破壊的なサンプリングの標本へのアクセスのためのミズーリの植物園とサンプリング標本、援助のクリスティーナ Zudock ヨルダン Teisher、テイラー AuBuchon-高齢者を感謝いたします。この作品は全米科学財団 (DEB-1457748) からの助成金による支援です。

<ol>
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著者は、彼らが競合する利益あるを宣言します。

ここで提示されたプロトコルは、DNA の隔離および植物の乾燥標本からのライブラリの準備をシーケンス処理の包括的かつ堅牢な方法です。メソッドとそれを変更する必要は最小限の一貫性は、それは大規模な標本に基づくシーケンス プロジェクトのスケーラブルな標本の品質確認に基づいています。低降伏点ライブラリのオプション reamplification ステップを含めることは、低品質、低量、珍...

標本コレクションは、種と系統1,2,3, 集団遺伝学4,5, 保全を含む研究のためのゲノムの多様性の両方の可能性がある貴重な情報源生物学6、外来種生物学7、および形質進化8。種、人口、地理的な場所、タイム ポイントの豊かな多様性を取得する機能は、標本は、「宝箱」9を強調表示します。歴史的に、標本由来 DNA の劣化した自然が多い領域の葉緑体ゲノムや、リボソームの内部転写スペーサー (ITS) などの高いコピー内で見つかったマーカーのみを使用する研究者を追いやって、PCR ベースのプロジェクトを妨げています。RNA。標本と DNA の品質によって異なります広く保全9,10二本鎖切断と損傷の最も一般的な形であること、作成の乾燥工程で使用される熱から断片化の手法、いわゆる 90 %dna ロックアップ PCR ベースの研究11を妨害しています。断片化、別標本のゲノムの第 2 最も一般的な問題は汚染、生菌13から派生する菌類コレクション後、標本12にもマウントする前に死後を取得したようにこの問題は、右真菌データベース (下記参照) を与えられた解決の bioinformatically をすることができます。11押し葉標本の低い (〜 0.03%) と推定されている、3 番目、およびより少なく共通の問題はシトシン脱アミノ化 (C/G→T/A)14日シーケンス変更です。短いリードとシーケンス深さ12,15、低品質の多数の標本から遺伝子レベルのデータ集録が可能高スループット シーケンス (HTS) の出現により、断片化の問題を克服できます。DNA と時々 全ゲノム シーケンス15を許可します。
標本サンプルはより頻繁に使用されるなる、系統のプロジェクト16の大きなコンポーネントです。標本を用いた高温超伝導体の現在の課題が個々 のための方法を最適化することがなく、タイムリーに多くの種から十分な二本鎖 DNA、シーケンス プロトコルは、必要な前提条件を一貫して取得します。標本。本稿では、既存の手段を活用して、高速かつ複製可能な結果を可能にするそれらを変更に DNA の抽出と標本のライブラリの準備のためのプロトコルが示されています。この方法は 13 h、8 h のハンズオン タイム、または 16 h、9 h、ハンズオン時間、オプション reamplification ステップが必要な場合 24 サンプルのライブラリに試験片から処理が完了すること。多くのサンプルの同時処理は、制限要因は遠心力と技術的なスキルが達成可能で。プロトコルは DNA をせん断してだけ典型的な実験装置 (たち、遠心とマグネット スタンド) ネブライザー、超音波発生装置などの特殊な機器ではなくを必要とする設計されています。
DNA の品質、フラグメント サイズと数量は、高スループット シーケンス実験の標本の使用のための要因を制限しています。標本の DNA を分離し、高スループット シーケンス ライブラリを作成する他の方法は 10 として少し使用の有用性を実証している DNA16; ngただし実験的 PCR の最適な数を決定する必要なライブラリの準備のために必要なサイクルです。これは、いくつか標本が単一のライブラリの準備のため十分な DNA のみを生成するよう実行可能なダブルの非常に小さな金額を扱う鎖 DNA (dsDNA)、非現実的ななります。ライブラリの最適化の手順で DNA は失われませんので、ここで紹介した方法は単一サンプルの質に関係なくサイクル数を使用します。代わりに、ライブラリは、シーケンスに必要な最低限の量を満たしていない場合に、reamplification ステップが呼び出されます。多くの植物標本サンプルはまれであり、多くの場合有害なサンプリングを正当化するが難しく少し材料を所有しています。これを対抗するため提案するプロトコルにより、dsDNA 入力サイズを 1.25 未満 ng のライブラリの準備プロセス、高スループット シーケンスと供試体の破壊のサンプリングのための必要性を最小限に抑えるのための実行可能なサンプルの範囲を拡大します。
次のプロトコルを草用に最適化されており、我々 はプロトコルことができます他の多くの植物のグループに適用されることを期待するものの、標本サンプルから別の種の何百ものテストします。低品質または珍しい標本を保存するために使用できる省略可能な回復手順が含まれています。テスト 200 以上の標本を基に、このプロトコルは、最小限の破壊的なサンプリングによって珍しい標本の保存を可能にする低組織入力と品質、標本で動作します。ここでこのプロトコルがゲノミクス ベース プロジェクトのシーケンスことができます高品質ライブラリを提供できることを示す.

