Name: purification of high molecular weight genomic dna from powdery mildew for long-read sequencing
Uploaded: 2017-03-31T13:00:00.000+00:00
Duration: 6 min 56 s
Description: Watch this Scientific Journal Video about Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing at JoVE.com

This protocol was developed in support of a Joint Genome Institute-Community Sequencing Project (#1657). The work was supported in part by the Philomathia Foundation, the National Science Foundation (#0929226) and the Energy Biosciences Institute to S. Somerville; and by Swiss National Science Foundation (#310030_163260) to B. Keller. We would like to thank our colleagues, M. Figureroa and M. Miller (University of Minnesota), R. Panstruga (RWTH Aachen University), C. Pedersen (University of Copenhagen), P. Spanu (Imperial College), J. Taneja (University of California Berkeley), M. Wildermuth (University of California Berkeley), R. Wise (Iowa State University) and S. Xiao (University of Maryland) for their generous advice and for volunteering their protocols as we developed the protocol described here.

真菌材料の作製<ol><li>成長うどんこ病<ol><li>以下の条件で、成長チャンバ内の植物を育てる：22°C日の温度、20℃の夜間温度、相対湿度80％、14時間の日長および125μE/ m 2 / sの光強度（蛍光灯によって提供）。 </li><li> 18、29を説明するようにGolovinomyces cichoracearum株 UCSC1とキュウリ植物（Cucumisにサティバ品種ブッシュチャンピオン）に感染。 </li></ol></li><li>収穫うどんこ病分生子<ol><li>分生子が蓄積たインラインフィルター（11ミクロン）を有する小型真空を用いて接種後10日 - 7に感染した植物から収穫分生子。穏やかな圧力を適用し、分生子を収集するために、葉の表面に沿って真空ノズルを実行します。 注：G.のcichoracearumの平均収率は、高濃度infeから分生子の20 mgで地域に約130 cm 2のキュウリの葉をCTED。 </li><li>清潔な紙のシート上のフィルタから（7-8感染したキュウリの葉から約375μLの体積、又は分生子）分生子の約150mgを磨きます。ここから、（液体窒素で）は、2つの32分の5インチ径予冷鋼粉砕ボールを2mLのクライオバイアルに分生子を移します。直ちに液体窒素にチューブを戻します。 -80°Cで保存チューブ。 </li></ol></li><li>ボールミルによりオープン分生子を破ります<ol><li> Flashには、液体窒素中でボールミルのアルミニウム板を凍結します。ロードは、ボールミルラックにクライオバイアル。激しく30サイクル/秒（室温）で30秒間ボールミルで分生子及びスチールボールとクリオバイアルを振ります。直ちに液体窒素で凍結バイアルを再凍結。 注：最初に、研削中の細胞壁破壊の効率を100倍の倍率で顕微鏡で分生子のサンプルにアクセスします。 B、30サイクル/ sの30秒の2回 - 必要に応じて、追加の1のために挽きますUTは最小限にラウンド数を保ちます。最適な条件が確立されていたら、この段階での顕微鏡による分生子破損のルーチン評価は不要です。 </li></ol></li></ol> 2.ゲノムDNA精製<ol><li>分生子溶解<ol><li>解凍分生子を避けるために素早く作業を、磁石とのクライオバイアルから鋼球を取り除きます。スラリーが形成されるまで10秒- 700μL65℃の溶解緩衝液（ 表1）、ボルテックス5を加えます。 </li><li> 300μL（65℃）で予熱した5％（v / v）のサルコシルを添加し、穏やかに混合するために5回（3秒ごとに1反転）を反転させます。 65°Cで30分間チューブをインキュベートします。 10、20及び30分で穏やかに3回転倒。ワイドボアピペットチップを使用して、新しい2 mlのマイクロ遠心チューブに全溶液を移します。すべての後続のステップで、穏やかな、ゆっくりし、反転により混合し、ワイドボアピペットチップを用いてDNA含有溶液を移します。 </li></ol></li><li> DNAの抽出I <ol><li>クロロの1つのボリュームを追加形式：イソアミルアルコール（24：1 v / v）の溶解液に、穏やかに5回混合する反転します。優しくインキュベーションの終わりに、再び中間点で5回混ぜるとする反転、室温で10分間インキュベートします。 