The authors thank Colleen Van Kirk for mouse retina and cerebellum samples, and Peter Gregory for figure generation. The authors also thank Dr. Allison Gillaspy and the Laboratory for Molecular Biology and Cytometry Research core for access to the MiSeq. This work was supported by NIH grants DA029405, EY021716, and AG026607 and the Donald W. Reynolds Foundation.

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The authors have nothing to disclose.

BSASサンプルとターゲットの数の両方数の高スループット能力を有する焦点、正確なDNAメチル化の定量を可能にする。さらに、トランスポ媒介tagmentationを使用して、このライブラリー生成は、より少ないインプットDNAを必要とし、より迅速で、伝統的なせん断およびその後のアダプターライゲーション技術より少ないステップが含まれています。既存の方法に比べてこれらの改善は、任意の目的の標的の正確な基本レベルのDNAメチル化解析を可能にする。また、BSASは全ゲノムバイサルファイト22またはキャプチャ23アプローチを用いて同定されている関心領域（差別的メチル化領域（DMRに）、CpGアイランド、調節領域）の確認のための新しい方法を提供する。直交確認のアプローチや、より定量的に正確な方法を使用すると、エピゲノム研究の分析的厳密さを向上させます。 BSASで達成高い読み取り深度再狭い信頼区間で正確な定量を可能にする正確な定量16にまで一貫した。さらに、単一の実験で多数のサンプルを分析する能力は、統計的検出力を増加させる全ゲノム法の確認のためのサンプルサイズを増加させることができる。現在の多くのエピジェネティック研究の弱さ。重要なことは、BSASはエピジェネティックな研究のための検証可能な仮説の生成を可能にする塩基特異的CpGメチル化の定量を、提供します。特定の応答エレメントでのそのような増加したメチル化のような仮説は、特定の転写因子の結合の減少をもたらすであろう。対のmRNA発現データと結合BSASは、組織または細胞集団の単一片内のエピジェネティックな調節およびmRNA発現の両方を得るための方法を提供する。規制と表現の対の測定値も、調査結果の科学的な厳格さとインパクトを高める。 BSASプロトコル内の重要なステップは、/プライマー設計、アンプリコンの最適化、およびライブラリ生成、QC。プライマーは、適切に設計され、異なる効率8で増幅されていない場合、PCRバイアスが導入される。そのような酵素的に生成された100％および0％のシトシンのメチル化の標準としてメチル化規格の使用は、もしあれば、メチル化定量バイアスの、程度を決定するために使用され、メチル化16を定量化する際に、適切な方法論的な制御であることができる。単一の高品質なアンプリコンを生成するためのPCR反応を最適化する場合も同様に、注意が払われるべきである。何リコンプロトコルで詳述示唆ガイドラインを使用して存在しない場合、最適化ステップが増加PCRサイクル数またはbsDNA入力量を含む。プライマー·ダイマーを含む複数のPCR産物は、存在する場合、最適化ステップは、bsDNA投入量を減少させるPCRプライマーのモル入力濃度を減少させ、アニール温度を増加させる、またはPCRのサイクル数を減少させることが含まれる。これらの最適化の手順のいずれかまたは組み合わせは、単一のHIGを生成するために、十分な結果が得られ時間品質のアンプリコン。また、プライマーを再設計することは、単一のアンプリコンを生じないプライマーセットのための良いアプローチです。再設計が適切なまたは不可能であるこれらの領域については、シトシンのチミンのフォワードプライマーおよびアデニンの中のCpG部位でのグアニンのリバースプライマー中のCpG部位での両方を含む縮重プライマーセットが動作する可能性があります。しかし、これらのプライマーセットは、PCRバイアスは、このプロトコルの範囲外のプロセスを生じていないことを確認するためにさらなる最適化を必要としています。品質ライブラリは、成功のために極めて重要である。 QC対策がシークエンシングの前にライブラリの大きさ、質と量を決定するために行われていることを確認します。配列決定反応は、低品質のライブラリーの入力に失敗しがちである。メチル化の定量におけるバイアスがメチル化頻度が前方の両方に存在し、シークエンシング読み取り逆にすることによって軽減することができる。さらに、CpG部位に合わせて拠点の品質スコアが高いCOのために≥Q30であるべき定量でnfidence。他のものは、上記の測定基準を確保し、重亜硫酸変換したDNA 19のtagmentation反応におけるバイアスなしに、以前にはほとんど示されているが、低バイアスメチル化定量を確認することができます。 BSASは現在、しかし、この方法の将来の拡張は、亜硫酸水素塩変換24の前にカリウム過ルテニウムを導入するなどの5-MCと5-HMCを区別する実験工程を追加してのCpG 5-HMCの対の測定値を可能にし、CpG部位でのメチル化を測定します。これは、DNAメチル化の遺伝子発現調節の役割を決定するために、対のメチル化を可能にすることにより、5-mCと5-HMC、および発現データの両方をBSASユーティリティの影響を増加させる。 PCRアンプリコンのトランスポ媒介ライブラリ調製物は増幅された領域の端部で低下し、配列決定深度をもたらすない。したがって、高い関心領域は、アンプリコンの中央に存在するように設計されるべきである。どうやってBSASが生成するので、今までに、シークエンシング&gt;は千X、増幅された領域の端部付近の測定は高いまま5-MCの定量的な精度の深さを読み取る。

