US 12,319,959 B2
Methods for nucleic acid analysis
Gilad Almogy, Palo Alto, CA (US); Florian Oberstrass, Menlo Park, CA (US); Omer Barad, Mazkeret Batya (IL); and Chandan Shee, Newark, CA (US)
Assigned to Ultima Genomics, Inc., Fremont, CA (US)
Filed by Ultima Genomics, Inc., Newark, CA (US)
Filed on Aug. 5, 2021, as Appl. No. 17/394,692.
Application 17/394,692 is a continuation of application No. PCT/US2020/017491, filed on Feb. 10, 2020.
Claims priority of provisional application 62/916,683, filed on Oct. 17, 2019.
Claims priority of provisional application 62/890,240, filed on Aug. 22, 2019.
Claims priority of provisional application 62/804,082, filed on Feb. 11, 2019.
Prior Publication US 2022/0042072 A1, Feb. 10, 2022
Int. Cl. C12Q 1/686 (2018.01)
CPC C12Q 1/686 (2013.01) [C12Q 2563/107 (2013.01); C12Q 2563/149 (2013.01); C12Q 2563/159 (2013.01)] 22 Claims
OG exemplary drawing
 
1. A method for nucleic acid processing, comprising
(a) generating a plurality of droplets with a plurality of beads and a plurality of nucleic acid molecules, wherein a droplet of said plurality of droplets comprises (i) a bead of said plurality of beads, wherein said bead comprises a first primer comprising a first primer sequence, wherein said droplet does not comprise any other beads, (ii) at least two nucleic acid molecules of said plurality of nucleic acid molecules, comprising a first nucleic acid molecule and a second nucleic acid molecule, wherein said first nucleic acid molecule and said second nucleic acid molecule have different nucleic acid sequences and each of said first nucleic acid molecule and said second nucleic acid molecule comprises a first adaptor sequence, wherein said first primer sequence does not have sequence complementarity with said first adaptor sequence, and (iii) one or more reagents comprising a second primer within said droplet, said second primer not attached to any beads and comprising a first portion and a second portion, wherein said first portion is configured to hybridize to said first adaptor sequence, and wherein said second portion comprises a second primer sequence that corresponds to said first primer sequence, wherein a ratio of a concentration of said second primer within said droplet to a concentration of said first primer in said droplet is on an order of 10-1 or less;
(b) in said droplet, with said second primer hybridized to said first adaptor sequence of said first nucleic acid molecule, (i) using said one or more reagents to generate one or more extension products of said first nucleic acid molecule comprising said second primer sequence or reverse complement thereof, (ii) attaching an extension product of said one or more extension products to said bead by annealing said second primer sequence or reverse complement thereof to said first primer sequence and extending said first primer, and (iii) generating amplification products of said first nucleic acid molecule attached to said bead, thereby monoclonally amplifying said first nucleic acid molecule on said bead;
(c) recovering said bead from said droplet; and
(d) assaying an amplification product of said amplification products attached to said bead, to identify a sequence of said first nucleic acid molecule.