Large scale parallelization of single cell phenomics

• Project/Area Number: 20K06584
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 43040:Biophysics-related
• Research Institution: The University of Tokyo
• Principal Investigator: Ohnuki Shinsuke 東京大学, 大学院新領域創成科学研究科, 特任助教 (80739756)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2022: ¥130,000 (Direct Cost: ¥100,000、Indirect Cost: ¥30,000); Fiscal Year 2021: ¥390,000 (Direct Cost: ¥300,000、Indirect Cost: ¥90,000); Fiscal Year 2020: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000)
• Keywords: 出芽酵母 / 細胞形態 / iIACS / FACS / CalMorph / Bar-seq / 次世代シーケンサ / 大規模解析 / DNA barcode / DNAバーコード / 顕微鏡画像 / 遺伝子破壊株 / プールド培養液 / シミュレーション / 一細胞フェノーム / 遺伝的相互作用

Outline of Research at the Start

生命活動は複数の遺伝子が互いに協調し相互作用することで可能となる。出芽酵母では、遺伝的相互作用が1800万株に及ぶ二重遺伝子破壊株の増殖表現型を網羅的に観察することで明らかにされた。本研究では、高次元形態表現型において遺伝的相互作用を明らかにすることが可能な技術の開発を行う。細胞形態の定量は、一株ずつ顕微鏡撮影する必要があったため、スループットが圧倒的に不足している。本研究で提案する新たな形態解析法は、最新のセルソーター技術と次世代シーケンサー技術を組み合わせることで一度に数千株の形態情報を取得可能である。本研究では、提案手法を実現するシステムの実用レベルの実装と、その実用可能性の実証を行う。

Outline of Final Research Achievements

Living systems function through the orchestration and interaction of genes. In Saccharomyces cerevisiae, the genetic interaction network was comprehensively clarified by the growth phenotypes of 18 million double disruption mutants. In this research, we will develop a technology that can thoroughly clarify genetic interactions using high-dimensional morphological information. The method proposed in this study combines the latest cell sorter technology and next-generation sequencing technology to enable acquisition of morphological information on thousands of strains at once. Here, as a proof-of-concept study, a practical level system was implemented by combining FACS and a next-generation sequencer, and the feasibility of the proposed method was demonstrated.

Academic Significance and Societal Importance of the Research Achievements

増殖表現型が生育機能を反映する一方で、高次元形態表現型は様々な細胞内プロセスを高解像度に反映する。生命活動の実態を遺伝子間の複雑な相互作用からより深く理解するためには、遺伝子間相互作用ネットワークを従来の増殖表現型だけでなく高次元形態表現型に拡張する必要がある。本研究では、そのための基盤的な技術開発の基礎を築いており、複雑な生物学的現象を理解するための足がかりとなる。遺伝子間相互は複雑で予想不可能な遺伝病や有害な薬理作用の源泉となっており、本研究が遺伝子間相互作用ネットワークのより深い理解に貢献することで、原因不明の難病や希少疾患を克服する機会につながることを期待できる。