それは自然にシトシンメチル化1の形態のDNAの変更を発生するの最初の報告以来、半世紀以上となっている。シトシンのメチル化は、ベースリング上の5 番目の炭素にメチル基（5-mCと）は、最も頻繁に、哺乳動物ゲノム中のCpGジヌクレオチドモチーフで生じる有する修飾シトシンヌクレオチド塩基である。不在は、転写活性2に関連している遺伝子のプロモーター中の5-mCとの機能的な存在は、一般に、転写抑制と関連している。 驚異的な進歩が開発3、エピジェネティックなプロファイル4、癌の病因5,6、および他の研究領域の数のtransgeneration伝搬におけるDNAメチル化の役割についての我々の理解がなされている。新しい方法論は、DNAメチル化7をプロファイリングするために開発されているようにこれらの進歩の多くは、過去数年間で来ている。出現でと次世代シーケンシング（NGS）技術の普及は現在、全ゲノムまたはゲノム8の大きな領域全体にDNAメチル化をプロファイリングするための方法がいくつかあります。これらのアプローチは、DNAメチル化パターン及びDNAメチル化の差異を定量発見研究の可能性を開く。これらの仮説生成のアプローチに加えて、標的/仮説テストのDNAメチル化の定量法のための重要な必要性がある。 パイロシーケンシング、メチル化特異的PCR、および重亜硫酸変換したDNAの直接のサンガー配列決定は、対象地域を分析するための最も使用される方法であった（ すなわち 、単一遺伝子のプロモーター領域またはCpGアイランド）9-12。メチル化シトシンのはそのまま13,14ままこれらの方法の全ては、亜硫酸水素塩変換、非メチル化シトシンのウラシルのに変換され、化学的脱アミノ化反応に依存している。 bisulfite-のPCR増幅差動メチル化塩基の違いとして読み出すことを可能チミン15とウラシルの交換で変換されたDNAをもたらす。非常に有用であるが、これらの方法には限界が低い定量的精度、短い読み取り長、および低いサンプルスループットを含む。これらの制限に対処するために我々は（BSAS）16重亜硫酸リコンシーケンシングと呼ばれるている方法を開発した。この方法の目的は、高い定量性を有する多数のサンプル中の目的の標的ゲノム領域を分析することができることである。 BSASは、既存の方法（重亜硫酸塩変換および領域特異的PCR増幅）の要素を使用して、単純な次世代ライブラリー構築とそれらを組み合わせた（トランスポ媒介）17及びベンチNGS 18（ 図1）。このアプローチは、目的の任意の領域におけるシトシンのメチル化を調べるためのより定量的な、高スループット方法を提供する。ここでは、提示詳細方法とトラブルシューティングと品質はBSASプロセスをチェックするためのアプローチを説明します。

