US 12,448,643 B2
Sequencing of nucleic acids via barcoding in discrete entities
Adam R. Abate, Daly City, CA (US); John R. Haliburton, San Francisco, CA (US); Freeman Lan, San Francisco, CA (US); and Adam R. Sciambi, San Francisco, CA (US)
Assigned to The Regents of the University of California, Oakland, CA (US)
Filed by The Regents of the University of California, Oakland, CA (US)
Filed on Jun. 22, 2023, as Appl. No. 18/212,868.
Application 15/940,850 is a division of application No. 15/015,015, filed on Feb. 3, 2016, granted, now 11,111,519, issued on Sep. 7, 2021.
Application 18/212,868 is a continuation of application No. 15/940,850, filed on Mar. 29, 2018, granted, now 11,732,287.
Claims priority of provisional application 62/112,075, filed on Feb. 4, 2015.
Prior Publication US 2024/0150811 A1, May 9, 2024
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6804 (2018.01); B01F 25/433 (2022.01); B01F 33/302 (2022.01); B01L 3/00 (2006.01); C12N 15/10 (2006.01); C12Q 1/68 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/6874 (2018.01); B01F 21/00 (2022.01); B01L 7/00 (2006.01)
CPC C12Q 1/6804 (2013.01) [B01F 25/4338 (2022.01); B01F 33/3021 (2022.01); B01L 3/502784 (2013.01); C12N 15/1096 (2013.01); C12Q 1/68 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6874 (2013.01); B01F 21/00 (2022.01); B01L 3/502707 (2013.01); B01L 3/502715 (2013.01); B01L 3/502761 (2013.01); B01L 7/52 (2013.01); B01L 2200/0647 (2013.01); B01L 2200/0652 (2013.01); B01L 2300/021 (2013.01); B01L 2300/0645 (2013.01); B01L 2300/0864 (2013.01); B01L 2300/0867 (2013.01); B01L 2300/123 (2013.01); B01L 2400/0409 (2013.01); B01L 2400/0415 (2013.01); B01L 2400/0424 (2013.01); B01L 2400/043 (2013.01)] 15 Claims
 
1. 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.