MULTI-OMIC ANALYSIS OF EXTRACELLULAR VESICLES IN MONODISPERSE DROPLETS
This disclosure provides methods and systems for single-extracellular (EV), multi-omic analysis of target EVs without microfluidic devices. The disclosed methods involve the use of template particles to template the formation of monodisperse droplets to generally capture a single target EV from a population of EVs in an encapsulation, derive a plurality of distinct mRNA molecules from the single target EV, and quantify the distinct mRNA molecules to generate an expression profile. Nucleic-acid-tagged antibody conjugates are used for simultaneous proteomic analysis along with the gene expression profiling, which enables classification of an EV in a sample.
1 . A method for single cell analysis, the method comprising:
preparing a mixture comprising extracellular vesicles, target-specific antibodies linked to index oligonucleotides, and template particles comprising capture oligonucleotides;
introducing a partitioning oil to the mixture;
shearing the mixture to form a plurality of water-in-oil partitions, wherein individual extracellular vesicles are (i) isolated in one of the partitions with template particles and (ii) bound by at least one of the antibodies;
lysing the extracellular vesicles to release nucleic acid within each partition; and
analyzing the index oligonucleotides and released nucleic acid to determine one or more characteristics of the cell.
2 . The method of claim 1 , wherein the index oligonucleotides contain a barcode sequence that identifies a protein to which the antibody binds.
3 . The method of claim 2 , wherein the capture oligonucleotides of each template particle comprise a partition barcode unique to each template particle.
4 . The method of claim 3 , further comprising creating a sequencing library containing copies of the index oligonucleotide barcodes, the partition barcode, and the released nucleic acid.
5 . The method of claim 4 , further comprising sequencing the library to produce sequence reads.
6 . The method of claim 5 , further comprising identifying from the sequence reads proteins and nucleic acids present in the EVs.
7 . The method of claim 6 , further comprising using the partition barcodes in the sequence reads to identify proteins and nucleic acids of at least one individual EV.
8 . The method of claim 7 , further comprising using the index oligonucleotide barcodes and/or the released nucleic acid to identify an extracellular vesicle subclass of the individual EVs.
9 . The method of claim 8 , wherein the subclass is one of an exosome, a microvesicle, an apoptotic body, an oncosome, and an exomere.
10 . The method of claim 6 , wherein the released nucleic acid is RNA.
11 . The method of claim 10 , further comprising reverse transcribing the released RNA captured by the capture oligonucleotides to produce a cDNA library.
12 . The method of claim 11 , wherein the released RNA is selected from one or more of mRNA, microRNA, ncRNA, tRNA, snRNA, and vault RNA.
13 . The method of claim 12 , wherein the released RNA is mRNA.
14 . The method of claim 7 , wherein the EVs in the aqueous mixture are from a sample from a subject.
15 . The method of claim 14 , further comprising assessing a pathology in the subject using extracellular vesicle subclass of one or more individual EVs in the sample.
16 . The method of claim 15 , wherein assessing further comprises quantifying amounts of individual EVs in the sample of a particular extracellular vesicle subclass.
17 . The method of claim 15 , wherein assessing further comprises analyzing the nucleic acids and/or proteins identified in the EVs.
18 . The method of claim 15 , wherein the target-specific antibodies are a panel of target-specific antibodies, and each antibody of the panel binds to a different protein.
19 . The method of claim 18 , wherein panel comprises an antibody the specifically binds to a protein selected from CD63, CD9, C3b TSP, Annexin V, Phosphatidylserine, CD40L, an integrin, and ARF6.
20 . The method of claim 15 , wherein the pathology is cancer and the extracellular vesicle subclass is an oncosome.