METHODS FOR THE ANALYSIS OF CIRCULATING MICROPARTICLES

TECHNICAL FIELD

The present invention relates to the analysis of cell free biomolecules (e.g. cell free nucleic acid molecules and cell free polypeptides). In particular, it relates to the analysis of cell free biomolecules contained within or derived from circulating microparticles. Provided are reagents and methods for analysing biomolecules of circulating microparticles including reagents and methods for analysing biomolecules of single circulating microparticles.

BACKGROUND

Cell-free DNA (cfDNA) in the circulation is typically fragmented (typically in the range of 100-200 base pairs in length), and thus methods for cfDNA analysis have traditionally focused upon biological signals that can be found with these short DNA fragments. For example, detecting single-nucleotide variants within individual molecules, or performing‘molecular counting’ across a large number of sequenced fragments to indirectly infer the presence of large-scale chromosomal abnormalities e.g. tests for foetal chromosomal trisomies that assess foetal DNA within the maternal circulation (a form of so-called‘non-invasive prenatal testing’, or NIPT).

A large variety of methods to analyse circulating cell-free DNA have been described previously. Depending upon the specific application area, these assays may employ different terminology for a broadly similar set of sample types and technical methods, such as circulating tumour DNA (ctDNA), cell-free foetal DNA (cffDNA), and/or liquid biopsy, or non-invasive prenatal testing. In general, these methods comprise a laboratory protocol to prepare samples of circulating cell-free DNA for sequencing, a sequencing reaction itself, and then an informatic framework to analyse the resulting sequences to detect a relevant biologic signal. The methods involve a DNA purification and isolation step prior to sequencing, which means that the subsequent analysis must rely solely on the information contained in the DNA itself. Following sequencing, such methods generally employ one or more informatic or statistical frameworks to analyse various aspects of the sequence data, such as detecting specific mutations therein, and/or detecting selective enrichment or selective depletion of particular chromosomes or sub-chromosomal regions (for example, which might be indicative of a chromosomal aneuploidy in a developing foetus).

Many of these methods are for use in NIPT (e.g. in US patents 6258540 B1 , 8296076 B2, 8318430 B2, 8195415 B2, 9447453 B2, and 8442774 B2). The most common methods for performing non-invasive prenatal testing for the detection of foetal chromosomal abnormalities (such as trisomies, and/or sub-chromosomal abnormalities such as microdeletions) involve sequencing a large number of molecules of cfDNA, mapping the resulting sequences to the genome (i.e. to determine which chromosome and/or which part of a given chromosome the

sequence derive from), and then, for one or more such chromosomal or sub-chromosomal regions, determining the amount of sequence that maps thereto (e.g. in the form of absolute numbers of reads or relative numbers of reads) and then comparing this to one or more normal or abnormal threshold or cutoff values, and/or performing a statistical test, to determine whether said region(s) may be overrepresented in amount of sequence (which may, for example, correspond to a chromosomal trisomy) and/or whether said region(s) may be underrepresented in amount of sequence (which may, for example, correspond to a microdeletion).

A variety of additional or modified approaches to analyzing cell free DNA using data from unlinked, individual molecules have also been described (e.g. WO2016094853 A1 ,

US2015344970 A1 and US20150105267 A1 ).

Despite the existance of such a wide range of methods, there remains a need for new methods of analysing cfDNA that would allow the reliable detection of long-range genetic information (e.g. phasing) and also for methods with greater sensitivity. For example, in the case of NIPT, foetal cfDNA only represents a minor fraction of the overall cfDNA in pregnant individuals (the majority of circulating DNA being normal maternal DNA). Therefore, a considerable technical challenge for NIPT revolves around differentiating foetal cfDNA from maternal DNA. Similarly, in a patient with cancer, cfDNA only represents a tiny fraction of the overall circulating DNA. Therefore, a similar technical challenge exists in relation to the use of cfDNA analysis for the diagnosis or monitoring of cancer.

Separately, methods have also been described that allow the isolation of cell type-specific apoptotic bodies by fluorescence-activated cell sorting (FACS) (Atkin-Smith et al., 2017. Scientific Reports 7, 39846) and that allow the multiplexed profiling of protein markers in single extracellular vesicles (Lee et al., 2018. ACS Nano. 23, 12(1 ), 494-503).

