CAPSULE ARRAY DEVICES AND METHODS OF USE

• US 20140155295A1
• Filed: 08/13/2013
• Published: 06/05/2014
• Est. Priority Date: 08/14/2012
• Status: Abandoned Application

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10x Technology Inc.
• Inventors: Saxonov, Serge, Hindson, Benjamin
• Application Number:
  US13/966,150
• Publication Number:
  US 20140155295A1
• Time in Patent Office: Days
• US Class Current: 506/16
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2400/0677 phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/508 rigid containers not provided for above
• CPC Class Codes: B01L 3/523 with means for closing or opening
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• 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

Abstract

This disclosure provides microwell capsule array devices. The microwell capsule array devices are generally capable of performing one or more sample preparation operations. Such sample preparation operations may be used as a prelude to one more or more analysis operations. For example, a device of this disclosure can achieve physical partitioning and discrete mixing of samples with unique molecular identifiers within a single unit in preparation for various analysis operations. The device may be useful in a variety of applications and most notably nucleic-acid-based sequencing, detection and quantification of gene expression and single-cell analysis.

Claims

1-77. -77. (canceled)

78. A composition comprising a first microcapsule, wherein:

a) said first microcapsule is degradable upon the application of a stimulus to said first microcapsule; and

b) said first microcapsule comprises an oligonucleotide barcode and a chemical cross-linker.

79. The composition of claim 78, wherein said chemical cross-linker is a disulfide bond.

80. The composition of claim 78, further comprising a polymer gel.

81. The composition of claim 80, wherein said polymer gel is a polyacrylamide gel.

82. The composition of claim 78, wherein said first microcapsule comprises a bead.

83. The composition of claim 82, wherein said bead is a gel bead.

84. The composition of claim 78, wherein said stimulus is selected from the group consisting of a biological, chemical, thermal, electrical, magnetic, or photo stimulus, and combination thereof.

85. The composition of claim 78, wherein said chemical stimulus is selected from the group consisting of a change in pH, a change in ion concentration, and a reducing agent.

86. The composition of claim 85, wherein said reducing agent is dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).

87. The composition of claim 78, wherein a second microcapsule comprises said first microcapsule.

88. The composition of claim 87, wherein said second microcapsule is a droplet.

89. The composition of claim 78, further comprising a nucleic acid that comprises said oligonucleotide barcode, wherein said nucleic acid comprises a deoxyuridine triphosphate (dUTP).

90. The composition of claim 78, further comprising a polymerase unable to accept a deoxyuridine triphosphate (dUTP).

91. The composition of claim 78, further comprising a target analyte.

92. The composition of claim 91, wherein said target analyte is a nucleic acid.

93. The composition of claim 92, wherein said nucleic acid is selected from the group consisting of DNA, RNA, dNTPs, ddNTPs, amplicons, synthetic nucleotides, synthetic polynucleotides, polynucleotides, oligonucleotides, peptide nucleic acids, cDNA, dsDNA, ssDNA, plasmid DNA, cosmid DNA, High Molecular Weight (MW) DNA, chromosomal DNA, genomic DNA, viral DNA, bacterial DNA, mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, ribozyme, riboswitch and viral RNA.

94. The composition of claim 93, wherein said nucleic acid is genomic DNA (gDNA).

95. The composition of claim 78, wherein the density of said oligonucleotide barcodes is at least about 1,000,000 oligonucleotide barcodes per said first microcapsule.

96. The composition of claim 78, wherein said oligonucleotide barcode is coupled to said microcapsule via said chemical cross-linker.

97. The composition of claim 96, wherein said chemical cross-linker is a disulfide bond.

98. A composition comprising a degradable gel bead, wherein the degradable gel bead comprises at least about 1,000,000 oligonucleotide barcodes.

99. The composition of claim 98, wherein the 1,000,000 oligonucleotide barcodes are identical.

100. A method for sample preparation, comprising:

a) combining a microcapsule comprising an oligonucleotide barcode and a target analyte into a partition, wherein said microcapsule is degradable upon the application of a stimulus to said microcapsule; and

b) applying said stimulus to said microcapsule to release said oligonucleotide barcode to said target analyte.

101. The method of claim 100, wherein said partition is a well.

102. The method of claim 100, wherein said partition is a droplet.

103. The method of claim 100, wherein said microcapsule comprises a polymer gel.

104. The method of claim 103, wherein said polymer gel is a polyacrylamide.

105. The method of claim 100, wherein said microcapsule comprises a bead.

106. The method of claim 105, wherein said bead is a gel bead.

107. The method of claim 100, wherein said microcapsule comprises a chemical cross-linker.

108. The method of claim 107, wherein said chemical cross-linker is a disulfide bond.

109. The method of claim 100, wherein said stimulus is selected from the group consisting of a biological, chemical, thermal, electrical, magnetic, photo stimulus, and a combination thereof.

