Name: optimization for sequencing and analysis of degraded ffpe-rna samples
Uploaded: 2020-06-08T15:00:00
Description: Watch this Scientific Journal Video about Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples at JoVE.com

ダニエル・キャリック博士(国立がん研究所がん対策人口科学部門)は、特にこの研究を始め、サンプルを提供し、データ分析中に役立つ提案をしてくれたことに感謝しています。フレデリック国立がん研究研究所のCCRシーケンシング施設のメンバー全員が、サンプル調製とシーケンシング中の支援、特にサンプルQC、オクサナドイツ語の図書館QC、テクサイダーを運営するためのタチアナ・スミルノワの支援に心から感謝します。また、シーケンシング・ファシリティ・バイオインフォマティクス・グループのツァイ・ウェイ・シェンとアシュリー・ウォルトンが、データ分析とRNA-seqパイプラインの実装を支援してくれたことに感謝します。また、RNaseq分析パイプラインおよびベストプラクティス開発に関する支援に対するCCBRおよびNCBRに感謝します。

1. RNA量と品質評価
<ol><li>事前定義された基準に従ってFFPEサンプルを選択し、適切な方法(例えば、FFPE-核酸抽出キット、材料表)を用いてRNAを抽出する。 注:FFPE-RNA抽出には、非常に少ない組織で動作し、良質のRNA12、13、14を抽出できる新しいマイクロディスセクション法を含む、13,14いくつかの異なる方法が...

<ol>
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ここで説明する方法は、FFPE-RNAサンプルから良好な配列データを得るために必要な主なステップを概説する。この方法で考慮すべき主なポイントは、(1)サンプルの取り扱いおよび凍結および解凍のサイクルを最小限に抑えることによって、抽出後にRNAを可能な限り保存することを確認することです。別々のQCアリコートは非常に便利です。(2) 所定のサンプル・セットに最適な QC メトリックを?...

次世代シーケンシングアプローチを利用することで、さまざまな種類のサンプルから豊富な情報を収集することができました。ただし、古くて保存が不十分なサンプルは、一般的に使用されるシーケンスデータの生成方法では動作しないため、確立されたプロトコルを変更する必要が生じることが多い。FFPE,組織は、臨床検体1、2、32に広く利用されてきたこのようなサンプル型を表す。13FFPE保存は組織形態を維持するが、FFPE組織の核酸は通常、広範囲の損傷および分解を示し、様々な障害の根底にある分子メカニズムに関する重要な洞察につながる可能性のあるゲノム情報を取り出すことを困難にする。
RNAシーケンシングによって生成される遺伝子発現データは、疾患や抵抗のメカニズムの研究に役立ち、DNA変異解析を補完することがよくあります。しかし、RNAは分解の影響を受けやすく、FFPE組織から正確な遺伝子発現データを生成することがより困難になります。さらに、シーケンシングの幅広い可用性と手頃な価格が比較的最近であるため、古い標本はRNAの完全性を維持するために必要な条件で保存されなかったことが多かった。FFPEサンプルの問題のいくつかは、パラフィンに埋め込むことによるRNAの分解、シーケンシングに必要な酵素プロセスに対する断片化または屈折性につながるRNAの化学修飾、およびポリA尾の喪失、逆転写酵素4のプライマーとしてのオリゴ-dTの適用性を制限する。もう一つの課題は、最適でない条件下でのFFPEサンプルの取り扱い/保存であり、組織5におけるRNAなどの不安定分子のさらなる分解につながる可能性がある。これは、RNAシーケンシングによる遺伝子発現解析がサンプルに対して予想されていなかった時期に収集された可能性のある古いサンプルに特に関連しています。これらのすべては、有用な配列データを生成するために利用可能な抽出されたRNAの品質と量の減少につながります。成功の可能性が低く、シーケンシングのコストが高いと相まって、多くの研究者が潜在的に有用なFFPEサンプルから遺伝子発現データを生成して分析しようとすることを妨げている。近年のいくつかの研究では、遺伝子発現解析,,2、6、7、8、96に対2するFFPE組織の使用可能性が実証されていますが、最近のサンプルの数が少ない、および/またはより最近のサンプルの場合はあります。,789
実現可能性調査として、我々は、3つの残留組織リポジトリから得られたFFPE腫瘍組織標本から抽出されたRNAを使用して、監視、疫学、および末期結果(SEER)癌登録をRNAシーケンシングおよび遺伝子発現解析10に用いた。臨床病理ラボから調達した、高等度卵巣漿液腺癌からのFFPE組織は、RNA抽出前に様々な条件下で7〜32年保存された。ほとんどの場合、これらのブロックは、将来的に敏感な遺伝子解析を期待することなく、何年も異なる場所に保存されていたので、核酸を保存するためにあまり注意が払われていなかった。したがって、サンプルのほとんどは、細菌で汚染されたサンプルの大部分を有する、質の悪いRNAを示した。それにもかかわらず、遺伝子定量を行い、遺伝子被覆の均一性と連続性を測定し、生物学的複製物間でピアソン相関解析を行い、再現性を測定することができました。主要な遺伝子パネルのセットに基づいて、我々は研究のサンプルを癌ゲノムアトラス(TCGA)データと比較し、サンプルの約60%が同等の遺伝子発現プロファイル11を有することを確認した。各種QC結果とサンプルメタデータとの相関関係に基づいて、我々は、使用可能なシーケンスデータ11を生成する可能性が高いサンプルを同定するための良好な予測値を有する主要なQC指標を同定した。
ここでは、FFPE-RNAの品質評価に用いる方法論、抽出されたRNAサンプルから始まるシーケンシングライブラリの生成、シーケンシングデータのバイオインフォマティクス解析について説明します。

