この作品は、(許可番号 41/11) - コア ・ プログラム、大腸炎の組織 (ECCO) とイスラエル科学財団 (グラント第 908/15) ヨーロッパのクローンの部分で支えられました。

シバ ローカル研究倫理委員会の研究のための倫理的な承認が付与され、関連するガイドラインと規制に従ってすべての方法を行った。プロトコルは、患者の同意の例外を受け取ったローカル倫理審査委員会から使用された糞便材料から、すでに提出された微生物学コア以外の年齢、個人の患者情報なし臨床精密検査の一部として性別、および微生物の結果。書かれた、健常者からインフォームド コンセントを得たし、治験審査委員会承認研究。これらのサンプルのいくつかは、以前の分析1で既に含まれています。
1. 検体の取扱い
<ol><li>収集、約 5 mm2スミア (大体鉛筆の消しゴムのサイズ) テストで滅菌綿棒で新鮮な糞便サンプルから管 (材料の表を参照してください)。-80 ° C 24 h 内で糞便を含むすべての綿棒を格納します。糞便の綿棒は、さらなる処理までそこに残ることができます。</li>
</ol>2. DNA の抽出
<ol><li>室温で抽出し、希釈のソリューションを解凍 (材料の表を参照してください)。</li>
	<li>転送空 2 mL コレクションに糞便の綿棒チューブ (材料の表を参照してください)。最小交差汚染と管閉鎖を有効にするきれいなはさみを使用して切断することによって綿棒スティック サイズを調整します。糞便の綿棒とミックスする渦を含む各コレクション チューブに抽出液の 250 μ L を追加します。</li>
	<li>沸騰水浴中で 10 分間のサンプルを加熱 (95-100° C)。各サンプルと混合する渦に 250 μ L の希釈液を追加します。</li>
	<li>DNA を抽出し、4 ° C で綿棒を含む 2 mL チューブに格納します。</li>
</ol>3. PCR およびライブラリの準備
3.1 と 3.2 の手順、テンプレートと私アンプリコン無料環境クリーンで PCR のワークステーションで作業します。
<ol><li>プライマー (表 1) によると、バーコードのラベルを付けます。50 μ M 濃度、-20 ° C でストアに二重蒸留水 (DDW) で各プライマーを希釈します。</li>
	<li>PCR の反作用の 96 ウェル プレートを使用します。各プレートは、別のインデックスのプライマーでタグ付けされて 32 の異なるサンプルを含めることができます。前方のプライマーおよび室温 32 逆プライマーを解凍し、5 μ M に希釈します。</li>
	<li>(各ウェルの最終的なボリュームは 20 μ l になります) 100 の反応の 5 μ M 前方プライマー、1 mL 2 X PCR マスター ミックス、および 400 μ L DDW の 100 μ L を混合することによって PCR 反応混合物を準備します。この PCR ミックスの 15 μ L を各ウェル (96 ウェルの合計) に入れてください。3 さまざまな坑井 (トリプリケートで 32 の異なるプライマーは、96 ウェルの合計を与える) に各 5 μ M の逆インデックス付きプライマーの 1 μ L を追加します。</li>
	<li>前 PCR 専用ゾーンのテンプレート、無料、フリー、私アンプリコン、クリーン ベンチを意味反応混合物に各抽出した DNA サンプルの 4 μ L を追加 (各抽出された DNA サンプルを 3 通で増幅-96 ウェル プレートあたり 32 サンプル)。</li>
	<li>次の設定で PCR を実行: 初期変性 94 ° C、3 分。変性 94 ° C で 1 分、55 ° C 1 分と 1 分の 72 ° C で拡張子で焼鈍 35 サイクルが続く10 分のための 72 ° C で最終的な拡張子。</li>
	<li>ポスト PCR 専用ゾーンとして定義されます、別のベンチの単一の容積の管 (サンプルあたり 60 μ L) に各帳票の PCR の反作用を組み合わせます。</li>
