進化する次世代シーケンサーによる腸内細菌叢の解析
次世代シーケンサー（NGS）が登場し，自然界の複雑な微生物コミュニティーのプロファイリングが実現可能となった．100兆個100種を超える細菌がひしめく腸内フローラの研究にも今やNGSは必須のアイテムとなった．しかし，NGSは進化を続け世代交代の時期を迎えている．それにともないNGSを用いる菌叢解析のプラットフォームも変更の必要性が生じる．ここでは，これまで腸内細菌叢研究に最も多く用いられてきたロシュ社454ピロシーケンサーと，近年発展の目覚しいイルミナ社MiSeqのデータを比較検討した．また，シーケンスする16S rRNAの可変領域の検討もin silicoと実際のサンプルデータの両者を用いて...
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| Published in | Journal of Intestinal Microbiology Vol. 29; no. 3; pp. 135 - 144 |
|---|---|
| Main Authors | , |
| Format | Journal Article |
| Language | Japanese |
| Published |
公益財団法人 腸内細菌学会
2015
日本ビフィズス菌センター The Intestinal Microbiology Society |
| Subjects | |
| Online Access | Get full text |
| ISSN | 1343-0882 1349-8363 |
| DOI | 10.11209/jim.29.135 |
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| Abstract | 次世代シーケンサー（NGS）が登場し，自然界の複雑な微生物コミュニティーのプロファイリングが実現可能となった．100兆個100種を超える細菌がひしめく腸内フローラの研究にも今やNGSは必須のアイテムとなった．しかし，NGSは進化を続け世代交代の時期を迎えている．それにともないNGSを用いる菌叢解析のプラットフォームも変更の必要性が生じる．ここでは，これまで腸内細菌叢研究に最も多く用いられてきたロシュ社454ピロシーケンサーと，近年発展の目覚しいイルミナ社MiSeqのデータを比較検討した．また，シーケンスする16S rRNAの可変領域の検討もin silicoと実際のサンプルデータの両者を用いて行った．その結果，系統解析にはV3-V4が最も良好な結果を与えた．定量性はV6-V8が全体的に良好な結果を示したが，ユニバーサルプライマーによる一部の細菌グループに対する増幅効率のバイアスがどの領域でも見られた．しかし，UniFrac-PCoA解析にて示される菌叢の全体的な傾向はどのデータでも同様に観察され，NGSによる腸内細菌叢解析の堅牢性が示された．数あるNGSの中において，MiSeqはランニングコストや操作性という観点からも腸内細菌叢解析に適しており，今後本分野の研究に頻用されていくであろう． |
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| AbstractList | 次世代シーケンサー（NGS）が登場し，自然界の複雑な微生物コミュニティーのプロファイリングが実現可能となった．100兆個100種を超える細菌がひしめく腸内フローラの研究にも今やNGSは必須のアイテムとなった．しかし，NGSは進化を続け世代交代の時期を迎えている．それにともないNGSを用いる菌叢解析のプラットフォームも変更の必要性が生じる．ここでは，これまで腸内細菌叢研究に最も多く用いられてきたロシュ社454ピロシーケンサーと，近年発展の目覚しいイルミナ社MiSeqのデータを比較検討した．また，シーケンスする16S rRNAの可変領域の検討もin silicoと実際のサンプルデータの両者を用いて行った．その結果，系統解析にはV3-V4が最も良好な結果を与えた．定量性はV6-V8が全体的に良好な結果を示したが，ユニバーサルプライマーによる一部の細菌グループに対する増幅効率のバイアスがどの領域でも見られた．しかし，UniFrac-PCoA解析にて示される菌叢の全体的な傾向はどのデータでも同様に観察され，NGSによる腸内細菌叢解析の堅牢性が示された．数あるNGSの中において，MiSeqはランニングコストや操作性という観点からも腸内細菌叢解析に適しており，今後本分野の研究に頻用されていくであろう． 「要旨」次世代シーケンサー(NGS)が登場し, 自然界の複雑な微生物コミュニティーのプロファイリングが実現可能となった. 100兆個100種を超える細菌がひしめく腸内フローラの研究にも今やNGSは必須のアイテムとなった. しかし, NGSは進化を続け世代交代の時期を迎えている. それにともないNGSを用いる菌叢解析のプラットフォームも変更の必要性が生じる. ここでは, これまで腸内細菌叢研究に最も多く用いられてきたロシュ社454ピロシーケンサーと, 近年発展の目覚しいイルミナ社MiSeqのデータを比較検討した. また, シーケンスする16S rRNAの可変領域の検討もin silicoと実際のサンプルデータの両者を用いて行った. その結果, 系統解析にはV3-V4が最も良好な結果を与えた. 定量性はV6-V8が全体的に良好な結果を示したが, ユニバーサルプライマーによる一部の細菌グループに対する増幅効率のバイアスがどの領域でも見られた. しかし, UniFrac-PCoA解析にて示される菌叢の全体的な傾向はどのデータでも同様に観察され, NGSによる腸内細菌叢解析の堅牢性が示された. 