<table border="0" cellpadding="0" cellspacing="0" width="840">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:304px;">Name</td>
			<td style="width:255px;">Company</td>
			<td style="width:97px;">Cat #</td>
			<td style="width:185px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agilent 2100 Bioanlyzer</td>
			<td>Agilent</td>
			<td>G29393AA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agilent RNA 6000 Nano kit</td>
			<td>Agilent</td>
			<td>5067-1511</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agilent High Sensitivity DNA kit</td>
			<td>Agilent</td>
			<td>5067-4626</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Typhoon 9200</td>
			<td>Amersham/GE Healthcare Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agencourt AMPure XP - PCR Purification</td>
			<td>Beckman Coulter</td>
			<td>A63880</td>
			<td>5 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PowerPac Basic Power Supply</td>
			<td>Bio Rad</td>
			<td>164-5050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">twin.tech PCR plate 96&#160;</td>
			<td>Eppendorf</td>
			<td>951020362</td>
			<td>semi-skirted</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heatsealing Film</td>
			<td>Eppendorf</td>
			<td>0030127838</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heatsealing Foil</td>
			<td>Eppendorf</td>
			<td>0030127854</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heat Sealer</td>
			<td>Eppendorf</td>
			<td>5390000.024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.1-10ul Tips</td>
			<td>Eppendorf</td>
			<td>0030073.002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-200ul Tips</td>
			<td>Eppendorf</td>
			<td>0030073.045</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50-1000ul Tips</td>
			<td>Eppendorf</td>
			<td>0030073.100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MixMate&#160;</td>
			<td>Eppendorf</td>
			<td>5353000.014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 mL Microcentrifuge Tubes</td>
			<td>Eppendorf</td>
			<td>0030121.023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge 5424</td>
			<td>Eppendorf</td>
			<td>5424000.010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2.0 mL Microcentrifuge Tube</td>
			<td>Eppendorf</td>
			<td>022363352</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nextera XT DNA Sample Preparation Kit</td>
			<td>Illumina</td>
			<td>FC-131-1024</td>
			<td>24 rxn</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nextera XT Index Kit</td>
			<td>Illumina</td>
			<td>FC-131-1001</td>
			<td>24 indexes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MiSeq Desktop Sequencer</td>
			<td>Illumina</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MiSeq Reagent Kit v2</td>
			<td>Illumina</td>
			<td>MS-102-2002</td>
			<td>300cycle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KAPA SYBR FAST Universal qPCR kit</td>
			<td>KAPA Biosystems</td>
			<td>KK4824</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Quant-iT PicoGreen dsDNA Assay kit</td>
			<td>Life Technologies</td>
			<td>P7589</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Quant-iT RiboGreen RNA Assay kit</td>
			<td>Life Technologies</td>
			<td>R11490</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ProFlex 96-well PCR system</td>
			<td>Life Technologies</td>
			<td>4484075</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Novex 6% TBE DNA gel</td>
			<td>Life Technologies</td>
			<td>EC6261BOX</td>
			<td>10-well</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Novex TBE Running Buffer</td>
			<td>Life Technologies</td>
			<td>LC6675</td>
			<td>5X (1 L)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Novex Hi-Density TBE Sample Buffer</td>
			<td>Life Technologies</td>
			<td>LC6678</td>
			<td>5X (10 mL)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Kb Plus DNA Ladder</td>
			<td>Life Technologies</td>
			<td>10787-018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xcell SureLock Mini-Cell</td>
			<td>Life Technologies</td>
			<td>EI0001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UltraPure 10mg/mL Ethidium Bromide</td>
			<td>Life Technologies</td>
			<td>15585-011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">7900HT Fast Real-Time PCR system</td>
			<td>Life Technologies</td>
			<td>4329001</td>
			<td>384well block</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DynaMag-96 Side Skirted</td>
			<td>Life Technologies</td>
			<td>12027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SpectroMax M2 Multi-Mode Microplate Reader</td>
			<td>Molecular Devices/VWR</td>
			<td>89429-532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AllPrep DNA/RNA Mini Kit</td>
			<td>Qiagen</td>
			<td>80204</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stainless Steel Beads</td>
			<td>Qiagen</td>
			<td>69989</td>
			<td>5 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CLC Genomics Workbench</td>
			<td>Qiagen</td>
			<td></td>
			<td>Software for methylation pipeline</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAQuick PCR Purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>50 rxn</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TissueLyser II</td>
			<td>Retsch/Qiagen</td>
			<td>85300</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuclease-Free Water</td>
			<td>Sigma</td>
			<td>W4502</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TRIS hydrochloride</td>
			<td>Sigma</td>
			<td>PHG0002-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tween-20</td>
			<td>Sigma</td>
			<td>P9416-50ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NanoDrop 2000</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">384-well Full-Skirted Plate, Standard</td>
			<td>Thermo Scientific</td>
			<td>AB-1384</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Absolute qPCR Seal</td>
			<td>Thermo Scientific</td>
			<td>AB-1170</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EZ DNA Methylation-Lightning kit</td>
			<td>Zymo Research</td>
			<td>D5030</td>
			<td>50 rxn</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ZymoTaq DNA Polymerase</td>
			<td>Zymo Research</td>
			<td>E2001</td>
			<td>50 rxn</td>
		</tr>
	</tbody>
</table>

