High-throughput single-cell sequencing with reduced amplification bias
AssigneesIllumina, Inc., University of Washington
InventorsFrank J. Steemers, Jay Shendure, Yi Yin
Life of this patent
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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 methods include linear amplification of the nucleic acids. In one embodiment, the sequencing library includes whole genome nucleic acids from the plurality of single cells. In one embodiment, the nucleic acids include three index sequences. Also provided herein are compositions, such as compositions that include the nucleic acids having three index sequences.
Claims33
A method for preparing a sequencing library comprising nucleic acids from a plurality of single nuclei or cells, the method comprising:
- providing a plurality of isolated nuclei or cells in a first plurality of compartments,
- wherein each compartment comprises a subset of isolated nuclei or cells, and
- wherein nuclei or cells comprise nucleic acid fragments;
- introducing a linear amplification mediator to the cells or nuclei;
- amplifying the nucleic acid fragments by linear amplification;
- processing each subset of nuclei or cells to generate indexed nuclei or cells,
- wherein the processing comprises adding to nucleic acid fragments present in the isolated nuclei or cells a first compartment specific index sequence to result in indexed nucleic acids present in isolated nuclei or cells,
- wherein the processing comprises ligation, primer extension, hybridization, amplification, or transposition;
- combining the indexed nuclei or cells to generate pooled indexed nuclei or cells, thereby producing a sequencing library from the plurality of nuclei or cells.
A method for preparing a sequencing library comprising nucleic acids from a plurality of single nuclei or cells, the method comprising:
- providing a plurality of isolated nuclei or cells,
- wherein nuclei or cells comprise nucleic acid fragments;
- introducing a linear amplification mediator to the isolated nuclei or cells;
- distributing the isolated nuclei or cells into a first plurality of compartments,
- wherein each compartment comprises a subset of isolated nuclei or cells;
- amplifying the nucleic acid fragments by linear amplification;
- processing each subset of isolated nuclei or cells to generate indexed nuclei or cells,
- wherein the processing comprises adding to nucleic acid fragments present in the isolated nuclei or cells a first compartment specific index sequence to result in indexed nucleic acids present in isolated nuclei or cells,
- wherein the processing comprises ligation, primer extension, amplification, or transposition;
- combining the indexed nuclei to generate pooled indexed nuclei or cells, thereby producing a sequencing library from the plurality of nuclei or cells.
A method for preparing a sequencing library comprising nucleic acids from a plurality of single nuclei or cells, the method comprising:
- providing a plurality of isolated nuclei or cells in a first plurality of compartments,
- wherein each compartment comprises a subset of isolated nuclei or cells, and
- wherein nuclei or cells comprise nucleic acid fragments;
- processing each subset of nuclei or cells to generate indexed nuclei or cells,
- wherein the processing comprises adding to nucleic acid fragments present in the isolated nuclei or cells (i) a first compartment specific index sequence to result in indexed nucleic acids present in isolated nuclei or cells and (ii) a nucleotide sequence recognized by a linear amplification mediator,
- wherein the processing comprises ligation, primer extension, hybridization, amplification, or transposition;
- introducing a linear amplification mediator to the cells or nuclei;
- amplifying the nucleic acid fragments by linear amplification;
- combining the indexed nuclei or cells to generate pooled indexed nuclei or cells, thereby producing a sequencing library from the plurality of nuclei or cells.
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Dependencies are inferred from the claim text.