DESCRIPTION

The invention provides methods for the analysis of samples comprising circulating microparticles (or samples derived from circulating microparticles) such as apoptotic bodies. The invention is based on multi-parametric measurement of different types of biomolecules comprised within or derived from single circulating microparticles. In particular, the invention allows the measurement of linked signals corresponding to the presence, absence and/or level of two or more types of target biomolecule in the same circulating microparticle. As illustrated in Figure 30, signals corresponding to the levels of fragments of genomic DNA may be produced (e.g. by partitioning, barcoding and sequencing) and a signal corresponding to the level of a target polypeptide may be produced (e.g. using a barcoded affinity probe). In addition, a signal corresponding to the level of a modified nucleotide (e.g. a nucleotide comprising 5-methylcytosine) may be produced (e.g. by an affinity-based enrichment approach such as one that uses an enrichment probe that is specific for or preferentially binds 5-methylcytosine in fragments of genomic DNA). These measurements and associated techniques thus produce a series of linked signals corresponding to the physical and biological state of a circulating microparticle.

The multi-parametric methods provided herein adds additional layers of information to the the earlier inventions provided by the inventor in PCT/GB2017/053820, PCT/GB2017/053812, and PCT/GB2017/053816.

In PCT/GB2017/053820, the inventor previously provided methods for the analysis of nucleic acid fragments in circulating microparticles (or microparticles originating from blood). That invention is based on a linked-fragment approach in which fragments of nucleic acid from a single microparticle are linked together. This linkage enables the production of a set of linked sequence reads (i.e. set of linked signals) corresponding to the sequences of fragments from a single microparticle.

The linked-fragment approach provides highly sensitive cfDNA analysis and also enables the detection of long-range genetic information. The approach is based on a combination of insights. Firstly, the methods take advantage of the insight that individual circulating microparticles (for example, an individual circulating apoptotic body) will contain a number of fragments of genomic DNA that have been generated from the same individual cell (somewhere in the body) which has undergone apoptosis. Secondly, a fraction of such fragments of genomic DNA within an individual microparticle will preferentially comprise sequences from one or more specific chromosomal regions. Cumulatively, such circulating microparticles thus serve as a data-rich and multi-feature ‘molecular stethoscope’ to observe what may be quite complex genetic events occurring in a limited somatic tissue space somewhere in the body; importantly, since such microparticles in large part enter the circulation prior to clearance or metabolism, they may be detected noninvasively. The invention describes experimental and informatic methods of using these ‘stethoscopes’ i.e. sets of linked fragments and linked sequence reads (either in the form of single, individual microparticles, or, in many embodiments, complex samples comprising a large number of single circulating microparticles) to perform analytic and diagnostic tasks.

The present invention advances the concept of the‘molecular stethoscope’ by harnessing the data provided by the co-localisation of, for example, non-nucleic acid molecules (e.g. target polypeptides) with nucleic acid molecules (e.g. fragments of genomic DNA) in single circulating microparticles. This advance is based on the discovery that rather than being singular and freely diffusible in the blood, many biomolecules (e.g. nucleic acid molecules and polypeptides) comprised within the circulation are biophysically retained within circulating microparticles. The invention exploits this rich source of information by measuring signals corresponding to the presence, absence and/or level of a plurality of target biomolecules of a circulating microparticle to produce a set of (informatically) linked signals for the circulating microparticle. In addition, by including in this set one or more signals corresponding to one or more target biomolecules that is/are characteristic of a particular cell or tissue type, the cellular origin of a particular set of linked signals, derived from a single circulating microparticle, can be determined. This provides the set of linked signals with a‘cellular context’ providing a much richer source of information than currently available methods. In so doing, the invention provides methods of analysis 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, and NIPT.

The inventor has previously provided reagents and methods related to barcoding. In

WO2016/207639, the inventor provided a wide range of reagents, kits and methods for molecular barcoding including multimeric barcoding reagents. In PCT/GB2017/053812, the inventor provided further methods and reagents for molecular barcoding. In PCT/GB2017/053816, the inventor provided reagents and methods for molecular barcoding of nucleic acids of single cells.

The entire content of WO2016/207639, PCT/GB2017/053812, PCT/GB2017/053816 and PCT/GB2017/053820 is incorporated herein by reference.

The invention provides a method of analysing a sample comprising a circulating microparticle or a sample derived from a circulating microparticle, wherein the circulating microparticle comprises at least two target molecules, wherein the at least two target molecules are biomolecules, and wherein the method comprises measuring a signal corresponding to the presence, absence and/or level of each of the target molecules to produce a set of at least two (informatically) linked signals for the circulating microparticle, wherein at least one of the linked signals corresponds to the presence, absence and/or level of a first biomolecule in the sample and at least one of the linked signals corresponds to the presence, absence and/or level of a second biomolecule in the sample.

