode reagents, affinity probes, barcoded affinity probes, etc.) to one or more cyclic micron particles.

[0307] In the method, any one or more of the reagents described herein can be transferred into one or more circulating micron particles by complexing with a transfection reagent or a lipid carrier (such as liposomes or micelles). The transfection reagent can be a lipid transfection reagent, such as a cationic lipid transfection reagent. Optionally, the cationic lipid transfection reagent contains at least two alkyl chains. Optionally, the cationic lipid transfection reagent can be a commercially available cationic lipid transfection reagent, such as Lipofectamine.

[0308] In the method, the reagent for analyzing the first circulating micron particles can be included in the first lipid carrier, and the reagent for analyzing the second circulating micron particles can be included in the second lipid carrier. The lipid carrier can be liposomes or micelles.

[0309] Before the transfer step, the method can include a step of crosslinking biomolecules (such as genomic DNA and / or fragments of the target polypeptide) in the micron particles. Before the transfer step, and optionally after the crosslinking step, the method can further include a step of permeabilizing the micron particles.

[0310] After the step of releasing the target biomolecule(s) from one or more circulating micron particles, any one or more target biomolecules can be measured and / or analyzed. One or more target biomolecules can be released from the circulating micron particles by steps of dissolving, permeabilizing, and / or lysing the circulating micron particles. The method of the present invention can include releasing target biomolecules from one or more circulating micron particles (such as by dissolving, permeabilizing, and / or lysing one or more circulating micron particles). This release step can be carried out by a high-temperature incubation step, and / or by incubation with a molecular solvent or a chemical surfactant.

[0311] After the step of purifying and / or separating and / or processing any one or more target biomolecules from one or more circulating micron particles, any one or more target biomolecules can be measured and / or analyzed. The method of the present invention can include one or more steps of processing, purifying, fractionating, and / or separating any or all of the target biomolecules and / or other components of the circulating micron particles before, and / or during, and / or after any step of analyzing the sample. The method can include a step of purifying and / or separating nucleic acids (such as DNA molecules and / or RNA molecules). The method can include a step of purifying and / or separating polypeptides (such as proteins and / or post-translationally modified proteins).

[0312] Any one or more target biomolecules can be measured and / or analyzed after the step of binding and / or attaching any one or more of the target biomolecules and / or target nucleic acid molecules to a carrier, such as a solid carrier, and / or a semi-solid carrier and / or a gel carrier.

[0313] The method may comprise the step of attaching one or more molecules (such as any one or more nucleic acid molecules, such as DNA molecules and / or RNA molecules, and / or any polypeptide molecule, such as a protein or a post-translationally modified protein) to a carrier. Any number or fraction of such molecules in a sample comprising one or more cyclic microparticles may be attached to one or more carriers; optionally, at least 0.01%, at least 0.1%, at least 1%, at least 10%, at least 50% or 100% of such molecules may be attached to one or more carriers.

[0314] Any one or more such molecules may be linked to any form of carrier (such as a macromolecule, a solid carrier or a semi-solid carrier or a dendrimer). Any carrier may be a bead (such as a gel bead, an agarose bead, a silica bead, a polystyrene foam bead, a gel bead (such as those available from 10x purchased), an antibody-binding bead, an oligo-dT-binding bead, an avidin bead or a magnetic bead (such as a superparamagnetic bead). Any bead may have any size and / or molecular structure (such as a diameter of 10 nanometers to 100 micrometers, a diameter of 100 nanometers to 10 micrometers or a diameter of 1 micrometer to 5 micrometers). The molecule may be directly or indirectly (such as via a linking molecule) linked to the carrier. The molecule may be linked by binding to the carrier and / or by binding or adhering to a linking molecule bound to the carrier. The molecule may bind to the carrier (or to the linking molecule) by a covalent bond, a non-covalent bond (such as a protein-protein interaction or an avidin-biotin bond) or nucleic acid hybridization. The linking molecule may be a biopolymer (such as a nucleic acid molecule) or a synthetic polymer. The linking molecule may comprise one or more ethylene glycol and / or poly(ethylene glycol) (such as hexaethylene glycol or pentaethylene glycol) units. The linking molecule may comprise one or more ethyl groups, such as a C3 (three-carbon) spacer, a C6 spacer, a C12 spacer or a C18 spacer. Any carrier may be functionalized to enable the linking of two or more molecules. Such functionalization may be achieved by adding a chemical moiety (such as a carboxylated group, an alkyne, an azide, an acrylate group, an amino group, a sulfate group or a succinimidyl group) and / or a protein-based moiety (such as avidin, streptavidin or protein G).

[0315] The molecule may be linked by a macromolecule by binding to and / or by adhering to the macromolecule. The macromolecule may be a nucleic acid comprising two or more nucleotides each capable of binding to a barcode molecule. Additionally or alternatively, the nucleic acid may comprise two or more regions each capable of hybridizing to a barcode molecule. The macromolecule may be a synthetic polymer (such as a dendrimer) or a biopolymer, such as a nucleic acid (such as a single-stranded nucleic acid, such as single-stranded DNA), a peptide, a polypeptide or a protein (such as a multimeric protein). The dendrimer may comprise at least 2, at least 3, at least 5 or at least 10 generations.

[0316] The method may comprise attaching one or more cyclic microparticles to a carrier by a method comprising: (a) attaching a coupling molecule comprising one or more biotin moieties to a target molecule (e.g., a target nucleic acid molecule or a target polypeptide molecule), and / or attaching a biotin-binding affinity probe to the target molecule, to produce a biotin-binding target molecule, and (b) attaching the biotin-binding target molecule to one or more streptavidin-binding carriers (e.g., one or more streptavidin-binding beads). Optionally, before and / or during step (b), the biotin-binding target molecule is partitioned into two or more compartments.

[0317] After the step of partitioning a sample into two or more partitions, any one or more target biomolecules can be measured and / or analyzed. The method can include partitioning a sample into two or more partitions. Optionally, each partition can contain one or more carriers, wherein molecules from the micron particles assigned to each partition are respectively attached to the carriers contained within the same compartment. Optionally, a sample containing any number of micron particles (e.g., at least 1000 micron particles, at least 1,000,000 micron particles, or at least 100,000,000 micron particles) can be attached by this method. Optionally, any number and / or average number of micron particles can be partitioned into each partition (e.g., 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 0.0001, less than 0.00001, or less than 0.000001 micron particles can be partitioned into each partition). Each partition can contain or on average contain any number of carriers, such as an average of 0.1 carrier, an average of 0.5 carrier, an average of 1 carrier, an average of 2 carriers, an average of 5 carriers, an average of 10 carriers, or an average of 100 carriers. Optionally, after any method of attaching molecules from a sample containing two or more cyclic micron particles to carriers within a partition, all or any part of the solution contained in any portion and / or all of the partitions can be combined together to form a single de-partitioned carrier-attached reaction mixture, wherein the de-partitioned carrier-attached reaction mixture contains carriers to which molecules from the sample have been attached as such. Optionally, the de-partitioned carrier-attached reaction mixture can then be used in any method for analyzing a sample containing two or more cyclic micron particles, such as any method for measuring fragments of genomic DNA, any method for measuring modified nucleotides or nucleobases, and / or any method for measuring one or more target polypeptides. Optionally, two or more target molecules attached to carriers within the de-partitioned carrier-attached reaction mixture (e.g., two or more molecules from the same cyclic micron particle, such as two or more fragments of genomic DNA, and / or two or more polypeptides bound to barcoded affinity probes) can be attached to the same barcode sequence or different barcode sequences within a set of barcode sequences to link the two or more target molecules. Optionally, any such method of attaching barcode sequences can include attaching two or more barcoded oligonucleotides from a polybarcode reagent to two or more target molecules, the two or more target molecules being attached to the same carrier within the de-partitioned carrier-attached reaction mixture.Optionally, any of the methods of attaching a barcode sequence may comprise contacting the partitioned carrier-attached reaction mixture with at least 2, at least 100, at least 1000, at least 10,000, at least 1,000,000, at least 10,000,000 or at least 1,000,000,000 polynucleotide barcode reagents, and attaching the barcoded oligonucleotides contained within the polynucleotide barcode reagents to target molecules that have been attached to carriers within the partitioned carrier-attached reaction mixture. Any one or more of the partitioned carrier-attached reaction mixtures may comprise a sample derived from one or more circulating micron-sized particles for use in any one or more of the methods described herein. Optionally, for any such method, any number of partitions (e.g., at least 10, at least 1000, at least 1,000,000 or at least 1,000,000,000 partitions), any type of partition (e.g., reaction tubes, or aqueous droplets, or aqueous droplets within an emulsion) and / or any volume of partition (e.g., 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, such as any partition number, type or volume described herein and / or in PCT / GB2017 / 053820, the contents of which are incorporated herein by reference.

[0318] 4. By barcode ligation

[0319] The present invention provides a method for preparing a sample for sequencing, wherein the sample comprises circulating micron-sized particles (or micron-sized particles derived from blood), wherein the micron-sized particles contain at least two fragments of a target nucleic acid (e.g., genomic DNA), and wherein the method comprises attaching at least two fragments of the target nucleic acid of the micron-sized particles to different barcode sequences in a barcode sequence or a set of barcode sequences to produce a set of ligated fragments of the target nucleic acid.

[0320] The present invention provides a method for preparing a sample for sequencing, wherein the sample comprises circulating micron-sized particles, wherein the circulating micron-sized particles contain at least two fragments of a target nucleic acid (e.g., genomic DNA), and wherein the method comprises attaching at least two fragments of the target nucleic acid of the circulating micron-sized particles to a barcode sequence, or to different barcode sequences in a set of barcode sequences, to produce a set of ligated fragments of the target nucleic acid.

[0321] Before the step of attaching at least two fragments of the target nucleic acid of the micron particles to a barcode sequence or to different barcode sequences in a set of barcode sequences, the method may comprise attaching a coupling sequence to each fragment of the target nucleic acid (e.g., genomic DNA) of the micron particles, where the coupling sequence is then attached to the barcode sequence or to different barcode sequences in a set of barcode sequences to produce a set of ligated fragments of the target nucleic acid.

[0322] In the method, the sample may comprise first and second micron particles derived from blood, where each micron particle comprises at least two fragments of a target nucleic acid (e.g., genomic DNA), and where the method may comprise attaching at least two fragments of the target nucleic acid of the first micron particle to a first barcode sequence or to different barcode sequences in a set of first barcode sequences to produce a first set of ligated fragments of the target nucleic acid, and attaching at least two fragments of the target nucleic acid of the second micron particle to a second barcode sequence or to different barcode sequences in a set of second barcode sequences to produce a second set of ligated fragments of the target nucleic acid.

[0323] The first barcode sequence may be different from the second barcode sequence. The barcode sequences in the set of first barcode sequences may be different from the barcode sequences in the set of second barcode sequences.

[0324] In the method, the sample may comprise n micron particles derived from blood, where each micron particle contains at least two fragments of a target nucleic acid (e.g., genomic DNA), and where the method comprises performing step (a) to produce n sets of ligated fragments of the target nucleic acid, each of the n micron particles producing one set.

[0325] In the 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, at least 10,000,000,000 or at least 100,000,000,000. Preferably, n is at least 100,000 micron particles.

