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WO 2017/014762 Al 26 January 2017 (26.01.2017) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2017/014762 Al 26 January 2017 (26.01.2017) P O P C T (51) International Patent Classification: DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, C12Q 1/68 (2006.01) HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, (21) International Application Number: MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PCT/US201 5/0414 15 PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (22) International Filing Date: SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, 2 1 July 20 15 (21 .07.2015) TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (26) Publication Language: English GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (71) Applicant: OMNIOME, INC. [US/US]; 4225 Executive TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, Square, Suite 440, La Jolla, California 92037 (US). TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (72) Inventors: VIJAYAN, Kandaswamy; 4465 Vision Drive, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, Unit 6, San Diego, California 92121 (US). TU, Eugene; SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, 5327 Lark Street, San Diego, California 92103 (US). GW, KM, ML, MR, NE, SN, TD, TG). BERNARD, Mark A.; 14574 Springvale Street, Poway, California 92064 (US). Declarations under Rule 4.17 : — as to applicant's entitlement to apply for and be granted a (74) Agents: THOMAS, Tiffany B. et al; Kilpatrick Town- patent (Rule 4.1 7(H)) send & Stockton LLP, Two Embarcadero Center, Eighth Floor, San Francisco, California 941 11 (US). Published: (81) Designated States (unless otherwise indicated, for every — with international search report (Art. 21(3)) kind of national protection available): AE, AG, AL, AM, — with sequence listing part of description (Rule 5.2(a)) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, (54) Title: NUCLEIC ACID SEQUENCING METHODS AND SYSTEMS (57) Abstract: The present disclosure provides compositions, methods and systems for sequencing a template nucleic acid using a polymerase based, nucleic acid binding reaction involving examination of the interaction between a polymerase and template nucleic acid in the presence of one or more unlabeled nucleotides. The methods rely, in part, on identifying a base of a template nucleic acid during nucleic acid synthesis by controlling the sequencing reaction conditions. Template nucleic acid bases may be identified dur ing an examination step followed by an optional incorporation step. NUCLEIC ACID SEQUENCING METHODS AND SYSTEMS BACKGROUND [0001] The determination of nucleic acid sequence information is an important part of biological and medical research. The sequence information is helpful for identifying gene associations with diseases and phenotypes, identifying potential drug targets, and understanding the mechanisms of disease development and progress. Sequence information is an important part of personalized medicine, where it is can be used to optimize the diagnosis, treatment, or prevention of disease in a specific subject. SUMMARY [0002] Provided herein are methods for sequencing a template nucleic acid molecule. The method generally includes an examination step prior to incorporation of an unlabeled nucleotide. More specifically, the examination step includes providing a template nucleic acid molecule primed with a primer, contacting the primed template nucleic acid molecule with a first reaction mixture that includes a polymerase and at least one unlabeled nucleotide molecule, monitoring the interaction of the polymerase with the primed template nucleic acid molecule in the presence of the unlabeled nucleotide molecule, without chemical incorporation of the nucleotide molecule into the primed template nucleic acid, and identifying a next base in the template nucleic acid based on the monitored interaction of the polymerase with the primed template nucleic acid molecule in the presence of the unlabeled nucleotide molecule. BRIEF DESCRIPTION OF THE DRAWINGS [0003] Figure 1 is a graph showing the results of an experiment using non-labeled optical detection methods where magnesium was present or absent during the binding or examination step. [0004] Figure 2 is a graph showing sequencing using Bst enzyme binding kinetics for determining the correct base using Bst2.0 enzyme and dNTPs. [0005] Figure 3 is a graph showing the effects of salt concentration on match and mismatch base discrimination effects using biolayer interferometry on a FORTEBIO® octet instrument (Menlo Park, CA). [0006] Figure 4 is a graph showing base discrimination during the wash