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.

Mantar Malzemesinin 1. Hazırlanması <ol><li> Külleme Büyüyen <ol><li> Aşağıdaki koşullara büyüme odalarına bitkilerin büyümesi: 22 ° C gündüz sıcaklığı, 20 ° C gece sıcaklığı,% 80 nispi nem, 14 saat gün uzunluğu ve 125 uE / m2 / s &#39;lik bir ışık yoğunluğu (floresan aydınlatma sağladığı ). </li><li> 18, 29 tarif edilen şekilde Golovinomyces cichoracearum gerilme UCSC1 salatalık bitkileri (Cucumis sativa çeşitli Bush Champion) Infect. </li></ol></li><li> Hasat Küllemesi Konidyumlar <ol><li> conidia toplandığı bir in-line filtre (11 um) ile bir küçük vakum kullanılarak aşılamadan sonra 10 gün - 7 enfekte bitkilerden hasat conidia. hafif basınç uygulanması, konıdi toplamak için yaprağın yüzeyi boyunca vakum memesi çalıştırın. Not: G. cichoracearum ortalama verimi ağır INFE gelen konidia 20 mg olanbölgede yaklaşık 130 cm2 CTED salatalık yaprağı. </li><li> temiz bir kağıt tabakası üzerine filtreden (7-8 enfekte salatalık yapraklarına yaklaşık 375 uL hacmi veya conidia) conidia yaklaşık 150 mg bir fırça. Buradan (sıvı azot içinde) önceden soğutulmuş iki 5/32 inç çapında çelik öğütme topları ile 2 ml cryovials içine konıdi aktarın. Hemen sıvı nitrojen tüpleri döndürür. -80 ° C&#39;de saklayın borular. </li></ol></li><li> Topu Mamüller tarafından Breaking Açık Konidyumlar <ol><li> Sıvı nitrojen içinde bilyalı değirmen alüminyum plakalar dondurma. Yük bilyeli değirmen rafa cryovials. Şiddetle (oda sıcaklığında) 30 döngü / s&#39;de 30 s için bilyeli değirmende conidia ve çelik topları ile cryovials sallayın. Hemen sıvı azot içinde cryovials dondurursam. NOT: Başlangıçta, öğütme sırasında hücre duvarı kopma verimliliği için 100X büyütme mikroskobu ile Konidyumların bir örneğe erişmek. 30 döngü / S, B 30 saniyelik 2 mermi - Gerekirse, ek 1 öğütmekut minimuma mermi sayısını tutmak. Optimal koşullar kurulduktan sonra bu aşamada mikroskopla konidial kırılma Rutin değerlendirme gereksizdir. </li></ol></li></ol> 2. genomik DNA saflaştırma <ol><li> Konidyumlar Liziz <ol><li> hızla çalışarak, çözdürme konidianıri önlemek bir mıknatısla cryovials çelik topları kaldırmak için. Karışım bir bulamaç elde edilinceye kadar 10 s - 700 ul 65 ° C lizis tamponu (Tablo 1) ve bir girdap 5 ekleyin. </li><li> 300 uL (65 ° C) önceden ısıtılmış% 5 (h / h) sarkosil ekleyin ve yavaşça karıştırmak için beş kez (3 s başına 1 inversiyon) ters çevirin. 65 ° C&#39;de 30 dakika boyunca inkübe edin; 10, 20 ve 30 dakika yavaşça 3 kez ters çevirin. Geniş çaplı bir pipet kullanarak, yeni 2 mL mikrosantrifüj tüpüne tüm çözüm aktarın. takip eden tüm adımlar da, yumuşak, yavaş ters çevirme ile karıştırmak ve geniş çaplı bir pipet uçları olan bir DNA-içeren çözeltiler aktarın. </li></ol></li><li> DNA Ekstraksiyon I <ol><li> kloro 1 hacim eklemeformu: izoamil alkol: yavaşça lizis çözeltisine (24 1 hacim / hacim) ve beş kez çevrilerek karıştırılmıştır. yavaşça inkübasyonun sonunda tekrar orta noktada beş kez karıştırmak ve tersini, oda sıcaklığında 10 dakika boyunca inkübe edin. 14,000 x g &#39;de 15 dakika süre ile oda sıcaklığında santrifüje. Geniş çaplı bir pipet kullanarak dikkatli bir şekilde yeni bir 2 ml mikrosantrifüj tüpüne Sulu tabaka transferi; arayüzünden madde içeren kaçının. </li></ol></li><li> DNA, yağış I <ol><li> 1 hacim oda sıcaklığında% 100 izopropanol (yaklaşık 750 uL) ilave edilir ve yavaşça, altı kez çevrilerek karıştırılmıştır. 14,000 x g &#39;de 15 dakika süre ile oda sıcaklığında santrifüje. Süpernatantı dikkatlice çıkarın ve atın. </li><li> 450 uL önceden soğutulmuş (-20 ° C)% 70 etanol ekleme. 14.000 x g&#39;de oda sıcaklığında 5 dakika boyunca santrifüje. Süpernatantı dikkatlice çıkarın ve atın. dikkatli bir şekilde ince bir pipet ucu ile süpernatantı ve atın, 3 s 1,000 x g&#39;de santrifüjleyin. 