	<li>PCR 産物の品質を評価、エチジウム ブロマイド染色 1% の agarose のゲルの各組み合わせ PCR 反応の 4 μ L を実行します。260 の UV 波長 [nm、正 amplicons 375-425 bp の期待されるバンド サイズで表示されます。以降の手順でこれらの産物のみが表示されます。 注: 各サンプル帳票、意味 3 別の PCR の反作用で各サンプルを増幅することで増幅されています。スケール アップしません。</li>
</ol>4. ライブラリの定量化とクリーニング
<ol><li>すべての PCR のサンプルのモル濃度プールを得るために二本鎖 DNA によってそれぞれの増幅を定量化 (dsDNA 定量化試薬) 蛍光核酸染色 (材料の表を参照)、大規模な同時定量に適してサンプルの量。 注: 私アンプリコンのサイズの範囲は曖昧なため、ナノグラムの実際の量はモル濃度を読み込む使用します。</li>
	<li>単一生殖不能の管に各サンプルの組み合わせ 500 ng。ミックスする渦。</li>
	<li>エチジウム ブロマイド染色 1% アガロースゲルでプールされたライブラリの 200 μ L を実行します。製造元の指示に従ってゲル抽出キットを使用してゲルから 375-425 bp バンドを抽出 (材料表参照) 80 μ L DDW でプールされたライブラリを溶出し、。 注: プールされたライブラリは、宿主の DNA から非特異的増幅製品を減らすために選択したサイズをする必要があります。</li>
	<li>製造元の指示に従ってキットの検出感度が高い dsDNA を使用して最終的なライブラリ濃度の測定 (材料の表を参照してください)。製造元の指示に従って DNA ライブラリの高感度分離分析キットを使用してライブラリの正確なサイズを測定 (材料の表を参照してください)。</li>
</ol>5. シーケンス
<ol><li>20% コントロール ライブラリの追加 19 にプールされたライブラリを希釈 (材料の表を参照)、シーケンス コンピューター プロトコルに従って。</li>
	<li>シーケンス、使用カスタム デザイン無料 (表 1参照) V4 増幅プライマーでプライマーを参照してください。</li>
	<li>追加のバーコードを読み取り使用 3 カスタム設計されたシーケンス プライマー (表 1参照) や、メーカーの仕様によると、各方向の長さで基地の 175 のペアエンド リードを生成します。</li>
	<li>シークエンシング マシンを実行し、製造元のプロトコルに従って FASTQ ファイルを取得します。</li>
</ol>6 データ処理
<ol><li>一緒にステッチし、QIIME 2 2017.7.0 バージョン17で実装されたデータのキュレーション パイプラインで重複するペアエンド FASTQ ファイルを処理します。サンプルの特定のバーコードによると読み取りをデマルチプレックスします。</li>
	<li>品質管理とシーケンスのバリアント (SVs) 検出するため DADA218を使用します。2 以上の予想されるエラーと破棄を読み取り最後 5' から 3' 末端から 13 基地と 15 で読み取りが切り捨てられます。識別し、コンセンサスを用いたキメラを削除-キメラが個別にサンプルで検出され、シーケンス サンプルの十分な割合でキメラが見つかりましたが削除されます。</li>
	<li>合うナイーブベイズ識別器を用いた SVs 分類学的分類を実行し、2013 年 8 月に訓練 99% のアイデンティティ Greengenes データベース6175 の長い読み取りと設定フォワード/リバースのプライマー。</li>
	<li>2,146 の系列の深さにすべてのサンプルを希薄にします。</li>
	<li>サンプル サイズの影響を避けるために、希薄サンプル (サンプル多様性19,20) の間 β 多様性測定のため加重 UniFrac を使用します。</li>
	<li>主座標分析 (PCoA) を実行するのに結果の距離行列を使用します。 注: データ処理のスクリプトおよびマッピング ファイル (補足資料 1と2) の補足資料として提供されます。</li>
</ol>