14,000×gで15分間、室温で遠心分離。ワイドボアピペットチップを使用して、慎重に新しい2 mlのマイクロ遠心チューブに水層を移します。インターフェースからの材料を含む避けます。 </li></ol></li><li> DNA降水I <ol><li> 1つの体積室温の100％イソプロパノール（約750μL）を加え、穏やかに6回混合する反転します。 14,000×gで15分間、室温で遠心分離。上清を慎重に取り除き、捨てます。 </li><li> 450μL予備冷却（-20°C）70％エタノールを追加します。 14,000×gで室温で5分間遠心。上清を慎重に取り除き、捨てます。注意深く微細ピペットチップで上清を除去し、廃棄し、3秒1,000×gで遠心します。 15分aのペレットを空気乾燥トンの室温。より長い15分間乾燥させないでください。 </li><li> 300μLのTE（10mMのトリス-Cl、1mMのエチレンジアミン四酢酸、pH8.0）中にペレットを再懸濁します。 4℃で一晩インキュベートします。静か液に行かなかった任意の残留物を再懸濁するためにフリック。 </li></ol></li><li> RNA汚染を除去するために、10μLのRNase A（10mg / ml）を加えます。ゆっくり3回を混在させる反転。 3秒1,000×gで遠心します。 2時間37℃でインキュベートします。 </li><li> DNA抽出II <ol><li> 300μLを、フェノールを追加：クロロホルム：イソアミルアルコール（25：24：1 v / v）を、穏やかに5回混合する反転します。優しくインキュベーションの終わりに、再び中間点で5回混ぜるとする反転、室温で10分間インキュベートします。 14,000×gで室温で遠心15分。ワイドボアピペットチップを使用して、新しい1.5 mlのマイクロ遠心チューブに上清を移します。 </li></ol></li><li> DNA降水II <ol><li> 0.01体積の3M酢酸ナトリウムpHが5.2（approximatelを追加Y 3μL）、そっと5回を混在させる反転。 2.5容量予め冷却（-20°C）100％エタノール（約750μL）を加えます。ゆっくり5回を混在させる反転。 -20℃で一晩インキュベート</li><li>遠心14,000×gで4℃で30分間。上清を慎重に取り除き、捨てます。 </li><li> 450μL予備冷却（-20°C）70％エタノールを追加します。 14,000×gで4℃で5分間遠心。上清を慎重に取り除き、捨てます。 3秒1,000×gで遠心します。慎重に細いピペットチップと廃棄で上清を除去します。 </li><li>室温で30〜60分間、ペレットを空気乾燥させます。 27.5μLTEでペレットを再懸濁。 4℃で一晩インキュベートします。静か液に行かなかった任意の残留物を再懸濁するためにフリック。 </li><li>品質管理試験のために22.5μLTE（最終容量25μL）にアリコート2.5μL（下記参照）。シークエンシングのために提出するまで4℃で保存DNAサンプル。長期保存、店のサンプルについてそして-80℃でせん断を防ぐために、凍結融解サイクルを最小限に抑えます。 注：適切にピペットすることが困難な場合が高分子量のゲノムDNAとして、この段階でピペッティングする際には注意してください、そして、「QCアリコート」は、ゲノムDNAサンプルの正確な表現であることを確認することが重要です。 </li></ol></li></ol> 3.品質管理<ol><li>サンプル純度<ol><li> TEブランクを用いて、小容量の分光光度計QCアリコートの1μLをロードすることによって、DNAのサンプルの純度を評価します。録音A260 / A280およびA260 / A230の測定値。純粋なDNAは、1.8と2.2の間、約1.8のA260 / A280比およびA260 / A230比を有することになります。 </li></ol></li><li>ゲノムDNAの定量化<ol><li>製造者の指示に従って商業的二本鎖DNA定量アッセイキットを用いて、QCアリコートの定量化を行います。 </li></ol></li><li> DNA品質評価I <ol><li>負荷が約60 ngのゲノムDNAと1X TAE（40mMのトリス、20mMの酢酸、および1mM EDTA、pH8.0）中0.7％アガロースゲル上のDNAラダー。ゲノムDNAのバンドがラダーでよく、バンド分離から約2cmで明らかであるようになるまで45分 - 40、70 Vでゲル装置を実行します。これらの条件は、利用可能なゲル電気泳動装置に基づいて調整される必要があるかもしれません。 </li></ol></li><li> DNA品質評価II <ol><li>負荷は約60 0.5×TBE（44.5 mMトリス、44.5 mMのホウ酸、1mMのEDTA、pHは8.3）と1％アガロースゲル上でゲノムDNAと（パルスフィールドゲル電気泳動に適した）高分子量DNAラダーをngの16時間80 kbpの波形パルスフィールドゲル電気泳動プロトコル - 5を用いて0.5×TBEランニングバッファーで実行されます。 </li></ol></li><li>細菌、真菌、および植物の汚染物質の評価<ol><li>プライマー及び増幅条件のリストを使用してリボソーム遺伝子の適切な内部転写スペーサー（ITS）領域を増幅するためのポリメラーゼ連鎖反応（PCR）を行います表2に編。ゲル上で得られた反応のロード10μL。 注：うどんこ病ITS領域を表すバンドは、真菌のITSプライマーを用いて増幅されるべきです。このアンプリコンおよび細菌、又は植物プライマーを用いて生成されたアンプリコンからのDNAは、汚染の起源を決定するために配列決定されるべきです。唯一のうどんこ病のITSシーケンスは、真菌アンプリコンに記載されていなければなりません。 </li></ol></li></ol>

