Establishment of genome analyses methods for non-model organisms

• Project Area: Genetic bases for the evolution of complex adaptive traits
• Project/Area Number: 22128008
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Kanazawa University
• Principal Investigator: Nishiyama Tomoaki 金沢大学, 学際科学実験センター, 助教 (50390688)
• Co-Investigator(Kenkyū-buntansha): SHIGENOBU Shuji 基礎生物学研究所, 生物機能解析センター, 特任准教授 (30399555) ; KADOTA Koji 東京大学, 大学院農学生命科学研究科, 特任准教授 (60392221)
• Co-Investigator(Renkei-kenkyūsha): KASAHARA Masahiro 東京大学, 新領域創成科学研究科, 講師 (60376605)
• Research Collaborator: YAMAGUCHI Katsushi
• Project Period (FY): 2010-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥110,240,000 (Direct Cost: ¥84,800,000、Indirect Cost: ¥25,440,000); Fiscal Year 2014: ¥20,670,000 (Direct Cost: ¥15,900,000、Indirect Cost: ¥4,770,000); Fiscal Year 2013: ¥21,580,000 (Direct Cost: ¥16,600,000、Indirect Cost: ¥4,980,000); Fiscal Year 2012: ¥22,620,000 (Direct Cost: ¥17,400,000、Indirect Cost: ¥5,220,000); Fiscal Year 2011: ¥22,360,000 (Direct Cost: ¥17,200,000、Indirect Cost: ¥5,160,000); Fiscal Year 2010: ¥23,010,000 (Direct Cost: ¥17,700,000、Indirect Cost: ¥5,310,000)
• Keywords: アセンブリー / ゲノム / トランスクリプトーム / 遺伝子発現比較 / ゲノムアセンブリ / 遺伝学的地図 / RNA-seq / 計数比較 / ゲノムアセンブリー / 単分子シーケンサー / トランスクリプトーム解析 / 発現定量 / 一塩基多 / ヒメツリガネゴケ / アブラムシ / 遺伝子発現変動

Outline of Final Research Achievements

Genome and transcriptome sequencing, assembly, and annotation methods and expression profile analysis methods were developed to facilitate identification of genes responsible for complex adaptive traits in non-model organisms. Long mate-pair library construction enabled over Mb scaffolds and assembly utilizing long reads with single molecule real-time sequencing technology by Pacificbiosciences enabled over 100-kb contigs in multiple species. A novel robust normalization method for digital gene expression analyses were implemented and distributed in the R package TCC.

Research Products

• [Journal Article] Cockroach GABA-B receptor subtypes: Molecular characterization, pharmacological properties and tissue distribution. (2015)
  • Author(s): Blankenburg S, Balfanz S, Hayashi Y, Shigenobu S, Miura T, Baumann O, Baumann A, Blenau W
  • Journal Title: Neuropharmacology Volume: 88 Pages: 134-144
  • DOI: 10.1016/j.neuropharm.2014.08.022
  • Peer Reviewed
• [Journal Article] The evolutionary history of termites as inferred from 66 mitochondrial genomes. (2015)
  • Author(s): Bourguignon T, Lo N, Cameron SL, Sobotnik J, Hayashi Y, Shigenobu S, Watanabe D, Roisin Y, Miura T, Evans TA
  • Journal Title: Mol Biol Evol Volume: 32 Issue: 2 Pages: 406-421
  • DOI: 10.1093/molbev/msu308
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] A plant U-box protein, PUB4, regulates asymmetric cell division and cell proliferation in the root meristem. (2015)
  • Author(s): Kinoshita, A, Hove, C.A., Tabata, R., Yamada, M., Shimizu, N., Ishida, T., Yamaguchi K., Shigenobu, S., Takebayashi, Y., Iuchi, S., Kobayashi, M., Kurata, T., Wada, T., Seo, M., Hasebe, M., Bilou, I., Fukuda, H., Scheres, B., Heidstra, R., Sawa, S.
