Patent SG-11201808775P-A (SG)
Title: Noninvasive diagnostics by sequencing 5-hydroxymethylated cell-free dna
Inventor: QUAKE STEPHEN R
IPC: G01N33/50
Filed: 2017-04-03
Abstract: INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property —, Organization 111111110111101110101011111010111110111010111111101101111101111101111011111 International Bureau ... ..... ..Yi j ..... ..... (10) International Publication Number (43) International Publication Date WO 2017/176630 Al 12 October 2017(12.10.2017) WIPO I PCT (51) International Patent Classification: Office of the General Counsel, Building 170, 3rd Floor, C12N 15/00 (2006.01) C12Q 1/68 (2006.01) Main Quad, P.O. Box 20386, Stanford, California 94305- C12N 15/07 (2006.01) G01N 33/50 (2006.01) 2038 (US). (21) International Application Number: (72) Inventor; and PCT/US2017/025735 (71) Applicant (for US only): QUAKE, Stephen R. [US/US]; 318 Campus Drive, Stanford, California 94305 (US). (22) International Filing Date: 3 April 2017 (03.04.2017) (72) Inventor: SONG, Chunxiao; Ludwig Institute, University of Oxford, Old Road Campus Research Building, Heading- (25) Filing Language: English ton, Oxford OX3 7DQ (GB). (26) Publication Language: English (74) Agent: KEDDIE, James S.; 201 Redwood Shores Park- (30) Priority Data: way, Suite 200, Redwood City, California 94065 (US). 62/319,702 7 April 2016 (07.04.2016) US (81) Designated States (unless otherwise indicated, for every 62/444,122 9 January 2017 (09.01.2017) US kind of national protection available): AE, AG, AL, AM, 62/461,712 21 February 2017 (21.02.2017) US AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (71) Applicant: THE BOARD OF TRUSTEES OF THE LE- BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, LAND STANFORD JUNIOR UNIVERSITY [US/US]; DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, [Continued on next page] = (54) Title: NONINVASIVE DIAGNOSTICS BY SEQUENCING 5-HYDROXYMETHYLATED CELL-FREE DNA (57) : Provided herein is a method of sequencing hydroxy- - _ ciDNA methyated cell-free DNA. In some embodiments, the method com- tag to hydroxymethyated DNA _ prises adding an affinity only mo- - a 5h mc 9 _.2____ lecules in a sample of cfDNA, enriching for the DNA molecules that are tagged with the affinity tag; and sequencing the enriched end repair &A-tailing DNA molecules. adapter ligation -,__ illurnina \" — adapter , -/' • 9 J , — ¨ , , , selective labeling of 5hmC with biotin , \----e-------, / • El biotin • \ . \ • _ • • — / I capture 5hmC + extensive wash M 1-1 strepdavidin beads • q • PCR from beads ir 0 final libary M IN FIG, 1 A ,—, IN ,—, © ei O WO 2017/176630 Al MUNRO 111111111111111111111111113111111111111111 Ell 0111110 111110111111111111 HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). Declarations under Rule 4.17: TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. — of inventorship (Rule 4.17(iv)) (84) Designated States (unless otherwise indicated, for every Published: kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, — with international search report (Art. 21(3)) with sequence listing part of description (Rule 5.2(a)) TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE,