Single cell whole genome libraries and combinatorial indexing methods of making thereof
AssigneesIllumina, Inc., Oregon Health & Science University
InventorsAndrew C. Adey, Sarah A. Vitak, Frank J. Steemers
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Abstract
Provided herein are methods for preparing a sequencing library that includes nucleic acids from a plurality of single cells. In one embodiment, the sequencing library includes whole genome nucleic acids from the plurality of single cells. In one embodiment, the method includes generating nucleosome-depleted nuclei by chemical treatment while maintaining integrity of the nuclei. Also provided herein are compositions, such as compositions that include chemically treated nucleosome-depleted isolated nuclei.
Claims14
A method of preparing a sequencing library comprising nucleic acids from a plurality of single cells, the method comprising, in the following order:
- (a) providing cross-linked isolated nuclei;
- (b) subjecting the cross-linked isolated nuclei to a chemical treatment to unbind nucleosomes from genomic DNA to generate nucleosome-depleted nuclei, while maintaining nuclear integrity of the isolated nuclei, wherein the chemical treatment comprises a treatment with a detergent comprising sodium dodecyl sulfate (SDS) which disrupts nucleic acid-protein interactions;
- (c) distributing subsets of the nucleosome-depleted nuclei into a first plurality of compartments and contacting each subset with a plurality of transposome complexes,
- wherein the transposome complexes in each compartment of the first plurality of compartments comprises a transposase and a first index sequence, and
- wherein the first index sequence in each compartment of the first plurality of compartments comprises a nucleotide sequence that is different from the nucleotide sequence of first index sequences in the other compartments of the first plurality of compartments;
- (d) fragmenting nucleic acids in the subsets of nucleosome-depleted nuclei into a plurality of nucleic acid fragments and incorporating the first index sequences into at least one strand of the nucleic acid fragments to generate indexed nuclei comprising indexed nucleic acid fragments,
- wherein the indexed nucleic acid fragments remain attached to the transposases,
- wherein the nucleic acid fragments are genomic DNA, and
- wherein the nucleosome depletion within the crosslinked isolated nuclei results in genome-wide uniform incorporation of the first index sequences that is not restricted to sites of chromatin accessibility;
- (e) combining the indexed nuclei to generate pooled indexed nuclei;
- (f) distributing subsets of the pooled indexed nuclei into a second plurality of compartments;
- (g) dissociating the transposases from the indexed nucleic acid fragments;
- (h) reversing the cross-links within the isolated nuclei;
- (i) incorporating into the indexed nucleic acid fragments in each compartment of the second plurality of compartments a second index sequence to generate dual-index fragments, wherein the second index sequence in each compartment comprises a nucleotide sequence that is different from the nucleotide sequence of second index sequences in the other compartments of the second plurality of compartments;
- and
- (j) combining the dual-index fragments, thereby producing a sequencing library from the plurality of single cells.
Dependencies are inferred from the claim text.