[0326] Preferably, each set of ligated sequence reads (i.e., set of ligation signals) is ligated with a different barcode sequence or a different set of barcode sequences. Each barcode sequence in a set of barcode sequences can be different from the barcode sequences in at least 1, at least 4, at least 9, at least 49, at least 99, at least 999, at least 9,999, at least 99,999, at least 999,999, at least 9,999,999, at least 99,999,999, at least 999,999,999, at least 9,999,999,999, at least 99,999,999,999, or at least 999,999,999,999 other sets of barcode sequences in the library. Each barcode sequence in a set of barcode sequences can be different from the 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 the barcode sequences in at least 9 other sets of barcode sequences in the library.

[0327] The present invention provides a method for analyzing a sample, the sample comprising micron-sized particles derived from blood, wherein the micron-sized particles contain at least two fragments of a target nucleic acid, and wherein the method comprises: (a) preparing a sample for sequencing, the sample comprising attaching at least two fragments of the target nucleic acid (e.g., genomic DNA) of the micron-sized particles to a barcode sequence to produce a set of ligated fragments of the target nucleic acid; and (b) sequencing each ligated fragment in the set to produce at least two ligated sequence reads, wherein the at least two ligated sequence reads are ligated by the barcode sequence.

[0328] The barcode sequence can contain a unique sequence. Each barcode sequence can comprise 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 comprises at least 5 nucleotides. Preferably, each barcode sequence comprises deoxyribonucleotides, and optionally, all nucleotides in the barcode sequence are deoxyribonucleotides. One or more deoxyribonucleotides can be modified deoxyribonucleotides (e.g., deoxyribonucleotides modified with a biotin moiety or deoxyuridine nucleotides). The barcode sequence can comprise one or more degenerate nucleotides or sequences. The barcode sequence can not comprise any degenerate nucleotides or sequences.

[0329] In the method, before the step of attaching at least two fragments of the target nucleic acid of the micron-sized particles to a barcode sequence, the method can comprise attaching a coupling sequence to each fragment of the nucleic acid of the micron-sized particles, wherein the coupling sequence is then attached to the barcode sequence to produce a set of ligated fragments.

[0330] In the method, the sample can comprise first and second micron particles derived from blood, wherein each micron particle contains at least two fragments of a target nucleic acid (e.g., genomic DNA), and wherein the method comprises performing step (a) to generate a first set of ligated fragments of the target nucleic acid for the first micron particle and a second set of ligated fragments of the target nucleic acid for the second micron particle, and performing step (b) to generate a first set of ligated sequence reads (i.e., a set of ligation signals) for the first micron particle and a second set of ligated sequence reads (i.e., a set of ligation signals) for the second micron particle, wherein at least two ligated sequence reads of the first micron particle are linked to at least two ligated sequence reads of the second micron particle by different barcode sequences.

[0331] The first set of ligated fragments can be linked to the second set of ligated fragments by different barcode sequences.

[0332] In the method, the sample can comprise n micron particles derived from blood, wherein each micron particle contains at least two fragments of a target nucleic acid (e.g., genomic DNA), and wherein the method comprises performing step (a) to generate n sets of ligated fragments of the target nucleic acid, each of the n micron particles generating one set, and performing step (b) to generate n sets of ligated sequence reads (i.e., a set of ligation signals), each of the n micron particles generating one set.

[0333] In the method, n can 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, at least 10,000,000,000 or at least 100,000,000,000. Preferably, n is at least 100,000 micron particles.

[0334] Preferably, each set of ligated sequence reads (i.e., a set of ligation signals) is linked by different barcode sequences.

[0335] In the method, different barcode sequences can be provided as a library of barcode sequences. The library used in the method can comprise 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, at least 100,000,000,000 or at least 1,000,000,000,000 different barcode sequences. Preferably, the library used in the method comprises at least 1,000,000 different barcode sequences.

[0336] In the method, each barcode sequence in the library can be attached to a fragment of only a single micron particle.

[0337] The method can be deterministic, i.e., a barcode sequence can be used to identify sequence reads from a single micron particle, or it can be probabilistic, i.e., a barcode sequence can be used to identify sequence reads that may be from a single micron particle. In some embodiments, a barcode sequence can be attached to fragments of genomic DNA from two or more micron particles.

[0338] The method can include: (a) preparing a sample for sequencing that includes attaching each of at least two fragments of a target nucleic acid (e.g., genomic DNA) of a micron particle to a different barcode sequence in a set of barcode sequences to produce a set of ligated fragments of the target nucleic acid; and (b) sequencing each ligated fragment in the set to produce at least two ligated sequence reads, wherein the at least two ligated sequence reads are linked by the set of barcode sequences.

[0339] In the method, prior to the step of attaching each of at least two fragments of a target nucleic acid of a micron particle to different barcode sequences, the method can include attaching a coupling sequence to each fragment of the target nucleic acid of the micron particle, wherein each of at least two fragments of the target nucleic acid of the micron particle is attached to a different barcode sequence in the set of barcode sequences via its coupling sequence.

[0340] In the method, the sample can include first and second micron particles derived from blood, wherein each micron particle contains at least two fragments of a target nucleic acid (e.g., genomic DNA), and wherein the method can include performing step (a) to produce a first set of ligated fragments of the target nucleic acid for the first micron particle and a second set of ligated fragments of the target nucleic acid for the second micron particle, and performing step (b) to produce a first set of ligated sequence reads (i.e., a set of ligation signals) for the first micron particle and a second set of ligated sequence reads (i.e., a set of ligation signals) for the second micron particle, wherein the first set of ligated sequence reads is linked to the second set of ligated sequence reads by different sets of barcode sequences.

[0341] In the method, the sample can include n micron particles derived from blood, wherein each micron particle contains at least two fragments of a target nucleic acid (e.g., genomic DNA), and wherein the method can include performing step (a) to produce n sets of ligated fragments of the target nucleic acid, each of the n micron particles producing one set, and performing step (b) to produce n sets of ligated sequence reads (i.e., a set of ligation signals), each of the n micron particles producing one set.

[0342] In the method, n can 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, at least 10,000,000,000 or at least 100,000,000,000. Preferably, n is at least 100,000 micron particles.

[0343] Preferably, each set of ligated sequence reads (i.e., the set of ligation signals) is ligated by a different set of barcode sequences.

[0344] In the method, different sets of barcode sequences can be provided as a library of sets of barcode sequences. The library used in the method can 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, at least 100,000,000,000 or at least 1,000,000,000,000 different sets of barcode sequences. Preferably, the library used in the method contains at least 1,000,000 different sets of barcode sequences.

[0345] Each barcode sequence in a set of barcode sequences can be different from the barcode sequences in at least 1, at least 4, at least 9, at least 49, at least 99, at least 999, at least 9,999, at least 99,999, at least 999,999, at least 9,999,999, at least 99,999,999, at least 999,999,999, at least 9,999,999,999, at least 99,999,999,999 or at least 999,999,999,999 other sets of barcode sequences in the library. Each barcode sequence in a set of barcode sequences can be different from the barcode sequences in all other sets of barcode sequences in the library. Preferably, each barcode sequence in the barcode sequences is different from the barcode sequences in at least 9 other sets of barcode sequences in the library.

[0346] In the method, the barcode sequences from one set of barcode sequences from the library can be attached only to the fragments from a single micron particle.

[0347] The method can be deterministic, i.e., one set of barcode sequences can be used to identify the sequence reads from a single micron particle, or it can be probabilistic, i.e., one set of barcode sequences can be used to identify the sequence reads that may be from a single micron particle.

[0348] The method may comprise preparing first and second samples for sequencing, wherein each sample comprises at least one blood-derived micron particle, wherein each micron particle comprises at least two fragments of target nucleic acid (e.g., genomic DNA), and wherein the barcode sequences each comprise a sample identifier region, and wherein the method comprises: (i) performing step (a) on each sample, wherein the barcode sequence attached to the target nucleic acid fragment from the first sample has a different sample identifier region from the barcode sequence attached to the target nucleic acid fragment from the second sample; (ii) performing step (b) on each sample, wherein each ligated sequence read comprises the sequence of the sample identifier region; and (iii) determining the sample from which each ligated sequence read is obtained by the sample identifier region.

[0349] In the method, prior to, during, and / or after the step of attaching the barcode sequence and / or the coupling sequence, the method may comprise the step of crosslinking the fragments of genomic DNA in the micron particles.

[0350] In the method, prior to, during, and / or after the step of attaching the barcode sequence and / or the coupling sequence, and / or optionally after the step of crosslinking the fragments of genomic DNA in the micron particles, the method may comprise the step of permeabilizing the micron particles. Prior to the transfer step, and optionally after the crosslinking step, the method comprises permeabilizing the micron particles.

[0351] The barcode sequence may be contained within a barcoded oligonucleotide in a barcoded oligonucleotide solution; for example, such barcoded oligonucleotides may be single-stranded, double-stranded, or single-stranded with one or more double-stranded regions. The barcoded oligonucleotides may be ligated to the fragments of the target nucleic acid in a single-stranded or double-stranded ligation reaction. The barcoded oligonucleotides may comprise a single-stranded 5' or 3' region capable of ligating to the fragments of the target nucleic acid. Each barcoded oligonucleotide may be ligated to the fragments of the target nucleic acid in a single-stranded ligation reaction. Alternatively, the barcoded oligonucleotides may comprise blunt, recessed, or protruding 5' or 3' regions capable of ligating to the fragments of the target nucleic acid. Each barcoded oligonucleotide may be ligated to the fragments of the target nucleic acid in a double-stranded ligation reaction.

[0352] In some methods, the ends of the fragments of the target nucleic acid can be converted to blunt double-stranded ends in a blunting reaction, and the barcoded oligonucleotides can contain blunt double-stranded ends. Each barcoded oligonucleotide can be ligated to a fragment of the target nucleic acid in a blunt-end ligation reaction. In some methods, the ends of the fragments of the target nucleic acid can convert their ends to blunt double-stranded ends in a blunting reaction and then convert their ends to a form with a single 3'-adenosine overhang, and wherein the barcoded oligonucleotide contains a double-stranded end with a single 3'-thymidine overhang that is capable of annealing to the single 3'-adenosine overhang of the fragment of the target nucleic acid. Each barcoded oligonucleotide can be ligated to a fragment of the target nucleic acid in a double-stranded A / T ligation reaction.

[0353] In some methods, the barcoded oligonucleotide contains a target region at its 3' or 5' end that is capable of annealing to a target region in the target nucleic acid and / or the coupling sequence, and the barcode sequence can be attached to the target nucleic acid by annealing the barcoded oligonucleotide to the target nucleic acid and / or the coupling sequence and optionally extending and / or ligating the barcoded oligonucleotide to the nucleic acid target and / or the coupling sequence.

[0354] In some methods, the coupling sequence can be attached to a fragment of genomic DNA before attaching the barcoded oligonucleotide.

[0355] The method can include a step of partitioning the nucleic acid sample into at least two different reaction volumes before the attachment step.