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The authors have nothing to disclose.

偏biotrophicウドンコ病菌類から純粋な、高分子量のゲノムDNAを得るために、以前に記載された方法の修正版30を開発しました。この最適化されたプロトコルを使用して平均収量は150mgの分生子あたり7μgのDNAを、従来のプロトコルを用いて得られた収量の倍増です。また、平均粒径は48.5 kbpの上に約20 kbpのから増加しました。このプロトコルはcucumber- G.のcichoracearumシステム</e...

<html>うどんこ病は、絶対biotrophic真菌植物のグループが一緒になったときに、植物病害全世界1の最大の原因である、という病原体です。分類学的に家族Erisyphaceae 2内の5つの種族に分類されているうどん粉病の900以上述べた種が、あります。彼らの経済的重要性と、彼らは彼らのホストと発展親密な関係の両方により、うどんこ病は&gt; 100年間の研究の対象とされてきました。感染すると、うどんこ病は、この病原体の利益のために、細胞構造、代謝およびそのホストの分子生物学の急激な変化を誘発します。 しかし 、うどんこ病の研究は、それらの偏ライフスタイルに特に困難である、と純粋培養中の真菌の成長はまだ3に記載されていない、4、5&quot;外部参照&quot;&gt; 6、7、8。一過性の形質転換は、いくつかの種9、10で報告されているが、うどんこ病の信頼性の高い、安定した遺伝的形質転換はまた、まだ達成されていません。 うどんこ病のゲノムのシーケンシングおよびアセンブリが原因ゲノム自体の機能の数に困難であることが判明しました。ウドンコ病ゲノムは、大きい（120から180 MBP）他の真菌ゲノムに対して、及び60から構成- 90％の均一に分布反復エレメント11。これらの要素は、非長末端反復だけでなく、未分類の反復要素を含みます。単一うどんこ病種、 ブルメリア・グラミニス f の二formae speciales。 SP。 hordeiと、f。 SP。 トリチシ（BGHそれぞれBGT）ならびにブドウうどんこ病エリシフェの鉤虫、蜂を有しますN配列決定し、そしていくつかの他のもののためのドラフトゲノムは12、13、14を完了しました。ゲノムの反復性は、アセンブリ困難になっており、完成BGHゲノムは2メガビット12のL50と6989 supercontigsに組み立てました。 大規模なゲノムにもかかわらず、うどんこ病は、それぞれ、BGHとBGTで予測5845および6540の遺伝子と、タンパク質をコードする遺伝子の数が少ないように見えます。配列決定された粉末状のカビも生存11、12、13、14のためにそれらの宿主植物上の真菌の依存性と一致している他の真菌において必須である少なくとも99のコア遺伝子を欠いているように見えます。 テロマー、セントロメア、リボソームRN近く反復配列遺伝子配列および転移因子に富む領域が不十分ショートリード配列決定戦略から組み立てられ、アンダー表さゲノムアセンブリ15であることが多いされています。そのような領域は、ゲノム配列において生じるギャップの多くの原因であると考えられ、これは、反復エレメント16のそれらの広範な拡大にうどんこ病に適用されています。高可塑性ゲノム領域は、多くの場合、このような反復領域3に記載されています。これらは、染色体再配列の部位として機能し、しばしばそのようなエフェクタータンパク質をコードする遺伝子と二次代謝の酵素をコードする遺伝子のような強い選択圧の下で遺伝子をコードします。単一分子の長いリード配列決定技術の進歩は、ゲノム15の繰り返し領域の両端の配列決定のための潜在的な解決策を提供します。例えば、Faino ら。 （2015）を含む長い配列は技術や光Mを読み取ることがわかっappingは、遺伝子注釈の数を増加させる（及び部分的または欠落遺伝子注釈の数を減らす、ゲノム中の反復DNA配列の割合が3倍、それらは真菌植物病原体のVerticilliumのダリアの二つの株のためにギャップのないゲノム配列を生成することができ）と暴露のゲノム再編成17。 &gt; 20 kbpの最小限のサイズでこれらの長い読みシーケンシング技術、高品質のゲノムDNAの高濃度を採用するために、必要とされています。ここでは、分生子から分生子コレクション、高分子量のDNAを精製するための私達の方法を説明し、うどんこ病種Golovinomyces cichoracearumを使用して、当社の品質管理評価はキュウリ18上に成長させました。このプロトコルはB.ケラー実験群（チューリッヒ大学、チューリッヒ、スイス）13、19に開発されたプロトコルに基づいていますそして、増加DNA収量につながったいくつかの修正およびサイズでDNA&gt; 48.5 kbpのより高い割合を含みます。プロトコルはまた、エネルギー共同ゲノム研究所20、21、22の米国部門からの推薦に基づき、品質管理工程を含みます。 ヘンリー（2008）23に記載されるように溶解緩衝液に含まれる各試薬の機能、及び各精製工程のための理論的根拠は、です。植物および微生物組織から高分子量のゲノムDNAの単離のための利用可能な公開されたプロトコルは、このプロトコル24、25、26、27、28の設計時に相談しました。