The invention provides a method of analysing a sample comprising a circulating microparticle or a sample derived from a circulating microparticle, wherein the circulating microparticle comprises at least two target molecules, wherein the at least two target molecules are biomolecules, and wherein the method comprises measuring a signal corresponding to the presence, absence and/or level of each of the target molecules to produce a single signal for the circulating microparticle, wherein the single signal corresponds to the presence, absence and/or level of the biomolecules in the sample.

The first biomolecule may be a fragment of a target nucleic acid (e.g. a fragment of genomic DNA) and second biomolecule may be a target (or predefined) non-nucleic acid biomolecule (e.g.

a target polypeptide). Optionally, the fragment of a target nucleic acid may comprise at least one modified nucleotide or nucleobase.

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

The first biomolecule may be a polypeptide and the second target biomolecule may be a fragment of a target nucleic acid (e.g. genomic DNA) comprising an epigenetic modification (e.g. 5-hydroxy-methylcytosine DNA or 5-methylcytosine DNA).

The first biomolecule may be 5-hydroxy-methylcytosine DNA and the second target biomolecule may be a fragment of RNA.

The first biomolecule may be 5-methylcytosine DNA and the second target biomolecule may be a fragment of RNA.

The first biomolecule may be 5-hydroxy-methylcytosine DNA and the second target biomolecule may be a biomolecule selected from Biomolecule group 1 .

The first biomolecule may be 5-methylcytosine DNA and the second target biomolecule may be a biomolecule selected from Biomolecule group 1 .

The first and second biomolecules may be selected from Biomolecule group 1 .

The invention provides a method of analysing a sample comprising a circulating microparticle or a sample derived from a circulating microparticle, wherein the circulating microparticle comprises 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 a fragment of RNA, and wherein the method comprises measuring a signal corresponding to the presence, absence and/or level of each of the target molecules to produce a set of at least two (informatically) linked signals for the circulating microparticle, wherein at least one of the linked signals corresponds to the presence, absence and/or level of the fragments of genomic DNA in the sample and at least one of the linked signals corresponds to the presence, absence and/or level of the fragment of RNA in the sample.

The invention provides a method of analysing a sample comprising a circulating microparticle or a sample derived from a circulating microparticle, wherein the circulating microparticle comprises 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 a fragment of RNA, and wherein the

method comprises measuring a signal corresponding to the presence, absence and/or level of each of the target molecules to produce a single signal for the circulating microparticle, wherein the single signal corresponds to the presence, absence and/or level of the fragments of genomic DNA and the fragment of RNA in the sample.

The invention provides a method of analysing a sample comprising a circulating microparticle or a sample derived from a circulating microparticle, wherein the circulating microparticle comprises 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 a signal corresponding to the presence, absence and/or level of each of the target molecules to produce a set of at least three (informatically) linked signals for the circulating microparticle, wherein each of at least two of the linked signals corresponds to the presence, absence and/or level of one of the fragments of the target nucleic acid (e.g. genomic DNA) in the sample and at least one of the linked signals corresponds to the presence, absence and/or level of the target biomolecule (e.g. the target polypeptide) in the sample.

The invention provides a method of analysing a sample comprising a circulating microparticle or a sample derived from a circulating microparticle, wherein the circulating microparticle comprises 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 a signal corresponding to the presence, absence and/or level of each of the target molecules to produce a set of at least two (informatically) linked signals for the circulating microparticle, wherein at least one of the linked signals corresponds to the presence, absence and/or level of the fragments of the target nucleic acid (e.g. genomic DNA) in the sample and at least one of the linked signals corresponds to the presence, absence and/or level of the target biomolecule (e.g. the target polypeptide) in the sample.

The invention provides a method of analysing a sample comprising a circulating microparticle or a sample derived from a circulating microparticle, wherein the circulating microparticle comprises 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 a signal corresponding to the presence, absence and/or level of each of the target molecules to produce a single signal for the circulating microparticle, wherein the single signal corresponds to the presence, absence and/or level of the fragments of the target nucleic acid (e.g. genomic DNA) and the target biomolecule (e.g. the target polypeptide) in the sample.