[0356] 5. Using a polybarcode reagent for barcode ligation

[0357] The present invention provides a method for preparing a sample for sequencing, wherein the sample contains circulating microparticles (i.e., microparticles derived from blood), and wherein the microparticles contain at least two fragments of a target nucleic acid (e.g., genomic DNA), and wherein the method comprises the steps of: (a) contacting the sample with a polybarcode reagent, wherein the polybarcode reagent contains first and second barcode regions linked together, and wherein each barcode region contains a nucleic acid sequence; and (b) attaching the barcode sequence 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, wherein the first barcoded target nucleic acid molecule contains the nucleic acid sequence of the first barcode region, and the second barcoded target nucleic acid molecule contains the nucleic acid sequence of the second barcode region.

[0358] The present invention provides a method for preparing a sample for sequencing, wherein the sample comprises micron-sized particles derived from blood, and wherein the micron-sized particles contain at least two fragments of a target nucleic acid (e.g., genomic DNA), and wherein the method comprises the steps of: (a) contacting the sample with a polybarcoded reagent, wherein the polybarcoded reagent comprises a first and a second barcoded oligonucleotide linked together, and wherein the barcoded oligonucleotides each comprise a barcode region; and (b) ligating or linking the first and second barcoded oligonucleotides to the first and second fragments of the target nucleic acid of the micron-sized particles to produce first and second barcoded target nucleic acid molecules.

[0359] The present invention provides a method for preparing a sample for sequencing, wherein the sample comprises first and second micron-sized particles derived from blood, and wherein each micron-sized particle contains at least two fragments of a target nucleic acid (e.g., genomic DNA), and wherein the method comprises the steps of: (a) contacting the sample with a library comprising at least two polybarcoded reagents, wherein each polybarcoded reagent comprises a first and a second barcode region linked together, wherein each barcode region comprises a nucleic acid sequence, and wherein the first and second barcode regions of the first polybarcoded reagent of the library are different from the first and second barcode regions of the second polybarcoded reagent; and (b) attaching barcode sequences to each of the first and second fragments of the target nucleic acid of the first micron-sized particle to produce first and second barcoded target nucleic acid molecules for the first micron-sized particle, wherein the first barcoded target nucleic acid molecule comprises the nucleic acid sequence of the first barcode region of the first polybarcoded reagent, and the second barcoded target nucleic acid molecule comprises the nucleic acid sequence of the second barcode region of the first polybarcoded reagent, and attaching barcode sequences to each of the first and second fragments of the target nucleic acid of the second micron-sized particle to produce first and second barcoded target nucleic acid molecules for the second micron-sized particle, wherein the first barcoded target nucleic acid molecule comprises the nucleic acid sequence of the first barcode region of the second polybarcoded reagent, and the second barcoded target nucleic acid molecule comprises the nucleic acid sequence of the second barcode region of the second polybarcoded reagent.

[0360] The present invention provides a method for preparing a sample for sequencing, wherein the sample comprises first and second micron-sized particles derived from blood, and wherein each micron-sized particle contains at least two fragments of a target nucleic acid (e.g., genomic DNA), and wherein the method comprises the steps of: (a) contacting the sample with a library comprising at least two polybarcoded reagents, wherein each polybarcoded reagent comprises a first and a second barcoded oligonucleotide linked together, wherein the barcoded oligonucleotides each comprise a barcode region, and wherein the barcode regions of the first and second barcoded oligonucleotides of the first polybarcoded reagent of the library are different from the barcode regions of the first and second barcoded oligonucleotides of the second polybarcoded reagent of the library; and (b) ligating or linking the first and second barcoded oligonucleotides of the first polybarcoded reagent to the first and second fragments of the target nucleic acid of the first micron-sized particle to produce first and second barcoded target nucleic acid molecules, and ligating or linking the first and second barcoded oligonucleotides of the second polybarcoded reagent to the first and second fragments of the target nucleic acid of the second micron-sized particle to produce first and second barcoded target nucleic acid molecules.

[0361] The barcoded oligonucleotides can be ligated to the fragments of the target nucleic acid in a single-stranded or double-stranded ligation reaction.

[0362] In the method, the barcoded oligonucleotides can comprise a single-stranded 5' or 3' region capable of ligating to a fragment of the target nucleic acid. Each barcoded oligonucleotide can be ligated to a fragment of the target nucleic acid in a single-stranded ligation reaction.

[0363] In the method, the barcoded oligonucleotides can comprise blunt, recessed, or protruding 5' or 3' regions capable of ligating to a fragment of the target nucleic acid. Each barcoded oligonucleotide can be ligated to a fragment of the target nucleic acid in a double-stranded ligation reaction.

[0364] In the method, the ends of the fragments of the target nucleic acid can be converted to blunt double-stranded ends in a blunting reaction, and the barcoded oligonucleotides can comprise blunt double-stranded ends. Each barcoded oligonucleotide can be ligated to a fragment of the target nucleic acid in a blunt-end ligation reaction.

[0365] In the method, the ends of the fragments of the target nucleic acid can be converted to blunt double-stranded ends in a blunting reaction and then converted to a form having a single 3' adenosine overhang, and wherein the barcoded oligonucleotides comprise double-stranded ends having a single 3' thymine overhang that can anneal to the single 3' adenosine overhang of the fragment of the target nucleic acid. Each barcoded oligonucleotide can be ligated to a fragment of the target nucleic acid in a double-stranded A / T ligation reaction.

[0366] In the method, the ends of the fragments of the target nucleic acid may be contacted with a restriction enzyme, where the restriction enzyme digests each fragment at a restriction site to create a ligation junction at these restriction sites, and where the barcoded oligonucleotides contain ends that are compatible with these ligation junctions. Each barcoded oligonucleotide may be ligated to a fragment of the target nucleic acid at the ligation junction in a double-stranded ligation reaction. Optionally, the restriction enzyme may be EcoRI, HindIII, or BglII.

[0367] In the method, prior to the step of ligating or joining the first and second barcoded oligonucleotides to the first and second fragments of the target nucleic acid, the method may include attaching a coupling sequence to each fragment of the target nucleic acid, where the first and second barcoded oligonucleotides are then ligated or joined to the coupling sequences of the first and second fragments of the target nucleic acid.

[0368] In the method, step (b) may include: (i) ligating the first and second barcoded oligonucleotides of a first polybarcoding reagent to the first and second fragments of the target nucleic acid of a first micron particle, and ligating the first and second barcoded oligonucleotides of a second polybarcoding reagent to the first and second fragments of the target nucleic acid of a second micron particle; and

[0369] (ii) extending the first and second barcoded oligonucleotides of the first polybarcoding reagent to generate first and second differently barcoded target nucleic acid molecules, and extending the first and second barcoded oligonucleotides of the second polybarcoding reagent to generate first and second differently barcoded target nucleic acid molecules, where each barcoded target nucleic acid molecule contains at least one nucleotide synthesized from a fragment of the target nucleic acid that serves as a template.

[0370] The method may comprise: (a) contacting a sample with a library comprising at least two polybarcoded reagents, wherein each polybarcoded reagent comprises a first and a second barcoded oligonucleotide linked together, wherein the barcoded oligonucleotides each comprise a target region and a barcode region in the 5' to 3' direction, wherein the barcode regions of the first and second barcoded oligonucleotides of the first polybarcoded reagent of the library are different from the barcode regions of the first and second barcoded oligonucleotides of the second polybarcoded reagent of the library, and wherein the sample is further contacted with the first and second target primers of each polybarcoded reagent; and (b) performing the following steps on each micron particle: (i) annealing the target region of the first barcoded oligonucleotide to a first subsequence of a first fragment of the target nucleic acid (e.g., genomic DNA) of the micron particle, and annealing the target region of the second barcoded oligonucleotide to a first subsequence of a second fragment of the target nucleic acid (e.g., genomic DNA) of the micron particle, (ii) annealing the first target primer to a second subsequence of the first fragment of the target nucleic acid of the micron particle, wherein the second subsequence is 3' to the first subsequence, and annealing the second target primer to a second subsequence of the second fragment of the target nucleic acid of the micron particle, wherein the second subsequence is 3' to the first subsequence, (iii) extending the first target primer using the first fragment of the target nucleic acid of the micron particle as a template until it reaches the first subsequence to produce a first extended target primer, and extending the second target primer using the second fragment of the target nucleic acid of the micron particle until it reaches the first subsequence to produce a second extended target primer, and (iv) ligating the 3' end of the first extended target primer to the 5' end of the first barcoded oligonucleotide to produce a first barcoded target nucleic acid molecule, and ligating the 3' end of the second extended target primer to the 5' end of the second barcoded oligonucleotide to produce a second barcoded target nucleic acid molecule, wherein the first and second barcoded target nucleic acid molecules are different and each comprises at least one nucleotide synthesized from the target nucleic acid used as a template.

[0371] Each polybarcoded reagent may comprise: (i) a first and a second hybridization molecule linked together, wherein each hybridization molecule contains a nucleic acid sequence comprising a hybridization region; and (ii) a first and a second barcoded oligonucleotide, wherein the first barcoded oligonucleotide is annealed to the hybridization region of the first hybridization molecule, and wherein the second barcoded oligonucleotide is annealed to the hybridization region of the second hybridization molecule.

[0372] Each polybarcoded reagent may comprise: (i) a first and a second barcode molecule linked together, wherein each barcode molecule contains a nucleic acid sequence comprising a barcode region; and (ii) a first and a second barcoded oligonucleotide, wherein the first barcoded oligonucleotide comprises a barcode region annealed to the barcode region of the first barcode molecule, and wherein the second barcoded oligonucleotide comprises a barcode region annealed to the barcode region of the second barcode molecule.

[0373] The present invention provides a method for preparing a sample for sequencing, wherein the sample comprises at least two blood-derived micron-sized particles, wherein each micron-sized particle comprises at least two fragments of a target nucleic acid, and wherein the method comprises the steps of: (a) contacting the sample with a library comprising first and second polynucleotide barcode reagents, wherein each polynucleotide barcode reagent comprises first and second barcode molecules linked together, and wherein each barcode molecule comprises a nucleic acid sequence which optionally comprises a barcode region and an adaptor region in the 5' to 3' direction;

[0374] (b) attaching a coupling sequence to first and second fragments of the target nucleic acid (e.g., genomic DNA) of the first and second micron-sized particles; (c) for each polynucleotide barcode reagent, ligating the coupling sequence of the first fragment to the adaptor region of the first barcode molecule and ligating the coupling sequence of the second fragment to the adaptor region of the second barcode molecule; and (d) for each polynucleotide barcode reagent, attaching a barcode sequence to each of at least two fragments of the target nucleic acid of the micron-sized particle to produce first and second different barcoded target nucleic acid molecules, wherein the first barcoded target nucleic acid molecule comprises the nucleic acid sequence of the barcode region of the first barcode molecule and the second barcoded target nucleic acid molecule comprises the nucleic acid sequence of the barcode region of the second barcode molecule.

[0375] In the method, each barcode molecule may comprise a nucleic acid sequence comprising a barcode region and an adaptor region in the 5' to 3' direction, and wherein for each polynucleotide barcode reagent, step (d) comprises: using the barcode region of the first barcode molecule as a template to extend the coupling sequence of the first fragment to produce a first barcoded target nucleic acid molecule, and using the barcode region of the second barcode molecule as a template to extend the coupling sequence of the second fragment to produce a second barcoded target nucleic acid molecule, wherein the first barcoded target nucleic acid molecule comprises a sequence complementary to the barcode region of the first barcode molecule and the second barcoded target nucleic acid molecule comprises a sequence complementary to the barcode region of the second barcode molecule.