<table>
	<tbody>
		<tr>
			<td>2100 Bioanalyzer</td>
			<td>Agilent</td>
			<td>G2939BA</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent DNA 7500 Kit</td>
			<td>Agilent</td>
			<td>5067-1506</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent High Sensitivity DNA Kit</td>
			<td>Agilent</td>
			<td>5067-4626</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent RNA 6000 Nano Kit</td>
			<td>Agilent</td>
			<td>5067-1511</td>
			<td></td>
		</tr>
		<tr>
			<td>AllPrep DNA/RNA FFPE Kit</td>
			<td>Qiagen</td>
			<td>80234</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX96 Touch System</td>
			<td>Bio-Rad</td>
			<td>1855195</td>
			<td></td>
		</tr>
		<tr>
			<td>Library Quantification kit v2-Illumina</td>
			<td>KapaBiosystems</td>
			<td>KK4824</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBNext Ultra II Directional RNA Library Prep Kit for Illumina</td>
			<td>New England Biolabs</td>
			<td>E7765S</td>
			<td>https://www.neb.com/protocols/2017/02/07/protocol-for-use-with-ffpe-rna-nebnext-rrna-depletion-kit</td>
		</tr>
		<tr>
			<td>NEBNext rRNA Depletion Kit (Human/Mouse/Rat)</td>
			<td>New England Biolabs</td>
			<td>E6310L</td>
			<td></td>
		</tr>
		<tr>
			<td>NextSeq 500 Sequencing System</td>
			<td>Illumina</td>
			<td>SY-415-1001</td>
			<td>NextSeq 500 System guide: https://support.illumina.com/content/dam/illumina-support/documents/documentation/system_documentation/nextseq/nextseq-500-system-guide-15046563-06.pdf</td>
		</tr>
		<tr>
			<td>NextSeq PhiX Control Kit</td>
			<td>Illumina</td>
			<td>FC-110-3002</td>
			<td></td>
		</tr>
		<tr>
			<td>NSQ 500/550 Hi Output KT v2.5 (150 CYS)</td>
			<td>Illumina</td>
			<td>20024907</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Genomics Magnetic Separator</td>
			<td>10X Genomics</td>
			<td>120250</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotator Multimixer</td>
			<td>VWR</td>
			<td>13916-822</td>
			<td></td>
		</tr>
		<tr>
			<td>C1000 Touch Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td>1851197</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencing reagent kit</td>
			<td>Illumina</td>
			<td>20024907</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cell package</td>
			<td>Illumina</td>
			<td>20024907</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer cartridge and the reagent cartridge</td>
			<td>Illumina</td>
			<td>20024907</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide solution (0.2N)</td>
			<td>Millipore Sigma</td>
			<td>SX0607D-6</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS-HCL Buffer 1.0M, pH 7.0</td>
			<td>Fisher Scientific</td>
			<td>50-151-871</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimization for sequencing and analysis of degraded ffpe-rna samples