<table><tbody><tr><td>MM400 Ball Mill</td><td>Retsch</td><td>20.745.0001</td><td>Or equivalent ball mill</td></tr><tr><td>2 mL tubes for ball milling</td><td>Sarstedt&amp;nbsp;</td><td>72.694.007</td><td></td></tr><tr><td>5/32&quot; stainless steel milling balls</td><td>OPS Diagnostics</td><td>GBSS 165-5000-01</td><td></td></tr><tr><td>Microprocessor controlled 280 Series Water Bath Preset to 65 &amp;deg;C&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>2825</td><td>Or equivalent water bath</td></tr><tr><td>Microprocessor controlled 280 Series Water Bath Preset to 37 &amp;deg;C</td><td>Thermo Fisher Scientific</td><td>2825</td><td>Or equivalent water bath</td></tr><tr><td>Eppendorf 5417R refrigerated microcentrifuge</td><td>Krakeler Scientific&amp;nbsp;</td><td>38-022621807</td><td>Or equivalent microcentrifuge</td></tr><tr><td>Chlorform:IAA (24:1 v/v)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>C0549</td><td>Hazardous in case of skin contact or inhalation. Wear nitrile gloves, protective eyewear, lab coat and work in chemical hood</td></tr><tr><td>Phenol:Chloroform:IAA&lt;br /&gt; (25:24:1 v/v)</td><td>Thermo Fisher Scientific</td><td>15593031</td><td>Hazardous in case of skin contact or inhalation. Wear nitrile gloves, protective eyewear, lab coat and work in chemical hood</td></tr><tr><td>100% Isopropanol</td><td>Sigma-Aldrich&amp;nbsp;</td><td>W292907</td><td></td></tr><tr><td>100% Ethanol</td><td>Sigma-Aldrich&amp;nbsp;</td><td>34923</td><td>Chill to -20 &amp;deg;C prior use</td></tr><tr><td>Sodium Acetate</td><td>Sigma-Aldrich&amp;nbsp;</td><td>S2889</td><td></td></tr><tr><td>Rnase, Dnase-free (10 mg/mL)</td><td>Thermo Fisher Scientific</td><td>EN0531</td><td></td></tr><tr><td>Potassium metabisulfite</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P2522</td><td></td></tr><tr><td>Sodium Lauryl Sacroinate</td><td>Sigma-Aldrich&amp;nbsp;</td><td>L9150</td><td></td></tr><tr><td>Tris base</td><td>Sigma-Aldrich&amp;nbsp;</td><td>10708976001</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>EDS</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>S9888</td><td></td></tr><tr><td>Cetyltrimethyl ammonium bromide (CTAB)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>H6269</td><td></td></tr><tr><td>Uvex by Honeywell Futura Goggles S345C, Uvextreme</td><td>Staples</td><td>423263</td><td>Or equivalent eyewear</td></tr><tr><td>High Five COBALT nitrile gloves, LARGE</td><td>Neta Scientific</td><td>HFG-N193</td><td>Or equivalent gloves</td></tr><tr><td>GelRed Nucleic Acid Gel Stain 10,000x in water</td><td>Biotium</td><td>41003</td><td></td></tr><tr><td>Ethidium Bromide 10 mg/mL</td><td>Biotium</td><td>40042</td><td>Hazardous in case of skin contact. Wear nitrile gloves and protective eyewear</td></tr><tr><td>Agarose Ultrapure Bioreagent</td><td>VWR International LLC</td><td>JT4063</td><td></td></tr><tr><td>GeneRuler 1 kb Plus DNA Ladder</td><td>Thermo Fisher Scientific</td><td>FERSM1332</td><td></td></tr><tr><td>8 - 48 kb CHEF DNA size standards</td><td>Bio-Rad</td><td>170-3707</td><td></td></tr><tr><td>Pippin Prep</td><td>Life Technologies Corporation</td><td>4472172</td><td>Or equivalent pulse-field gel aparatus</td></tr><tr><td>Whatman qualitative filter paper, Grade 1</td><td>Sigma-Aldrich&amp;nbsp;</td><td>WHA1001042</td><td>Or equivalent growth chamber</td></tr><tr><td>Percival Growth Chamber</td><td>Percival</td><td>AR66LXC9</td><td></td></tr><tr><td>NanoDrop 8000 UV-Vis Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>ND-8000-GL</td><td>Or equivalent spectrophotometer</td></tr><tr><td>Quant-iT PicoGreen dsDNA Assay Kit</td><td>Thermo Fisher Scientific</td><td>P11496</td><td></td></tr><tr><td>TAE Buffer (Tris-acetate-EDTA) (50x)</td><td>Thermo Fisher Scientific</td><td>B49</td><td>Dilute 1/50 with water before use (to 1x)</td></tr><tr><td>TE Buffer</td><td>Thermo Fisher Scientific</td><td>12090015&amp;nbsp;</td><td></td></tr><tr><td>Tris-Borate-EDTA, 10x Solution (Electrophoresis)</td><td>Thermo Fisher Scientific</td><td>BP13334</td><td>Dilute 1/10 with water before use (to 1x)</td></tr><tr><td>Liquid Nitrogen</td><td></td><td></td><td>Liquid nitrogen (-196 &amp;deg;C) is a freezing hazard. It will also expand rapidly upon warming and should not keep in a tightly closed container</td></tr></tbody></table>