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著者が明らかに何もありません。

16S rRNA 増幅とメタゲノム ショットガン ・ シークエンシング法は、臨床微生物学アプリケーション21,-2223で人気を得ています。これらのテクニック、人為的・非人為的分類群、病原性菌の能力をより正確にモデルの感染を識別するために相対的な豊かさについてのデータを提供することをキャプチャする能力を向上に有利であります。指紋の<sup clas...

力を注いではマイクロバイ多様性と違いと健康および病理学的条件における個体間の類似点をキャプチャの別の側面としての豊かさを理解するためになされました。年齢2,3、地理学4、ライフ スタイル5,6、および病気5腸内細菌の叢の組成に関連付けられていることが示されたが、多くの条件や人口がまだ完全特徴とします。最近、治療への応用7,8,9マイクロバイを変更できることが報告されています。したがって、いろいろな生理的条件と微生物の組成の関係に追加の洞察力は、潜在的な将来の変更の最適化への第一歩です。
伝統的な微生物培養法低利回り10,11, に制限は、細菌がいずれかのバイナリの状態として概念化、腸内に存在しない、または。ハイスループット DNA ベースのシーケンスは、微生物生態学、微生物のコミュニティのすべてのメンバーのキャプチャを有効にするをもたらしました。ただし、シーケンスは、長さと質に残る正確な分類割り当て12への重要な障壁を読み取る。さらに、高スループットに基づいて実験の測定は非生物学的または非科学的な変数13によって影響を受ける、バッチの効果に苦しむことがあります。近年、アメリカの腸プロジェクト、アメリカ合衆国 (米国) 人間マイクロバイ プロジェクト、およびイギリス (英国) の MetaHIT プロジェクトを含む人間のマイクロバイを勉強するいくつかのプログラムを設けています。これらの取り組みは、彼らのアプローチに一貫性の欠如のため容易に対等ではないデータの膨大な量を生成しています。さまざまな国際人間マイクロバイ コンソーシアム、国際人間マイクロバイ基準プロジェクトと米国商務省標準技術局 (NIST) などの国際プロジェクト14 これらの問題の一部に対処しようとしています。、信頼性の高い生殖の結果の達成を可能にする必要がありますマイクロバイ測定のための基準を開発しました。ここで説明は 16S rRNA 高スループット シーケンス (seq 16S) 開始データ分析を通じて糞便サンプルのコレクションからのいくつかの広く使用されている方法15,16の統合されたプロトコルです。プロトコル記述列無料 PCR アプローチ、植物 DNA16の直接抽出用に設計された、高品質で比較的短時間でサンプル増幅 DNA 糞便の多数の同時処理を有効にする対象のシーケンスの共通プラットホームを微生物可変 V4 領域のシーケンス。このプロトコルは、堅牢な生殖、使いやすい、詳細な方法で 16S rRNA 増幅シーケンスの使用を開始するのに興味がある重要なコントロールを使用して科学者のガイドを目指しています。ガイドと詳細なステップバイ ステップ プロトコルを持つバッチ効果を最小限に抑えることができるし、ラボ間比較可能なシーケンス結果がこのようになります。

<table><tbody><tr><td>Primers</td><td>Integrated DNA Technologies (IDT)</td><td></td><td></td></tr><tr><td>Extraction solution</td><td>Sigma-Aldrich</td><td>E7526</td><td></td></tr><tr><td>Dilution solution</td><td>Sigma-Aldrich</td><td>D5688</td><td></td></tr><tr><td>Kapa HiFi HotStart ReadyMix PCR Kit</td><td>KAPABIOSYSTEMS</td><td>KK2601</td><td>PCR Master mix</td></tr><tr><td>Quant-iT PicoGreen dsDNA Reagent kit</td><td>Invitrogen</td><td>P7589</td><td>dsDNA quantify reagent</td></tr><tr><td>MinElute Gel extraction kit</td><td>Qiagen</td><td>28606</td><td></td></tr><tr><td>Agarose</td><td>Amresco</td><td>0710-250G</td><td></td></tr><tr><td>Ultra Pure Water Dnase and Rnase Free</td><td>Biological Industries</td><td>01-866-1A</td><td></td></tr><tr><td>Qubit dsDNA HS assay kit</td><td>Molecular probes</td><td>Q32854</td><td>dsDNA detecting kit</td></tr><tr><td>High Sensitivity D1000</td><td>Agilent Technologies</td><td>Screen Tape 5067-5582</td><td>separation and analysis</td></tr><tr><td>Screen Tape Assay</td><td>Agilent Technologies</td><td>Reagents 5067-5583</td><td>for DNA libraries</td></tr><tr><td>PhiX Control v3</td><td>Illumina</td><td>15017666</td><td>control library</td></tr><tr><td>MiSeq Reagent Kit v2 (500 cycle)</td><td>Illumina</td><td>MS-102-2003</td><td></td></tr><tr><td>Ethidium Bromide</td><td>Amresco</td><td>E406-10mL-TAM</td><td></td></tr><tr><td>2 mL collection tubes</td><td>SARSTEDT</td><td>72.695.400</td><td>Safe Seal collection tubes</td></tr><tr><td>Plastic stick swab in PP test tube</td><td>STERILE INTERIOR</td><td>23117</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PCR Machine</td><td>Applied Biosystems</td><td>2720 Thermal Cycler</td><td></td></tr><tr><td>Sequncing Machine</td><td>Illumina</td><td>Miseq</td><td></td></tr><tr><td>PCR workstation</td><td>Biosan</td><td>UV-cleaner</td><td></td></tr><tr><td>scissors</td><td></td><td></td><td></td></tr><tr><td>vortexer</td><td>Scientific Industries</td><td>Vortex-Genie 2</td><td></td></tr></tbody></table>

guided protocol for fecal microbial characterization by 16s rrna-amplicon sequencing