[0376] In the method, each barcode molecule may comprise a nucleic acid sequence that, in the 5' to 3' direction, comprises an adaptor region and a barcode region, wherein for each multiplex barcode reagent, step (d) comprises: (i) using the barcode region of the first barcode molecule as a template to ligate and extend a first extension primer to produce a first barcoded oligonucleotide, and using the barcode region of the second barcode molecule as a template to ligate and extend a second extension primer to produce a second barcoded oligonucleotide, wherein the first barcoded oligonucleotide comprises a sequence complementary to the barcode region of the first barcode molecule, and the second barcoded oligonucleotide comprises a sequence complementary to the barcode region of the second barcode molecule,

[0377] (ii) ligating the 3' end of the first barcoded oligonucleotide to the 5' end of the coupling sequence of the first fragment to produce a first barcoded target nucleic acid molecule, and ligating the 3' end of the second barcoded oligonucleotide to the 5' end of the coupling sequence of the second fragment to produce a second barcoded target nucleic acid molecule.

[0378] In the method, each barcode molecule may comprise a nucleic acid sequence that, in the 5' to 3' direction, comprises an adaptor region, a barcode region, and a priming region, wherein for each multiplex barcode reagent, step (d) comprises: (i) ligating a first extension primer to the priming region of the first barcode molecule and using the barcode region of the first barcode molecule as a template to extend the first extension primer to produce a first barcoded oligonucleotide, and ligating a second extension primer to the priming region of the second barcode molecule and using the barcode region of the second barcode molecule as a template to extend the second extension primer to produce a second barcoded oligonucleotide, wherein the first barcoded oligonucleotide comprises a sequence complementary to the barcode region of the first barcode molecule, and the second barcoded oligonucleotide comprises a sequence complementary to the barcode region of the second barcode molecule, and (ii) ligating the 3' end of the first barcoded oligonucleotide to the 5' end of the coupling sequence of the first fragment to produce a first barcoded target nucleic acid molecule, and ligating the 3' end of the second barcoded oligonucleotide to the 5' end of the coupling sequence of the second fragment to produce a second barcoded target nucleic acid molecule.

[0379] The method may comprise: (a) contacting a sample with a library comprising first and second polynucleotide barcode reagents, wherein each polynucleotide barcode reagent comprises first and second barcode molecules linked together, wherein each barcode molecule comprises a nucleic acid sequence that, in the 5' to 3' direction, comprises a barcode region and an adapter region, wherein the sample is further contacted with first and second adapter oligonucleotides of each polynucleotide barcode reagent, wherein the first and second adapter oligonucleotides each comprise an adapter region, and (b) ligating the first and second adapter oligonucleotides of the first polynucleotide barcode reagent to first and second fragments of a target nucleic acid of a first microparticle, and ligating the first and second adapter oligonucleotides of the second polynucleotide barcode reagent to first and second fragments of a target nucleic acid of a second microparticle; (c) for each polynucleotide barcode reagent, ligating the adapter region of the first adapter oligonucleotide to the adapter region of the first barcode molecule, and ligating the adapter region of the second adapter oligonucleotide to the adapter region of the second barcode molecule; and (d) for each polynucleotide barcode reagent, using the barcode region of the first barcode molecule as a template to extend the first adapter oligonucleotide to produce a first barcoded target nucleic acid molecule, and using the barcode region of the second barcode molecule as a template to extend the second adapter oligonucleotide to produce a second barcoded target nucleic acid molecule, wherein the first barcoded target nucleic acid molecule comprises a sequence complementary to the barcode region of the first barcode molecule, and the second barcoded target nucleic acid molecule comprises a sequence complementary to the barcode region of the second barcode molecule.

[0380] The method may comprise the following steps: (a) contacting a sample with a library comprising first and second polybarcode reagents, wherein each polybarcode reagent comprises: (i) first and second barcode molecules linked together, wherein each barcode molecule comprises a nucleic acid sequence which optionally comprises an adaptor region and a barcode region in the 5' to 3' direction, and (ii) first and second barcoded oligonucleotides, wherein the first barcoded oligonucleotide comprises a barcode region bonded to the barcode region of the first barcode molecule, wherein the second barcoded oligonucleotide comprises a barcode region bonded to the barcode region of the second barcode molecule, and wherein the barcode regions of the first and second barcoded oligonucleotides of the first polybarcode reagent of the library are different from the barcode regions of the first and second barcoded oligonucleotides of the second polybarcode reagent of the library; wherein the sample is further contacted with first and second adaptor oligonucleotides of each polybarcode reagent, wherein the first and second adaptor oligonucleotides each comprise an adaptor region; (b) bonding or ligating the first and second adaptor oligonucleotides for the first polybarcode reagent to first and second fragments of a target nucleic acid (e.g., genomic DNA) of a first micron particle, and bonding or ligating the first and second adaptor oligonucleotides for the second polybarcode reagent to first and second fragments of a target nucleic acid (e.g., genomic DNA) of a second micron particle; (c) for each polybarcode reagent, bonding the adaptor region of the first adaptor oligonucleotide to the adaptor region of the first barcode molecule, and bonding the adaptor region of the second adaptor oligonucleotide to the adaptor region of the second barcode molecule; and (d) for each polybarcode reagent, ligating the 3' end of the first barcoded oligonucleotide to the 5' end of the first adaptor oligonucleotide to produce a first barcoded target nucleic acid molecule, and ligating the 3' end of the second barcoded oligonucleotide to the 5' end of the second adaptor oligonucleotide to produce a second barcoded target nucleic acid molecule.

[0381] In the method, step (b) can include ligating first and second adapter oligonucleotides of a first polybarcode reagent to first and second fragments of a target nucleic acid (e.g., genomic DNA) of a first micron particle, and ligating first and second adapter oligonucleotides of a second polybarcode reagent to first and second fragments of a target nucleic acid (e.g., genomic DNA) of a second micron particle, and wherein: (i) for each polybarcode reagent, step (d) includes ligating the 3'-end of a first barcoded oligonucleotide to the 5'-end of the first adapter oligonucleotide to produce a first barcoded adapter oligonucleotide, and ligating the 3'-end of a second barcoded oligonucleotide to the 5'-end of the second adapter oligonucleotide to produce a second barcoded adapter oligonucleotide, and extending the first and second barcoded adapter oligonucleotides to produce first and second different barcoded target nucleic acid molecules, each of the target nucleic acid molecules comprising at least one nucleotide synthesized from a fragment of the target nucleic acid as a template, or (ii) for each polybarcode reagent, prior to step (d), the method includes extending the first and second adapter oligonucleotides to produce first and second different target nucleic acid molecules, each of the target nucleic acid molecules comprising at least one nucleotide synthesized from a fragment of the target nucleic acid as a template.

[0382] In the method, prior to the step of ligating or connecting the first and second adapter oligonucleotides to the first and second fragments of the target nucleic acid, the method can include attaching a coupling sequence to each fragment of the target nucleic acid, wherein the first and second adapter oligonucleotides are then ligated or connected to the coupling sequences of the first and second fragments of the target nucleic acid.

[0383] In any of the methods described herein, the method can include a step of crosslinking fragments of a target nucleic acid (e.g., genomic DNA) in the micron particles. The step can be carried out with a chemical crosslinking agent such as formaldehyde, paraformaldehyde, glutaraldehyde, succinimidyl glutarate, ethylene glycol bis(succinimidyl succinate), homobifunctional crosslinking agents or heterobifunctional crosslinking agents. This step can be carried out as follows: before any permeabilization step, after any permeabilization step, before any partitioning step, before any step of attaching a coupling sequence, after any step of attaching a coupling sequence, before any step of attaching a barcode sequence (e.g., before step (b)), after any step of attaching a barcode sequence (e.g., after step (d)), while attaching a barcode sequence, or any combination thereof. For example, a sample containing micron particles can be crosslinked before contacting the sample containing micron particles with a library of two or more polybarcode reagents. Any such crosslinking step can be further ended by a quenching step, e.g., quenching a formaldehyde crosslinking step by mixing with a glycine solution. Any such crosslinking can be removed prior to a particular subsequent step of the protocol (e.g., before primer extension, PCR or nucleic acid purification steps).

[0384] In the method, during steps (b), (c) and / or (d) (i.e., the steps of attaching barcode sequences), the micron particles and / or fragments of the target nucleic acid may be included in any of the following: gels or hydrogels, such as agarose gels, polyacrylamide gels or any covalently crosslinked gels, such as covalently crosslinked poly(ethylene glycol) gels, or covalently crosslinked gels comprising a mixture of thiol-functionalized poly(ethylene glycol) and acrylate-functionalized poly(ethylene glycol).

[0385] In any method described herein, optionally after any crosslinking step, the method may include permeabilizing the micron particles. The micron particles may be permeabilized by an incubation step. The incubation step may be carried out in the presence of a chemical surfactant. Optionally, this permeabilization step may be carried out before attaching the barcode sequence (e.g., before step (b)), after attaching the barcode sequence (e.g., after step (d)), or before and after attaching the barcode sequence. The incubation step may be performed at a temperature of at least 20 °C, at least 30 °C, at least 37 °C, at least 45 °C, at least 50 °C, at least 60 °C, at least 65 °C, at least 70 °C or at least 80 °C. 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 carried out as follows: after any crosslinking step, before any permeabilization step, after any permeabilization step, before any partitioning step, before any step of attaching a coupling sequence, after any step of attaching a coupling sequence, before any step of attaching a barcode sequence (e.g., before step (b)), after any step of attaching a barcode sequence (e.g., after step (d)), while attaching the barcode sequence, or any combination thereof. For example, before contacting a sample comprising micron particles with a library of two or more polybarcode reagents, the sample comprising micron particles may be crosslinked and then permeabilized in the presence of a chemical surfactant.

[0386] In any of the methods described herein, the micron particle sample can be digested with a protease digestion step, for example, digested with Proteinase K. Optionally, this protease digestion step can 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 6 hours long, at least 12 hours long, or at least 24 hours long. This step can be carried out as follows: after any cross-linking step, before any permeabilization step, after any permeabilization step, before any partitioning step, before any step of attaching a coupling sequence, after any step of attaching a coupling sequence, before any step of attaching a barcode sequence (e.g., before step (b)), after any step of attaching a barcode sequence (e.g., after step (d)), simultaneously with attaching a barcode sequence, or any combination thereof. For example, before contacting a sample containing micron particles with a library of two or more polybarcode reagents, the sample containing micron particles can be cross-linked and then partially digested with a Proteinase K digestion step.

[0387] In the method, steps (a) and (b), and optionally (c) and (d) can be carried out on at least two micron particles in a single reaction volume.

[0388] The method can further comprise a step of partitioning the nucleic acid sample into at least two different reaction volumes before step (b).

