MASSIVELY PARALLEL SINGLE CELL ANALYSIS

• US 20150299784A1
• Filed: 08/28/2014
• Published: 10/22/2015
• Est. Priority Date: 08/28/2013
• Status: Active Grant

• Current Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Fodor, Stephen P.A., Fu, Glenn, Wilhelmy, Julie, Fan, Christina, Facer, Geoffrey Richard
• Granted Patent: US 9,567,645 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The disclosure provides for methods, compositions, and kits for multiplex nucleic acid analysis of single cells. The methods, compositions and systems may be used for massively parallel single cell sequencing. The methods, compositions and systems may be used to analyze thousands of cells concurrently. The thousands of cells may comprise a mixed population of cells (e.g., cells of different types or subtypes, different sizes).

Claims

1-163. -163. (canceled)

164. A method comprising:

a) contacting a sample comprising a plurality of RNA molecules with a plurality of oligonucleotide tags to produce a labelled-RNA molecule, wherein;

i) the plurality of RNA molecules comprises at least 2 mRNA molecules of different sequences;

ii) the plurality of oligonucleotide tags comprises at least 2 oligonucleotide tags of different sequences; and

iii) an oligonucleotide tag of the plurality of oligonucleotide tags comprises an oligodT sequence and a unique identifier region;

b) conducting a first strand synthesis reaction by contacting the labelled-RNA molecule with a reverse transcriptase enzyme to produce a single-stranded labelled-cDNA molecule;

c) amplifying the single-stranded labelled-cDNA molecule to produce a double-stranded labelled-cDNA molecule;

d) conducting a nested PCR reaction on the double-stranded labelled-cDNA molecule to produce a nested PCR labelled-amplicon; and

e) detecting at least a portion of the nested PCR labelled-amplicon.

165. The method of claim 164, wherein the sample is from a single cell.

166. The method of claim 164, wherein the sample is from less than 50 cells.

167. The method of claim 164, wherein said conducting said first strand synthesis reaction is performed on a bead surface.

168. The method of claim 164, wherein said detecting comprises determining the sequence of at least a portion of the oligonucleotide tag, at least a portion of the single-stranded labelled-cDNA molecule, at least a portion of the double-stranded labelled-cDNA molecule, at least a portion of the nested PCR labelled-amplicon, a complement thereof, a reverse complement thereof, or any combination thereof.

169. The method of claim 164, wherein said detecting comprises using an array detector, fluorescent reader, non-fluorescent detector, CR reader, sequencer, or scanner.

170. The method of claim 164, wherein said detecting comprises hybridizing said labelled-amplicon to a solid support.

171. The method of claim 170, further comprising determining the sequence of at least a portion of said labelled-amplicon.

172. A method comprising:

a. contacting a sample comprising a plurality of RNA molecules with a plurality of oligonucleotide tags to produce a labelled-RNA molecule, wherein;

i. the plurality of RNA molecules comprises at least 2 mRNA molecules of different sequences;

ii. the plurality of oligonucleotide tags comprises at least 2 oligonucleotide tags of different sequences; and

iii. an oligonucleotide tag of the plurality of oligonucleotide tags comprises an oligodT sequence and a unique identifier region;

b. producing a single-stranded labelled-cDNA molecule comprising a 3′

-homopolymer tail by conducting a first strand synthesis reaction on the labelled-RNA molecule;

c. producing a double-stranded labelled-cDNA molecule by conducting a amplification reaction on the single-stranded labelled-cDNA molecule; and

d. detecting the double-stranded labelled-cDNA molecule.

173. The method of claim 172, wherein said producing further comprises treating said double-stranded labelled-cDNA with a restriction enzyme.

174. The method of claim 172, wherein the sample is from a single cell.

175. The method of any claim 172, wherein detecting the double-stranded labelled-cDNA molecule comprises hybridizing at least a portion the double-stranded labelled-cDNA molecule to a solid support.

176. The method of claim 175, further comprising determining the sequence of at least a portion of said double-stranded labelled-cDNA.

177. The method claim 175, wherein the solid support is an array.

178. The method claim 175, wherein the solid support is an addressable array.

179. The method claim 175, wherein the solid support is a bead.

180. The method of claim 172, further comprising amplifying the double-stranded labelled-cDNA molecule to produce a double-stranded labelled-amplicon.

181. The method of claim 180, wherein detecting the double-stranded labelled-cDNA comprises detecting the double-stranded labelled-amplicon.