人間の腸内マイクロバイ損傷から細胞を保護する上で、エネルギーと栄養分の処理に、免疫を促進する上で中心的な役割を果たしています。健全な細菌叢の組成 (dysbiosis) と考えられるものからの逸脱は、病的な状態につながる重要な機能を損なう可能性があります。最近、継続的な研究努力は微生物組成と人間の健康と病気の間の関連付けのキャラクタリゼーション向かっていた。
高スループット シーケンス技術の進歩は、腸内細菌の組成の特性を有効にします。16S rRNA 増幅配列には散弾銃の配列が含まれます。16S rRNA 増幅シーケンスを使用して、ショットガン ・ シークエンシング法遺伝子の予言と機能アノテーションに関する追加情報を提供しますが、分類学的組成をプロファイルします。16S rRNA 遺伝子の可変領域のターゲット シーケンスのメソッドを使用しての利点は、ショットガン ・ シークエンシング法と比較して大幅に低コストです。16S rRNA 遺伝子配列の差異は、異なる分類群の個々 のサンプル内の定量化微生物指紋として使用されます。
主要な国際的な取り組みは 16S rRNA 増幅配列のための標準を入隊しています。ただし、いくつかの研究は、バッチの効果によって引き起こされる変化の一般的な原因を報告します。サンプル コレクションの制服を着たプロトコルは、この効果を最小限に抑えるため、処理、およびシーケンスを実装する必要があります。このプロトコルは、糞便のサンプル コレクションからデータ解析に広く使用されているプロトコルの統合を提案します。このプロトコルには、同時処理および V4 領域の PCR の拡大と共に、糞便の多数の DNA 抽出できるよう、柱のないダイレクト PCR アプローチが含まれています。さらに、プロトコル解析パイプラインをについて説明し、QIIME (QIIME 2 バージョン 2017.7.0 と DADA2) の最新バージョンを使用してスクリプトを提供します。このステップバイ ステップのプロトコルは、堅牢な生殖、使いやすい、詳細な方法で 16S rRNA 増幅シーケンスの使用を開始するのに興味のある方をガイドする対象です。

プロトコルの模式図を図 1に示します。
前向きが疑われる伝染性下痢症入院患者から便のサンプルを収集しました。これらのサンプルは、された前述12 月と 2015 年 5 月間のシェバ医療センターで臨床微生物学研究室に送信されました。便のサンプルを受けた臨床微生物学研究室で実...

Watch this Scientific Journal Video about Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing at JoVE.com

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

本稿では、高スループット 16S rRNA 増幅シーケンスの詳細な標準化されたプロトコルについて説明します。プロトコルには、データ分析を通じて糞便サンプル コレクションから統合、制服を着た、実現可能な安価なプロトコルが導入されています。このプロトコルには、厳格な基準とサンプルの多数の分析といくつかのコントロールができます。

16S rRNA 増幅配列による糞便の微生物評価ガイドのプロトコル

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. ...

guided-protocol-for-fecal-microbial-characterization-16s-rrna

Research

JoVE Journal

Biology

19.4K ビュー.  Sheba Medical Center. 本稿では、高スループット 16S rRNA 増幅シーケンスの詳細な標準化されたプロトコルについて説明します。プロトコルには、データ分析を通じて糞便サンプル コレクションから統合、制服を着た、実現可能な安価なプロトコルが導入されています。このプロトコルには、厳格な基準とサンプルの多数の分析といくつかのコントロールができます。

ビデオ: Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

探索ルート マイクロバイ: 土、根、およびルート Endosphere から細菌群集のデータを抽出

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

遺伝学

Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous catalytic enzymes and are naturally capable of metabolizing hydrocarbons. Scientists harness biodiversity in aquatic ecosystems to isolate bacteria with biodegradation and bioremediation potential. Such isolates from the environment provide a rich set of metabolic pathways and enzymes, which can be further utilized to scale up the degradation process at an industrial scale. In this article, we outline the general process of isolation, propagation, and identification of bacterial species from aquatic habitats and screen their ability to utilize hydrocarbons as the sole carbon source in vitro using simple techniques. The present protocol describes the isolation of various bacterial species and their subsequent identification using the 16S rRNA analysis. The protocol also presents steps for characterizing the hydrocarbon degrading potential of bacterial isolates. This protocol will be useful for researchers trying to isolate bacterial species from environmental habitats for their biotechnological applications.