targeted dna methylation analysis by next-generation sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

BSASは正しく図5のようになり、変換参照配列に整列読み出す。CpGジヌクレオチドを明確に識別することができ、メチル化状態は、マッピングされた読み取りにおけるCpG部位でのベースコールを観察することによって推定することができる。例えば、メチル化コントロールと、0％メチル化は、すべてのTの（ 図5A）を含むCpG部位にマッピングされた読み取りをもたらす。 100％のメチル化コントロールがマップされて読み込まれるすべてのCの（ 図5B）を含むCpG部位に読み込むことになります。 CpG部位のシトシン周波数（C / C + T）の定量は、元の試料中のメチル化頻度をもたらす。全ゲノムメチル化コントロールた（n = 3 /メチル化率）から生成された代表的な標準曲線は、それぞれ、メチル化率（ 図6）で定量におけるメチル化の定量の直線性並びに精度を示している。合計で、この方法の一例として、RNAおよびDNA共イソた<html>マウスの小脳および網膜（n = 4 /群）からlated。選択的に、網膜組織において発現ロドプシン発現は、定量PCRを用いて測定した。発現は、唯一の網膜（ 図7A）で検出された。 BSASを使用して、CpGメチル化のレベルはロドプシンプロモーター領域で定量した。プロモーター領域を横切る累積メチル化レベルは、網膜の&lt;15％に（p &lt;0.001、パラメトリックt検定）（ 図7B）と比較して小脳に&gt; 80％であった。部位特異的メチル化の定量からBSASメチル化定量の利点、及びメチル化レベルは、任意のゲノム領域を横切るCpG特異的に基づいて比較することができる。網膜サンプルた（p &lt;0.001、各々のCpG部位におけるパラメトリックt検定）（ 図7C）と比較した場合、ロドプシンプロモーター渡ってCpGメチル化のレベルは、小脳の試料において有意に高かった。 1highres.jpg &quot;/&gt; 図1：BSAS方法の概略は、この方法では、ゲノムDNAを重亜硫酸塩がウラシルに非メチル化シトシンを修飾するために変換される。その後、PCRの間にこれらのウラシルはチミンに変更されます。 PCR増幅は、関心領域に向けられた、高度にだけこれらの配列について富化される。得られたPCRアンプリコンは、単純なtagmentationプロセスを通じてデュアルインデックス化ライブラリに作られています。その後、ライブラリは、ベンチトップ次世代シーケンサー上で配列され、配列決定は、シトシンのは、塩基特異的方法で決定されたのシリコ変換参照配列とパーセントメチル化にマッピングされて読み込みます。 <img alt="図2" src="/files/ftp_upload/52488/52488fig2highres.jpg" /> 図2：関心領域のシリコ亜硫酸水素塩変換では 、任意のゲノム基準からの関心の任意の領域は、セレクことができますシリコ亜硫酸水素変換におけるためのテッド。非CpGシトシンがチミンの参照配列中に置換されます。のCpGシトシンは重亜硫酸塩変換された参照内のシトシンのまま。 <img alt="図3" src="/files/ftp_upload/52488/52488fig3highres.jpg" /> 図3：プライマー配置亜硫酸塩PCRプライマーは、 インシリコ亜硫酸変換参照配列におけるに対して設計されている。プライマーは、それらがCGジヌクレオチドを​​重複しないように設計またはCGジヌクレオチドに隣接するように設計されるべきである。 <img alt="図4" src="/files/ftp_upload/52488/52488fig4highres.jpg" /> 図4：高および低品質のPCRアンプリコンとシーケンシングライブラリーの例代表電気泳動図トレースとゲルショー（A）単一の高とトランスポ媒介ライブラリ生成のための理想的なアンプリコン。濃縮製品。このアンプリコンから生成された（B）ライブラリ（C）低品質のPCRアンプリコンのサイズ、濃度が低くてもよい。高濃度、さらにはサイズ分布を示し、複数の製品および/ ​​またはプライマー二量体を含む。これらの低品質のアンプリコンは、（D）につながる低濃度と低サイズ分布を有するライブラリー生成に失敗しました。 <img alt="図5" src="/files/ftp_upload/52488/52488fig5highres.jpg" /> 図5：メチル化コントロールからシーケンシング出力の例。 インシリコ （A）は、CpG部位を有する基準シングが赤で強調表示に変換。マップされた0％のメチル化制御はCpG部位でのチミン（貪欲ハイライト）マッピングを示す選択された領域に読み込みます。（B）100％のメチル化制御マッピングされたがCpG部位のシトシンの（青のハイライト）のマッピングを示して読み込みます。 図6：メチル化コントロールの範囲にわたるメチル化定量 0％から100％までのメチル化比率た（n = 3 /比）で混合した全ゲノムメチル化コントロールはBSASを用いて定量した。定量化に対する期待の定量をプロットすると、メチル化制御定量の直線性を示している。すべてのコントロールを越え、高い精度がメチル化定量にあった。 <img alt="図7" src="/files/ftp_upload/52488/52488fig7highres.jpg" /> 図7：組織サンプルからの対のDNAメチル化および遺伝子発現分析の例。 （A）マウス小脳および網膜組織からロドプシン相対的mRNA発現。（B）小脳および網膜DNA（***はp &lt;0.001、パラメトリックt検定）との間にBSASにより定量化ロドプシン平均プロモーターのメチル化。 &lt;マウスの小脳および網膜DNA中の転写開始部位[TSS]）に対してロドプシンプロモーター（6へ-244、全体で強い&gt; C）のCpG部位特異的メチル化レベル。