182. The method of claim 181, wherein detecting the double-stranded labelled-amplicon comprises hybridizing at least a portion of the double-stranded labelled-amplicon to a solid support.

183. The method of claim 182, further comprising determining the sequence of at least a portion of said labelled-amplicon.

184. The method of claim 172, wherein conducting the amplification reaction comprises conducting a cDNA synthesis reaction or a polymerase chain reaction.

185. The method of claim 172, wherein said amplification reaction comprises strand displacement amplification.

186. The method of claim 172, wherein said amplification reaction is isothermal.

187. A method comprising:

a. contacting a sample comprising a plurality of RNA molecules with a plurality of oligonucleotide tags to produce a labelled-RNA molecule, wherein;

i. the plurality of RNA molecules comprises at least 2 mRNA molecules of different sequences;

ii. the plurality of oligonucleotide tags comprises at least 2 oligonucleotide tags of different sequences; and

iii. an oligonucleotide tag of the plurality of oligonucleotide tags comprises an oligodT sequence and a unique identifier region;

b. producing a single-stranded labelled-cDNA molecule by conducting a first strand synthesis reaction on the labelled-RNA molecule, wherein conducting the strand synthesis reaction comprises using a strand-switch oligonucleotide;

c. producing a double-stranded labelled-cDNA molecule by conducting an amplification reaction on the single-stranded labelled-cDNA; and

d. detecting the double-stranded labelled-cDNA molecule.

188. The method of claim 187, wherein said producing said single-stranded labelled-cDNA molecule is performed on a bead surface.

189. The method of claim 187, wherein the sample is from a single cell.

190. The method of any claim 187, wherein detecting the double-stranded labelled-cDNA molecule comprises hybridizing at least a portion the double-stranded labelled-cDNA molecule to a solid support.

191. The method of claim 190, further comprising determining the sequence of at least a portion of said double-stranded labelled-cDNA.

192. A method for amplifying an RNA molecule comprising:

a. contacting a sample comprising a plurality of RNA molecules with a plurality of oligonucleotide tags to produce a labelled-RNA molecule, wherein;

i. the plurality of RNA molecules comprises at least 2 mRNA molecules of different sequences;

ii. the plurality of oligonucleotide tags comprises at least 2 oligonucleotide tags of different sequences; and

iii. an oligonucleotide tag of the plurality of oligonucleotide tags comprises an oligodT sequence and a unique identifier region;

b. producing a single-stranded labelled-cDNA by conducting a first strand synthesis reaction on the labelled-RNA molecule;

c. amplifying said single stranded labelled cDNA molecule using a primer comprising universal primer binding site and a random multimer sequence, thereby generating a double-stranded labelled-cDNA molecule; and

d. detecting at least a portion of said double-stranded labelled cDNA molecule.

193. The method of claim 192, wherein said random multimer sequence hybridizes to said single-stranded labelled cDNA molecule randomly.

194. The method of claim 192, wherein said amplifying further comprises amplifying with a universal primer.

195. The method of claim 194, wherein said universal primer hybridizes to a universal primer binding site on said oligonucleotide tag.

196. The method of claim 192, further comprising treating the sample with an enzyme.

197. The method of claim 196, wherein the enzyme is uracil DNA glycosylase (UDG).

198. The method of claim 196, wherein treating the sample with the enzyme occurs after contacting the sample with the plurality of oligonucleotide tags.

199. The method of claim 196, wherein treating the sample with the enzyme occurs prior to the detecting step.

200. The method of claim 192, wherein said detecting comprises conducting a sequencing reaction to determine the sequence of at least a portion of said oligonucleotide tag, at least a portion of the labelled cDNA molecule, a complement thereof, a reverse complement thereof, or any combination thereof.

201. The method of claim 192, wherein said at least 2 mRNA molecules are labelled stochastically.

202. The method of claim 192, wherein said detecting comprises hybridizing at least a portion of said labelled-cDNA molecule to a solid support.

203. The method of claim 202, further comprising determining the sequence of at least a portion of said labelled-cDNA amplicon.

204. The method of claim 202, wherein said solid support comprises a plurality of probes.

205. The method of claim 202, wherein said solid support comprises an array.

206. The method of claim 192, wherein said amplifying comprises strand displacement amplification.

207. The method of claim 192, wherein said amplifying is isothermal.

208. The method of claim 192, wherein said sample is from a single cell.

209. The method of claim 192, wherein said sample is from less than 50 cells.