110. The method of claim 109, wherein said chemical stimulus is selected from the group consisting of a change in pH, change in ion concentration, and a reducing agent.

111. The method of claim 110, wherein said reducing agent is dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).

112. The method of claim 100, wherein a nucleic acid comprises said oligonucleotide barcode and wherein said nucleic acid comprises a deoxyuridine triphosphate (dUTP).

113. The method of claim 100, wherein said partition comprises a polymerase unable to accept a deoxyuridine triphosphate (dUTP).

114. The method of claim 100, further comprising attaching said oligonucleotide barcode to said target analyte.

115. The method of claim 114, wherein said attaching is completed with a nucleic acid amplification reaction.

116. The method of claim 100, wherein said target analyte is a nucleic acid.

117. The method of claim 116, wherein said nucleic acid is selected from the group consisting of DNA, RNA, dNTPs, ddNTPs, amplicons, synthetic nucleotides, synthetic polynucleotides, polynucleotides, oligonucleotides, peptide nucleic acids, cDNA, dsDNA, ssDNA, plasmid DNA, cosmid DNA, High Molecular Weight (MW) DNA, chromosomal DNA, genomic DNA, viral DNA, bacterial DNA, mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, ribozyme, riboswitch and viral RNA.

118. The method of claim 117, wherein said nucleic acid is genomic DNA (gDNA).

119. The method of claim 100, wherein said oligonucleotide barcode is coupled to said microcapsule via a chemical cross-linker.

120. A device comprising a plurality of partitions, wherein:

a) at least one partition of the plurality of partitions comprises a microcapsule comprising an oligonucleotide barcode; and

b) said microcapsule is degradable upon the application of a stimulus to said microcapsule.

121. The device of claim 120, wherein said partition is a well.

122. The device of claim 120, wherein said partition is a droplet.

123. The device of claim 120, wherein said microcapsule comprises a chemical cross-linker.

124. The device of claim 123, wherein said chemical cross-linker is a disulfide bond.

125. The device of claim 120, wherein said microcapsule further comprises a polymer gel.

126. The device of claim 125, wherein said polymer gel is a polyacrylamide gel.

127. The device of claim 120, wherein said microcapsule comprises a bead.

128. The device of claim 127, wherein said bead is a gel bead.

129. The device of claim 120, wherein said stimulus is selected from the group consisting of a biological, chemical, thermal, electrical, magnetic, or photo stimulus, and a combination thereof.

130. The device of claim 129, wherein said chemical stimulus is selected from the group consisting of a change in pH, change in ion concentration, and a reducing agent.

131. The device of claim 130, wherein said reducing agent is dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).

132. The device of claim 120, wherein a nucleic acid comprises said oligonucleotide barcode and wherein said nucleic acid comprises a deoxyuridine triphosphate (dUTP).

133. The device of claim 120, wherein said partition comprises a polymerase unable to accept a deoxyuridine triphosphate (dUTP).

134. The device of claim 120, wherein said partition comprises a target analyte.

135. The device of claim 134, wherein said target analyte is a nucleic acid.

136. The device of claim 135, wherein said nucleic acid is selected from the group consisting of DNA, RNA, dNTPs, ddNTPs, amplicons, synthetic nucleotides, synthetic polynucleotides, polynucleotides, oligonucleotides, peptide nucleic acids, cDNA, dsDNA, ssDNA, plasmid DNA, cosmid DNA, High Molecular Weight (MW) DNA, chromosomal DNA, genomic DNA, viral DNA, bacterial DNA, mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, ribozyme, riboswitch and viral RNA.

137. The device of claim 136, wherein said nucleic acid is genomic DNA (gDNA).

138. The device of claim 120, wherein said oligonucleotide barcode is coupled to said microcapsule via a chemical cross-linker.

139. The device of claim 138, wherein said chemical cross-linker is a disulfide bond.

140. A composition comprising a first microcapsule, wherein:

a) said first microcapsule is degradable upon the application of a stimulus to said first microcapsule; and

b) said first microcapsule comprises an oligonucleotide barcode and a polymer gel.

141. The composition of claim 140, wherein said polymer gel is a polyacrylamide gel.

142. The composition of claim 140, wherein said first microcapsule comprises a bead.

143. The composition of claim 142, wherein said bead is a gel bead.

144. The composition of claim 140, wherein said stimulus is selected from the group consisting of a biological, chemical, thermal, electrical, magnetic, or photo stimulus, and combination thereof.