  • Journal Title: Development Volume: 142 Issue: 3 Pages: 444-453
  • DOI: 10.1242/dev.113167
  • Peer Reviewed / Open Access
• [Journal Article] Mutations in plastidial 5-aminolevulinic acid biosynthesis genes suppress pleiotropic defect in shoot development of mitochondrial GABA shunt mutant in Arabidopsis (2015)
  • Author(s): Toyokura, K., Yamaguchi, K., Shigenobu, S., Fukaki, H., Tatematsu, K., and Okada, K.
  • Journal Title: Plant and Cell Physiology Volume: 56 Issue: 6 Pages: 1229-1238
  • DOI: 10.1093/pcp/pcv050
  • Peer Reviewed
• [Journal Article] NMDA receptor activation upstream of methyl farnesoate signaling for short day-induced male offspring production in the water flea, Daphnia pulex (2015)
  • Author(s): Kenji Toyota, Hitoshi Miyakawa, Katsushi Yamaguchi, Shuji Shigenobu, Yukiko Ogino, Norihisa Tatarazako, Shinichi Miyagawa, Taisen Iguchi
  • Journal Title: BMC Genomics Volume: 16 Issue: 1 Pages: 186-186
  • DOI: 10.1186/s12864-015-1392-9
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] An Arabidopsis thaliana copper-sensitive mutant suggests a role of phytosulfokine in ethylene production (2015)
  • Author(s): Tao Wu, Takehiro Kamiya, Hiroko Yumoto, Naoyuki Sotta, Yamaguchi Katsushi, Shuji Shigenobu, Yoshikatsu Matsubayashi and Toru Fujiwara
  • Journal Title: Journal of experimental botany Volume: 66 Issue: 13 Pages: 3657-3667
  • DOI: 10.1093/jxb/erv105
  • Peer Reviewed / Open Access
• [Journal Article] Performance comparison of second- and third-generation sequencers using a bacterial genome with two chromosomes. (2014)
  • Author(s): Miyamoto M, Motooka D, Gotoh K, Imai T, Yoshitake K, Goto N, Iida T, Yasunaga T, Horii T, Arakawa K, Kasahara M, Nakamura S.
  • Journal Title: BMC Genomics Volume: 15 Issue: 1 Pages: 699-699
  • DOI: 10.1186/1471-2164-15-699
  • Peer Reviewed / Open Access
• [Journal Article] Methylome diversification through changes in DNA methyltransferase sequence specificity. (2014)
  • Author(s): Yoshikazu Furuta, Hiroe Namba-Fukuyo, Tomoko F. Shibata, Tomoaki Nishiyama, Shuji Shigenobu, Yutaka Suzuki, Sumio Sugano, Mitsuyasu Hasebe, Ichizo Kobayashi
  • Journal Title: PLoS Genetics Volume: 10(4) Issue: 4 Pages: e1004272-e1004272
  • DOI: 10.1371/journal.pgen.1004272
  • Peer Reviewed / Open Access
• [Journal Article] Comparative genome sequencing reveals genomic signature of desiccation tolerance in the anhydrobiotic midge (2014)
  • Author(s): Gusev O. Okuda T, Kikawada T etal.,
  • Journal Title: Nat. Commun Volume: 5 Issue: 1 Pages: 1-9
  • DOI: 10.1038/ncomms5784
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Development of a PCR method for the detection of Helicobacter suis in gastric biopsy specimens. (2014)
  • Author(s): Matsui H, Takahashi T, Yamagata-Murayama S, Uchiyama I, Yamaguchi K, Shigenobu S, Matsumoto T, Kawakubo M, Ota H, Osaki T, Kamiya S, Takahashi S, Nakamura S, Nakamura M
  • Journal Title: Helicobacter Volume: in press Issue: 4 Pages: 260-271
  • DOI: 10.1111/hel.12127
  • Peer Reviewed
• [Journal Article] WOX13-like genes are required for reprogramming of leaf and protoplast cells into stem cells in the moss Physcomitrella patens. (2014)
  • Author(s): Keiko Sakakibara, Pascal Reisewitz, Tsuyoshi Aoyama, Thomas Friedrich, Sayuri Ando, Yoshikatsu Sato, Yosuke Tamada, Tomoaki Nishiyama, Yuji Hiwatashi, Tetsuya Kurata, Masaki Ishikawa, Hironori Deguchi, Stefan A. Rensing, Wolfgang Werr, Takashi Murata, Mitsuyasu Hasebe, Thomas Laux
  • Journal Title: Development Volume: 141 Issue: 8 Pages: 1660-1670
  • DOI: 10.1242/dev.097444
  • Peer Reviewed / Open Access
• [Journal Article] Whole-genome sequence of Burkholderia sp. strain RPE67, a bacterial gut symbiont of the bean bug Riptortus pedestris (2014)
  • Author(s): Takeshita K., Shibata T. F., Nikoh N., Yamaguchi K., Nishiyama T., Hasebe M., Fukatsu T., Shigenobu S., Kikuchi Y.
