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><tbody><tr><td>Agilent 2100 Bioanlyzer</td><td>Agilent</td><td>G29393AA</td><td></td></tr><tr><td>Agilent RNA 6000 Nano kit</td><td>Agilent</td><td>5067-1511</td><td></td></tr><tr><td>Agilent High Sensitivity DNA kit</td><td>Agilent</td><td>5067-4626</td><td></td></tr><tr><td>Typhoon 9200</td><td>Amersham/GE Healthcare Life Sciences</td><td></td><td></td></tr><tr><td>Agencourt AMPure XP - PCR Purification</td><td>Beckman Coulter</td><td>A63880</td><td>5 ml</td></tr><tr><td>PowerPac Basic Power Supply</td><td>Bio Rad</td><td>164-5050</td><td></td></tr><tr><td>twin.tech PCR plate 96&amp;nbsp;</td><td>Eppendorf</td><td>951020362</td><td>semi-skirted</td></tr><tr><td>Heatsealing Film</td><td>Eppendorf</td><td>0030127838</td><td></td></tr><tr><td>Heatsealing Foil</td><td>Eppendorf</td><td>0030127854</td><td></td></tr><tr><td>Heat Sealer</td><td>Eppendorf</td><td>5390000.024</td><td></td></tr><tr><td>0.1-10 &amp;#956;l Tips</td><td>Eppendorf</td><td>0030073.002</td><td></td></tr><tr><td>2-200 &amp;#956;l Tips</td><td>Eppendorf</td><td>0030073.045</td><td></td></tr><tr><td>50-1,000 &amp;#956;l Tips</td><td>Eppendorf</td><td>0030073.100</td><td></td></tr><tr><td>MixMate&amp;nbsp;</td><td>Eppendorf</td><td>5353000.014</td><td></td></tr><tr><td>1.5 ml Microcentrifuge Tubes</td><td>Eppendorf</td><td>0030121.023</td><td></td></tr><tr><td>Centrifuge 5424</td><td>Eppendorf</td><td>5424000.010</td><td></td></tr><tr><td>2.0 ml Microcentrifuge Tube</td><td>Eppendorf</td><td>022363352</td><td></td></tr><tr><td>&lt;a href=&quot;http://www.illumina.com/products/nextera_xt_dna_library_prep_kit.html&quot;&gt;Nextera XT DNA Sample Preparation Kit&lt;/a&gt;</td><td>Illumina</td><td>FC-131-1024</td><td>24 rxn</td></tr><tr><td>&lt;a href=&quot;http://www.illumina.com/products/nextera_xt_dna_library_prep_kit.html&quot;&gt;Nextera XT Index Kit&lt;/a&gt;</td><td>Illumina</td><td>FC-131-1001</td><td>24 indexes</td></tr><tr><td>&lt;a href=&quot;http://www.illumina.com/systems/miseq.html&quot;&gt;MiSeq Desktop Sequencer&lt;/a&gt;</td><td>Illumina</td><td></td><td></td></tr><tr><td>&lt;a href=&quot;http://www.illumina.com/products/miseq_reagent_kit_v2.html&quot;&gt;MiSeq Reagent Kit v2&lt;/a&gt;</td><td>Illumina</td><td>MS-102-2002</td><td>300 cycles</td></tr><tr><td>KAPA SYBR FAST Universal qPCR kit</td><td>KAPA Biosystems</td><td>KK4824</td><td></td></tr><tr><td>Quant-iT PicoGreen dsDNA Assay kit</td><td>Life Technologies</td><td>P7589</td><td></td></tr><tr><td>Quant-iT RiboGreen RNA Assay kit</td><td>Life Technologies</td><td>R11490</td><td></td></tr><tr><td>ProFlex 96-well PCR system</td><td>Life Technologies</td><td>4484075</td><td></td></tr><tr><td>Novex 6% TBE DNA gel</td><td>Life Technologies</td><td>EC6261BOX</td><td>10-well</td></tr><tr><td>Novex TBE Running Buffer</td><td>Life Technologies</td><td>LC6675</td><td>5x (1 L)</td></tr><tr><td>Novex Hi-Density TBE Sample Buffer</td><td>Life Technologies</td><td>LC6678</td><td>5x (10 ml)</td></tr><tr><td>1 Kb Plus DNA Ladder</td><td>Life Technologies</td><td>10787-018</td><td></td></tr><tr><td>Xcell SureLock Mini-Cell</td><td>Life Technologies</td><td>EI0001</td><td></td></tr><tr><td>UltraPure 10 mg/ml Ethidium Bromide</td><td>Life Technologies</td><td>15585-011</td><td></td></tr><tr><td>7900HT Fast Real-Time PCR system</td><td>Life Technologies</td><td>4329001</td><td>384-well block</td></tr><tr><td>DynaMag-96 Side Skirted</td><td>Life Technologies</td><td>12027</td><td></td></tr><tr><td>SpectroMax M2 Multi-Mode Microplate Reader</td><td>Molecular Devices/VWR</td><td>89429-532</td><td></td></tr><tr><td>AllPrep DNA/RNA Mini Kit</td><td>Qiagen</td><td>80204</td><td></td></tr><tr><td>Stainless Steel Beads</td><td>Qiagen</td><td>69989</td><td>5 mm</td></tr><tr><td>CLC Genomics Workbench</td><td>Qiagen</td><td></td><td>Software for methylation pipeline</td></tr><tr><td>QIAQuick PCR Purification kit</td><td>Qiagen</td><td>28104</td><td>50 rxn</td></tr><tr><td>TissueLyser II</td><td>Retsch/Qiagen</td><td>85300</td><td></td></tr><tr><td>Nuclease-Free Water</td><td>Sigma</td><td>W4502</td><td></td></tr><tr><td>TRIS hydrochloride</td><td>Sigma</td><td>PHG0002-100G</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P9416-50ML</td><td></td></tr><tr><td>NanoDrop 2000</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>384-well Full-Skirted Plate, Standard</td><td>Thermo Scientific</td><td>AB-1384</td><td></td></tr><tr><td>Absolute qPCR Seal</td><td>Thermo Scientific</td><td>AB-1170</td><td></td></tr><tr><td>EZ DNA Methylation-Lightning kit</td><td>Zymo Research</td><td>D5030</td><td>50 rxn</td></tr><tr><td>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