The fragments of the target nucleic acid (e.g. genomic DNA) may comprise a specific sequence of nucleotides and/or the fragments of the target nucleic acid (e.g. genomic DNA) may comprise at least one modified nucleotide or nucleobase. The fragments of the target nucleic acid may not comprise a specific sequence of nucleotides. The fragments of the target nucleic acid may comprise untargeted and/or unknown and/or randomly-selected and or randomly-sampled sequences of nucleotides. For example, the modified nucleotide or nucleobase may be 5-methylcytosine or 5-hydroxy-methylcytosine. The fragments of the target nucleic acid (e.g.

genomic DNA) may comprise one or microsattelite sequences and/or microsattelite genomic regions (i.e. short tandem repeats).

A 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 an acetylated amino acid residue and/or a methylated amino acid residue (for example, a specific acetylated amino acid residue on/within a specific polypeptide and/or a specific methylated amino acid residue on/within a specific polypeptide).

The method may comprise measuring the signal corresponding to the presence, absence and/or level of each of the target molecules of the circulating microparticle to produce a set of at least three (informatically) linked signals for the circulating microparticle, wherein one of the linked signals corresponds to the presence, absence and/or level of a first fragment of a target nucleic acid (e.g. genomic DNA) of the circulating microparticle, one of the linked signals corresponds to the presence, absence and/or level of a second fragment of a target nucleic acid (e.g. genomic DNA) of the circulating microparticle, and one of the linked signals corresponds to the presence, absence and/or level of the target biomolecule (e.g. the target polypeptide) of the circulating microparticle.

The step of measuring a signal corresponding to the presence, absence and/or level of the fragments of a target nucleic acid (e.g. genomic DNA) may comprise analysing a sequence of each of at least two of the at least two fragments of the target nucleic acid (e.g. genomic DNA), optionally wherein the step of measuring a signal corresponding to the presence, absence and/or level of the fragments of the target nucleic acid (e.g. genomic DNA) comprises sequencing at least a portion of each of at least two of the at least two fragments of the target nucleic acid (e.g. genomic DNA) to produce at least two (informatically) linked sequence reads.

The step of measuring a signal corresponding to the presence, absence and/or level of the fragments of a target nucleic acid (e.g. genomic DNA) may comprise: (a) linking at least two of the at least two fragments of the target nucleic acid (e.g. genomic DNA) to produce a set of at least two linked fragments of the target nucleic acid (e.g. genomic DNA); and, optionally, (b) analysing a sequence of each of at least two of the linked fragments in the set. Step (b) may comprise

sequencing at least a portion of each of at least two of the linked fragments in the set to produce at least two (informatically) linked sequence reads.

The step of measuring a signal corresponding to the presence, absence and/or level of the fragments of a target nucleic acid (e.g. genomic DNA) may comprise: (a) appending each of at least two of the at least two fragments of the target nucleic acid (e.g. genomic DNA) of the circulating microparticle to a barcode sequence to produce a set of linked fragments of the target nucleic acid (e.g. genomic DNA); and, optionally, (b) analysing a sequence of each of at least two of the linked fragments in the set. Step (b) may comprise sequencing at least a portion of each of at least two of the linked fragments in the set to produce at least two (informatically) linked sequence reads, wherein the at least two linked sequence reads are linked by the barcode sequence. Optionally, each of at least two of the at least two fragments of the target nucleic acid may comprise the same barcode sequence.

The step of measuring a signal corresponding to the presence, absence and/or level of the fragments of a target nucleic acid (e.g. genomic DNA) may comprise: (a) appending each of at least two of the at least two fragments of the target nucleic acid (e.g. genomic DNA) of the circulating microparticle to a different barcode sequence of a set of barcode sequences to produce a set of linked fragments of the target nucleic acid (e.g. genomic DNA); and, optionally, (b) analysing a sequence of each of at least two of the linked fragments in the set. Step (b) may comprise sequencing at least a portion of each of at least two of the linked fragments in the set to produce at least two (informatically) linked sequence reads. The at least two linked sequence reads may be linked by the set of barcode sequences (i.e. the barcode sequence appended to a first fragment of the target nucleic acid and the barcode sequence appended to a second fragment of the target nucleic acid link the two sequence reads to each other by being present within the same set of barcode sequences).

The step of measuring a signal corresponding to the presence, absence and/or level of the fragments of a target nucleic acid (e.g. genomic DNA) may comprise: (a) appending 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 appending 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 a different barcode sequence of a set of barcode sequences; and, optionally, (b) analysing a sequence of each of the first and second barcoded target nucleic acid molecules. Step (b) may comprise sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules to produce at least two (informatically) linked sequence reads. The at least two linked sequence reads may be linked by the same barcode sequence or the set of barcode sequences. Step (b) may comprise sequencing all or at

least a portion of each of the first and second barcode sequences appended to the first and second fragments of the target nucleic acid.