[0389] The present invention provides a method for analyzing a sample, the sample comprising micron particles derived from blood, wherein the micron particles contain at least two fragments of a target nucleic acid (e.g., genomic DNA), and wherein the method comprises: (a) preparing a sample for sequencing, comprising: (i) contacting the sample with a polybarcode reagent comprising a first and a second barcode region linked together, wherein each barcode region comprises a nucleic acid sequence, and (ii) attaching the barcode sequence to each of at least two fragments of the target nucleic acid of the micron particles to produce first and second different barcoded target nucleic acid molecules, wherein the first barcoded target nucleic acid molecule comprises the nucleic acid sequence of the first barcode region, and the second barcoded target nucleic acid molecule comprises the nucleic acid sequence of the second barcode region; and (b) sequencing each barcoded target nucleic acid molecule to produce at least two linked sequence reads.

[0390] In the method, before attaching the barcode sequence to each of at least two fragments of the genomic DNA of the micron particles, the method can comprise attaching a coupling sequence to each fragment of the genomic DNA of the micron particles, wherein then the barcode sequence is attached to the coupling sequence of each of at least two fragments of the genomic DNA of the micron particles to produce first and second different barcoded target nucleic acid molecules.

[0391] During step (a), the micron particles and / or fragments of the target nucleic acid can be included in any of the following: gels or hydrogels, such as agarose gels, polyacrylamide gels, or any covalently crosslinked gels, such as covalently crosslinked poly(ethylene glycol) gels, or covalently crosslinked gels containing a mixture of thiol-functionalized poly(ethylene glycol) and acrylate-functionalized poly(ethylene glycol).

[0392] The micron particle sample can be digested with a protease digestion step, such as digestion with Proteinase K enzyme. Optionally, this protease digestion step can 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 6 hours long, at least 12 hours long, or at least 24 hours long. This step can be performed as follows: before permeabilization, after permeabilization, before attaching the barcode sequence (e.g., before step (a)(ii)), after attaching the barcode sequence (e.g., after step (a)(ii)), simultaneously with attaching the barcode sequence, or any combination thereof.

[0393] Step (a) of the method can be performed by any method for preparing a sample (or nucleic acid sample) for sequencing described herein.

[0394] The method can include preparing first and second samples for sequencing, where each sample includes at least one micron particle derived from blood, where each micron particle contains at least two fragments of the target nucleic acid (e.g., genomic DNA), and where the barcode sequences each include a sample identifier region, and where the method includes: (i) performing step (a) on each sample, where the barcode sequence attached to the target nucleic acid fragments from the first sample has a different sample identifier region than the barcode sequence attached to the target nucleic acid fragments from the second sample; (ii) performing step (b) on each sample, where each ligated sequence read includes the sequence of the sample identifier region; and (iii) determining the sample from which each ligated sequence read was obtained by the sample identifier region. The method can include preparing first and second samples for sequencing, where each sample includes at least one micron particle derived from blood, where each micron particle contains the target nucleic acid (e.g., genomic DNA) at least two fragments, and where the barcode sequences each include a sample identifier region, and where the method includes: (i) performing step (a) on each sample, where the barcode sequence attached to the target nucleic acid fragments from the first sample has a different sample identifier region than the barcode sequence attached to the target nucleic acid fragments from the second sample; (ii) performing step (b) on each sample, where each sequence read includes the sequence of the sample identifier region; and (iii) determining the sample from which each ligated sequence read was obtained by the sample identifier region.

[0395] The method may include analyzing a sample comprising at least two blood-derived micron particles, wherein each micron particle contains at least two fragments of a target nucleic acid (e.g., genomic DNA), and wherein the method comprises the steps of: (a) preparing a sample for sequencing, comprising: (i) contacting the sample with a library of polymeric barcoding reagents, the library comprising a polymeric barcoding reagent for each of two or more micron particles, wherein each polymeric barcoding reagent is as defined herein; and (ii) attaching a barcode sequence to each of at least two fragments of the target nucleic acid of each micron particle, wherein at least two barcoded target nucleic acid molecules are produced from each of at least two micron particles, and wherein at least two barcoded target nucleic acid molecules produced from a single micron particle each contain a nucleic acid sequence from the barcode region of the same polymeric barcoding reagent; and (b) sequencing each barcoded target nucleic acid molecule to generate at least two contiguous sequence reads for each micron particle.

[0396] The barcode sequence may be attached to a fragment of the genomic DNA of the micron particle in a single reaction volume, i.e., step (a) of the method may be performed in a single reaction volume.

[0397] Prior to the attachment step (step (a)(ii)), the method may further comprise the step of dividing the sample into at least two different reaction volumes.

[0398] In any method, prior to the step of attaching the barcode sequence, the polymeric barcoding reagent may be separated, fractionated or solubilized into two or more components, e.g., releasing barcoded oligonucleotides.

[0399] In any method, the concentration of the polymeric barcoding reagent may be less than 1.0 femtomole, less than 10 femtomoles, less than 100 femtomoles, less than 1.0 picomole, less than 10 picomoles, less than 100 picomoles, less than 1 nanomole, less than 10 nanomoles, less than 100 nanomoles or less than 1.0 micromole.

[0400] 6. Ligation by ligating the fragments together

[0401] The present invention provides a method for analyzing a sample comprising blood-derived micron particles, wherein the micron particles contain at least two fragments of a target nucleic acid (e.g., genomic DNA), and wherein the method comprises: (a) preparing a sample for sequencing, comprising ligating together at least two fragments of the target nucleic acid of the micron particle to produce a single nucleic acid molecule comprising the sequences of at least two fragments of the target nucleic acid; and (b) sequencing each fragment in the single nucleic acid molecule to generate at least two contiguous sequence reads.

[0402] At least two fragments of the target nucleic acid (e.g., genomic DNA) may be contiguous in the single nucleic acid molecule.

[0403] At least two ligated sequence reads can be provided within a single original sequence read.

[0404] The method can include attaching a coupling sequence to at least one fragment of a target nucleic acid (e.g., genomic DNA) prior to a ligation step and then ligating together at least two fragments of the target nucleic acid through the coupling.

[0405] Fragments of a target nucleic acid (e.g., genomic DNA) can be ligated together via a solid support, where two or more fragments are ligated to the same solid support (directly or indirectly, e.g., via a coupling sequence). Optionally, the solid support is a bead, such as a polystyrene foam bead, a superparamagnetic bead, or an agarose bead.

[0406] Fragments of a target nucleic acid (e.g., genomic DNA) can be ligated together via a ligation reaction, such as a double-strand ligation reaction or a single-strand ligation reaction.

[0407] The ends of fragments of a target nucleic acid can be converted to blunt ligatable double-strand ends in a blunting reaction, and the method can include ligating two or more fragments to each other via a blunt-end ligation reaction.

[0408] The ends of fragments of a target nucleic acid can be contacted with a restriction enzyme, where the restriction enzyme digests the fragments at restriction sites to create ligation junctions at these restriction sites, and where the method can include ligating two or more of the fragments to each other via a ligation reaction at the ligation junctions. Any target nucleic acid can be contacted with a restriction enzyme, where the restriction enzyme digests the fragment at a restriction site to create a ligation junction at this restriction site, and where the method can include ligating two or more fragments to each other via a ligation reaction at the ligation junction. Optionally, the restriction enzyme can be EcoRI, HindIII, or BglII.

[0409] Prior to ligating the fragments together, a coupling sequence can be attached to two or more fragments of a target nucleic acid. Optionally, two or more different coupling sequences are attached to a group of fragments of a target nucleic acid.

[0410] The coupling sequence can include a ligation junction at at least one end, and where a first coupling sequence is attached to a first fragment of a target nucleic acid, and where a second coupling sequence is attached to a second fragment of a target nucleic acid, and where the two coupling sequences are ligated to each other, thereby ligating together two fragments of the target nucleic acid.

[0411] The coupling sequences may comprise an adhesion region at at least one 3'-end, and wherein a first coupling sequence is attached to a first fragment of the target nucleic acid, and wherein a second coupling sequence is attached to a second fragment of the target nucleic acid, and wherein the two coupling sequences are complementary and adhere to each other along a segment of at least one nucleotide in length, and wherein a DNA polymerase is used to extend at least one of the 3'-ends of the first coupling sequence of at least one nucleotide into the sequence of the second fragment of the target nucleic acid, thereby ligating two fragments of the target nucleic acid (e.g., genomic DNA) together.

[0412] Prior to ligating at least two fragments together, the method may further comprise the step of crosslinking the micron particles, for example, with a chemical crosslinking agent such as formaldehyde, paraformaldehyde, glutaraldehyde, succinimidyl glutarate, ethylene glycol bis(succinimidyl succinate), homobifunctional crosslinking agent or heterobifunctional crosslinking agent.

[0413] Prior to ligating at least two fragments together, the method may further comprise dividing the micron particles into two or more partitions.

[0414] The method may further comprise permeabilizing the micron particles during the incubation step. This step may be performed before the partitioning (if performed), after the partitioning (if performed), before ligating the fragments together and / or after ligating the fragments together.

[0415] The incubation step may be performed in the presence of a chemical surfactant such as Triton X-100 (C 14 H 22 O(C2H4O) n (n = 9-10)), NP-40, Tween 20, Tween 80, saponin, digitonin or sodium dodecyl sulfate.

[0416] The incubation step is performed at a temperature of at least 20 °C, at least 30 °C, at least 37 °C, at least 45 °C, at least 50 °C, at least 60 °C, at least 65 °C, at least 70 °C, at least 80 °C, at least 90 °C or at least 95 °C.

[0417] The incubation step is 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.

[0418] The method may include a protease digestion step, such as proteinase K digestion, to digest the micron particle sample. Optionally, this protease 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 6 hours long, at least 12 hours long or at least 24 hours long. This step may be performed before partitioning (if performed), after partitioning (if performed), before joining the fragments together and / or after joining the fragments together.

[0419] The method may include amplifying (original) fragments of the target nucleic acid and then joining together two or more resulting nucleic acid molecules.

[0420] The step of joining the fragments together may produce a concatemerized nucleic acid molecule comprising 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 that have been attached to each other to form a single continuous nucleic acid molecule.

[0421] The method can be used to generate 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, at least 10,000,000,000 or at least 100,000,000,000 ligated sequence reads of micron particles.

[0422] The sample may include at least two blood-derived micron particles, where each micron particle contains at least two fragments of the target nucleic acid (e.g., genomic DNA), and where the method includes performing step (a) to generate a single nucleic acid molecule comprising the sequences of at least two fragments of the target nucleic acid for each micron particle, and performing step (b) to generate ligated sequence reads for each micron particle.

[0423] Before, during and / or after the step of joining together at least two fragments of the target nucleic acid (e.g., genomic DNA), the method may include a step of crosslinking the fragments of the target nucleic acid in the micron particles. The crosslinking step may be performed with a chemical crosslinker such as formaldehyde, paraformaldehyde, glutaraldehyde, succinimidyl glutarate, ethylene glycol bis(succinimidyl succinate), homobifunctional crosslinkers or heterobifunctional crosslinkers.

[0424] Prior to, during, and / or after the step of ligating together at least two fragments of a target nucleic acid (e.g., genomic DNA), and / or optionally after the step of crosslinking fragments of the target nucleic acid in the micron particles, the method comprises the step of permeabilizing the micron particles.

[0425] Prior to step (a), the method may further comprise the step of partitioning a nucleic acid sample into at least two different reaction volumes.

