US 12,286,622 B2
Genomics-based identification and characterization of rare cell types
Christopher Armour, Palo Alto, CA (US); and Pek Yee Lum, Palo Alto, CA (US)
Assigned to Auransa Inc., Palo Alto (CA)
Appl. No. 17/419,422
Filed by Auransa Inc., Palo Alto, CA (US)
PCT Filed Dec. 30, 2019, PCT No. PCT/US2019/068898
§ 371(c)(1), (2) Date Jun. 29, 2021,
PCT Pub. No. WO2020/142409, PCT Pub. Date Jul. 9, 2020.
Claims priority of provisional application 62/786,828, filed on Dec. 31, 2018.
Prior Publication US 2022/0056434 A1, Feb. 24, 2022
Int. Cl. C12N 15/10 (2006.01); C12Q 1/6876 (2018.01); G16B 30/10 (2019.01)
CPC C12N 15/1065 (2013.01) [C12Q 1/6876 (2013.01); G16B 30/10 (2019.02)] 17 Claims
OG exemplary drawing
 
1. A method of identifying the presence of a rare cell type in a biological sample, comprising steps of:
(a) generating, for each of a plurality of nucleic acid subsets of the biological sample, a subset genomic library comprising barcoded double-stranded genomic DNA (gDNA) constructs, wherein the gDNA constructs comprise a first gDNA strand and a second gDNA strand, wherein the first gDNA strand comprises, from 5′ to 3′:
(i) a first universal next generation sequencing (NGS) primer comprising, from 5′ to 3′, a first flow cell adapter sequence; and the nucleotide sequence SEQ ID NO:56;
(ii) a gDNA sequence of the rare cell type;
(iii) a sequencing primer for a nucleic acid subset-specific molecular barcode;
(iv) the nucleic acid subset-specific molecular barcode; and
(v) a sequence complementary to a second flow cell adapter sequence present on the second gDNA strand;
(b) pooling the subset genomic libraries to form a combined sequencing library;
(c) obtaining DNA sequencing reads from the combined sequencing library;
(d) identifying by means of the nucleic acid subset-specific molecular barcode a nucleic acid subset comprising the gDNA sequence of the rare cell type, thereby identifying the presence of the rare cell type in the biological sample.