37.0K 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

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

Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during cancer development and progression. Despite this, the global epigenetic regulation of lncRNAs and repetitive sequences in cancer has not been well investigated, particularly in chronic lymphocytic leukemia (CLL). This study focuses on a unique approach: the immunoprecipitation-based capture of double-stranded, methylated DNA fragments using methyl-binding domain (MBD) proteins, followed by next-generation sequencing (MBD-seq). CLL patient samples belonging to two prognostic subgroups (5 IGVH mutated samples + 5 IGVH unmutated samples) were used in this study. Analysis revealed 5,800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal healthy controls. Importantly, these results identified several CLL-specific, differentially methylated lncRNAs, repetitive elements, and protein-coding genes with potential prognostic value. This work outlines a detailed protocol for an MBD-seq and bioinformatics pipeline developed for the comprehensive analysis of global methylation profiles in highly CpG-rich regions using CLL patient samples. Finally, a protein-coding gene and an lncRNA were validated using pyrosequencing, which is a highly quantitative method to analyze CpG methylation levels to further corroborate the findings from the MBD-seq protocol.

This work describes an optimized methyl-CpG-binding domain (MBD) sequencing protocol and a computational pipeline to identify differentially methylated CpG-rich regions in chronic lymphocytic leukemia (CLL) patients.

慢性リンパ球性白血病患者におけるメチル-CpG結合ドメイン捕捉に基づく包括的DNAメチル化解析

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

ミトコンドリア DNA のメチル化の正確な検出のための方法論

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these animal models. The rat is one particular model animal where an epigenetic tool can complement many pharmacological and behavioral studies to provide insightful mechanistic information. To this end, we adapted the SureSelect Target Capture System (referred to as Methyl-Seq) for the rat, which can assess DNA methylation levels across the rat genome. The rat design targeted promoters, CpG islands, island shores, and GC-rich regions from all RefSeq genes. 
To implement the platform on a rat experiment, male Sprague Dawley rats were exposed to chronic variable stress for 3 weeks, after which blood samples were collected for genomic DNA extraction. Methyl-Seq libraries were constructed from the rat DNA samples by shearing, adapter ligation, target enrichment, bisulfite conversion, and multiplexing. Libraries were sequenced on a next-generation sequencing platform and the sequenced reads were analyzed to identify DMRs between DNA of stressed and unstressed rats. Top candidate DMRs were independently validated by bisulfite pyrosequencing to confirm the robustness of the platform.
Results demonstrate that the rat Methyl-Seq platform is a useful epigenetic tool that can capture methylation changes induced by exposure to stress.

Here, we describe the protocol and implementation of Methyl-Seq, an epigenomic platform, using a rat model to identify epigenetic changes associated with chronic stress exposure. Results demonstrate that the rat Methyl-Seq platform is capable of detecting methylation differences that arise from stress exposure in rats.

ストレス曝露に伴うエピジェネティックな変化を識別するためにラット メチル Seq プラットフォーム

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these ...

