(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 2015/021080 A2 12 February 2015 (12.02.2015) P O P C T (51) International Patent Classification: (74) Agents: BAILEY, F., Pinar et al; Wilson Sonsini C12N 15/10 (2006.01) B01J 19/00 (2006.01) Goodrich & Rosati, 650 Page Mill Road, Palo Alto, CA 94304-1050 (US). (21) International Application Number: PCT/US20 14/049834 (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (22) International Filing Date: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, 5 August 2014 (05.08.2014) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, (25) Filing Language: English DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, (26) Publication Language: English KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, (30) Priority Data: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 61/862,445 5 August 2013 (05.08.2013) OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, 61/862,457 5 August 2013 (05.08.2013) SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, (71) Applicant: TWIST BIOSCIENCE CORPORATION ZW. [US/US]; 455 Mission Bay Blvd., South, Suite 545, San Francisco, CA 94158 (US). (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (72) Inventors: BANYAI, William; 738 Wayland Street, San GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, Francisco, CA 94134 (US). PECK, Bill, James; 3086 Car- UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, leton Place, Santa Clara, CA 9505 1 (US). FERNANDEZ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, Andres; 400 Beale Street # 1406, San Francisco, CA EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, 94105 (US). CHEN, Siyuan; 1021 Coriander Walkway, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, San Jose, CA 95 133 (US). INDERMUHLE, Pierre; 1817- TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, A Oregon Street, Berkeley, CA 94703 (US). KM, ML, MR, NE, SN, TD, TG). [Continued on nextpage] (54) Title: DE NOVO SYNTHESIZED GENE LIBRARIES Figure 4 O igo Wafer Reactor s 13 r pitch < © o00 © (57) Abstract: De novo synthesized large libraries of nucleic acids are provided herein with low error rates. Further, devices for the o manufacturing of high-quality building blocks, such as oligonucleotides, are described herein. Longer nucleic acids can be synthes ized in parallel using microfluidic assemblies. Further, methods herein allow for the fast construction of large libraries of long, high- quality genes. Devices for the manufacturing of large libraries of long and high-quality nucleic acids are further described herein. WO 2015/021080 A2 llll II II 11III II Published: — without international search report and to be republished upon receipt of that report (Rule 48.2(g)) DE NOVO SYNTHESIZED GENE LIBRARIES CROSS-REFERENCE [0001] This application claims the benefit of U.S. Provisional Application No. 61/862445, filed August 5, 2013 and U.S. Provisional Application No. 61/862457, filed August 5, 2013, which applications are incorporated herein by reference. BACKGROUND OF THE INVENTION [0002] Highly efficient chemical gene synthesis with high fidelity and low cost has a central role in biotechnology and medicine, and in basic biomedical research. [0003] De novo gene synthesis is a powerful tool for basic biological research and biotechnology applications. While various methods are known for the synthesis of relatively short fragments in a small scale, these techniques suffer from scalability, automation, speed, accuracy, and cost. There is a need for devices for simple, reproducible, scalable, less error- prone and cost-effective methods that guarantee successful synthesis of desired genes and are amenable to automation. SUMMARY OF THE INVENTION [0004] As noted above, there exists a pressing need for methods, devices and systems that can quickly synthesize large gene libraries or relatively longer oligonucleotide fragments efficiently with less error. Similarly, there is also a need for methods that can partition and mix liquid reagents in a microfluidic scale for large numbers of individually addressable reactions in parallel. The present invention addresses these needs and provides related advantages as well. [0005] In one aspect, the present invention provides a gene library as described herein. The gene library comprises a collection of genes. In some embodiments, the collection comprises at least 100 different preselected synthetic genes that can be of at least 0.5 kb length with an error rate of less than 1 in 3000 bp compared to predetermined sequences comprising the genes. In another aspect, the present invention also provides a gene library that comprises a collection of genes. The collection may comprise at least 100 different preselected synthetic genes that can be each of at least 0.5 kb length. At least 90% of the preselected synthetic genes may comprise an error rate of less than 1 in 3000 bp compared to predetermined sequences comprising the genes. Desired predetermined sequences may be supplied by any method, typically by a user, e.g. a user entering data using a computerized system. In various embodiments, synthesized nucleic acids are compared against these predetermined sequences, in some cases by sequencing at least a portion of the synthesized nucleic acids, e.g. using next-generation sequencing methods. In some embodiments related to any of the gene libraries described herein, at least 90% of the preselected synthetic genes comprise an error rate of less than 1 in 5000 bp compared to predetermined sequences comprising the genes. In some embodiments, at least 0.05% of the preselected synthetic genes are error free. In some embodiments, at least 0.5% of the preselected synthetic genes are error free. In some embodiments, at least 90% of the preselected synthetic genes comprise an error rate of less than 1 in 3000 bp compared to predetermined sequences comprising the genes. In some embodiments, at least 90% of the preselected synthetic genes are error free or substantially error free. In some embodiments, the preselected synthetic genes comprise a deletion rate of less than 1 in 3000 bp compared to predetermined sequences comprising the genes. In some embodiments, the preselected synthetic genes comprise an insertion rate of less than 1 in 3000 bp compared to predetermined sequences comprising the genes. In some embodiments, the preselected synthetic genes comprise a substitution rate of less than 1 in 3000 bp compared to predetermined sequences comprising the genes. In some embodiments, the gene library as described herein further comprises at least 10 copies of each synthetic gene. In some embodiments, the gene library as described herein further comprises at least 100 copies of each synthetic gene. In some embodiments, the gene library as described herein further comprises at least 1000 copies of each synthetic gene. In some embodiments, the gene library as described herein further comprises at least 1000000 copies of each synthetic gene. In some embodiments, the collection of genes as described herein comprises at least 500 genes. In some embodiments, the collection comprises at least 5000 genes. In some embodiments, the collection comprises at least 10000 genes. In some embodiments, the preselected synthetic genes are at least lkb. In some embodiments, the preselected synthetic genes are at least 2kb. In some embodiments, the preselected synthetic genes are at least 3kb. In some embodiments, the predetermined sequences comprise less than 20 bp in addition compared to the preselected synthetic genes. In some embodiments, the predetermined sequences comprise less than 15 bp in addition compared to the preselected synthetic genes. In some embodiments, at least one of the synthetic genes differs from any other synthetic gene by at least 0.1%. In some embodiments, each of the synthetic genes differs from any other synthetic gene by at least 0.1%. In some embodiments, at least one of the synthetic genes differs from any other synthetic gene by at least 10%. In some embodiments, each of the synthetic genes differs from any other synthetic gene by at least 10%. In some embodiments, at least one of the synthetic genes differs from any other synthetic gene by at least 2 base pairs. In some embodiments, each of the synthetic genes differs from any other synthetic gene by at least 2 base pairs. In some embodiments, the gene library as described herein further comprises synthetic genes that are of less than 2kb with an error rate of less than 1 in 20000 bp compared to preselected sequences of the genes. In some embodiments, a subset of the deliverable genes is covalently linked together. In some embodiments, a first subset of the collection of genes encodes for components of a first metabolic pathway with one or more metabolic end products. In some embodiments, the gene library as described herein further comprises selecting of the one or more metabolic end products, thereby constructing the collection of genes. In some embodiments, the one or more metabolic end products comprise a biofuel. In some embodiments, a second subset of the collection of genes encodes for components of a second metabolic pathway with one or more metabolic end products. In some embodiments, the gene library is in a space that is less than 100 m3 .