We present the process of isolating, propagating, and characterizing hydrocarbon-degrading bacteria from aquatic habitats. The protocol outlines bacterial isolation, identification by the 16S rRNA method, and testing of their hydrocarbon-degrading potential. This article would help researchers in characterizing microbial biodiversity in environmental samples, and specifically screen for microbes with bioremediation potential.

水生生物生息地からの炭化水素代謝特性を有する細菌種の単離・増殖・同定

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous ...

環境学

Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its complex nature, and its diverse association with mammalian components. The goal of this protocol is to culture the gut microbiota in vitro, which allows for the study of the gut microbiota dynamics, without having to consider the contribution of the mammalian milieu. Using in vitro culturing technology, the physiological conditions of the gastro intestinal tract are simulated, including parameters such as pH, temperature, anaerobiosis, and transit time. The intestinal surface of the colon is simulated by adding mucin-coated carriers, creating a mucosal phase, and adding further dimension. The gut microbiota is introduced by inoculating with the human fecal material. Upon inoculation with this complex mixture of bacteria, specific microbes are enriched in the different longitudinal (ascending, transverse and descending colons) and transversal (luminal and mucosal) environments of the in vitro model. It is crucial to allow the system to reach a steady state, in which the community and the metabolites produced remain stable. The experimental results in this manuscript demonstrate how the inoculated gut microbiota community develops into a stable community over time. Once steady state is achieved, the system can be used to analyze bacterial interactions and community functions or to test the effects of any additives on the gut microbiota, such as food, food components, or pharmaceuticals.

Here, we present a protocol for culturing the gut microbiota of the colon in vitro, using a series of bioreactors that simulate the physiological conditions of the gastro intestinal tract.

体外養殖人間の腸内細菌を研究する技術を適用する高度な

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its ...

生物工学

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are needed to decipher the utilization of microbiome modulators for human health benefits. However, the large-scale screening and optimization of modulators to validate microbiome modulation and predict related health benefits may be practically difficult due to the need for large number of animals and/or human subjects. To this end, in vitro or ex vivo models can facilitate preliminary screening of microbiome modulators. Herein, it is optimized and demonstrated an ex vivo fecal microbiota culture system that can be used for examining the effects of various interventions of gut microbiome modulators including probiotics, prebiotics and other food ingredients, aside from nutraceuticals and drugs, on the diversity and composition of the human gut microbiota. Inulin, one of the most widely studied prebiotic compounds and microbiome modulators, is used as an example here to examine its effect on the healthy fecal microbiota composition and its metabolic activities, such as fecal pH and the fecal levels of organic acids including lactate and short-chain fatty acids (SCFAs). The protocol may be useful for studies aimed at estimating the effects of different interventions of modulators on fecal microbiota profiles and at predicting their health impacts.

This protocol describes an in vitro batch-culture fermentation system of human fecal microbiota, using inulin (a well-known prebiotic and one of the most widely studied microbiota modulators) to demonstrate the use of this system in estimating effects of specific interventions on fecal microbiota composition and metabolic activities.

ヒトの胎児微生物叢に対する介入レジメンの影響を推定するインビトロバッチ培養モデル

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are ...

免疫・感染学

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

16SリボソームRNA遺伝子アンプリコンの次世代シーケンシング

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

生物学

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

細菌群集の特徴付けのための効率的な核酸抽出および16S rRNAの遺伝子配列決定

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

次世代の 16S rRNA 増幅シーケンスによる目盛りマイクロバイ評価

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

2段階PCRと次世代16S rRNA遺伝子シーケンシングを用いての微生物叢解析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

Fecal Microbiota Transplantation and Detection of Prevalence of IgA-Coated Bacteria in the Gut

Gut microbiota exert pleiotropic roles in human health and disease. Fecal microbiota transplantation (FMT) is an effective method to investigate the biological function of intestinal bacteria as a whole or at the species level. Several different FMT methods have been published. Here, we present an FMT protocol that successfully depletes gut microbiota in a matter of days, followed by transplantation of fecal microbiota from fresh or frozen donor intestinal contents to conventional mice. Real time-PCR is applied to test the efficacy of bacterial depletion. Sequencing of the 16S ribosomal RNA (rRNA) is then applied to test the relative abundance and identity of gut microbiota in recipient mice. We also present a flow cytometry-based detection method of immunoglobulin A (IgA)-coated bacteria in the gut.

