US 12,241,059 B2
Tiered ligation oligos
Robert Meltzer, Belmont, MA (US); Kristina Fontanez, Arlington, MA (US); and Yi Xue, Shrewsbury, MA (US)
Assigned to Illumina, Inc., San Diego, CA (US)
Filed by ILLUMINA, INC., San Diego, CA (US)
Filed on Jul. 15, 2021, as Appl. No. 17/376,709.
Claims priority of provisional application 63/052,073, filed on Jul. 15, 2020.
Prior Publication US 2022/0017892 A1, Jan. 20, 2022
Int. Cl. C12N 15/10 (2006.01); C12Q 1/6853 (2018.01)
CPC C12N 15/1065 (2013.01) [C12Q 1/6853 (2013.01)] 19 Claims
 
1. A method for creating a target capture reagent, the method comprising:
dividing a plurality of beads linked to initial oligos into a first set of partitions;
ligating partition-specific first duplexes comprising first barcodes to the initial oligos to form ligation products;
pooling the ligation products into a pool;
splitting the pool into a second set of partitions;
ligating partition-specific second duplexes comprising second barcodes to the ligation products to form multi-part oligonucleotides each comprising (i) one of the initial oligos, (ii) one of the first barcodes, and (iii) one of the second barcodes;
repeating the pooling, splitting, and ligating steps at least twice with additional sets of partition-specific duplexes and barcodes, wherein each successive repetition comprises a number of partition-specific duplexes and barcodes that is one greater than the immediate prior round of pooling, splitting, and ligating, and wherein a penultimate repeating step comprises a partition-specific duplex having two overhangs on the same strand.