[0426] In one embodiment of a method of ligating together at least two fragments of a target nucleic acid of a circularized micron particle to produce a single nucleic acid molecule comprising the sequences of at least two fragments of the target nucleic acid, a sample comprising at least one circularized micron particle (e.g., wherein the sample is obtained and / or purified by any method disclosed herein) is crosslinked in a 1% formaldehyde solution at room temperature for 10 minutes and then the formaldehyde crosslinking step is quenched with glycine. The micron particles are pelleted by a centrifugation step (e.g., centrifugation at 3000 x G for 5 minutes) and resuspended in 1×NEBuffer 2 (New England Biolabs) containing 1.0% sodium dodecyl sulfate (SDS) and incubated at 45 degrees Celsius for 10 minutes to permeabilize the micron particles. The SDS is quenched by adding Triton X-100 and the solution is incubated overnight at 37 degrees Celsius with AluI (New England Biolabs) to generate blunt, ligatable ends. The enzyme is inactivated by adding SDS to a final concentration of 1.0% and incubated at 65 degrees Celsius for 15 minutes. The SDS is quenched by adding Triton X-100 and the solution is diluted at least 10-fold in 1×T4 DNA ligase buffer and the total concentration of DNA is at most 1.0 ng DNA / μL. The diluted solution is incubated overnight at 16 degrees Celsius with T4 DNA ligase to ligate together the fragments from the circularized micron particles. The crosslinking is then reversed and the protein component is degraded by incubating overnight at 65 degrees Celsius 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). Then Illumina sequencing adapter sequences are attached using the Nextera in vitro transposition method (Illumina; according to the manufacturer's protocol), an appropriate number of PCR cycles are performed to amplify the ligated material; and then the amplified and purified DNA of appropriate size is sequenced on an Illumina sequencer (e.g., an Illumina NextSeq 500 or MiSeq), with paired-end reads of at least 50 bases each. Each end of the paired-end sequences is independently mapped to a reference human genome to elucidate the ligated sequence reads (e.g., reads wherein both ends contain sequences of different fragments of genomic DNA from a single circularized micron particle).

[0427] Methods of joining together at least two fragments of a target nucleic acid of a micron particle to produce a single nucleic acid molecule comprising the sequences of at least two fragments of the target nucleic acid can have a variety of unique properties and characteristics, making them a desired method for joining sequences from one or more circulating micron particles. In one aspect, such methods can join sequences from circulating micron particles without the need for complex instrumentation (e.g., microfluidics for partition-based methods). Additionally, the methods are (broadly) capable of being performed in a single, separate reaction, which can contain a large number of circulating micron particles (e.g., hundreds, thousands, or greater numbers), and thus are capable of processing a large number of circulating micron particles without the need for multiple reactions that might otherwise be required in combinatorial indexing methods. Further, since the methods do not necessarily require the use of barcodes and / or polybarcode reagents, they are not limited by the size of the barcode library (and / or polybarcode reagent library) to enable useful molecular measurements of the joined sequences from circulating micron particles.

[0428] 7. Ligation by partitioning

[0429] The method can be performed on a nucleic acid sample comprising at least two micron particles that have been divided into at least two different reaction volumes (or partitions).

[0430] In any method, a nucleic acid sample comprising at least two micron particles can be divided into at least two different reaction volumes (or partitions). The different reaction volumes (or partitions) can be provided by different reaction vessels (or different physical reaction vessels). The different reaction volumes (or partitions) can be provided by different aqueous droplets, such as different aqueous droplets within an emulsion or different aqueous droplets on a solid support (e.g., a glass slide).

[0431] For example, the nucleic acid sample can be partitioned before attaching a barcode sequence to a fragment of the target nucleic acid of the micron particle. Alternatively, the nucleic acid sample can be partitioned before joining together at least two fragments of the target nucleic acid of the micron particle.

[0432] For any method involving a partitioning step, any step of the method after the partitioning step can be performed independently on each partition, such as any step of attaching a barcode sequence or an attachment conjugate sequence, or any step of ligation, bonding, primer extension, or PCR. Reagents (e.g., oligonucleotides, enzymes, and buffers) can be added directly to each partition. In methods where the partition comprises an aqueous droplet in an emulsion, such addition steps can be carried out via a process of merging the aqueous droplets in the emulsion, such as using a microfluidic droplet merger conduit, and optionally using mechanical or thermal mixing steps.

[0433] The partition contains different droplets of an aqueous solution within an emulsion, where the emulsion is a water-in-oil emulsion, and where the droplets are generated by a physical shaking or vortexing step, or where the droplets are generated by combining an aqueous solution and an oil solution within a microfluidic conduit or junction.

[0434] For methods where the partition contains aqueous droplets within an 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).

[0435] The partition can contain different physically non-overlapping spatial volumes within a gel or hydrogel, such as an agarose gel, a polyacrylamide gel, or any covalently cross-linked gel, such as a covalently cross-linked poly(ethylene glycol) gel, or a covalently cross-linked gel containing a mixture of thiol-functionalized poly(ethylene glycol) and acrylate-functionalized poly(ethylene glycol).

[0436] The micron particle sample can be divided into a total of 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, at least 100,000,000, or at least 1,000,000,000 partitions. Preferably, the micron particle solution is divided into a total of at least 1000 partitions.

[0437] The micron particle sample can be divided into multiple partitions such that there is an average of less than 0.0001 micron particles, less than 0.001 micron particles, less than 0.01 micron particles, less than 0.1 micron particles, less than 1.0 micron particles, less than 10 micron particles, less than 100 micron particles, less than 1000 micron particles, less than 10,000 micron particles, less than 100,000 micron particles, less than 1,000,000 micron particles, less than 10,000,000 micron particles, or less than 100,000,000 micron particles per partition. Preferably, there is an average of less than 1.0 micron particles per partition.

[0438] The micron particle solution can be divided into multiple partitions such that each partition has an average of less than 1.0 attogram of DNA, less than 10 attograms of DNA, less than 100 attograms 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 partition has less than 10 picograms of DNA.

[0439] The volume of the partition can be 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.

[0440] A barcode sequence can be provided in each partition. For each of two or more partitions containing a barcode sequence, the barcode sequence contained therein can contain multiple copies of the same barcode sequence or different barcode sequences from the same set of barcode sequences.

[0441] After dividing the micron particles into two or more partitions, the micron particles can be permeabilized by a culturing step by any method described herein.

[0442] The micron particle sample can be digested with a protease digestion step, such as digestion with Proteinase K. Optionally, this protease digestion step can 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 6 hours long, at least 12 hours long, or at least 24 hours long. This step can be performed before partitioning, after partitioning, before attaching the barcode sequence, after attaching the barcode sequence, and / or simultaneously with attaching the barcode sequence.

[0443] Attaching sequences by the combinatorial barcode process

[0444] The method of attaching a barcode sequence can include at least two steps of the combinatorial barcode process, wherein a first barcode step is performed, wherein the micron particle sample is divided into two or more partitions, wherein each partition contains a different barcode sequence or a different set of barcode sequences, which are then attached to the sequences of target nucleic acids (such as genomic DNA) fragments from the micron particles contained within the partition, and wherein the barcoded nucleic acid molecules of at least two partitions are combined into a second sample mixture, and wherein this second sample mixture is then divided into two or more new partitions, wherein each new partition contains a different barcode sequence or a different set of barcode sequences, which are then attached to the sequences of target nucleic acids (such as genomic DNA) fragments from the micron particles contained within the two or more new partitions.

[0445] Optionally, the combinatorial barcoding process may comprise a first barcoding step, wherein: A) a first sample mixture comprising at least first and second cyclic microparticles is divided into at least first and second original partitions (e.g., wherein at least first cyclic microparticles from the sample are divided into a first original partition and wherein at least second cyclic microparticles from the sample are divided into a second original partition), wherein the first original partition comprises a barcode sequence (or set of barcode sequences) different from the barcode sequence (or set of barcode sequences) contained within the second original partition, and wherein the barcode sequence (or barcode sequence from the set of barcode sequences) contained within the first original partition is attached to at least first and second fragments of the target nucleic acid of the first cyclic microparticle, and wherein the barcode sequence (or barcode sequence from the set of barcode sequences) contained within the second original partition is attached to at least first and second fragments of the target nucleic acid of the second cyclic microparticle; and wherein at least one cyclic microparticle contained within the first original partition and at least one cyclic microparticle contained within the second original partition are combined to produce a second sample mixture, and a second barcoding step, wherein: B) the microparticles contained within the second sample are divided into at least first and second new partitions (e.g., wherein at least first cyclic microparticles from the second sample mixture are divided into a first new partition and wherein at least second cyclic microparticles from the second sample mixture are divided into a second new partition), wherein the first new partition comprises a barcode sequence (or set of barcode sequences) different from the barcode sequence (or set of barcode sequences) contained within the second new partition, and wherein the barcode sequence (or barcode sequence from the set of barcode sequences) contained within the first new partition is attached to at least first and second fragments of the target nucleic acid of the first cyclic microparticle, and wherein the barcode sequence (or barcode sequence from the set of barcode sequences) contained within the second new partition is attached to at least first and second fragments of the target nucleic acid of the second cyclic microparticle.

[0446] An alternative process for the combinatorial barcoding process is described in PCT / GB2017 / 053820, which is incorporated herein by reference.

[0447] Optionally, in any combinatorial barcoding process, one or more chemical cross-linking steps may be performed before and / or after any step in any combinatorial barcoding process.

[0448] Optionally, in any combinatorial barcoding process, in a step after the chemical cross-linking step, the cross-linked microparticles may be permeabilized. Other details are provided in PCT / GB2017 / 053820, which is incorporated herein by reference.

[0449] Optionally, in any combinatorial barcoding process, at any one or more steps after the chemical crosslinking step, the crosslinking can be partially or fully reversed. Other details are provided in PCT / GB2017 / 053820, which is incorporated herein by reference.

[0450] Optionally, in any combinatorial barcoding process, barcode sequences can be attached by any one or more of the methods described herein (e.g., single-strand ligation, double-strand ligation, blunt-end ligation, A-tailing, sticky-end-mediated ligation, hybridization, hybridization and extension, hybridization and extension and ligation and / or transposition).

[0451] Optionally, at 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 5000, at least 10,000, at least 50,000, at least 100,000, at least 500,000, or at least 1,000,000 cyclic microparticles can be included within a partition (and / or within each of at least a first and a second partition; and / or within any greater number of partitions). Preferably, at least 50 cyclic microparticles can be included within a partition (and / or within each of at least a first and a second partition; and / or within any greater number of partitions).

[0452] Optionally, at 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 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 partitions can be employed (e.g., the cyclic microparticles can be divided into the number of partitions). Preferably, at any step of any combinatorial barcoding process, at least 24 partitions can be employed (e.g., the cyclic microparticles can be divided into the number of partitions).

[0453] Optionally, at any step of any combinatorial barcoding process, the micron particle sample can be divided into multiple partitions such that there are on average fewer than 0.0001 micron particles, fewer than 0.001 micron particles, fewer than 0.01 micron particles, fewer than 0.1 micron particles, fewer than 1.0 micron particles, fewer than 10 micron particles, fewer than 100 micron particles, fewer than 1000 micron particles, fewer than 10,000 micron particles, fewer than 100,000 micron particles, fewer than 1,000,000 micron particles, fewer than 10,000,000 micron particles, or fewer than 100,000,000 micron particles in each partition. Preferably, there are on average fewer than 1.0 micron particles in each partition.