The step of measuring a signal corresponding to the presence, absence and/or level of the fragments of a target nucleic acid (e.g. genomic DNA) may comprise: (a) appending (e.g.

annealing or ligating) 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 appending (e.g. annealing or ligating) 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 comprise the same barcode sequence, or each comprise a different barcode sequence of a set of barcode sequences; and, optionally, (b) analysing a sequence of each of the first and second barcoded target nucleic acid molecules. Step (b) may comprise sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules to produce at least two (informatically) linked sequence reads. The at least two linked sequence reads may be linked by the same barcode sequence or the set of barcode sequences. Step (b) may comprise sequencing all or at least a portion of each of the first and second barcoded oligonucleotides appended to the first and second fragments of the target nucleic acid.

The step of measuring a signal corresponding to the presence, absence and/or level of the fragments of a target nucleic acid (e.g. genomic DNA) may comprise: (a) contacting the sample with a multimeric barcoding reagent, wherein the multimeric barcoding reagent comprises first and second barcode regions linked together, wherein each barcode region comprises a nucleic acid sequence; and (b) appending barcode sequences to each of first and second fragments of the target nucleic acid of the microparticle to produce first and second barcoded target nucleic acid molecules for the microparticle, 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 may each comprise the same barcode sequence, or the first and second barcode regions may comprise a different barcode sequence of a set of barcode sequences. The method may further comprise (c) analysing a sequence of each of the first and second barcoded target nucleic acid molecules. Step (c) may comprise sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules to produce at least two (informatically) linked sequence reads. The at least two linked sequence reads may be linked by the same barcode sequence or by the set of barcode sequences.

The step of measuring a signal corresponding to the presence, absence and/or level of the fragments of a target nucleic acid (e.g. genomic DNA) may comprise: (a) contacting the sample with a multimeric barcoding reagent, wherein the multimeric barcoding reagent comprises first

and second barcoded oligonucleotides linked together, and wherein the barcoded

oligonucleotides each comprise a barcode region; and (b) appending (e.g. annealing or ligating) the first and second barcoded oligonucleotides to first and second fragments of the target nucleic acid of the microparticle to produce first and second barcoded target nucleic acid molecules. The barcode regions of the first and second barcoded oligonucleotides may each comprise the same barcode sequence, or the barcode regions of the first and second barcoded oligonucleotides may each comprise a different barcode sequence of a set of barcode sequences. The method may further comprise (c) analysing a sequence of each of the first and second barcoded target nucleic acid molecules. Step (c) may comprise sequencing at least a portion of each of the first and second barcoded target nucleic acid molecules to produce at least two (informatically) linked sequence reads. The at least two linked sequence reads may be linked by the same barcode sequence or the set of barcode sequences.

The fragments of the target nucleic acid (e.g. genomic DNA) may 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 the fragments of the target nucleic acid (e.g. genomic DNA) may comprise measuring a signal corresponding to the presence, absence and/or level of the epigenetic modification (e.g. the modified nucleotide or nucleobase) of the fragments of the target nucleic acid (e.g. genomic DNA). For example, the modified nucleotide or nucleobase may comprise 5-methylcytosine or 5-hydroxy-methylcytosine.

The invention provides a method of analysing a sample comprising a circulating microparticle or a sample derived from a circulating microparticle, wherein the circulating microparticle comprises at least two target molecules, wherein at least one of the target molecules is a fragment of a target nucleic acid (e.g. genomic DNA) comprising an epigenetic modification and at least one of the target molecules 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 of the target molecules to produce a set of at least two (informatically) linked signals for the circulating microparticle, wherein at least one of the linked signals corresponds to the presence, absence and/or level of the epigenetic modification in the sample and at least one of the linked signals corresponds to the presence, absence and/or level of the target biomolecule (e.g. the target polypeptide) in the sample.

The invention provides a method of analysing a sample comprising a circulating microparticle or a sample derived from a circulating microparticle, wherein the circulating microparticle comprises at least two target molecules, wherein at least one of the target molecules is a fragment of a target nucleic acid (e.g. genomic DNA) comprising an epigenetic modification and at least one of the target molecules 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 of the target molecules to produce a single signal for the circulating microparticle, 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. the target polypeptide) in the sample.

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

An epigenetic modification may comprise a modified nucleotide e.g. 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 e.g. 5-methylcytosine or 5-hydroxy-methylcytosine. The fragment of a target nucleic acid (e.g. genomic DNA) comprising an epigenetic modification may comprise 5-methylcytosine DNA or 5-hydroxy-methylcytosine DNA.

