Transposition of Native Chromatin for Personal Epigenomics

• US 20190309362A1
• Filed: 05/21/2019
• Published: 10/10/2019
• Est. Priority Date: 05/23/2013
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Giresi, Paul, Buenrostro, Jason D., Chang, Howard Y., Greenleaf, William J.
• Granted Patent: US 10,619,207 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 method for generating a sequencing library from a plurality of cells, comprising:

a) lysing a plurality of cells to provide a plurality of cell nuclei, wherein the plurality of cell nuclei comprises chromatin;

b) contacting a cell nucleus of the plurality of cell nuclei with a transposase complex such that polynucleotides of the cell nucleus are tagmented at regions of open chromatin to produce a plurality of tagged fragments; and

c) performing one or more nucleic acid reactions on the tagged fragment to produce a sequencing library.

99. The method of claim 98, further comprising sequencing the sequencing library.

100. The method of claim 98, wherein the one or more nucleic acid reactions comprise a nucleic acid amplification reaction.

101. The method of claim 100, wherein said nucleic acid amplification reaction is configured to add one or more functional sequences to said tagged nucleic acid molecules or derivatives thereof, wherein said one or more functional sequences are compatible with a selected next generation sequencing platform.

102. The method of claim 98, wherein a tagged fragment of the plurality of tagged fragments comprises a nucleotide sequence corresponding to the tagged fragment.

103. The method of claim 102, wherein the tagged fragment further comprises a primer sequence.

104. The method of claim 98, wherein the transposase complex comprises a Tn5 transposase or a transposase derived from a Tn5 transposase.

105. The method of claim 104, wherein said Tn5 transposase is a hyperactive Tn5 transposase.

106. The method of claim 98, wherein said transposase complex comprises a first nucleic acid insert element comprising a first adapter sequence.

107. The method of claim 106, wherein said first adapter sequence comprises a first sequencing adapter sequence.

108. The method of claim 106, wherein said first adapter sequence comprises a barcode sequence.

109. The method of claim 106, wherein said first adapter sequence comprises a first primer sequence.

110. The method of claim 98, wherein said transposase complex comprises a second nucleic acid insert element comprising a second adapter sequence.

111. The method of claim 110, wherein said second adapter sequence comprises a comprises a second sequencing adapter sequence.

112. The method of claim 110, wherein said second adapter sequence comprises a barcode sequence.

113. The method of claim 110, wherein said first adapter sequence comprises a second primer sequence.

114. The method of claim 98, further comprising using the sequencing library to determine chromatin accessibility of the polynucleotides of the cell nuclei.

115. The method of claim 98, further comprising using the sequencing library to identify transcriptionally active sites on the polynucleotides of the cell nuclei.

116. The method of claim 98, further comprising using the sequencing library to determine epigenetic features of the polynucleotides of the cell nuclei.

117. The method of claim 98, further comprising using the sequencing library to determine transcription start sites.

118. The method of claim 98, further comprising using the sequencing library to determine positioning of nucleosomes in the chromatin.

119. The method of claim 98, wherein the transposase complex does not comprise an antibody specific to a protein that is part of chromatin.

120. A method for generating a sequencing library from a plurality of cells, comprising:

a) lysing a plurality of cells to isolate a plurality of cell nuclei, wherein the plurality of cell nuclei comprise chromatin;

b) contacting a cell nucleus of the plurality of cell nuclei with a Tn5 transposase complex such that polynucleotides of the cell nucleus are tagmented at regions of open chromatin to produce a plurality of tagged fragments,

wherein the Tn5 transposase complex comprises a first sequencing adapter sequence and a second sequencing adapter sequence,

wherein the Tn5 transposase complex does not comprise an antibody specific to a protein that is part of chromatin,

wherein a tagged fragment of the plurality of tagged fragment comprises

i. a nucleotide sequence corresponding to a region of open chromatin,

ii. the first sequencing adapter sequence, and

iii. the second sequencing adapter sequence; and

c) performing one or more nucleic acid reactions on the tagged fragment to produce a sequencing library.

121. The method of claim 120, further comprising sequencing the sequencing library.

122. The method of claim 120, further comprising using the sequencing library to determine chromatin accessibility of the polynucleotides of the cell nuclei.

123. The method of claim 120, further comprising using the sequencing library to identify transcriptionally active sites on the polynucleotides of the cell nuclei.

124. The method of claim 120, further comprising using the sequencing library to determine epigenetic features of the polynucleotides of the cell nuclei.

125. The method of claim 120, further comprising using the sequencing library to determine transcription start sites (TSSs).

126. The method of claim 120, further comprising using the sequencing library to determine positioning of nucleosomes in the chromatin.