[0454] Optionally, at any step of any combinatorial barcoding process, the micron particle solution can be divided into multiple partitions such that there is less than 1.0 attogram of DNA, less than 10 attograms of DNA, less than 100 attograms 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 in each partition. Preferably, there is less than 10 picograms of DNA in each partition.

[0455] Optionally, at any step of any combinatorial barcoding process, the volume of the partition can be 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.

[0456] Optionally, any combinatorial barcoding process can 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 barcoding step can be as described herein for the first and second barcoding steps.

[0457] Optionally, in any combinatorial barcoding process, any one or more dispensing steps can include random features - for example, an estimated number (rather than an exact or precise number) of circulating micron particles can be divided into one or more partitions; that is, the number of the circulating micron particles in each partition can be affected by statistical or probabilistic uncertainties (e.g., affected by Poisson loading and / or distribution statistics).

[0458] Optionally, in any combinatorial barcoding process, a set of barcodes attached to a specific sequence (e.g., a sequence attached to a fragment of genomic DNA; e.g., a set comprising 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) can be used to link sequences from a single micron particle and / or link sequences from a set of two or more micron particles. Optionally, in any combinatorial barcoding process, the same set of two (or more than two) barcodes can be attached to a specific sequence from two or more cycling micron particles (e.g., a sequence attached to a fragment of genomic DNA) (e.g., where the two or more cycling micron particles are each divided into the same series of first and second partitions during first and second barcoding steps, respectively). Optionally, in any combinatorial barcoding process, the same set of two (or more than two) barcodes can be attached to a specific sequence from only one cycling micron particle (e.g., a sequence attached to a fragment of genomic DNA) (e.g., where only one cycling micron particle is divided into a specific series of first and second partitions during first and second barcoding steps, respectively).

[0459] Optionally, in any combinatorial barcoding process, the number of partitions employed in any one or more barcoding steps, and the number of different barcoding steps can be combined in a combinatorial manner such that, on average, each set of two (or more) barcodes is attached to a sequence from only one cycling micron particle. Other details are provided in PCT / GB2017 / 053820, which is incorporated herein by reference.

[0460] The combinatorial barcoding process can offer advantages over alternative barcoding processes in the form of reduced need for precision and / or complex equipment to obtain a large number of potential identifying barcode sets for attaching barcodes to sequences from cycling micron particles (e.g., from fragments of genomic DNA). For example, a combinatorial barcoding process using 96 different partitions across two different barcoding steps (e.g., easily implemented in the case of a standard 96-well plate widely used in molecular biology) can obtain a net of (96 × 96 =) 9216 different barcode sets; this significantly reduces the amount of partitions required to perform such indexing compared to alternative non-combinatorial methods. By increasing the number of barcoding steps and / or increasing the number of partitions employed in one or more such barcoding steps, significantly higher levels of combinatorial indexing resolution can be additionally achieved. Furthermore, the combinatorial barcoding process can obviate the need for complex instrumentation (e.g., microfluidic instrumentation (e.g., the 10X Genomics Chromium system)) used for alternative barcoding methods.

[0461] 8. Ligation by spatial sequencing or in situ sequencing or in situ library construction

[0462] The present invention provides a method for preparing a sample for sequencing, wherein the sample comprises micron-sized particles derived from blood, and wherein the micron-sized particles contain at least two fragments of a target nucleic acid (e.g., genomic DNA), and wherein the method comprises: (a) preparing a sample for sequencing, wherein at least two fragments of the target nucleic acid of the micron-sized particles are joined by their proximity to each other on a sequencing device to produce a collection of at least two joined fragments of the target nucleic acid; and (b) sequencing each joined fragment of the target nucleic acid using a sequencing device to produce at least two joined sequence reads.

[0463] The nucleic acid sample may comprise at least two micron-sized particles derived from blood, wherein each micron-sized particle contains at least two fragments of a target nucleic acid (e.g., genomic DNA), and wherein the method comprises performing step (a) to produce a collection of joined fragments of the target nucleic acid for each micron-sized particle, and wherein the fragments of the target nucleic acid of each micron-sized particle are spatially distinct on the sequencing device, and performing step (b) to produce joined sequence reads for each micron-sized particle.

[0464] At least two fragments from the micron-sized particles may maintain their physical proximity to each other within or on the sequencing device itself, and wherein this physical proximity is known or can be determined or observed by or during the operation of the sequencing device, and wherein this measure of physical proximity is used to join at least two sequences.

[0465] The method may comprise sequencing using an in situ library construction process. In the method, intact or partially intact micron-sized particles from the sample may be placed on a sequencer, and wherein two or more fragments of a target nucleic acid (e.g., genomic DNA) are processed into templates that can be sequenced within the sequencer, i.e., sequencing using an in situ library construction process. In situ library construction is described in Schwartz et al. (2012) Proceedings of the National Academy of Sciences of the United States of America (PNAS) 109(46):18749-54).

[0466] The method may comprise in situ sequencing. In the method, the sample may remain intact (e.g., mostly or partially intact), and fragments of a target nucleic acid (e.g., genomic DNA) within the micron-sized particles may be directly sequenced, e.g., using the 'FISSEQ' fluorescence in situ sequencing technique method as described in Lee et al. (2014) Science, 343, 6177, 1360-1363.

[0467] Optionally, the micron particle sample can be cross-linked with a chemical cross-linking agent and then placed within or on a sequencing device and then maintained in physical proximity to each other. Optionally, two or more fragments of a target nucleic acid (e.g., genomic DNA) from the micron particles placed within or on the sequencing device can then have all or part of their sequences determined by a sequencing process. Optionally, such fragments can be sequenced by fluorescence in situ sequencing techniques, where the sequence of the fragment is determined by an optical sequencing process. Optionally, one or more coupling sequences, adapter sequences, or amplification sequences can be attached to the fragments of the target nucleic acid. Optionally, the fragments can be amplified during an amplification process, where the amplification product remains in physical proximity or physical contact with the fragment that amplified it. Optionally, these amplification products are then sequenced by an optical sequencing process. Optionally, the amplification products are attached to a flat surface, such as a sequencing flow cell. Optionally, the amplification products generated from a single fragment each constitute a single cluster within the flow cell. Optionally, in any of the above methods, the distance between any two or more sequencing molecules is known a priori by the configuration within the sequencing device or can be determined or observed during the sequencing process. Optionally, each sequencing molecule is mapped within the area of a cluster or within an array of pixels, where the distance between any two or more sequencing molecules is determined by the distance between the clusters or pixels. Optionally, any measure or estimate of distance or proximity can be used to join any two or more determined sequences.

[0468] Optionally, the sequences determined by any of the above methods can be further evaluated, where a measure of the distance or proximity between two or more sequencing molecules is compared to one or more cut-off values or thresholds, and only the molecules within a particular range, or above or below a particular threshold or cut-off value, are determined to be informationally joined. Optionally, two or more such cut-off values or thresholds or a set of their ranges can be employed such that different degrees and / or classifications and / or categories of joining can be determined for any two or more sequencing molecules.

[0469] 9. Joining by a Separate Sequencing Process

[0470] The present invention provides a method for preparing a sample for sequencing, where the sample comprises micron particles derived from blood, and where the micron particles contain at least two fragments of a target nucleic acid (e.g., genomic DNA), and where the method comprises: (a) preparing a sample for sequencing, where at least two fragments of the target nucleic acid (e.g., genomic DNA) of each micron particle are joined by loading into a separate sequencing process to produce a set of at least two joined fragments of the target nucleic acid; and (b) sequencing each joined fragment of the target nucleic acid using a sequencing device to produce a set of at least two joined sequence reads (i.e., a set of at least two joined signals).

[0471] The sample may comprise at least two blood-derived micron particles, wherein each micron particle contains at least two fragments of a target nucleic acid (e.g., genomic DNA), and the method may comprise performing step (a) to generate a ligated fragment of the target nucleic acid of each micron particle, wherein at least two fragments of the target nucleic acid of each micron particle are ligated by loading into separate sequencing processes, and performing step (b) on each sequencing process to generate a ligated sequence read for each micron particle.

[0472] In the method, the fragments of the first single micron particle (or group of micron particles) can be sequenced independently of the fragments of other micron particles, and the resulting sequence reads are informationally ligated; the fragments contained in the second single micron particle (or group of micron particles) are sequenced independently of the first micron particle or group of micron particles, and the resulting sequence reads are informationally ligated.

[0473] Optionally, the first and second sequencing processes (in all sequencing processes) are performed on different sequencing instruments, and / or on the same sequencing instrument, but at two different times or in two different sequencing runs. Optionally, the first and second sequencing processes are performed on the same sequencing instrument, but in two different regions, partitions, compartments, conduits, flow cells, channels, nanopores, microcarriers, microcarrier arrays or integrated circuits of the sequencing instrument. Optionally, 3 or more, 10 or more, 1000 or more, 1,000,000 or more or 1,000,000,000 or more micron particles or groups of micron particles can be ligated by the above method.

[0474] 10. Amplify the original fragments before ligation

[0475] As will be understood by those skilled in the art, as used herein, the term 'fragment' (e.g., 'fragment of genomic DNA' or 'fragment of target nucleic acid' or 'fragment of genomic DNA belonging to / from a micron particle') refers to the original fragment present in the micron particle and its parts, copies or amplicons, including copies of only a part of the original fragment (e.g., its amplicon), and modified fragments or copies (e.g., fragments to which a coupling sequence has been attached). For example, the term fragment of genomic DNA refers to the original genomic DNA fragment present in the micron particle, and for example, a DNA molecule that can be prepared from the fragment of the original genomic DNA by primer extension reaction. As another example, the term mRNA fragment refers to the original mRNA fragment present in the micron particle, and for example, a cDNA molecule 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.

[0476] The method may further include, prior to the step of attaching the barcode sequence, a step of amplifying the original fragment of the target nucleic acid of the micron particles, for example, by a primer extension step or a polymerase chain reaction step. The barcode sequence may then be attached to the amplicon or copy of the original fragment of the target nucleic acid using any of the methods described herein.

[0477] The primer extension step or the polymerase chain reaction step may be carried out using one or more primers containing one or more degenerate base-containing fragments.

[0478] The primer extension step or the polymerase chain reaction step may be carried out using one or more primers specific for a particular target nucleic acid sequence (e.g., a particular target genomic DNA sequence).

[0479] The amplification step may be carried out by a strand displacement polymerase, such as Phi29 DNA polymerase, or Bst polymerase or Bsm polymerase, or a modified derivative of phi29, Bst or Bsm polymerase. The amplification may be carried out by a multiple displacement amplification reaction and a primer set containing a region with one or more degenerate bases. Optionally, random hexamers, random heptamers, random octamers, random nonamers or random decamers are used.

[0480] The amplification step may include extending a single-strand nick in the original target nucleic acid fragment by a DNA polymerase. The nick may be generated by an enzyme with single-stranded DNA cleavage behavior or a sequence-specific nicking restriction endonuclease.

