Sequencing of nucleic acids via barcoding in discrete entities
AssigneeThe Regents of the University of California
InventorsAdam R. Abate, John R. Haliburton, Freeman Lan, Adam R. Sciambi
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Abstract
Microfluidic methods for barcoding nucleic acid target molecules to be analyzed, e.g., via nucleic acid sequencing techniques, are provided. Also provided are microfluidic, droplet-based methods of preparing nucleic acid barcodes for use in various barcoding applications. The methods described herein facilitate high-throughput sequencing of nucleic acid target molecules as well as single cell and single virus genomic, transcriptomic, and/or proteomic analysis/profiling. Systems and devices for practicing the subject methods are also provided.
Claims15
A method comprising:
- encapsulating, within a discrete entity,
- a nucleic acid released from a single cell or a product of the released nucleic acid;
- an oligonucleotide including a first nucleic acid barcode serving as an indicator of an affinity reagent that was bound to a molecular target of the single cell;
- a plurality of primer sequences;
- a plurality of copies of a sequence comprising both a second nucleic acid barcode and a universal sequence,
- wherein the second nucleic acid barcode serves as an indicator of the discrete entity in which the oligonucleotide including the first nucleic acid barcode and the released nucleic acid or the product of the released nucleic acid are encapsulated;
- incorporating a first copy of the sequence comprising the second nucleic acid barcode and the universal sequence with the oligonucleotide including the first nucleic acid barcode by performing nucleic acid amplification using the universal sequence of the first copy and at least a first primer sequence;
- incorporating a second copy of the sequence comprising the second nucleic acid barcode and the universal sequence with the released nucleic acid or the product of the released nucleic acid by performing nucleic acid amplification using the universal sequence of the second copy and at least a second primer sequence;
- and
- sequencing the first nucleic acid barcode, the first copy of the sequence comprising the second nucleic acid barcode, and the second copy of the sequence comprising the second nucleic acid barcode.
Dependencies are inferred from the claim text.