数あるNGSの中において, MiSeqはランニングコストや操作性という観点からも腸内細菌叢解析に適しており, 今後本分野の研究に頻用されていくであろう. Next generation sequencer (NGS) has allowed the precise profiling of complex microbial communities in nature. This methodology has been introduced into research of gastrointestinal microbiota which is composed of 100 trillion cells of several hundred species. However, recent developments in NGS technology have obliged us to update the platform of microbiota analysis. In this study, we compared and validated the results obtained using 454 pyrosequencer (Roche), which has hitherto mainly been used for gut microbiota analysis, and a MiSeq (Illumina) which recently incorporated remarkable technological advances. We also validated the accuracy of taxonomic classification obtained from different variable regions of 16S ribosomal RNA genes. The V3-V4 region showed the best results for accuracy of taxonomic classification. In contrast, V6-V8 showed less bias for the bacterial composition of higher taxonomic levels, but all variable regions generated more or less bias for certain bacteria groups due to the universal primers used. Finally, UniFrac-PCoA data generated by different NGS data sets were compared using Procrustes analysis, and were shown to be robust regardless of variable regions and sequencers. Among a variety of NGSs, MiSeq is suitable for gut microbiota analysis in terms of running cost and operability, and can be more generally used for gastrointestinal tract microbial research. 次世代シーケンサー（NGS）が登場し，自然界の複雑な微生物コミュニティーのプロファイリングが実現可能となった．100兆個100種を超える細菌がひしめく腸内フローラの研究にも今やNGSは必須のアイテムとなった．しかし，NGSは進化を続け世代交代の時期を迎えている．それにともないNGSを用いる菌叢解析のプラットフォームも変更の必要性が生じる．ここでは，これまで腸内細菌叢研究に最も多く用いられてきたロシュ社454ピロシーケンサーと，近年発展の目覚しいイルミナ社MiSeqのデータを比較検討した．また，シーケンスする16S rRNAの可変領域の検討もin silicoと実際のサンプルデータの両者を用いて行った．その結果，系統解析にはV3-V4が最も良好な結果を与えた．定量性はV6-V8が全体的に良好な結果を示したが，ユニバーサルプライマーによる一部の細菌グループに対する増幅効率のバイアスがどの領域でも見られた．しかし，UniFrac-PCoA解析にて示される菌叢の全体的な傾向はどのデータでも同様に観察され，NGSによる腸内細菌叢解析の堅牢性が示された．数あるNGSの中において，MiSeqはランニングコストや操作性という観点からも腸内細菌叢解析に適しており，今後本分野の研究に頻用されていくであろう． |
| Author | 東, 佳那子 中山, 二郎 |
| Author_FL | HIGASHI Kanako NAKAYAMA Jiro |
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| Copyright | 2015 （公財）日本ビフィズス菌センター |
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| DocumentTitleAlternate | Profiling of Gut Microbiota Community by Renewed Next Generation Sequencer |
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| Snippet | 次世代シーケンサー（NGS）が登場し，自然界の複雑な微生物コミュニティーのプロファイリングが実現可能となった．100兆個100種を超える細菌がひしめく腸内フローラの研... 「要旨」次世代シーケンサー(NGS)が登場し, 自然界の複雑な微生物コミュニティーのプロファイリングが実現可能となった. 100兆個100種を超える細菌がひしめく腸内フローラの... Next generation sequencer (NGS) has allowed the precise profiling of complex microbial communities in nature. This methodology has been introduced into... |
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| Title | 進化する次世代シーケンサーによる腸内細菌叢の解析 |
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