A signal corresponding to the presence, absence and/or level of the epigenetic modification (e.g. the modified DNA or RNA nucleotide) may be measured using a barcoded affinity probe. 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 at least one nucleotide in length), and wherein the affinity moiety is capable of binding to a 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).

A signal corresponding to the presence, absence and/or level of the epigenetic modification (e.g. the modified DNA or RNA nucleotide) may be measured by flow cytometry and/or fluorescence-activated cell sorting using an optically-labelled affinity probe and/or a fluorescently-labelled affinity probe. The optically-labelled affinity probe and/or fluorescently-labelled affinity probe may be measured and/or detected using optical microscopy and/or fluorescence microscopy visualisation. For example, using a fluorescence microscope, and/or using fluorescent laser-based detection, and/or using a fluorescence-activated cell sorting (FACS) instrument. The optically-labelled affinity probe and/or fluorescently-labelled affinity probe may be measured and/or detected using a sorting process e.g. using fluorescence-activated cell sorting (FACS).

A signal corresponding to the presence, absence and/or level of the epigenetic modification (e.g. a modified DNA or RNA nucleotide) may be measured using a method comprising a molecular conversion step. In the case of a modified nucleotide (i.e. a nucleotide comprising a modified base such as 5-methylcytosine or 5-hydroxy-methylcytosine), the molecular conversion step may

be performed to convert said modified base(s) into a different modified or unmodified nucleotide which may be detected (e.g. using PCR or sequencing), providing the signal corresponding to the presence, absence and/or level of the epigenetic modification. This conversion step may comprise a bisulfite conversion step, an oxidative bisulfite conversion step, or any other molecular conversion step. The methods may be used to measure 5-methylcytosine in fragments of genomic DNA of a circulating microparticle.

The method may further comprise one or more steps of partitioning a sample comprising one or more circulating microparticles (or a sample derived from one or more circulating microparticles). Additionally or alternatively, the method may further comprise one or more steps of appending 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 may be provided by and/or comprised within one or more multimeric barcoding reagents as described herein.

A signal corresponding to the presence, absence and/or level of the non-nucleic acid biomolecule (e.g. target polypeptide) may be measured using a barcoded affinity probe. 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 at least one nucleotide in length), and wherein the affinity moiety is capable of binding to a target biomolecule (i.e. the target non-nucleic acid biomolecule (e.g. target polypeptide)). 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).

A signal corresponding to the presence, absence and/or level of the non-nucleic acid biomolecule (e.g. target polypeptide) may be measured by flow cytometry and/or fluorescence-activated cell sorting using an optically-labelled affinity probe and/or a fluorescently-labelled affinity probe. The optically-labelled affinity probe and/or fluorescently-labelled affinity probe may be measured and/or detected using optical microscopy and/or fluorescence microscopy visualisation. For example, using a fluorescence microscope, and/or using fluorescent laser-based detection, and/or using a fluorescence-activated cell sorting (FACS) instrument. The optically-labelled affinity probe and/or fluorescently-labelled affinity probe may be measured and/or detected using a sorting process e.g. using fluorescence-activated cell sorting (FACS).

A signal corresponding to the presence, absence and/or level of the non-nucleic acid biomolecule (e.g. target polypeptide) may be measured by supports labelled with an affinity probe. The supports labelled with an affinity probe may comprise beads (such as magnetic beads) labelled with affinity probes, for example labelled with antibodies specific for a target polypeptide. The presence, absence and/or level of the non-nucleic acid biomolecule (e.g. target polypeptide within a circulating microparticle) may be measured by incubating and/or binding said non-nucleic acid biomolecule to said affinity probe(s) on said supports, optionally wherein the support-bound fraction (ie the microparticle(s) comprising, and/or comprising high levels of the said non-nucleic acid biomolecule) is further isolated and/or processed (such as partitioned and/or barcoded and/or analysed by nucleic acid sequencing), and optionally wherein the support-unbound fraction (ie the microparticle(s) not comprising, and/or comprising low levels of the said non-nucleic acid biomolecule) is further isolated and/or processed (such as partitioned and/or barcoded and/or analysed by nucleic acid sequencing).

The signal corresponding to the presence, absence and/or level of the non-nucleic acid biomolecule (e.g. target polypeptide) may be measured separately from the signal corresponding to the presence, absence and/or level of the nucleic acid biomolecule. For example, the signal corresponding to the presence, absence and/or level of the non-nucleic acid biomolecule (e.g. target polypeptide) may be measured by FACS and the signal corresponding to the presence, absence and/or level of the nucleic acid biomolecule may be measured by sequencing.