purification of high molecular weight genomic dna from powdery mildew for long-read sequencing

うどんこ病菌は経済的に重要な真菌植物病原体のグループです。比較的少ないが原因よく発達し、遺伝とゲノムリソースの不足のため一部では、これらの病原体の分子生物学および遺伝学について知られています。これらの生物は、ゲノム配列決定およびアセンブリが極めて困難作った大規模な、反復ゲノムを、持っています。ここでは、1つのうどんこ病種、Golovinomyces cichoracearumから高分子量のゲノムDNAの採取、抽出、精製及び品質管理の評価のための方法について説明します。記載されたプロトコルは、最適化されたフェノール/クロロホルムゲノムDNAを抽出した胞子の機械的破壊を含みます。典型的な収量は、150mgの分生子あたり7μgのDNAをでした。この手順を用いて単離されたゲノムDNAは、長いリードシーケンシング（ すなわち 、&gt; 48.5 kbpの）に適しています。品質管理対策は、ゲノムDNAの大きさ、収率、および純度を確保するためでもありますこの方法で説明します。ここで説明する品質のゲノムDNAの配列決定は、組み立てと今度はより良い理解につながる複数のうどんこ病のゲノムの比較を可能にし、この農業病原体の制御を改善します。

ゲル電気泳動を使用し、パルスフィールドゲル電気泳動を用いてアガロースゲル上G.のcichoracearum実行から精製したゲノムDNAを60ngの代表的な例は、それぞれ、 図1および図 2に示されています。わずか品質管理に合格し、不合格、通過ゲノムDNA調製物が表されています。完全な品質管理に合格したゲノムDNA調製物...