Research Products

• [Journal Article] Prediction of ethanol fermentation under stressed conditions using yeast morphological data. (2023)
  • Author(s): Itto-Nakama K, Watanabe S, Ohnuki S, Kondo N, Kikuchi R, Nakamura T, Ogasawara W, Kasahara K, Ohya Y.
  • Journal Title: J Biosci Bioeng. Volume: 135 Issue: 3 Pages: 210-216
  • DOI: 10.1016/j.jbiosc.2022.12.008
  • Peer Reviewed
• [Journal Article] Intelligent sort‐timing prediction for image‐activated cell sorting (2022)
  • Author(s): Zhao Yaqi、Isozaki Akihiro、Herbig Maik、Hayashi Mika、Hiramatsu Kotaro、Yamazaki Sota、Kondo Naoko、Ohnuki Shinsuke、Ohya Yoshikazu、Nitta Nao、Goda Keisuke
  • Journal Title: Cytometry Part A Volume: 103 Issue: 1 Pages: 88-97
  • DOI: 10.1002/cyto.a.24664
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Assignment of unimodal probability distribution models for quantitative morphological phenotyping. (2022)
  • Author(s): Ghanegolmohammadi F, Ohnuki S, Ohya Y.
  • Journal Title: BMC Biol. Volume: 20 Issue: 1 Pages: 81-81
  • DOI: 10.1186/s12915-022-01283-6
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Deep imaging flow cytometry. (2022)
  • Author(s): Huang K, Matsumura H, Zhao Y, Herbig M, Yuan D, Mineharu Y, Harmon J, Findinier J, Yamagishi M, Ohnuki S, Nitta N, Grossman AR, Ohya Y, Mikami H, Isozaki A, Goda K.
  • Journal Title: Lab Chip. Volume: 22 Issue: 5 Pages: 876-889
  • DOI: 10.1039/d1lc01043c
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] High-throughput platform for yeast morphological profiling predicts the targets of bioactive compounds (2022)
  • Author(s): Ohnuki, S., Ogawa, I., Itto-Nakama, K., Lu, F., Ranjan, A., Kabbage, M., Gebre, AA., Yamashita, M., Li, SC., Yashiroda, Y., Yoshida, S., Usui, T., Piotrowski, JS., Andrews, BJ., Boone, C., Brown, GW., Ralph, J., Ohya, Y.
  • Journal Title: npj Systems Biology and Applications Volume: 8 Issue: 1 Pages: 3-3
  • DOI: 10.1038/s41540-022-00212-1
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Jerveratrum-Type Steroidal Alkaloids Inhibit β-1,6-Glucan Biosynthesis in Fungal Cell Walls (2022)
  • Author(s): Kubo Karen、Itto-Nakama Kaori、Ohnuki Shinsuke、Yashiroda Yoko、Li Sheena C.、Kimura Hiromi、Kawamura Yumi、Shimamoto Yasuhiro、Tominaga Ken-Ichi、Yamanaka Daisuke、Adachi Yoshiyuki、Takashima Shinichiro、Noda Yoichi、Boone Charles、Ohya Yoshikazu
  • Journal Title: Microbiology Spectrum Volume: 10 Issue: 1
  • DOI: 10.1128/spectrum.00873-21
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] AI-based forecasting of ethanol fermentation using yeast morphological data. (2021)
  • Author(s): Itto-Nakama K, Watanabe S, Kondo N, Ohnuki S, Kikuchi R, Nakamura T, Ogasawara W, Kasahara K, Ohya Y.
  • Journal Title: Biosci Biotechnol Biochem. Volume: 86 Issue: 1 Pages: 125-134
  • DOI: 10.1093/bbb/zbab188
  • Peer Reviewed / Open Access
• [Journal Article] Are droplets really suitable for single-cell analysis? A case study on yeast in droplets. (2021)
  • Author(s): Nakagawa Y, Ohnuki S, Kondo N, Itto-Nakama K, Ghanegolmohammadi F, Isozaki A, Ohya Y, Goda K.
  • Journal Title: Lab Chip. Volume: 21 Issue: 19 Pages: 3793-3803
  • DOI: 10.1039/d1lc00469g
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Defining Functions of Mannoproteins in Saccharomyces cerevisiae by High-Dimensional Morphological Phenotyping. (2021)
  • Author(s): Ghanegolmohammadi F, Okada H, Liu Y, Itto-Nakama K, Ohnuki S, Savchenko A, Bi E, Yoshida S, Ohya Y.
  • Journal Title: J Fungi (Basel). Volume: 7 Issue: 9 Pages: 769-769
  • DOI: 10.3390/jof7090769
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Poacic acid, a β-1,3-glucan-binding antifungal agent, inhibits cell-wall remodeling and activates transcriptional responses regulated by the cell-wall integrity and high-osmolarity glycerol pathways in yeast. (2021)
  • Author(s): Garcia R, Itto-Nakama K, Rodriguez-Pena JM, Chen X, Sanz AB, de Lorenzo A, Pavon-Verges M, Kubo K, Ohnuki S, Nombela C, Popolo L, Ohya Y, Arroyo J.
  • Journal Title: FASEB J. Volume: 10 Issue: 9 Pages: 1299-1299
  • DOI: 10.1096/fj.202100278r
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Genome Editing to Generate Sake Yeast Strains with Eight Mutations That Confer Excellent Brewing Characteristics. (2021)
  • Author(s): Chadani T, Ohnuki S, Isogai A, Goshima T, Kashima M, Ghanegolmohammadi F, Nishi T, Hirata D, Watanabe D, Kitamoto K, Akao T, Ohya Y.
  • Journal Title: Cells Volume: 10 Issue: 6 Pages: 1299-1299
  • DOI: 10.3390/cells10061299
  • Peer Reviewed / Open Access
• [Journal Article] Sequentially addressable dielectrophoretic array for high-throughput sorting of large-volume biological compartments (2020)
  • Author(s): A Isozaki, Y Nakagawa, M H Loo, Y Shibata, N Tanaka, D L Setyaningrum, J-W Park, Y Shirasaki, H Mikami, D Huang, H Tsoi, C T Riche, T Ota, H Miwa, Y Kanda, T Ito, K Yamada, O Iwata, K Suzuki, S Ohnuki, Y Ohya, Y Kato, T Hasunuma, S Matsusaka, M Yamagishi, M Yazawa, S Uemura, K Nagasawa, H Watarai, D Di Carlo, K Goda
  • Journal Title: Science Advances Volume: 29 Issue: 22 Pages: 1-11
  • DOI: 10.1126/sciadv.aba6712
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] 6 Virtual-freezing fluorescence imaging flow cytometry. (2020)
  • Author(s): Mikami H, Kawaguchi M, Huang CJ, Matsumura H, Sugimura T, Huang K, Lei C, Ueno S, Miura T, Ito T, Nagasawa K, Maeno T, Watarai H, Yamagishi M, Uemura S, Ohnuki S, Ohya Y, Kurokawa H, Matsusaka S, Sun CW, Ozeki Y, Goda K.
  • Journal Title: Nat Commun. Volume: 6 Issue: 1 Pages: 1162-1162
  • DOI: 10.1038/s41467-020-14929-2
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Intelligent image-activated cell sorting 2.0 (2020)
  • Author(s): Isozaki Akihiro, Hiraki Kei, et al.
  • Journal Title: Lab on a Chip Volume: 1 Issue: 13 Pages: 1-11
  • DOI: 10.1039/d0lc00080a
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] ストレス条件下における細胞形態からエタノール発酵量を予測するAIの開発 (2022)
  • Author(s): 一刀かおり、渡部峻、大貫慎輔、近藤直子、菊地亮太、中村徹、小笠原渉、笠原堅、大矢禎一
  • Organizer: 酵母遺伝学フォーラム
• [Presentation] 機械学習を用いた遺伝子機能に共通する形態特徴量の抽出 (2020)
  • Author(s): 大貫 慎輔、徐 聴涛、大矢 禎一
  • Organizer: 酵母遺伝学フォーラム