In the methods, a set of linked signals may be measured for the (or for each) circulating microparticle corresponding to the presence, absence and/or level of fragments of a target nucleic acid (e.g. genomic DNA), an epigenetic modfication (e.g. a modified nucleotide such as a modified nucleotide comprising 5-methylcytosine and/or 5-hydroxymethylcytosine) and a target non-nucleic acid biomolecule (e.g. the target polypeptide).

For example, in the methods, the target molecules of the circulating microparticle may comprise at least 2 (different) fragments of a target nucleic acid (e.g. genomic DNA), at least one fragment of a target nucleic acid (e.g. genomic DNA) comprising an epigenetic modification, and at least one target non-nucleic acid biomolecule (e.g. a target polypeptide). The method may comprise measuring a signal corresponding to the presence, absence and/or level of each of the target molecules to produce a set of linked signals for the circulating microparticle, The method may provide a (different) linked signal for each of the target molecules. In the method, each of at least two of the linked signals may correspond to the presence, absence and/or level of one of the fragments of the target nucleic acid (e.g. genomic DNA); at least one of the linked signals may correspond to the presence, absence and/or level of the epigenetic modficiation (e.g. a modififed nucleotide such as a modified nucleotide comprising 5-methylcytosine and/or 5-hydroxymethylcytosine); and at least one of the linked signals may correspond to the presence, absence and/or level of the target non-nucleic acid biomolecule (e.g. the target polypeptide).

The circulating microparticle may comprise at least 3, at least 4, at least 5, at least 10, at least 50, at least 1 00, at least 500, at least 1000, at least 5000, at least 1 0,000, at least 100,000, or at least 1 ,000,000 (different) target molecules, and optionally wherein the method comprises producing a set of at least 3, at least 4, at least 5, at least 10, at least 50, at least 1 00, at least 500, at least 1000, at least 5000, at least 10,000, at least 100,000, or at least 1 ,000,000 (different) linked signals for the circulating microparticle (i.e. a (different) linked signal for each of the target molecules of the circulating microparticle).

The target molecules of the circulating microparticle may 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 9,999, at least 99,999, or at least 999,999 (different) fragments of a target nucleic acid (e.g. genomic DNA), and at least one target non-nucleic acid biomolecule (e.g. a target polypeptide), optionally wherein the method comprises producing a set of at least 3, at least 4, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10,000, at least 100,000, or at least 1 ,000,000 (different) linked signals for the circulating microparticle (i.e. a (different) linked signal for each of the target molecules of the circulating microparticle).

The target molecules of the circulating microparticle may 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 9,999, at least 99,999, or at least 999,999 (different) target polypeptides, and at least one fragment of a target nucleic acid (e.g. genomic DNA), optionally wherein the method comprises producing a set of at least at least 3, at least 4, at least 5, at least 1 0, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10,000, at least 100,000, or at least 1 ,000,000 (different) linked signals for the circulating microparticle (i.e. a (different) linked signal for each of the target molecules of the circulating microparticle).

CLAIMS

1 . A method of analysing a sample comprising a circulating microparticle or a sample derived from a circulating microparticle, wherein the circulating microparticle is a membranous vesicle, wherein the circulating microparticle comprises 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 a target polypeptide, and wherein the method comprises measuring a signal corresponding to the presence, absence and/or level of each of the target molecules to produce a set of at least two linked signals for the circulating microparticle, wherein at least one of the linked signals corresponds to the presence, absence and/or level of the fragments of genomic DNA 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 a signal corresponding to the presence, absence and/or level of the fragments of genomic DNA comprises linking at least two of the at least two fragments of genomic DNA to produce a set of at least two linked fragments of genomic DNA.

2. The method of claim 1 , wherein the fragments of genomic DNA comprise a specific sequence of nucleotides and/or wherein the fragments of genomic DNA comprise at least one modified nucleotide or nucleobase, optionally wherein the modified nucleotide or nucleobase is 5- methylcytosine or 5-hydroxy-methylcytosine.

3. The method of claim 1 or claim 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 an acetylated amino acid residue and/or a methylated amino acid residue.