We established an efficient way to deplete intestinal bacteria in three days, and subsequently transplant fecal microbiota via gavage of fecal fluid prepared from fresh or frozen intestinal contents in mice. We also present an optimized method to detect the frequency of IgA-coated bacteria in the gut.

Gut microbiota exert pleiotropic roles in human health and disease. Fecal microbiota transplantation (FMT) is an effective method to investigate the ...

16S rRNA Sequencing: A PCR-based Technique to Identify Bacterial Species

Source: Ewa Bukowska-Faniband1, Tilde Andersson1, Rolf Lood1 
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund, Sweden
Planet Earth is a habitat for millions of bacterial species, each of which has specific characteristics. Identification of bacterial species is widely used in microbial ecology to determine biodiversity of environmental samples and medical microbiology to diagnose infected patients. Bacteria can be classified using conventional microbiology methods, such as microscopy, growth on specific media, biochemical and serological tests, and antibiotic sensitivity assays. In recent decades, molecular microbiology methods have revolutionized bacterial identification. A popular method is 16S ribosomal RNA (rRNA) gene sequencing. This method is not only faster and more accurate than conventional methods, but also allows identification of strains that are difficult to grow in laboratory conditions. Furthermore, differentiation of strains at the molecular level enables discrimination between phenotypically identical bacteria (1-4).
16S rRNA joins with a complex of 19 proteins to form a 30S subunit of the bacterial ribosome (5). It is encoded by the 16S rRNA gene, which is present and highly conserved in all bacteria due to its essential function in ribosome assembly; however, it also contains variable regions which may serve as fingerprints for particular species. These features have made the 16S rRNA gene an ideal genetic fragment to be used in identification, comparison, and phylogenetic classification of bacteria (6).
16S rRNA gene sequencing is based on the polymerase chain reaction (PCR) (7-8) followed by DNA sequencing (9). PCR is a molecular biology method used to amplify specific fragments of DNA through a series of cycles that include:
i) Denaturation of a double stranded DNA template 
ii) Annealing of primers (short oligonucleotides) that are complementary to the template 
iii) Extension of primers by the DNA polymerase enzyme, which synthesizes a new DNA strand
A schematic overview of the method is shown in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10510/10510fig1.jpg" /> 
Figure 1: Schematic overview of the PCR reaction. <a href="https://www.jove.com/files/ftp_upload/10510/10510fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
There are several factors that are important for a successful PCR reaction, one of which is quality of the DNA template. Isolation of chromosomal DNA from bacteria can be performed using standard protocols or commercial kits. Special care should be taken to obtain DNA that is free of contaminants that can inhibit the PCR reaction.
Conserved regions of the 16S rRNA gene permit the design of universal primer pairs (one forward and one reverse) that can bind to and amplify the target region in any bacterial species. The target region can vary in size. While some primer pairs can amplify most of the 16S rRNA gene, others amplify only parts of it. Examples of commonly used primers are shown in Table 1 and their binding sites are depicted in Figure 2.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Primer name</td>
			<td>Sequence (5&#39;&#8594;3&#39;)</td>
			<td>Forward/ reverse</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>8F b)</td>
			<td>AGAGTTTGATCCTGGCTCAG</td>
			<td>forward</td>
			<td>-1</td>
		</tr>
		<tr>
			<td>27F</td>
			<td>AGAGTTTGATCMTGGCTCAG</td>
			<td>forward</td>
			<td>-10</td>
		</tr>
		<tr>
			<td>515F</td>
			<td>GTGCCAGCMGCCGCGGTAA</td>
			<td>forward</td>
			<td>-11</td>
		</tr>
		<tr>
			<td>911R</td>
			<td>GCCCCCGTCAATTCMTTTGA</td>
			<td>reverse</td>
			<td>-12</td>
		</tr>
		<tr>
			<td>1391R</td>
			<td>GACGGGCGGTGTGTRCA</td>
			<td>reverse</td>
			<td>-11</td>
		</tr>
		<tr>
			<td>1492R</td>
			<td>GGTTACCTTGTTACGACTT</td>
			<td>reverse</td>
			<td>-11</td>
		</tr>
	</tbody>
</table>
Table 1: Examples of standard oligonucleotides used in amplification of 16S rRNA genes a). 
a) The expected lengths of the PCR product generated using the different primer combinations can be estimated by calculating the distance between the binding sites for the forward and the reverse primer (see Figure 2), e.g. the size of the PCR product using primer pair 8F-1492R is ~1500 bp, and for primer pair 27F-911R ~900 bp. 
b) also known as fD1
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10510/10510fig2.jpg" /> 
Figure 2: Representative figure of the 16S rRNA sequence and the primer-binding sites. Conserved regions are colored in grey and variable regions are filled with diagonal lines. To allow for the highest resolution, primer 8F and 1492R (name based on location on rRNA sequence) are used to amplify the whole sequence, allowing for the sequencing of several variable regions of the gene. <a href="https://www.jove.com/files/ftp_upload/10510/10510fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Cycling conditions for PCR (i.e. the temperature and time required for the DNA to be denatured, annealed with primers, and synthesized) are dependent on the type of polymerase that is used and the properties of the primers. It is recommended to follow the manufacturer&#39;s guidelines for a particular polymerase.
After the PCR program is completed, the products are analyzed by agarose gel electrophoresis. A successful PCR yields a single band of expected size. The product must be purified prior to sequencing to remove residual primers, deoxyribonucleotides, polymerase, and buffer which were present in the PCR reaction. The purified DNA fragments are usually sent for sequencing to commercial sequencing services; however, some institutions perform DNA sequencing at their own core facilities.
The DNA sequence is automatically generated from a DNA chromatogram by a computer and must be carefully checked for quality, as manual editing is sometimes needed. Following this step, the gene sequence is compared with sequences deposited in the 16S rRNA database. The regions of similarity are identified, and the most similar sequences are delivered.