<table border="0" cellpadding="0" cellspacing="0" width="733">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Veriti Thermal Cycler</td>
			<td style="width:180px;">Applied Biosystems</td>
			<td style="width:104px;">4452300</td>
			<td style="width:147px;">96 well&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Gel Imaging System</td>
			<td style="width:180px;">Azure Biosystems</td>
			<td style="width:104px;">c300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microfuge 20 Series</td>
			<td style="width:180px;">Beckman Coulter</td>
			<td style="width:104px;">B30137</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Digital Dry Bath</td>
			<td style="width:180px;">Benchmark Scientific</td>
			<td style="width:104px;">BSH1001</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Electrophoresis System</td>
			<td style="width:180px;">EasyCast</td>
			<td style="width:104px;">B2</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">PURELAB flex 2 (Ultra pure water)</td>
			<td style="width:180px;">ELGA&#160;</td>
			<td style="width:104px;">89204-092</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">DNA LoBind Tube&#160;</td>
			<td style="width:180px;">Eppendorf</td>
			<td>30108078</td>
			<td style="width:147px;">2 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Mini centrifuge</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:104px;">12-006-901</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Vortex-Genie 2</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:104px;">12-812</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Mortar</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:104px;">S02591</td>
			<td style="width:147px;">porcelain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pestle</td>
			<td style="width:180px;">fisher Scientific</td>
			<td style="width:104px;">S02595</td>
			<td style="width:147px;">porcelain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Centrifuge tubes</td>
			<td style="width:180px;">fisher Scientific</td>
			<td style="width:104px;">21-403-161</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microwave</td>
			<td style="width:180px;">Kenmore</td>
			<td style="width:104px;">405.7309231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Qubit Assay Tubes</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:104px;">Q32856</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">0.2 ml Strip tube and Cap for PCR</td>
			<td style="width:180px;">VWR</td>
			<td style="width:104px;">20170-004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Qubit 2.0 Fluorometer</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:104px;">Q32866</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Balance</td>
			<td style="width:180px;">Mettler Toledo</td>
			<td style="width:104px;">PM2000</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Liquid Nitrogen Short-term Storage</td>
			<td style="width:180px;">Nalgene</td>
			<td style="width:104px;">F9401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Magnetic-Ring Stand&#160;</td>
			<td style="width:180px;">ThermoFisher Scientific&#160;</td>
			<td style="width:104px;">AM10050</td>
			<td style="width:147px;">96 well&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Water Bath</td>
			<td style="width:180px;">VWR</td>
			<td style="width:104px;">89032-210</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Hot Plate Stirrers</td>
			<td style="width:180px;">VWR</td>
			<td style="width:104px;">97042-754</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Liquid Nitrogen</td>
			<td style="width:180px;">Airgas</td>
			<td style="width:104px;">UN1977</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">1 X TE Buffer</td>
			<td style="width:180px;">Ambion</td>
			<td style="width:104px;">AM9849</td>
			<td style="width:147px;">pH 8.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">CTAB</td>
			<td style="width:180px;">AMRESCO</td>
			<td style="width:104px;">0833-500G</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">2-MERCAPTOETHANOL</td>
			<td style="width:180px;">AMRESCO</td>
			<td style="width:104px;">0482-200ML</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Ribonuclease A</td>
			<td style="width:180px;">AMRESCO</td>