Watch this Scientific Journal Video about Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing at JoVE.com

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

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

ロングリード配列決定のためのうどんこ病から高分子量のゲノムDNAの精製

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

purification-high-molecular-weight-genomic-dna-from-powdery-mildew

Universidad San Sebastian

Research

JoVE Journal

Genetics

11.6K ビュー.  University of California Berkeley. Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome sequencing.

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

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 ...

遺伝学

Nanopore DNA Sequencing for Metagenomic Soil Analysis

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA sequences identified using computer software. Nanopore DNA sequencing is a flexible technique that allows for rapid microbial genome sequencing to identify bacterial and viral species, to characterize bacterial strains, and to detect genetic mutations that confer resistance to antibiotics. The advantages of nanopore sequencing (NS) for life sciences include its low complexity, reduced cost, and rapid real-time sequencing of purified genomic DNA, PCR amplicons, cDNA samples, or RNA. NS is an example of "strand sequencing" which involves sequencing DNA by guiding a single stranded DNA molecule through a nanopore that is inserted into a synthetic polymer membrane. The membrane has an electrical current applied across it, so as the individual bases pass through the nanopore the electrical current is disrupted to varying degrees by the four nucleotide bases. The identification of each nucleotide occurs by detecting the characteristic modulation of the electrical current by the different bases as they pass through the nanopore. The NS system consists of a handheld, USB powered portable device and a disposable flow cell that contains a nanopore array. The portable device plugs into a standard laptop computer that reads and records the DNA sequence using computer software.

Nanopore technology for sequencing biomolecules has wide applications in the life sciences, including identification of pathogens, food safety monitoring, genomic analysis, metagenomic environmental monitoring, and characterization of bacterial antibiotic resistance. In this article, the procedure for metagenomic soil DNA sequencing for species identification using the nanopore sequencing technology is demonstrated.

ナノポア DNA シーケンス メタゲノム土壌分析用

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA ...

環境学

Novel Sequence Discovery by Subtractive Genomics

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a larger genomic context. Subtractive genomics enables a researcher to isolate a target sequence of interest (T) by comprehensive sequencing and subtracting out known genetic elements (reference, R). The method can be used to identify novel sequences such as mitochondria, chloroplasts, viruses, or germline restricted chromosomes, and is particularly useful when T cannot be easily isolated from R. Beginning with the comprehensive genomic data (R + T), the method uses Basic Local Alignment Search Tool (BLAST) against a reference sequence, or sequences, to remove the matching known sequences (R), leaving behind the target (T). For subtraction to work best, R should be a relatively complete draft that is missing T. Since sequences remaining after subtraction are tested through quantitative Polymerase Chain Reaction (qPCR), R does not need to be complete for the method to work. Here we link computational steps with experimental steps into a cycle that can be iterated as needed, sequentially removing multiple reference sequences and refining the search for T. The advantage of subtractive genomics is that a completely novel target sequence can be identified even in cases in which physical purification is difficult, impossible, or expensive. A drawback of the method is finding a suitable reference for subtraction and obtaining T-positive and negative samples for qPCR testing. We describe our implementation of the method in the identification of the first gene from the germline-restricted chromosome of zebra finch. In that case computational filtering involved three references (R), sequentially removed over three cycles: an incomplete genomic assembly, raw genomic data, and transcriptomic data.

The purpose of this protocol is to use a combination of computational and bench research to find novel sequences that cannot be easily separated from a co-purifying sequence, which may be only partially known.

減法混色のゲノム解析による新規シーケンスの発見

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a ...