step, i.e., during dissociation of the polymerase, using phiX matchC and FP2 primer and klenow or Bst2.0 enzyme, and SrCl2. [0007] Figure 5 is a graph showing the effect of washing on the stabilization of nucleic acid, polymerase complex using varying concentrations of SrCl2 (0 mM-14 mM). [0008] Figure 6 is a graph showing the effect of 3'-5' exonuclease activity of DNA pol I on sequencing. [0009] Figures 7A and 7B are graphs showing sequencing of human ALK gatekeeper region using HIV-1 reverse transcriptase, NNRTI compound 7 and the indicated dNTPs. Figure 7A is a graph showing the time course for consecutive cycles of sequencing. Figure 7B is a graph showing cycles 1-12 in individual panels after subtracting background from the previous cycle. The expected sequence read was CAGCAGGA (SEQ ID NO:l) and the observed sequence read was CAGCAGG (SEQ ID NO:2). [0010] Figure 8 is a graph showing sequencing of human ALK gatekeeper region using HIV-1 reverse transcriptase, NNRTI compound 18 and the various dNTPs. [0011] Figure 9 is a sensorgram showing sequencing of the phiX matchA template using a SPRi biosensor. Grayed areas correspond to correct base calls. The dotted line indicates the intensity change threshold used to determine binding of the correct Klenow/dNTP combination. [0012] Figures 10A, 10B, and IOC are graphs showing sequencing of dsDNA by nick translation using Bst DNA Polymerase from Bacillus stearothermophilus. Double-stranded DNA with one base gap was treated with Bst DNA Pol with or without the indicated dNTP in Binding Buffer. Biosensors were transferred to Reaction Buffer for dNTP incorporation followed by transfer to Reaction Buffer containing Bst DNA Pol without dNTP for 5'-3' exonucleolytic cleavage of the non-template strand for 120 seconds (Fig. 10A) or 60 seconds (Fig. 10B). As a control, Bst DNA Pol was used for sequencing by binding a primed ssDNA template, dNTP incorporation followed by 5'-3' exonucleolytic processing for 60 seconds (Fig. IOC). [0013] Figures 11A, 1IB, and 1ID are graphs showing sequencing of dsDNA with 5'-flap by strand displacement using Klenow (3'→5' exo-) fragment of E. coli DNA polymerase. DNA templates were treated with Klenow exo- DNA Pol with or without the indicated dNTP in Binding Buffer without MgC12. Biosensors were transferred to Wash Buffer with MgC12 for catalysis followed by re-equilibration in Binding Buffer without enzyme or dNTP. Cycles were repeated for each individual dNTP as indicated. Figure 11A : Single-stranded DNA. Figure 11B: double-stranded DNA with one base gap. Figure 11C : double-stranded DNA with a 5'- oligo-dT flap downstream of a one base pair gap. [0014] Figures 12A, 12B, and 12C are graphs showing sequencing ssDNA by Klenow (3'→5' exo-) fragment of E. coli DNA polymerase are promoted by salt components. Binding Buffers contain 200 mM glutamate (Fig. 12A), 100 mM glutamate (Fig. 12B) and 50 mM glutamate (Fig. 12C). Reaction Buffers contain MgC12 without glutamate. The applied dNTP for each cycle ("dNTP") is shown in the top text row (SEQ ID NO: 14) of each of Figures 12A, 12B, and 12C. Binding of Klenow (exo-) indicates observed sequence ("Observed") in the second row of Figures 12A (SEQ ID NO: 15), 12B (SEQ ID NO: 17), and 12C (SEQ ID NO: 19). "Expected" sequence based on the template is shown in the third text row of Figures 12A (SEQ ID NO: 16), 12B (SEQ ID NO: 18), and 12C (SEQ ID NO:20). [0015] Figures 13A, 13B, and 13C are graphs of sequencing the sense strand of human ALK C4493A mutant in a background of ALK wild-type by Klenow exo- DNA polymerase. Figure 13 A is a sensorgram demonstrating sequencing in ssDNA mixtures of wild-type and C4493A mutant. Figure 13B is a graph showing, in ssDNA mixtures, linear quantitation of C4493A mutant shown in Cycle 4 (T), and linear quantitation of wild-type ALK shown in cycle 3 (G). Figure 13C is a graph showing, in dsDNA-flap mixtures, linear quantitation of C4493A mutant is shown in Cycle 4 (T), and roughly linear quantities of wild-type ALK are shown in cycle 3 (G). [0016] Figures 14A and 14B are graphs of divalent cation-mediated binding of Klenow exo- and dCTP to human ALK C4493A mutant and dissociation with or without catalytic metal (MgCl2). Figure 14A is a graph showing binding to the primer/template and dissociation in the presence of non-catalytic metals. Figure 14B is a graph showing binding to the primer/template and dissociation in the presence of catalytic metals. [0017] Figure 15 is a graph of Klenow exo- sequencing of human ALK C4493A mutant in which binding is mediated by low concentration of CoCl2, and incorporation is mediated by high concentration of CoCl2.
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