  • Journal Title: Genome Announcements Volume: 2(3) Issue: 3
  • DOI: 10.1128/genomea.00556-14
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Identification and Characterization of an Arabidopsis Mutant with Altered Localization of NIP5;1, a Plasma Membrane Boric Acid Channel, Reveals the Requirement for D-Galactose in Endomembrane Organization. (2014)
  • Author(s): Uehara, M., Wang, S., Kamiya, T., Shigenobu, S., Yamaguchi, K., Fujiwara, T., Naito, S., and Takano, J.
  • Journal Title: Plant Cell Physiology Volume: 55 Issue: 4 Pages: 704-714
  • DOI: 10.1093/pcp/pct191
  • Peer Reviewed / Open Access
• [Journal Article] Sex chromosome turnover contributes to genomic divergence between incipient stickleback species. (2014)
  • Author(s): Yoshida, K., Makino, T., Yamaguchi, K., Shigenobu, S., Hasebe, M., Kawata, M., Kume, M., Mori, S., Peichel, C.L., Toyoda, A., et al.
  • Journal Title: PLoS genetics Volume: 10 Issue: 3 Pages: e1004223-e1004223
  • DOI: 10.1371/journal.pgen.1004223
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Comparison of gene expression of Paramecium bursaria with and without Chlorella variabilis symbionts (2014)
  • Author(s): Kodama, Y., Suzuki, H., Dohra, H., Sugii, M., Kitazume, T., Yamaguchi, K., Shigenobu, S., and Fujishima, M.
  • Journal Title: BMC Genomics Volume: 15 Issue: 1 Pages: 183-183
  • DOI: 10.1186/1471-2164-15-183
  • NAID: 120005611450
  • Peer Reviewed / Open Access
• [Journal Article] TCC: an R package for comparing tag count data with robust normalization strategies. (2013)
  • Author(s): Sun, J., Nishiyama, T., Shimizu, K., and Kadota, K.
  • Journal Title: BMC Bioinformatics Volume: 14 Issue: 1 Pages: 219-219
  • DOI: 10.1186/1471-2105-14-219
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Complete genome sequence of Burkholderia sp. strain RPE64, bacterial symbiont of the bean bug Riptortus pedestris (2013)
  • Author(s): Shibata T., Maeda T., Nikoh N., Yamaguchi K., Oshima K., Hattori M., Nishiyama T., Hasebe M., Fukatsu T., Kikuchi Y., Shigenobu S.
  • Journal Title: Genome Announcements Volume: 1(4) Issue: 4
  • DOI: 10.1128/genomea.00441-13
  • Peer Reviewed / Open Access
• [Journal Article] Too much love, a novel Kelch repeat-containing F-box protein, functions in the long-distance regulation of the legume-Rhizobium symbiosis (2013)
  • Author(s): Takahara, M., Magori, S., Soyano, T., Okamoto, S., Yoshida, C., Yano, K., Sato, S., Tabata, S., Yamaguchi, K., Shigenobu, S., et al
  • Journal Title: Plant Cell Physiol Volume: 54 Issue: 4 Pages: 433-447
  • DOI: 10.1093/pcp/pct022
  • NAID: 10031163838
  • Peer Reviewed
• [Journal Article] The draft genomes of soft-shell turtle and green sea turtle yield insights into the development and evolution of the turtle-specific body plan. (2013)
  • Author(s): Wang, Z., Pascual-Anaya, J., Zadissa, A., Li, W., Niimura, Y., Huang, Z., Li, C., White, S., Xiong, Z., Fang, D., et al.
