[세상사 논문] 이호원 Ph.D.

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1.     이호원 Ph.D. Harvard Medical School, Genetics Department (Church Lab) Postdoc

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2.     Article

A high-throughput optomechanical retrieval method for sequence-verified clonal DNA from the NGS platform.

PMID: 25641679

http://www.nature.com/ncomms/2015/150202/ncomms7073/full/ncomms7073.html

 

3.     Abstract

Writing DNA plays a significant role in the fields of synthetic biology, functional genomics and bioengineering. DNA clones on next-generation sequencing (NGS) platforms have the potential to be a rich and cost-effective source of sequence-verified DNAs as a precursor for DNA writing. However, it is still very challenging to retrieve target clonal DNA from high-density NGS platforms. Here we propose an enabling technology called ‘Sniper Cloning’ that enables the precise mapping of target clone features on NGS platforms and non-contact rapid retrieval of targets for the full utilization of DNA clones. By merging the three cutting-edge technologies of NGS, DNA microarray and our pulse laser retrieval system, Sniper Cloning is a week-long process that produces 5,188 error-free synthetic DNAs in a single run of NGS with a single microarray DNA pool. We believe that this technology has potential as a universal tool for DNA writing in biological sciences.

 

4.     논문과 관련된 분야, 본 연구의 중요성 및 후속 연구계획

The rapid growth of synthetic biology and functional genomics has spurred the need to synthesize increasingly larger pieces of DNA. Writing such long DNA molecules depends on the step-wise assembly of purified oligonucleotides of well-defined sequences, which can be costly and error-prone when carried out by standard solid-phase oligonucleotide synthesis.

A major advance came with the development of programmable parallel oligonucleotide synthesis using DNA microarrays that can produce up to millions of sequences in a mixed pool from one array in a single run at significant cost and time savings. These advantages, however, were offset by the need to isolate the oligonucleotides with the correct sequences from a highly complex pool containing a significant fraction of oligonucleotides with sequence errors. Using the conventional method of in vivo cloning followed by Sanger sequencing was impractical given its very low throughput.

This was the problem that confronted Duhee Bang, a synthetic biologist at Yonsei University, and Sunghoon Kwon, an electrical engineer and bioengineer at Seoul National University. Five years ago, Bang was attempting genome-scale engineering of E. coli, and “had a huge problem with preparing high-quality precursor oligonucleotides for contamination-resistant E. coli gene modification,” said Kwon, who has long been interested in developing novel approaches to minimize the expense and time involved in conventional biotechnology methods. “Professor Bang brought up several technical problems in utilizing microarray DNA pools, and our group accepted that technical challenge.”

Bang and Kwon first tried selecting the oligonucleotides by adding different specific barcoded primers to the microarray-generated pool of oligonucleotides, followed by next-generation sequencing (NGS) verification of the oligonucleotide sequences. With this approach, it would be possible to selectively PCR amplify the desired error-free sequence using the appropriate matching pair of barcode primers. This method suffers, however, from missing and mixed sequences, and they found that the time and expense of designing and synthesizing the large numbers of barcoded primers with the required orthogonality was a major hindrance in scaling up the technique.

Fortunately, they came across a 2010 Nature Biotechnology paper (1) from George Church’s group describing megacloning, which uses NGS to directly select sequence-verified clones from a complex pool of mixed oligonucleotides after NGS without the need for subsequent selective PCR amplification. This was possible using Roche 454 Life Sciences’ GLS platform, where each individual bead in a well in the open-top 454-Picotiterplate (PTP) that corresponds to a sequenced oligonucleotide clone could be directly physically extracted with a micropipette using a robotic imaging and bead picking system.

“The concept of direct utilization of clones on the NGS substrate was a refreshing jolt,” said Kwon, but as an engineer, he felt that there was room for improvement to the method. The throughput was constrained by the need for physical contact-based retrieval of the beads, making the method inadequate for generating the large number of oligonucleotides necessary for writing megabase-sized DNA pieces.

