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.
Claims25
A method of preparing a sequencing library comprising nucleic acids from a plurality of single cells, the method comprising:
- (a) providing isolated nuclei from a plurality of cells;
- (b) subjecting the isolated nuclei to a chemical treatment to generate nucleosome-depleted nuclei, while maintaining integrity of the isolated nuclei;
- (c) distributing subsets of the nucleosome-depleted nuclei into a first plurality of compartments and contacting each subset with a transposome complex, wherein the transposome complex in each compartment comprises a transposase and a first index sequence that is different from first index sequences in the other 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;
- (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) incorporating into the indexed nucleic acid fragments in each compartment a second index sequence to generate dual-index fragments, wherein the second index sequence in each compartment is different from second index sequences in the other compartments;
- (h) combining the dual-index fragments, thereby producing a sequencing library comprising whole genome nucleic acids from the plurality of single cells.
A composition comprising chemically treated nucleosome-depleted isolated nuclei, wherein the isolated nuclei comprise indexed nucleic acid fragments.
Dependencies are inferred from the claim text.