  • Journal Title: Nature genetics Volume: 45 Issue: 6 Pages: 701-706
  • DOI: 10.1038/ng.2615
  • Peer Reviewed / Open Access
• [Journal Article] Construction and characterization of normalized cDNA libraries by 454 pyrosequencing and estimation of DNA methylation levels in three distantly related termite specie (2013)
  • Author(s): Hayashi Y, Shigenobu S, Watanabe D, Toga K, Saiki R, Shimada K, Bourguignon T, Lo N, Hojo M, Maekawa K, Miura T
  • Journal Title: PLOS ONE Volume: 8 Issue: 9 Pages: e76678-e76678
  • DOI: 10.1371/journal.pone.0076678
  • Peer Reviewed / Open Access
• [Journal Article] Aphids evolved novel secreted proteins for symbiosis with bacterial endosymbiont (2013)
  • Author(s): Shigenobu, S. and Stern, D
  • Journal Title: Proc. Royal Soc. B Volume: 280 Issue: 1750 Pages: 20121952-20121952
  • DOI: 10.1098/rspb.2012.1952
  • Peer Reviewed
• [Journal Article] A normalization strategy for comparing tag count data (2012)
  • Author(s): Kadota K., Nishiyama T., Shimizu K.
  • Journal Title: Algorithms Mol. Biol. Volume: 7 Issue: 1 Pages: 5-5
  • DOI: 10.1186/1748-7188-7-5
  • Peer Reviewed
• [Journal Article] Digital gene expression profiling by 5'-end sequencing of cDNAs during reprogramming in the moss Physcomitrella patens. (2012)
  • Author(s): Nishiyama T., Miyawaki K., Ohshima M., Thompson K., Nagashima A., Hasebe M., Kurata T
  • Journal Title: PLoS One Volume: 7 Issue: 5 Pages: e36471-e36471
  • DOI: 10.1371/journal.pone.0036471
  • Peer Reviewed
• [Journal Article] Exploration and characterization of genes involved in the synthesis of diterpene defence secretion in nasute termite soldiers. (2012)
  • Author(s): Hojo M, Maekawa K, Saitoh S, Shigenobu S, Miura T, Hayashi Y, Tokuda G, Maekawa H
  • Journal Title: Insect Mol Biol Volume: 21 Issue: 6 Pages: 545-557
  • DOI: 10.1111/j.1365-2583.2012.01162.x
  • Peer Reviewed
• [Journal Article] Identification of an EMS-induced causal mutation in a gene required for boron-mediated root development by low-coverage genome re-sequencing in Arabidopsis. (2012)
  • Author(s): Ryo Tabata, Takehiro Kamiya, Shuji Shigenobu, Katsushi Yamaguchi, Masashi Yamada, Mitsuyasu Hasebe, Toru Fujiwara, Shinichiro Sawa
  • Journal Title: Plant Signaling & Behavior Volume: 8 Issue: 1 Pages: e22534-e22534
  • DOI: 10.4161/psb.22534
  • Peer Reviewed
• [Journal Article] The Selaginella genome identifies genetic changes associated with the evolution of vascular plants (2011)
  • Author(s): Banks, et al
  • Journal Title: Science Volume: 332 Issue: 6032 Pages: 960-963
  • DOI: 10.1126/science.1203810
  • Peer Reviewed
• [Journal Article] Evaluating methods for ranking differentially expressed genes applied to MicroArray Quality Control data (2011)
  • Author(s): Kadota K and Shimizu K
  • Journal Title: BMC Bioinformatics Volume: 12 Issue: 1 Pages: 227-227
  • DOI: 10.1186/1471-2105-12-227
  • Peer Reviewed
• [Journal Article] Genomic revelations of a mutualism: the pea aphid and its obligate bacterial symbiont (2011)
  • Author(s): Shigenobu, S. and Wilson, A. C.
  • Journal Title: Cellular and Molecular Life Sciences Volume: 68 Issue: 8 Pages: 1297-1297
  • DOI: 10.1007/s00018-011-0645-2
  • Peer Reviewed
• [Journal Article] Physcomitrella cyclin-dependent kinase A links cell cycle reactivation to other cellular changes during reprogramming of leaf cells (2011)
  • Author(s): Ishikawa, M., Murata, T., Sato, Y., Nishiyama, T., Hiwatashi, Y., Imai, A., Kimura, M., Sugimoto, N., Akita, A., Oguri, Y. et al.