4. The method of any one of claims 1 -3, wherein the method comprises measuring the signal corresponding to the presence, absence and/or level of each of the target molecules of the circulating microparticle to produce a set of at least three linked signals for the circulating microparticle, wherein one of the linked signals corresponds to the presence, absence and/or level of a first fragment of genomic DNA of the circulating microparticle, one of the linked signals corresponds to the presence, absence and/or level of a second fragment of genomic DNA of the circulating microparticle, and one of the linked signals corresponds to the presence, absence and/or level of the target polypeptide of the circulating microparticle.

5. The method of any one of claims 1 -4, wherein the step of measuring a signal corresponding to the presence, absence and/or level of the fragments of genomic DNA comprises analysing a sequence of each of at least two of the at least two fragments of genomic DNA, optionally wherein the step of measuring a signal corresponding to the presence, absence and/or level

of the fragments of genomic DNA comprises sequencing at least a portion of each of at least two of the at least two fragments of genomic DNA.

6. The method of any one of claims 1 -5, wherein the step of measuring a signal corresponding to the presence, absence and/or level of the fragments of genomic DNA comprises sequencing at least a portion of each of at least two of the linked fragments in the set to produce at least two linked sequence reads.

7. The method of any one of claims 1 -6, wherein the step of measuring a signal corresponding to the presence, absence and/or level of the fragments of genomic DNA comprises:

(a) appending each of at least two of the at least two fragments of genomic DNA of the circulating microparticle to a barcode sequence to produce a set of linked fragments of genomic DNA; and, optionally,

(b) sequencing at least a portion of each of at least two of the linked fragments in the set to produce at least two linked sequence reads, wherein the at least two linked sequence reads are linked by the barcode sequence.

8. The method of any one of claims 1 -6, wherein the step of measuring a signal corresponding to the presence, absence and/or level of the fragments of genomic DNA comprises:

(a) appending each of at least two of the at least two fragments of genomic DNA of the circulating microparticle to a different barcode sequence of a set of barcode sequences to produce a set of linked fragments of genomic DNA; and, optionally,

(b) sequencing at least a portion of each of at least two of the linked fragments in the set to produce at least two linked sequence reads, wherein the at least two linked sequence reads are linked by the set of barcode sequences.

9. The method of any one of claims 1 -8, wherein the fragments of genomic DNA comprise at least one modified nucleotide or nucleobase and wherein the step of measuring a signal corresponding to the presence, absence and/or level of the fragments of genomic DNA comprises measuring a signal corresponding to the presence, absence and/or level of the modified nucleotide or nucleobase of the fragments of genomic DNA, optionally wherein the modified nucleotide or nucleobase is 5-methylcytosine or 5-hydroxy-methylcytosine.

10. The method of claim 9, wherein the signal corresponding to the presence, absence and/or level of the modified nucleotide or nucleobase is measured 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 modfified nucleotide or nucleobase, 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-labelled affinity probe and/or a fluorescently-labelled affinity probe, optionally wherein the signal is measured by flow cytometry and/or fluorescence-activated cell sorting.

1 1 . The method of any one of claims 1 -10, wherein the signal corresponding to the presence, absence and/or level of the target polypeptide is measured 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-labelled affinity probe and/or a fluorescently-labelled affinity probe, optionally wherein the signal is measured by flow cytometry and/or fluorescence-activated cell sorting.

12. The method of any one of claims 1 -1 1 , wherein the circulating microparticle comprises 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 target molecules, and wherein the method comprises producing 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 linked signals for the circulating microparticle.

13. The method of any one of claims 1 -12, wherein the target molecules 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 9,999, at least 99,999, or at least 999,999 fragments of genomic DNA, and optionally wherein the method comprises producing 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 linked signals for the circulating microparticle.

14. The method of any one of claims 1 -13, wherein the target molecules 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 9,999, at least 99,999, or at least 999,999 target polypeptides, and optionally wherein the method comprises producing 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 linked signals for the circulating microparticle.

15. The method of any one of claims 1 -14, wherein the sample comprises first and second

circulating microparticles, wherein each circulating microparticle comprises at least three target molecules as defined in any one of claims 1 -14, and wherein the method comprises performing the step of measuring in accordance with any one of claims 1 -14 to produce a set

of linked signals for the first circulating microparticle and performing the step of measuring in accordance with any one of claims 1 -14 to produce a set of linked signals for the second circulating microparticle; optionally wherein the sample comprises n circulating microparticles, wherein each circulating microparticle comprises at least three target molecules as defined in any one of claims 1 -14, and wherein the method comprises performing the step of measuring in accordance with any one of claims 1 -14 for each circulating microparticle to produce a set of linked signals for each circulating microparticle, optionally wherein n is at least 3, at least 5, at least 1 0, 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.