Watch this Scientific Journal Video about Targeted DNA Methylation Analysis by Next-generation Sequencing at JoVE.com

Targeted DNA Methylation Analysis by Next-generation Sequencing

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

次世代シーケンシングによりDNAメチル化解析をターゲット

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

targeted-dna-methylation-analysis-by-next-generation-sequencing

Research

JoVE Journal

Biology

36.8K ビュー.  University of Oklahoma College of Medicine. Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

ビデオ: Targeted DNA Methylation Analysis by Next-generation Sequencing

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

のインプリント遺伝子のDNAメチル化の定量シロイヌナズナ胚乳

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

生物学

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

DNAメチル化：重亜硫酸修飾と分析

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

蛍光顕微鏡スクリーニングと次世代シーケンシング：オルガネラの整合性に関与する遺伝子の同定のための有用なツール

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

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 ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

塩基対解像度でDNAメチル化の評価のための強化されたの減少表現重亜硫酸塩シークエンシング

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

レトロウイルス統合サイトの増幅、次世代シーケンシング、およびゲノムDNAのマッピング

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 &#946;-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called &#34;domainome&#34;, will be well expressed and soluble, making them suitable for structural/functional studies.
By cloning and displaying the &#34;domainome&#34; directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification.
The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

The protocols described allow the construction, characterization and selection (against the target of choice) of a &#34;domainome&#34; library made from any DNA source. This is achieved by a research pipeline that combines different technologies: phage display, a folding reporter and next generation sequencing with a web tool for data analysis.

インタラクトーム Seq: Domainome ライブラリーの構築、検証およびバクテリオファージの表示と次世代シーケンシングによる選択のプロトコル

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...

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 ...

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

次世代シーケンシングのための磁気ビーズベースの蚊DNA抽出プロトコル

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...