RNAシーケンシング(RNA-seq)による遺伝子発現解析により、臨床サンプルに対する独自の洞察が可能となり、さまざまな疾患の基礎や抵抗性や感受性のメカニズムを機械化的に理解する可能性があります。しかし、臨床検体における組織形態を保存する最も一般的な方法を表すFFPE組織は、遺伝子発現プロファイリング分析の最良の情報源ではありません。このようなサンプルから得られたRNAは、しばしば分解され、断片化され、化学的に修飾され、最適ではないシーケンシングライブラリにつながります。さらに、これらは、遺伝子発現解析や変異発見に信頼性がない可能性のある低品質のシーケンスデータを生成します。FFPEサンプルを最大限に活用し、低品質のサンプルから可能な限り最良のデータを得るためには、実験計画、シーケンスライブラリの準備、データ分析中に、一定の予防措置を講じる必要があります。これには、正確なサンプル品質管理(QC)、シーケンスライブラリ生成中のさまざまなステップに最適な方法の特定、および慎重なライブラリQCのための適切なメトリックの使用が含まれます。さらに、RNA-seqデータのアーチファクトを特定し、汚染や低品質の読み取り、遺伝子被覆の均一性を評価し、生物学的複製物間で遺伝子発現プロファイルの再現性を測定するためには、正しいソフトウェアツールと配列データ解析パラメータを適用することが重要です。これらのステップは非常に異質なRNAサンプルのプロファイリングのための高精度および再現性を保障できる。ここでは、サンプルQC、ライブラリ調製およびQC、シーケンシング、およびFFPE-RNA組織から得られるような低品質RNAから得られる有用なデータの量を増加させるデータ分析のための様々なステップを説明します。

上記の方法論は、7~32年間のさまざまな条件下で保存されていた67個のFFPEサンプルに適用されました(サンプル保存時間の中央値は17.5年でした)。ここで示したデータセットと解析結果は、Zhaoら11で既に説明および公開されています。先に説明したサンプル品質(例えば図2のトレース)をチェックする上で、DV100は、高分解RNAサンプルに対する小さ?...

Watch this Scientific Journal Video about Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples at JoVE.com

Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples

本手法はホルマリン固定パラフィン埋め込み(FFPE)RNAサンプルから得ることができる配列データの質と量を改善するステップを説明する。我々は、FFPE-RNAサンプルの品質をより正確に評価し、シーケンシングライブラリを準備し、FFPE-RNAサンプルからのデータを分析するための方法論を説明する。

分解されたFFPE-RNAサンプルのシーケンシングと分析の最適化

Gene expression analysis by RNA sequencing (RNA-seq) enables unique insights into clinical samples that can potentially lead to mechanistic understanding ...

当社のプロトコルは、FFPE由来のRNAサンプルから生成される遺伝子発現データの質と量を改善するために使用することができます, 特に最適でない品質サンプルで.この技術は、FFPE組織サンプル内のRNA品質を評価し、下流処理の最適化を可能にし、サンプルシーケンシングデータの量と品質を向上させます。この方法は、生物学的および臨床サンプルにおける転写産物の包括的な特徴付けを可能にし、開発および疾患におけるゲノムの機能的要素に関する洞察を提供し得る。サンプルQC、ライブラリ調製、およびデータ分析のための方法のFFPE-RNA分解および選択はすべて非常に難しい場合があります。適切なメソッドと適切なコントロールを選択する際には、細心の注意を払います。視覚的なデモンストレーションにより、特に保存状態の悪い FFPE サンプルの計画、評価、およびシーケンシング プロセス中に必要なわずかな変更と予防措置を観察できます。RNAサンプルの品質を確認するには、メーカーの指示に従ってRNA QCシステム上でサンプルを実行し、適切なバイオアナリシスソフトウェアを使用してサンプル内のRNA断片の分布を分析し、100ヌクレオチドと200ヌクレオチドより長い断片の割合を計算します。QCメトリックに基づいて、100ヌクレオチドより長いフラグメントの40%未満のサンプルを特定し、これらのサンプルを処理から除外できるようにします。シーケンシングの場合は、室温の水浴で実行パラメータに従って適切なシーケンシング試薬キットを解凍し、解凍後に試薬トレイを摂氏4度に置きます。フローセルパッケージを室温で30分間置きます。パッケージが温まるときに、Illumina 実験マネージャー アプリケーションを開き、[サンプル シートの作成] を選択します。使用するシーケンサを選択し、[次へ]をクリックします。試薬キットのバーコードと、その他の適切なワークフローパラメータを入力します。次に、イルミナシーケンス基準に従って作成したサンプルシートをアップロードします。サンプルライブラリとコントロールライブラリPhiXを調製するには、適切な濃度にライブラリを変性および希釈し、サンプルとPhiXコントロールライブラリを混合して5%PhiX制御体積比を得る。フローセルの準備ができたら、ラッピングを取り除き、糸くずのないアルコール拭き取りでガラス表面をきれいにし、低い糸くず実験室組織でガラスを乾燥させます。試薬カートリッジを4°Cのストレージから取り外し、カートリッジを5回反転させて試薬を混ぜます。ベンチのカートリッジを軽くタップして気泡を減らし、変性した希釈したサンプルを指定されたリザーバーの試薬カートリッジに積み込みます。フローセル、バッファーカートリッジ、試薬カートリッジをシステムにロードします。次に、自動チェックを実行し、出力を確認して、実行パラメーターがシステムチェックに合格していることを確認します。自動チェックが完了したら、[開始] を選択してシーケンス実行を開始します。前処理 QC および後アライメント QC の結果を視覚化するには、マルチ QC を実行して HTML 形式の集計レポートを生成します。バッチ効果を決定し、指定されたデータセットの品質マップを評価するには、R スクリプトを使用して主成分分析を実行します。サンプル相関分析は、次いで、異なるサンプル間のピアソン相関を使用して行うことができる。100ヌクレオチドより長い断片の割合の推定は、高分解RNAサンプルの小さな断片サイズの割合を正確に測定する場合に感度が高いため、200ヌクレオチドよりも長い断片の推定よりも有用です。サンプルセットの分解度が高いことを考えると、トータルRNAライブラリ調製方法が推奨されます。例えば、4つの異なるサンプルから作成されたシーケンスライブラリをここに示します。ここでは、Raw FastQ ファイルのシーケンス品質とサンプル アダプタの内容の概要を示します。FastQC スクリーニングは、サンプル内の細菌やマウスの汚染などの汚染を検出するのに役立ちます。STARアライメントを使用して、参照ゲノムにマッピングされた読み取りの割合、参照ゲノムに一意にマッピングされた読み取りの割合、マッピングされなかった読み取りの割合、または複数の遺伝子座にマッピングされた読み取りの割合を決定できます。カードおよびRSeQC統計は、マップされた読み取りに存在するメッセンジャーRNA、イントロニックおよびインタージェニックな塩基の割合を決定するために使用することができる。これらの代表的な分析では、一部の劣化したライブラリには、5つのプライムエンドよりも多くの読み取りが3つの素数に近いマッピングされる3つの主要なバイアスがありました。さらに、主成分分析を行い、各サンプルの生物学的複製に対して主成分によって捕捉された総ばらの割合を求めることができる。サンプルの劣化を避け、各サンプルに反復と複数のコートを準備し、適切なメトリックを使用してサンプルの品質を評価し、適切なライブラリ調製方法を使用してください。この方法論を用いて、より大きなNGS研究は、様々な保存時間と条件を持つ以前は使用できなかったFFPEサンプルで行われ、さまざまな疾患状態に関する洞察を得ることができます。この技術は、研究者が豊富な臨床情報を持つFFPEサンプルの既存の大規模なアーカイブを研究する道を開き、人口ベースの癌研究を大幅に強化することができます。

