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High-fidelity gene synthesis by retrieval of sequence-verified DNA identified using high-throughput pyrosequencing

Abstract

The construction of synthetic biological systems involving millions of nucleotides is limited by the lack of high-quality synthetic DNA. Consequently, the field requires advances in the accuracy and scale of chemical DNA synthesis and in the processing of longer DNA assembled from short fragments. Here we describe a highly parallel and miniaturized method, called megacloning, for obtaining high-quality DNA by using next-generation sequencing (NGS) technology as a preparative tool. We demonstrate our method by processing both chemically synthesized and microarray-derived DNA oligonucleotides with a robotic system for imaging and picking beads directly off of a high-throughput pyrosequencing platform. The method can reduce error rates by a factor of 500 compared to the starting oligonucleotide pool generated by microarray. We use DNA obtained by megacloning to assemble synthetic genes. In principle, millions of DNA fragments can be sequenced, characterized and sorted in a single megacloner run, enabling constructive biology up to the megabase scale.

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Figure 1: Coalescence of DNA reading and writing.
Figure 2: NGS-based comparison of untreated and megacloned oligonucleotide pools from microarray.

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Acknowledgements

We thank B.A. Roe, F.Z. Najar and D.D. White for sequencing support, J. Jäger for technical consulting, and D. Summerer, T. Brefort, S. Kosuri and D. Levner for discussions and comments.

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Authors

Contributions

M.M., P.F.S. and G.M.C. conceptualized the megacloning method and wrote the manuscript; M.M. designed and lead the study, wrote all algorithms for sequence design, data analysis, image conversion, image processing and microactuator control; M.M., N.K., N.S. acquired the used technology, set up the microactuator device and optical systems; N.S. designed the uidA genetic model; M.M., N.K., N.S., V.B. and P.H. designed and optimized molecular biological methods; C.F.S. and J.T.L. contributed to bead picking and engineering concepts; A.K. set up the statistical models and calculations; J.T.L. contributed to the design of molecular biological steps and the acquisition of sequencing samples; B.G. and F.B. evaluated and implemented necessary changes into the sample preparation and the sequencing process on the 454/Roche platform.

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Correspondence to George M Church.

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Competing interests

M.M., N.K., N.S., V.B., J.T.L., P.F.S., A.K., C.F.S. and P.H. have potentially competing financial interests in companies affiliated to the febit group.

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Matzas, M., Stähler, P., Kefer, N. et al. High-fidelity gene synthesis by retrieval of sequence-verified DNA identified using high-throughput pyrosequencing. Nat Biotechnol 28, 1291–1294 (2010). https://doi.org/10.1038/nbt.1710

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