Kwon and Bang’s improved version of the megacloning method, Sniper Cloning, was recently published in Nature Communications (2). Their key modification is the use of a fully automated pulse laser system for the non-contact and very rapid retrieval of the desired beads from the PTP. They cleverly took advantage of the structure of the PTP, where each well that holds one bead is formed by etching of the end of an individual fiber optic core that conducts the light released from the bead during pyrosequencing back to the CCD camera. By inverting the PTP, a low-energy laser pulse targeted to the back side (i.e. top) of a selected well is transmitted by the fiber optic core to the bead, pushing it out by radiation pressure down into the well of a 96-well microtiter plate positioned underneath the PTP. This approach greatly reduces the possibility of cross-contamination inherent to megacloning and, by using an automated motorized stage, is orders of magnitude faster than retrieving the selected beads from the PTP.

Sniper Cloning also uses an improved mapping algorithm to precisely locate each well in a PTP containing the bead corresponding to a desired oligonucleotide sequence. This mapping requires the precise alignment of the sequencing data pixel map obtained by the CCD with the image of the wells for the entire PTP. Bang and Kwon initially tried the mapping algorithm used in megacloning and found that “in a small area with distinctive natural markers, their algorithm showed very good results,” said Kwon. “But the algorithm could not be used as a general method to identify all useful clonal beads in the whole chip area. Imaging error and quantification error accumulates as the region of interest gets larger.” By developing a diffusion-based local mapping algorithm, it was possible to eliminate these sources of positional errors and correctly map the entire PTP.

By combining DNA microarray-directed oligonucleotide synthesis, NGS, and pulse laser retrieval, Sniper Cloning is able to generate 5188 error-free oligonucleotide sequences in 1 week from a single NGS run of a single microarray pool. The system is also applicable to non-bead-based NGS systems such Illumina where the DNA for sequencing is directly attached as clusters to the surface of the sequencing substrate. This method should prove to be a powerful tool for synthetic biologists in their quest to write megabase-scale DNA.

http://www.biotechniques.com/news/Sniper-Cloning-Targeting-Next-generation-Sequencing-to-Write-DNA/biotechniques-356987.html?service=print#.Vs4WDvnhDRY

References

1. Matzas M, Stähler PF, Kefer N, Siebelt N, Boisguérin V, Leonard JT, Keller A, Stähler CF, Häberle P, Gharizadeh B, Babrzadeh F, Church GM. 2010. High-fidelity gene synthesis by retrieval of sequence-verified DNA identified using high-throughput pyrosequencing. Nat Biotechnol. 2010 Dec;28(12):1291-4. doi: 10.1038/nbt.1710

2. Lee H, Kim H, Kim S, Ryu T, Kim H, Bang D, Kwon S. 2015. A high-throughput optomechanical retrieval method for sequence-verified clonal DNA from the NGS platform. Nat Commun. 2015 Feb 2;6:6073. doi: 10.1038/ncomms7073

 

5.     못다한 이야기들

제가 전기공학부 학부를 졸업하고 동 대학원에서 광학으로 석사를 전공할 때만 해도 제가 DNA를 다루는 일을 연구하게 될 줄은 몰랐습니다. 박사 과정 후반기에 와서야 비로소 DNA를 접하게 되고 PCR 과 같은 지극히 기본적인 분자생물학 실험을 하기 시작했습니다. 제가 현재까지도 몸 담고 있는 연구 주제는 전통적인 생명과학, 생명공학의 관점에서는 조금은 급진적이며 device와의 연관도도 높습니다. 그러나 오히려 이러한 부분이 전기공학을 전공한 저로서는 더 매력을 느낄 수 있는 계기가 되었습니다. 마지막으로 제가 박사학위를 받은 서울대 연구실의 mission 으로 제 글을 마무리 하고자 합니다.

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