生化学

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

脳の小、解剖学的に定義された領域からのDNAメチル化と遺伝子発現の最適化分析

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

神経科学

LINE-1 Methylation Analysis in Mesenchymal Stem Cells Treated with Osteosarcoma-Derived Extracellular Vesicles

Methylation-specific probe amplification (MSPA) is a simple and robust technique that can be used to detect relative differences in methylation levels of DNA samples. It is resourceful, requires small amounts of DNA, and takes around 4&#8211;5 h of hands-on work. In the presented technique, DNA samples are first denatured then hybridized to probes that target DNA at either methylated or reference sites as a control. Hybridized DNA is separated into parallel reactions, one undergoing only ligation and the other undergoing ligation followed by HhaI-mediated digestion at unmethylated GCGC sequences. The resultant DNA fragments are amplified by PCR and separated by capillary electrophoresis. Methylated GCGC sites are not digested by HhaI and produce peak signals, while unmethylated GCGC sites are digested and no peak signals are generated. Comparing the control-normalized peaks of digested and undigested versions of each sample provides the methylation dosage ratio of a DNA sample. Here, MSPA is used to detect the effects of osteosarcoma-derived extracellular vesicles (EVs) on the methylation status of long interspersed nuclear element-1 (LINE-1) in mesenchymal stem cells. LINE-1s are repetitive DNA elements that typically undergo hypomethylation in cancer and, in this capacity, may serve as a biomarker. Ultracentrifugation is also used as a cost-effective method to separate extracellular vesicles from biological fluids (i.e., when preparing EV-depleted fetal bovine serum [FBS] and isolating EVs from osteosarcoma conditioned media [differential centrifugation]). For methylation analysis, custom LINE-1 probes are designed to target three methylation sites in the LINE-1 promoter sequence and seven control sites. This protocol demonstrates the use of MSPA for LINE-1 methylation analysis and describes the preparation of EV-depleted FBS by ultracentrifugation.

Described here is the use of a methylation-specific probe amplification method to analyze methylation levels of LINE-1 elements in mesenchymal stem cells treated with osteosarcoma-derived extracellular vesicles. Ultracentrifugation, a popular procedure for separating extracellular vesicles from fetal bovine serum, is also demonstrated.

骨肉腫由来細胞外小胞で治療した間葉系幹細胞のメチル化解析

Methylation-specific probe amplification (MSPA) is a simple and robust technique that can be used to detect relative differences in methylation levels of ...

Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is a useful measure to provide an accurate analysis of the methylation status of gastrointestinal (GI) malignancies. Given the multiple potential translational uses of DNA methylation analysis, practicing clinicians and others new to DNA methylation studies need to be able to understand step by step how these genome-wide analyses are performed. The goal of this protocol is to provide a detailed description of how this method is used for the biomarker identification in GI malignancies. Importantly, we describe three critical steps that are needed to obtain accurate results during genome-wide analysis. Clearly and concisely written, these three methods are often lacking and not noticeable to those new to epigenetic studies. We used 48 samples of a GI malignancy (gastric cancer) to highlight practically how genome-wide DNA methylation analysis can be performed for GI malignancies.

Herein, we describe a procedure for genome-wide analysis of DNA methylation in gastrointestinal cancers. The procedure is of relevance to studies that investigate relationships between methylation patterns of genes and factors contributing to carcinogenesis in gastrointestinal cancers.

消化器癌におけるDNAメチル化のゲノムワイド解析

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is ...

がん研究

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNAメチルトランスフェラーゼ阻害剤をスクリーニングするための連続蛍光ベースのエンドヌクレアーゼ共役DNAメチル化アッセイ(英語)

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

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 ...

生物学

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 ...

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

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患者の組織のためのメチル結合DNA捕捉シーケンシング

慢性リンパ球性白血病患者におけるメチル-CpG結合ドメイン捕捉に基づく包括的DNAメチル化解析

ミトコンドリア DNA のメチル化の正確な検出のための方法論

ストレス曝露に伴うエピジェネティックな変化を識別するためにラットメチル Seq プラットフォーム

脳の小、解剖学的に定義された領域からのDNAメチル化と遺伝子発現の最適化分析

骨肉腫由来細胞外小胞で治療した間葉系幹細胞のメチル化解析

消化器癌におけるDNAメチル化のゲノムワイド解析

DNAメチルトランスフェラーゼ阻害剤をスクリーニングするための連続蛍光ベースのエンドヌクレアーゼ共役DNAメチル化アッセイ(英語)

DNAメチル化：重亜硫酸修飾と分析

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

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

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

慢性リンパ球性白血病患者におけるメチル-CpG結合ドメイン捕捉に基づく包括的DNAメチル化解析

ミトコンドリア DNA のメチル化の正確な検出のための方法論

ストレス曝露に伴うエピジェネティックな変化を識別するためにラット メチル Seq プラットフォーム

脳の小、解剖学的に定義された領域からのDNAメチル化と遺伝子発現の最適化分析

骨肉腫由来細胞外小胞で治療した間葉系幹細胞のメチル化解析

消化器癌におけるDNAメチル化のゲノムワイド解析

DNAメチルトランスフェラーゼ阻害剤をスクリーニングするための連続蛍光ベースのエンドヌクレアーゼ共役DNAメチル化アッセイ(英語)

DNAメチル化：重亜硫酸修飾と分析

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

ストレス曝露に伴うエピジェネティックな変化を識別するためにラットメチル Seq プラットフォーム