optimization-for-sequencing-and-analysis-of-degraded-ffpe-rna-samples

Research

JoVE Journal

Genetics

Flow-sorting and Exome Sequencing of the Reed-Sternberg Cells of Classical Hodgkin Lymphoma

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically comprise less than 1% of the tumor mass. Material derived from bulk tumor contains tumor content at a concentration insufficient for characterization. Therefore, fluorescence activated cell sorting using eight antibodies, as well as side- and forward-scatter, is described here as a method of rapidly separating and concentrating with high purity thousands of HRS cells from the tumor for subsequent study. At the same time, because standard protocols for exome sequencing typically require 100-1,000 ng of input DNA, which is often too high, even with flow sorting, we also provide an optimized, low-input library construction protocol capable of producing high-quality data from as little as 10 ng of input DNA. This combination is capable of producing next-generation libraries suitable for hybridization capture of whole-exome baits or more focused targeted panels, as desired. Exome sequencing of the HRS cells, when compared against healthy intratumor T or B cells, can identify somatic alterations, including mutations, insertions and deletions, and copy number alterations. These findings elucidate the molecular biology of HRS cells and may reveal avenues for targeted drug treatments.

Here, we describe a combined flow cytometric cell sorting and low-input, next-generation library construction protocol designed to produce high-quality, whole-exome data from the Hodgkin Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (CHL).

古典的なホジキンリンパ腫のリードステルベルグ細胞のフローソーティングとExome配列決定

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically ...

遺伝学

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

単一細胞のシーケンシングを使用したコピー数変化の検出

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

次世代シークエンシングに適した植物オルガネラDNA濃縮法の最適化と比較分析

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

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 ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

クロマチン免疫沈降 (チップ) プロトコル低豊富な胚サンプル

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

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

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 a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

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

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 ...

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 ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

次世代シーケンシングのフルレングスの HIV-1 Proviruses 近くの増幅

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60&#8722;80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2&#8722;4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.

Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.

無呼吸性遺伝子検査用半導体シーケンシング

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

ナノポアシーケンシングを用いた全血からの mRNA の配列決定

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...