  • Journal Title: Plant Cell Volume: 23 Issue: 8 Pages: 2924-2938
  • DOI: 10.1105/tpc.111.088005
  • Peer Reviewed
• [Journal Article] Genome expansion and differential expression of amino acid transporters at the aphid/Buchnera symbiotic interface (2011)
  • Author(s): Price, D. R., Duncan, R. P., Shigenobu, S. and *Wilson, A. C.
  • Journal Title: Molecular Biology and Evolution Volume: 28 Issue: 11 Pages: 3113-3126
  • DOI: 10.1093/molbev/msr140
  • Peer Reviewed
• [Presentation] 様々な実験デザインに対応した頑健なRNA-seq発現変動解析パイプラインTCCの開発 (2013)
  • Author(s): 孫建強, 西山智明, 清水謙多郎, 門田幸二
  • Organizer: 第36回日本分子生物学会年会
  • Place of Presentation: 神戸ポートアイランド
  • Year and Date: 2013-12-03 – 2013-12-06
• [Presentation] ゲノムに刻まれた食虫植物の進化 (2013)
  • Author(s): (12)福島健児, Likai Mao, 今井飛将, 西山智明, 野澤昌文, 住川直美, 笠原雅弘, 柴田朋子, 重信秀治, Xiaodong Fang, 長谷部光泰
  • Organizer: NGS現場の会第三回研究会
  • Place of Presentation: 神戸国際会議場
  • Year and Date: 2013-09-04 – 2013-09-05
• [Presentation] 第二世代シークエンサーによる高密度の遺伝学的地図作成法 ヒメツリガネゴケへの適用例 (2013)
  • Author(s): 鎌田寛彬, 笠原雅弘, 長谷部光泰, 西山智明
  • Organizer: NGS現場の会第三回研究会
  • Place of Presentation: 神戸国際会議場
  • Year and Date: 2013-09-04 – 2013-09-05
• [Presentation] 1分子シークエンサーを用いた非モデル生物の de novo ゲノム解析の現状 (2013)
  • Author(s): 柴田朋子, 西山智明, 重信秀治, 長谷部光泰
  • Organizer: NGS現場の会第三回研究会
  • Place of Presentation: 神戸国際会議場
  • Year and Date: 2013-09-04 – 2013-09-05
• [Presentation] TCC: 頑健な正規化法を実装した発現変動解析用 R パッケージ (2013)
  • Author(s): 孫建強, 西山智明, 清水謙多郎, 門田幸二
  • Organizer: NGS現場の会第三回研究会
  • Place of Presentation: 神戸国際会議場
  • Year and Date: 2013-09-04 – 2013-09-05
• [Presentation] Optimization of Procedures of Genomic DNA Preparation in Diverse Organisms for Single Molecule Real-Time Sequencing. (2013)
  • Author(s): Tomoko Shibata, Tomoaki Nishiyama, Shuji Shigenobu, Mitsuyasu Hasebe
  • Organizer: The 14th annual Advances in Genome Biology and Technology (AGBT) meeting
  • Place of Presentation: フロリダ (Marco Island Marriott Resort & Golf Club))
• [Presentation] Rでトランスクリプトーム解析 (2013)
  • Author(s): 門田幸二
  • Organizer: HPCI チュートリアルセミナー
  • Place of Presentation: 東京 (産総研臨海副都心センター別館８階)
  • Invited
• [Presentation] 食品機能解析研究とバイオインフォマティクス (2013)
  • Author(s): 門田幸二
  • Organizer: 日本農芸化学会2013年度大会
  • Place of Presentation: 仙台 (東北大学川内北キャンパス)
  • Invited
• [Presentation] Digital gene expression profiling by 5’-end sequencing of cDNAs during (2012)
  • Author(s): Tomoaki Nishiyama, Kaori Miyawaki, Masumi Ohshima, Kari Thompson, Akitomo Nagashima, Mitsuyasu Hasebe & Tetsuya Kurata
  • Organizer: moss2012
  • Place of Presentation: ニューヨーク (ニューヨーク植物園)