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription factors (TFs), chromatin regulators, and histone modifications, as well as detecting entire genomes for uncovering TF binding patterns and histone posttranslational modifications. Chromatin-modifying activities, such as histone methylation, are often recruited to specific gene regulatory sequences, causing localized changes in chromatin structures and resulting in specific transcriptional effects. The rice blast is a devastating fungal disease on rice throughout the world and is a model system for studying fungus-plant interaction. However, the molecular mechanisms in how the histone modifications regulate their virulence genes in Magnaporthe oryzae remain elusive. More researchers need to use ChIP-seq to study how histone epigenetic modification regulates their target genes. ChIP-seq is also widely used to study the interaction between protein and DNA in animals and plants, but it is less used in the field of plant pathology and has not been well developed. In this paper, we describe the experimental process and operation method of ChIP-seq to identify the genome-wide distribution of histone methylation (such as H3K4me3) that binds to the functional target genes in M. oryzae. Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

マグナペアテオリザエにおけるクロマチン免疫沈降シーケンシング法を用いたヒストン修飾分布のゲノム解析

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

土壌と堆積物から高分子量のゲノムDNAの抽出

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

生物学

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

単一細胞のゲノムの配列のための超高スループット マイクロ流体プラットフォーム

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

生物工学

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes and analysis of complex structural variants. Nanopore platforms perform single-molecule sequencing by directly measuring the current changes mediated by DNA passage through the pores and can generate hundreds of kilobase (kb) reads with minimal capital cost. This platform has been adopted by many researchers for a variety of applications. Achieving longer sequencing read lengths is the most critical factor to leverage the value of nanopore sequencing platforms. To generate ultra-long reads, special consideration is required to avoid DNA breakages and gain efficiency to generate productive sequencing templates. Here, we provide the detailed protocol of ultra-long DNA sequencing including high molecular weight (HMW) DNA extraction from fresh or frozen cells, library construction by mechanical shearing or transposase fragmentation, and sequencing on a nanopore device. From 20-25 &#181;g of HMW DNA, the method can achieve N50 read length of 50-70 kb with mechanical shearing and N50 of 90-100 kb read length with transposase mediated fragmentation. The protocol can be applied to DNA extracted from mammalian cells to perform whole genome sequencing for the detection of structural variants and genome assembly. Additional improvements on the DNA extraction and enzymatic reactions will further increase the read length and expand its utility.

Long-read sequences greatly facilitate the assembly of complex genomes and characterization of structural variation. We describe a method to generate ultra-long sequences by nanopore-based sequencing platforms. The approach adopts an optimized DNA extraction followed by modified library preparations to generate hundreds of kilobase reads with moderate coverage from human cells.

全 Genomic DNA 分析のための超長いリード シーケンス

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

バイオマットから高分子量DNAの抽出

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

複雑な動物に関連したサンプルからのシーケンシングメタゲノムとMetatranscriptomes：不純を精製する

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

真核細胞から体外の環状DNAのゲノムワイドな精製

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

ロングリード配列決定のためのうどんこ病から高分子量のゲノムDNAの精製

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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

ナノポア DNA シーケンスメタゲノム土壌分析用

減法混色のゲノム解析による新規シーケンスの発見

マグナペアテオリザエにおけるクロマチン免疫沈降シーケンシング法を用いたヒストン修飾分布のゲノム解析

土壌と堆積物から高分子量のゲノムDNAの抽出

単一細胞のゲノムの配列のための超高スループットマイクロ流体プラットフォーム

全 Genomic DNA 分析のための超長いリードシーケンス

バイオマットから高分子量DNAの抽出

複雑な動物に関連したサンプルからのシーケンシングメタゲノムとMetatranscriptomes：不純を精製する

真核細胞から体外の環状DNAのゲノムワイドな精製

ロングリード配列決定のためのうどんこ病から高分子量のゲノムDNAの精製

要約

さらに動画を探す

この動画の章

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

ナノポア DNA シーケンス メタゲノム土壌分析用

減法混色のゲノム解析による新規シーケンスの発見

マグナペアテオリザエにおけるクロマチン免疫沈降シーケンシング法を用いたヒストン修飾分布のゲノム解析

土壌と堆積物から高分子量のゲノムDNAの抽出

単一細胞のゲノムの配列のための超高スループット マイクロ流体プラットフォーム

全 Genomic DNA 分析のための超長いリード シーケンス

バイオマットから高分子量DNAの抽出

複雑な動物に関連したサンプルからのシーケンシングメタゲノムとMetatranscriptomes：不純を精製する

真核細胞から体外の環状DNAのゲノムワイドな精製

ナノポア DNA シーケンスメタゲノム土壌分析用

単一細胞のゲノムの配列のための超高スループットマイクロ流体プラットフォーム

全 Genomic DNA 分析のための超長いリードシーケンス