			<td style="width:104px;">E866-5ML</td>
			<td style="width:147px;">10 mg/ml solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Agencourt AMPure XP</td>
			<td style="width:180px;">Beckman Coulter</td>
			<td style="width:104px;">A63882</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Sodium Chloride</td>
			<td style="width:180px;">bio WORLD</td>
			<td style="width:104px;">705744</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Isopropyl Alcohol</td>
			<td style="width:180px;">bio WORLD</td>
			<td style="width:104px;">40970004-1</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Nuclease Free water</td>
			<td style="width:180px;">bio WORLD</td>
			<td style="width:104px;">42300012-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Isoamyl Alcohol</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:104px;">A393-500</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Sodium Acetate Trihydrate</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:104px;">s608-500</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">LE Agarose</td>
			<td style="width:180px;">GeneMate</td>
			<td style="width:104px;">E-3120-500</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">100bp PLUS DNA Ladder</td>
			<td style="width:180px;">Gold Biotechnology</td>
			<td style="width:104px;">D003-500</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">EDTA, Disodium Salt</td>
			<td style="width:180px;">IBI Scientific</td>
			<td style="width:104px;">IB70182</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Qubit dsDNA HS Assay Kit</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:104px;">Q32854</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">TRIS</td>
			<td style="width:180px;">MP Biomedicals</td>
			<td style="width:104px;">103133</td>
			<td style="width:147px;">ultra pure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Gel Loading Dye Purple (6 X)</td>
			<td style="width:180px;">New England BioLabs</td>
			<td style="width:104px;">B7024S</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">NEBNext dsDNA Fragmentase</td>
			<td style="width:180px;">New England BioLabs</td>
			<td style="width:104px;">M0348L</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">NEBNext Ultra II DNA Library Prep Kit for Illumina&#160;</td>
			<td style="width:180px;">New England BioLabs</td>
			<td style="width:104px;">E7645L</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">NEBNext Multiplex Oligos for Illumina</td>
			<td style="width:180px;">New England BioLabs</td>
			<td style="width:104px;">E7600S</td>
			<td style="width:147px;">Dual Index Primers Set 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">NEBNext Q5 Hot Start HiFi PCR Master Mix</td>
			<td style="width:180px;">New England BioLabs</td>
			<td style="width:104px;">M0543L</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Mag-Bind RXNPure Plus</td>
			<td style="width:180px;">Omega bio-tek</td>
			<td style="width:104px;">M1386-02</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">GelRed 10000 X</td>
			<td style="width:180px;">Pheonix Research</td>
			<td style="width:104px;">41003-1</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Phenol solution</td>
			<td style="width:180px;">SIGMA Life Science</td>
			<td style="width:104px;">P4557-400ml</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">PVP40</td>
			<td style="width:180px;">SIGMA-Aldrich</td>
			<td style="width:104px;">PVP40-50G</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Chloroform</td>
			<td style="width:180px;">VWR</td>
			<td style="width:104px;">EM8.22265.2500</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Ethanol</td>
			<td style="width:180px;">Koptec</td>
			<td style="width:104px;">V1016</td>
			<td style="width:147px;">200 Proof</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Silica sand</td>
			<td style="width:180px;">VWR</td>
			<td style="width:104px;">14808-60-7</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reamplification primers</td>
			<td colspan="2">Integrated DNA Technologies</td>
			<td>see text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Sequencher v.5.0.1</td>
			<td style="width:180px;">GeneCodes</td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

