MASSIVELY PARALLEL SINGLE CELL ANANLYSIS

• US 20160258012A2
• Filed: 08/28/2014
• Published: 09/08/2016
• Est. Priority Date: 08/28/2013
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Fan, Christina, Fodor, Stephen P.A., Fu, Glenn, Facer, Geoffrey Richard, Wilhelmy, Julie
• 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

1-102. -102. (canceled)

103. A method comprising:

(a) separating cells into individual wells on a substrate such that a plurality of said wells have a single cell, and wherein said substrate comprises at least 1,000 wells; and

(b) associating a single bead with a single well having said single cell.

104. The method of claim 103, wherein the method further comprises diluting said cells.

105. The method of claim 104, wherein said diluting comprises diluting said cells such that at most one cell is distributed to one well.

106. The method of claim 104, wherein said diluting comprises diluting said cells such that the number of cells is about 10% of the number of wells on said substrate.

107. The method of claim 103, wherein said separating comprises distributing a single cell to at least 10% of said wells.

108. The method of claim 103, wherein said separating is random.

109. The method of claim 103, wherein said cells comprise a cell selected from the group consisting of:

a rare cell, a tumor cell, a cell from a human, a cell from a tissue, a cell from a tumor, and a cell infected with viral polynucleotides, or any combination thereof.

110. The method of claim 103, wherein said cells comprise at least 2 different cell types.

111. The method of claim 103, wherein said associating occurs prior to lysing said cell.

112. The method of claim 103, wherein said associating occurs after lysing said cell.

113. The method of claim 103, further comprising removing said bead from said well.

114. The method of claim 113, wherein said removing comprises removing with a magnet.

115. The method of claim 103, wherein said bead comprises a plurality of oligonucleotides.

116. The method of claim 115, wherein said plurality of oligonucleotides comprises at least 100,000 oligonucleotides.

117. The method of claim 115, wherein said plurality of oligonucleotides each comprise a universal label, a cellular label, a molecular label, and a target-binding region.

118. The method of claim 115, wherein individual oligonucleotides of said plurality of oligonucleotides have different molecular labels.

119. The method of claim 115, wherein individual oligonucleotides of said plurality of oligonucleotides have the same cellular label.

120. The method of claim 103, wherein individual oligonucleotides of a plurality of oligonucleotides on beads in different wells of said plurality of wells have different cellular labels.

121. The method of claim 117, wherein said target-binding region comprises a sequence selected from the group consisting of:

an oligo-dT sequence, a gene-specific sequence, and a random multimer sequence.

122. The method of claim 103, further comprising stochastically labelling targets from said single cell.

123. The method of claim 122, wherein said labelling comprises hybridizing said targets to a plurality of oligonucleotides on said bead.

124. The method of claim 123, further comprising reverse transcribing said targets, thereby generating labelled target cDNAs.

125. The method of claim 124, wherein said reverse transcribing is done on pooled beads.

126. The method of claim 124, wherein said labelled target cDNAs are attached to said bead.

127. The method of claim 126, further comprising performing second strand synthesis on said labelled target cDNAs, thereby generating double-stranded labelled target polynucleotides.

128. The method of claim 127, further comprising amplifying said double-stranded labelled target polynucleotides, thereby generating labelled target amplicons.

129. The method of claim 128, wherein said amplifying is performed with a universal primer and a random multimer primer.

130. The method of claim 128, wherein said amplifying is performed with a universal primer and a gene-specific primer.

131. The method of claim 128, wherein said amplifying comprises conducting a nested peR reaction.

132. The method of claim 128, wherein said amplifying comprises amplifying the whole transcriptome of said one or more cells.

133. The method of claim 128, further comprising estimating the number of said targets.

134. The method of claim 133, wherein said estimating comprises determining the sequence of at least a portion said double-stranded labelled target amplicons.

135. The method of claim 133, wherein said estimating comprises determining the sequence of at least a portion said double-stranded labelled target amplicons and at least a portion of said oligonucleotide.

136. The method of claim 133, wherein said estimating comprises counting the number of unique oligonucleotides associated with a distinct target polynucleotide per cell.

137. A composition comprising:

a substrate comprising at least 1,000 wells wherein a plurality of wells comprise a single bead.

138. A method of diagnosis comprising:

(a) separating cells into individual wells on a substrate such that a plurality of said wells have a single cell;

(b) stochastically labeling nucleic acid targets from said cells with a plurality of oligonucleotide labels, thereby generating labelled nucleic acids;

(c) detecting said labelled nucleic acids; and

(d) estimating the number of labelled nucleic acid targets in each of said single cells.