16S rRNA Sequencing: Identifying Bacterial Species by PCR

16S rRNAシーケンシング:細菌種を同定するPCRベースの技術

Source: Ewa Bukowska-Faniband1, Tilde Andersson1, Rolf Lood1
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, ...

16S rRNA 増幅配列による糞便の微生物評価ガイドのプロトコル

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探索ルートマイクロバイ: 土、根、およびルート Endosphere から細菌群集のデータを抽出

水生生物生息地からの炭化水素代謝特性を有する細菌種の単離・増殖・同定

体外養殖人間の腸内細菌を研究する技術を適用する高度な

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16SリボソームRNA遺伝子アンプリコンの次世代シーケンシング

細菌群集の特徴付けのための効率的な核酸抽出および16S rRNAの遺伝子配列決定

次世代の 16S rRNA 増幅シーケンスによる目盛りマイクロバイ評価

2段階PCRと次世代16S rRNA遺伝子シーケンシングを用いての微生物叢解析

Fecal Microbiota Transplantation and Detection of Prevalence of IgA-Coated Bacteria in the Gut

16S rRNAシーケンシング:細菌種を同定するPCRベースの技術

16S rRNA 増幅配列による糞便の微生物評価ガイドのプロトコル

要約

さらに動画を探す

この動画の章

探索ルート マイクロバイ: 土、根、およびルート Endosphere から細菌群集のデータを抽出

水生生物生息地からの炭化水素代謝特性を有する細菌種の単離・増殖・同定

体外養殖人間の腸内細菌を研究する技術を適用する高度な

ヒトの胎児微生物叢に対する介入レジメンの影響を推定するインビトロバッチ培養モデル

16SリボソームRNA遺伝子アンプリコンの次世代シーケンシング

細菌群集の特徴付けのための効率的な核酸抽出および16S rRNAの遺伝子配列決定

次世代の 16S rRNA 増幅シーケンスによる目盛りマイクロバイ評価

2段階PCRと次世代16S rRNA遺伝子シーケンシングを用いての微生物叢解析

Fecal Microbiota Transplantation and Detection of Prevalence of IgA-Coated Bacteria in the Gut

16S rRNAシーケンシング:細菌種を同定するPCRベースの技術

探索ルートマイクロバイ: 土、根、およびルート Endosphere から細菌群集のデータを抽出