Transposition of Native Chromatin for Personal Epigenomics

• US 20190316196A1
• Filed: 05/21/2019
• Published: 10/17/2019
• Est. Priority Date: 05/23/2013
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Giresi, Paul, Buenrostro, Jason D., Chang, Howard Y., Greenleaf, William J.
• Granted Patent: US 10,738,357 B2
• Time in Patent Office: Days
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16H 50/20 for computer-aided diagnosis, e.g. based on medical expert systems
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

Provided herein is a method for analyzing polynucleotides such as genomic DNA. In certain embodiments, the method comprises: (a) treating chromatin isolated from a population of cells with an insertional enzyme complex to produce tagged fragments of genomic DNA; (b) sequencing a portion of the tagged fragments to produce a plurality of sequence reads; and (c) making an epigenetic map of a region of the genome of the cells by mapping information obtained from the sequence reads to the region. A kit for performing the method is also provided.

Claims

1-97. -97. (canceled)

98. A composition comprising:

a permeabilized cell nucleus comprising a plurality of polynucleotide molecules;

an insertional enzyme complex; and

an insert element comprising at least one nucleic acid comprising a predetermined sequence.

99. The composition of claim 98, further comprising a permeabilization agent selected from the group consisting of:

NP40, digitonin, tween, steptolysin, cationic lipids, and any combination thereof.

100. A composition comprising:

a permeabilized cell nucleus comprising a plurality of polynucleotide molecules, wherein the plurality of polynucleotide molecules comprises genomic DNA comprising a plurality of open chromatin regions;

an insertional enzyme complex; and

an insert element comprising at least one nucleic acid comprising a predetermined sequence.

101. The composition of claim 100, wherein the insertional enzyme complex comprises one or more enzymatic moieties.

102. The composition of claim 101, wherein the insertional enzyme complex comprises two or more enzymatic moieties.

103. The composition of claim 102, wherein the two or more enzymatic moieties are linked together.

104. The composition of claim 100, wherein the insertional enzyme complex comprises a nuclear localization signal.

105. The composition of claim 100, wherein the insertional enzyme complex is configured to insert a nucleic acid sequence into at least one of the polynucleotide molecules.

106. The composition of claim 105, wherein the insertional enzyme complex is configured to insert the nucleic acid sequence into at least one of the polynucleotide molecules in a substantially sequence independent manner.

107. The composition of claim 100, wherein the insertional enzyme complex comprises an enzyme selected from the group consisting of:

transposases, HERMES, and HIV integrases.

108. The composition of claim 107, wherein the insertional enzyme complex comprises a transposase.

109. The composition of claim 108, wherein the transposase is a Tn transposase.

110. The composition of claim 109, wherein the Tn transposase is selected from the group consisting of:

Tn3, Tn5, Tn7, Tn10, Tn552 and Tn903.

111. The composition of claim 110, wherein the transposase is a Tn5 transposase.

112. The composition of claim 100, wherein the insert element comprises one or more molecular tags.

113. The composition of claim 112, wherein one or more of the molecular tags comprises a sequencing adaptor.

114. The composition of claim 113, wherein one or more of the sequencing adaptors comprises a barcode label.

115. A composition comprising:

a permeabilized cell nucleus;

an insertional enzyme complex comprising a transposase enzyme; and

a plurality of tagged nucleic acid fragments, wherein each tagged nucleic acid fragment comprises a first sequencing adapter and a second sequencing adapter;

wherein each tagged nucleic acid fragment is derived from a region of open chromatin.

116. The composition of claim 115, wherein the plurality of tagged nucleic acid fragments correspond to two or more regions of a genome.

117. The composition of claim 115, wherein the plurality of tagged nucleic acid fragments correspond to at least 1,000 regions of a genome.

118. The composition of claim 117, wherein the plurality of tagged nucleic acid fragments correspond to genome-wide regions of open chromatin.

119. The composition of claim 117, wherein the transposase is a Tn transposase.

120. The composition of claim 119, wherein the Tn transposase is a Tn5 transposase.

121. A composition comprising:

a permeabilized cell nucleus;

an insertional enzyme complex comprising a transposase enzyme; and

a plurality of tagged nucleic acid fragments, wherein each tagged nucleic acid fragment comprises a first sequencing adapter and a second sequencing adapter;

wherein the insertional enzyme complex and the tagged nucleic acid fragments are bound to one or more regions of open chromatin within the cell nucleus in a substantially sequence independent manner to form a high affinity macromolecule complex.

122. The composition of claim 121, further comprising a plurality of divalent cations.

123. The composition of claim 122, wherein the divalent cations are not chelated.

124. The composition of claim 122, wherein the divalent cations are selected from the group consisting of:

magnesium, manganese, or any combination thereof.

125. The composition of claim 121, wherein the transposase is a Tn transposase.

126. The composition of claim 125, wherein the Tn transposase is selected from the group consisting of:

Tn3, Tn5, Tn7, Tn10, Tn552 and Tn903.

127. The composition of claim 126, wherein the transposase is a Tn5 transposase.