robust dna isolation and high-throughput sequencing library construction for herbarium specimens

標本は、さまざまな生物学的研究で使用できる植物材料の非常に貴重な源です。標本の使用はサンプル保存品質、劣化 DNA、珍しい標本の破壊的なサンプリングを含む課題の番号に関連付けられます。効果的に大規模なプロジェクトの標本材料を使用、するために DNA の分離およびライブラリの準備の信頼性と拡張性の高い方法が必要です。本稿は、DNA 分離、高速ライブラリ構築に標本から個々 のサンプルの変更を必要としない堅牢で、始めから終わりまでプロトコルを示します。このプロトコルは、組織研削、ライブラリ サイズ選択を変更して低収量ライブラリのオプション reamplification ステップを導入することを最適化することにより従来の低品質の乾燥植物素材とを利用に合わせて調整します。低収量 DNA ライブラリの reamplification は、かけがえのない、潜在的に貴重な標本、追加破壊サンプリングし、共通認識できるシーケンス バイアスを導入することがなく、必要性を否定することから派生したサンプルを救うことができます。系統のアプリケーション。プロトコル数百草種でテストされていますが確認後他の植物の系統での使用に適応します。このプロトコルは、フラグメントは目的のサイズの範囲では存在しない、非常に低下した DNA ときれいな DNA 分離を抑制する二次代謝産物いくつかの植物材料の存在によって制限することができます。全体的に、このプロトコルは DNA の隔離と最小限の変更でアクティブなハンズオン タイムの 8 時間だけで 13 h 未満の 24 サンプル調製されたライブラリでは、高速かつ包括的な方法を紹介します。

DNA の隔離および最終的なライブラリ収量 本研究では 1920 年からの最も古いと 2012 年 (表 2) から最年少 50 の異なるサンプルを使用して標本の DNA の分離および高品質シーケンス ライブラリの回復用のプロトコルの有効性を示した.各サンプルは、葉の組織の約 10 mg は DNA の隔離に使われました。場合は、環境に優しい葉組織が支持され、明?...

Watch this Scientific Journal Video about Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens at JoVE.com

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

堅牢な DNA の隔離および標本の高スループット シーケンス ライブラリーの構築

この記事では、DNA の隔離及び非常に質の悪い DNA の救助を含む標本材料から高スループット シーケンス ライブラリ構築のための詳しいプロトコルを示します。

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

robust-dna-isolation-high-throughput-sequencing-library-construction

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

10.5K ビュー.  Donald Danforth Plant Science Center. この記事では、DNA の隔離及び非常に質の悪い DNA の救助を含む標本材料から高スループット シーケンス ライブラリ構築のための詳しいプロトコルを示します。

ビデオ: Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

患者の組織のためのメチル結合DNA捕捉シーケンシング

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

遺伝学

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

ハイスループットプロトプラスト単離と変革のためのロボットプラットフォーム

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 &#181;g DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., &#62; 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.

Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome sequencing.

ロングリード配列決定のためのうどんこ病から高分子量のゲノムDNAの精製

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

ネイティブのクロマチン免疫沈降のヌクレオソーム密度解析ライブラリをシーケンスの生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