• [Presentation] １分子シークエンシングを用いた共生生物ゲノムのde novo アセンブリ (2012)
  • Author(s): 柴田朋子、前田太郎、西山智明、菊池義智、重信修治、深津武馬、長谷部光泰
  • Organizer: 日本進化学会第１４回東京大会
  • Place of Presentation: 東京 (首都大学東京)
• [Presentation] Closterium and Chara genome and transcriptome analyses (2012)
  • Author(s): Jun Abe, Hidetoshi Sakayama, Yutaka Suzuki, Atsushi Toyoda, Yuki Tsuchikane, Hiroyuki Sekimoto and Tomoaki Nishiyama
  • Organizer: Plant and Animal Genomes XX
  • Place of Presentation: Towns & Country, San Diego (USA)
  • Invited
• [Presentation] 次世代シーケンサーデータのde novo アセンブリー解析 (2012)
  • Author(s): 西山智明，阿部淳，土金勇樹，坂山英俊，関本弘之
  • Organizer: 第５３回植物生理学会年会
  • Place of Presentation: 京都産業大, 京都
• [Presentation] Next Generation Sequencing Technologies for Identification of Molecular Basis of Complex Adaptive Traits (2011)
  • Author(s): Tomoaki Nishiyama
  • Organizer: The 20th CDB Meeting : Molecular Bases for Evolution of Complex Traits
  • Place of Presentation: 理研神戸研究所(兵庫県)(招待講演)
  • Year and Date: 2011-02-24
• [Presentation] Land Plant Evolution Learnt from Moss and Lycophyte Genomes (2011)
  • Author(s): Tomoaki Nishiyama
  • Organizer: SMBE2011
  • Place of Presentation: Kyoto Univ, Kyoto
  • Invited
• [Presentation] RNAseqによる定量的解析とqPCR、マイクロアレイなどとの比較 (2010)
  • Author(s): 門田幸二
  • Organizer: 新学術領域研究「複合適応形質進化の遺伝子基盤解明」・複合適応形質進化インフォマティクスオープンセミナー
  • Place of Presentation: 金沢大学(石川県)
  • Year and Date: 2010-12-28
• [Presentation] Genomic revelations of a mutualism : the pea aphid and its obligate bacterial symbiont (2010)
  • Author(s): Shuji Shigenobu
  • Organizer: Memorial Symposium for the 26th International Prize for Biology "Biology of Symbiosis"-Celebrating Dr.Nancy Moran
  • Place of Presentation: つくば国際会議場(茨城県)(招待講演)
  • Year and Date: 2010-12-07
• [Presentation] 非モデル生物のゲノム解読と責任遺伝子同定の展望 (2010)
  • Author(s): 西山智明
  • Organizer: 公開シンポジウム「複合適応形質の進化」
  • Place of Presentation: 東京大学(東京都)
  • Year and Date: 2010-09-17
• [Presentation] トランスクリプトーム解析の一手段としての次世代シーケンサーデータ解析～実演を中心に～ (2010)
  • Author(s): 門田幸二
  • Organizer: 日本バイオインフォマティクス学会・第2回アグリバイオインフォマティクス研究会
  • Place of Presentation: 琉球大学(沖縄県)
  • Year and Date: 2010-09-17
• [Book] トランスクリプトーム解析 (2014)
  • Author(s): 門田幸二
  • Total Pages: 240
  • Publisher: 共立出版
• [Book] 「非モデル植物におけるNGSを用いたゲノム統合解析」(菅野純夫、鈴木穣 監『次世代シーケンサー』) (2012)
  • Author(s): 西山智明、長谷部光泰
  • Publisher: 学研メディカル秀潤社
• [Remarks] HOWTO
  • URL: http://koke.asrc.kanazawa-u.ac.jp/HOWTO/
• [Remarks] (Rで)塩基配列解析
  • URL: http://www.iu.a.u-tokyo.ac.jp/~kadota/r_seq.html
• [Remarks] (Rで)塩基配列解析
  • URL: http://www.iu.a.u-tokyo.ac.jp/~kadota/r_seq.html