[0481] The amplification step may include incorporating at least one or more dUTP nucleotides into a DNA strand, which is synthesized by replicating or amplifying at least a portion of one or more fragments of genomic DNA by a DNA polymerase, and wherein a nick is generated by a uracil excision enzyme, such as uracil DNA glycosylase.

[0482] The amplification step may include generating a primer sequence on a nucleic acid containing a fragment of genomic DNA, wherein the primer sequence is generated by a primase, such as Thermus Thermophilus PrimPol polymerase or TthPrimPol polymerase, and wherein the DNA polymerase is used to replicate at least one nucleotide of the sequence of the fragment of genomic DNA using this primer sequence as a primer.

[0483] The amplification step may be carried out by a linear amplification reaction, such as an RNA amplification process carried out by an in vitro transcription process.

[0484] The amplification step can be carried out by a primer extension step or a polymerase chain reaction step, and one or more primers used therein are universal primers corresponding to one or more universal primer sequences. The universal primer sequence can be attached to a fragment of genomic DNA by a ligation reaction, by primer extension or polymerase chain reaction, or by an in vitro transposition reaction.

[0485] 11. Attach the coupling sequence to the fragment before ligation

[0486] In any method, the barcode sequence can be attached directly or indirectly (e.g., by adhesion or ligation) to a fragment of the target nucleic acid (e.g., gDNA) of the micron particle. The barcode sequence can be attached to a coupling sequence (e.g., a synthetic sequence) that is attached to the fragment.

[0487] In a method that includes ligating together at least two fragments of the target nucleic acid of the micron particle to produce a single nucleic acid molecule, the coupling sequence can first be attached to each of the at least two fragments, and the fragments can then be ligated together through the coupling sequence.

[0488] The coupling sequence can be attached to the original fragment of the target nucleic acid of the micron particle or its copy or amplicon.

[0489] The coupling sequence can be added to the 5' end or 3' end of two or more fragments of the nucleic acid sample. In this method, the target region (of the barcoded oligonucleotide) can contain a sequence complementary to the coupling sequence.

[0490] The coupling sequence can be included within a double-stranded coupling oligonucleotide or a single-stranded coupling oligonucleotide. The coupling oligonucleotide can be attached to the target nucleic acid by a double-stranded ligation reaction or a single-stranded ligation reaction. The coupling oligonucleotide can contain a single-stranded 5' or 3' region capable of ligating to the target nucleic acid, and the coupling sequence can be attached to the target nucleic acid by a single-stranded ligation reaction.

[0491] The coupling oligonucleotide can contain a blunt, recessed, or protruding 5' or 3' region capable of ligating to the target nucleic acid, and the coupling sequence can be attached to the target nucleic acid by a double-stranded ligation reaction.

[0492] The ends of the target nucleic acid can be converted to blunt double-stranded ends in a blunting reaction, and the coupling oligonucleotide can contain blunt double-stranded ends, and wherein the coupling oligonucleotide can be ligated to the target nucleic acid in a blunt-end ligation reaction.

[0493] One or more ends of the target nucleic acid can be converted to blunt double-stranded ends in a blunting reaction and then converted to a form having one or more single 3' adenosine overhangs, and wherein the coupling oligonucleotide can contain a double-stranded end having a single 3' thymine overhang that is capable of adhering to the single 3' adenosine overhang of the target nucleic acid, and wherein the coupling oligonucleotide is ligated to the target nucleic acid in a double-stranded A / T ligation reaction.

[0494] The target nucleic acid can be contacted with a restriction enzyme, where the restriction enzyme digests the target nucleic acid at a restriction site to produce one or more ligation junctions at the restriction site, and where the coupled oligonucleotide contains ends that are compatible with the ligation junctions, and where the coupled oligonucleotide is then ligated to the target nucleic acid in a double-stranded ligation reaction.

[0495] The coupled oligonucleotide can be attached by primer extension or polymerase chain reaction steps.

[0496] The coupled oligonucleotide can be attached by primer extension or polymerase chain reaction steps using one or more oligonucleotides that contain a primer segment, and the primer segment contains one or more degenerate bases.

[0497] The coupled oligonucleotide can be attached by primer extension or polymerase chain reaction steps using one or more oligonucleotides that further contain a primer or hybridization segment that is specific for a particular target nucleic acid sequence.

[0498] The coupled sequence can be added by a polynucleotide tailing reaction. The coupled sequence can be added by a terminal transferase (such as terminal deoxynucleotidyl transferase). The coupled sequence can be attached by a polynucleotide tailing reaction with terminal deoxynucleotidyl transferase, and where the coupled sequence contains at least two consecutive nucleotides of a homopolymeric sequence.

[0499] The coupled sequence can contain a homopolymeric 3' tail (such as a poly(A) tail). Optionally, in such methods, the target region (of the barcoded oligonucleotide) contains a complementary homopolymeric 3' tail (such as a poly(T) tail).

[0500] The coupled sequence can be included in a synthetic transposome and can be attached by an in vitro transposition reaction.

[0501] The coupled sequence can be attached to the target nucleic acid, and where the barcoded oligonucleotide is attached to the target nucleic acid by at least one primer extension step or polymerase chain reaction step, and where the barcoded oligonucleotide contains a region that is complementary to the coupled sequence and that is at least one nucleotide in length. Optionally, this complementary region is at the 3' end of the barcoded oligonucleotide. Optionally, the length of this complementary region is at least 2 nucleotides, at least 5 nucleotides, at least 10 nucleotides, at least 20 nucleotides, or at least 50 nucleotides.

[0502] 12. Coupling molecules and methods for micron particle analysis using coupling molecules

[0503] The method...

Claims

1. A method for analyzing a sample comprising circulating micron-sized particles or a sample derived from circulating micron-sized particles, wherein the circulating micron-sized particles are membranous vesicles, wherein the circulating micron-sized particles comprise at least three target molecules, wherein at least two of the target molecules are fragments of RNA and at least one of the target molecules is a target polypeptide, and wherein the method comprises measuring signals corresponding to the presence, absence, and / or level of each of the target molecules to generate at least two sets of linked signals for the circulating micron-sized particles, wherein at least one of the linked signals corresponds to the presence, absence, and / or level of the fragment of RNA in the sample, and at least one of the linked signals corresponds to the presence, absence, and / or level of the target polypeptide in the sample, and wherein the step of measuring signals corresponding to the presence, absence, and / or level of the fragment of RNA comprises ligating at least two of at least two fragments of RNA to generate at least two sets of ligated RNA fragments.

2. The method according to claim 1, wherein the fragment of RNA is a fragment of mRNA.

3. The method according to claim 1 or 2, wherein the target polypeptide comprises a specific amino acid sequence, and / or wherein the target polypeptide comprises a post-translational modification, optionally, wherein the target polypeptide comprises acetylated amino acid residues and / or methylated amino acid residues.

4. The method according to any one of claims 1 to 3, wherein the method comprises measuring signals corresponding to the presence, absence, and / or level of each of the target molecules of the circulating micron-sized particles to generate at least three sets of linked signals for the circulating micron-sized particles, wherein one of the linked signals corresponds to the presence, absence, and / or level of a first fragment of RNA of the circulating micron-sized particles, one of the linked signals corresponds to the presence, absence, and / or level of a second fragment of RNA of the circulating micron-sized particles, and one of the linked signals corresponds to the presence, absence, and / or level of the target polypeptide of the circulating micron-sized particles.

5. The method according to any one of claims 1 to 4, wherein the step of measuring signals corresponding to the presence, absence, and / or level of the fragment of RNA comprises analyzing the sequence of each of at least two of at least two fragments of RNA, optionally, wherein the step of measuring signals corresponding to the presence, absence, and / or level of the fragment of RNA comprises sequencing at least a portion of each of at least two of at least two fragments of RNA.

6. The method according to any one of claims 1 to 5, wherein the step of measuring signals corresponding to the presence, absence, and / or level of the fragment of RNA comprises sequencing at least a portion of each of at least two of the ligated fragments in the set to generate at least two ligated sequence reads.

7. The method according to any one of claims 1 to 6, wherein the step of measuring signals corresponding to the presence, absence, and / or level of the fragment of RNA comprises: (a) Attaching each of at least two of the fragments of the at least two RNAs of the circulating micron particles to a barcode sequence to produce a collection of ligated RNA fragments; and optionally, (b) Sequencing at least a portion of each of at least two of the ligated fragments in the collection to produce at least two ligated sequence reads, wherein the at least two ligated sequence reads are linked by the barcode sequence.

8. 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 fragment of the RNA comprises: (a) Attaching each of at least two of the fragments of the at least two RNAs of the circulating micron particles to a different barcode sequence in a collection of barcode sequences to produce a collection of ligated RNA fragments; and optionally, (b) Sequencing at least a portion of each of at least two of the ligated fragments in the collection to produce at least two ligated sequence reads, wherein the at least two ligated sequence reads are linked by the collection of barcode sequences.

9. The method according to any one of claims 1 to 8, wherein a signal corresponding to the presence, absence, and / or level of the target polypeptide is measured as follows: using (i) a barcoded affinity probe, wherein the barcoded affinity probe comprises at least one affinity moiety linked to a barcoded oligonucleotide, wherein the barcoded oligonucleotide comprises at least one nucleotide, and wherein the affinity moiety is capable of binding to the target polypeptide, optionally, wherein the signal is measured by determining the presence, absence, and / or level of the barcoded oligonucleotide by sequencing; and / or (ii) an optically labeled affinity probe and / or a fluorescently labeled affinity probe, optionally, wherein the signal is measured by flow cytometry and / or fluorescence-activated cell sorting.

10. The method according to any one of claims 1 to 9, wherein the circulating micron particles comprise 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 target molecules, and wherein the method comprises generating a collection 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 ligation signals for the circulating micron particles.

11. The method according to any one of claims 1 to 10, wherein the target molecule comprises at least 3, at least 4, at least 9, at least 49, at least 99, at least 499, at least 999, at least 4999, at least 9,999, at least 99,999 or at least 999,999 fragments of RNA, and optionally, wherein the method comprises 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 ligation signals for the circulating microparticles.

12. The method according to any one of claims 1 to 11, wherein the target molecule comprises at least 2, at least 3, at least 4, at least 9, at least 49, at least 99, at least 499, at least 999, at least 4999, at least 9,999, at least 99,999 or at least 999,999 target polypeptides, and optionally, wherein the method comprises generating a set of at least 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 ligation signals for the circulating microparticles.

13. The method according to any one of claims 1 to 12, wherein the sample comprises first and second circulating microparticles, wherein each circulating microparticle comprises at least three target molecules according to any one of claims 1 to 12, and wherein the method comprises performing the measurement step according to any one of claims 1 to 12 to generate a set of ligation signals for the first circulating microparticle, and performing the measurement step according to any one of claims 1 to 12 to generate a set of ligation signals for the second circulating microparticle; optionally, wherein the sample comprises n circulating microparticles, wherein each circulating microparticle comprises at least three target molecules according to any one of claims 1 to 12, and wherein the method comprises, for each circulating microparticle, performing the measurement step according to any one of claims 1 to 12 to generate a set of ligation signals for each circulating microparticle, optionally, wherein 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.

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