大規模な gRNA ライブラリ生産 DNA の任意のソースからの普遍的なプロトコル

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

G2-seq: 高スループット シーケンス ベースを識別する方法後半ゲノムの領域を複製します。

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

ホルマリン固定パラフィン包埋 RNA 試料を用いた回顧マイクロ Rna シーケンス: 相補的な DNA ライブラリの準備のプロトコル

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

エラー修正 DNA ・ RNA シーケンスを用いた珍しいイベント検出

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state when water is supplied. The genomic sequencing of microscopic animals such as tardigrades risks bacterial contamination that sometimes leads to erroneous interpretations, for example, regarding the extent of horizontal gene transfer in these animals. Here, we provide an ultralow input method to sequence the genome of the tardigrade, Hypsibius dujardini, from a single specimen. By employing rigorous washing and contaminant exclusion along with an efficient extraction of the 50 ~ 200 pg genomic DNA from a single individual, we constructed a library sequenced with a DNA sequencing instrument. These libraries were highly reproducible and unbiased, and an informatics analysis of the sequenced reads with other H. dujardini genomes showed a minimal amount of contamination. This method can be applied to unculturable tardigrades that could not be sequenced using previous methods.

Contamination during the genomic sequencing of microscopic organisms remains a large problem. Here, we show a method to sequence the genome of a tardigrade from a single specimen with as little as 50 pg of genomic DNA without whole genome amplification to minimize the risk of contamination.

超低入力ヒトゲノム単一 Tardigrade の標本からのライブラリの準備

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

ハイスループット DNA の抽出およびフィン クリッピングが 3dpf ゼブラフィッシュ仔のジェノタイピング

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

堅牢な DNA の隔離および標本の高スループットシーケンスライブラリーの構築

要約

さらに動画を探す

この動画の章

患者の組織のためのメチル結合DNA捕捉シーケンシング

ハイスループットプロトプラスト単離と変革のためのロボットプラットフォーム

ロングリード配列決定のためのうどんこ病から高分子量のゲノムDNAの精製

ネイティブのクロマチン免疫沈降のヌクレオソーム密度解析ライブラリをシーケンスの生成

大規模な gRNA ライブラリ生産 DNA の任意のソースからの普遍的なプロトコル

G2-seq: 高スループットシーケンスベースを識別する方法後半ゲノムの領域を複製します。

ホルマリン固定パラフィン包埋 RNA 試料を用いた回顧マイクロ Rna シーケンス: 相補的な DNA ライブラリの準備のプロトコル

エラー修正 DNA ・ RNA シーケンスを用いた珍しいイベント検出

超低入力ヒトゲノム単一 Tardigrade の標本からのライブラリの準備

ハイスループット DNA の抽出およびフィンクリッピングが 3dpf ゼブラフィッシュ仔のジェノタイピング

堅牢な DNA の隔離および標本の高スループット シーケンス ライブラリーの構築

要約

さらに動画を探す

この動画の章

患者の組織のためのメチル結合DNA捕捉シーケンシング

ハイスループットプロトプラスト単離と変革のためのロボットプラットフォーム

ロングリード配列決定のためのうどんこ病から高分子量のゲノムDNAの精製

ネイティブのクロマチン免疫沈降のヌクレオソーム密度解析ライブラリをシーケンスの生成

大規模な gRNA ライブラリ生産 DNA の任意のソースからの普遍的なプロトコル

G2-seq: 高スループット シーケンス ベースを識別する方法後半ゲノムの領域を複製します。

ホルマリン固定パラフィン包埋 RNA 試料を用いた回顧マイクロ Rna シーケンス: 相補的な DNA ライブラリの準備のプロトコル

エラー修正 DNA ・ RNA シーケンスを用いた珍しいイベント検出

超低入力ヒトゲノム単一 Tardigrade の標本からのライブラリの準備

ハイスループット DNA の抽出およびフィン クリッピングが 3dpf ゼブラフィッシュ仔のジェノタイピング

堅牢な DNA の隔離および標本の高スループットシーケンスライブラリーの構築

G2-seq: 高スループットシーケンスベースを識別する方法後半ゲノムの領域を複製します。

ハイスループット DNA の抽出およびフィンクリッピングが 3dpf ゼブラフィッシュ仔のジェノタイピング