15 dakika a için pelet hava-kurutunt, oda sıcaklığı. daha uzun 15 dakika kurutmayın. </li><li> 300 uL TE pelet (10 mM Tris-CI, 1 mM etilen diamin tetra-asetik asit, pH 8.0) içinde yeniden süspanse edin. 4 ° C&#39;de gece boyunca inkübe edilir. Yavaşça çözelti içine gitmedi kalan malzemeyi yeniden süspanse etmek fiske. </li></ol></li><li> RNA kontaminasyonu temizlemek 10 mL RNaz A (10 mg / ml) ekleyin. Yavaşça üç kez karıştırmak için ters. 3 s 1,000 x g&#39;de santrifüjleyin. 2 saat boyunca 37 ° C&#39;de inkübe edilir. </li><li> DNA Ekstraksiyon II <ol><li> 300 ul fenol ekleyin: kloroform: izoamil alkol (25: 24: 1 h / h) ve yavaşça beş kez çevrilerek karıştırılmıştır. nazikçe inkübasyon sonunda yine yarım işaretinde beş kez karıştırın ve için tersini, oda sıcaklığında 10 dakika inkübe edin. 14.000 x g&#39;de oda sıcaklığında 15 dakika santrifüj. Geniş çaplı bir pipet kullanarak, yeni bir 1.5 mL mikrosantrifüj tüpüne süpernatant aktarın. </li></ol></li><li> DNA, yağış II <ol><li> 0.01 hacim 3 M sodyum asetat pH 5.2 (approximatel eklemey3 uL) yavaşça beş kez çevrilerek karıştırılmıştır. 2.5 hacim önceden soğutulmuş (-20 ° C),% 100 etanol (yaklaşık 750 uL) ilave edilir. Yavaşça beş kez karıştırmak için ters. -20 ° C de bir gece boyunca inkübe </li><li> Santrifüj 14,000 x g&#39;de 4 ° C&#39;de 30 dakika. Süpernatantı dikkatlice çıkarın ve atın. </li><li> 450 uL önceden soğutulmuş (-20 ° C)% 70 etanol ekleme. 14,000 x g&#39;da 4 ° C&#39;de 5 dakika boyunca santrifüje. Süpernatantı dikkatlice çıkarın ve atın. 3 s 1,000 x g&#39;de santrifüjleyin. Dikkatle ince pipet ucu ve tamamen yok olan süpernatant kaldırmak. </li><li> Oda sıcaklığında 30-60 dakika boyunca pelet hava-kurutun. 27.5 uL TE içinde pelletini. 4 ° C&#39;de gece boyunca inkübe edilir. Yavaşça çözelti içine gitmedi kalan malzemeyi yeniden süspanse etmek fiske. </li><li> Kısım kalite kontrol testleri için 22.5 uL TE (nihai hacim 25 ul) halinde 2.5 uL (aşağıya bakınız). dizileme için gönderilmeden önce 4 ° C&#39;de saklayın DNA örnekleri. Uzun süreli depolama, mağaza örnekleri içinve -80 ° C &#39;de yırtılmayı önlemek için donma-erime döngülerinden en aza indirir. Not: doğru pipetle zor olabilir, yüksek moleküler ağırlıklı genomik DNA olarak bu aşamada pipetleme dikkat ediniz, ve &quot;QC kısım&quot;, genomik DNA örneği doğru bir temsili olduğundan emin olmak için önemlidir. </li></ol></li></ol> 3. Kalite Kontrol <ol><li> Numune Saflık <ol><li> TE boş kullanarak, küçük hacimli spektrofotometrede QC alikot 1 uL yükleme ile DNA numunesi, saflığı analiz. Tutanak A260 / A280 ve A260 / A230 okumaları. Saf DNA, 1.8 ve 2.2 arasında, yaklaşık 1.8 ve A260 / A230 oranı olan bir A260 / A280 oranı olacaktır. </li></ol></li><li> Genomik DNA miktar belirlemesi, <ol><li> üreticinin talimatlarına göre bir ticari çift sarmallı DNA miktar deney kiti kullanılarak QC kısım ölçümü gerçekleştirmek. </li></ol></li><li> DNA Kalite Değerlendirmesi I <ol><li> Yük yaklaşık olarak 60 ng genomik DNA ve bir1x TAE (40 mM Tris, 20 mM asetik asit ve 1 mM EDTA, pH 8.0) içinde bir% 0.7 agaroz jeli üzerinde DNA merdiveni. 70 V 40 çalıştırmak jel tertibatına - genomik DNA bantları kadar, 45 dakika, yaklaşık merdiven iyi ve bant ayırma 2 cm belirgindir. Bu koşullar mevcut jel elektroforez aygıtı göre ayarlanması gerekebilir. </li></ol></li><li> DNA Kalite Değerlendirmesi II <ol><li> Yük, yaklaşık 60, 0.5 x TBE (44.5 mM Tris, 44.5 mM borik asit, 1 mM EDTA, pH 8.3) ve bir% 1 agaroz jeli üzerinde genomik DNA ve (değişken alanlı jel elektroforez için uygun), yüksek moleküler ağırlıklı DNA merdiveni ng 16 saat boyunca 80 kbp&#39;lik dalga palslı-alan jel elektroforezi protokolü - 5 ile 0.5x TBE tamponunda çalıştırın. </li></ol></li><li> Bakteri, mantar, ve Bitki Kirleticilerin Değerlendirilmesi <ol><li> primerler ve amplifikasyon koşulları listesini kullanarak ribozomal genlerinin uygun iç transkripsiyonlu aralayıcılar (ITS) bölgelerin yükseltilmesi için polimeraz zincir reaksiyonu (PCR) gerçekleştirmekTablo 2&#39;de ed. Bir jel üzerinde elde edilen reaksiyon yük 10 uL. Not: ITS bölgesi ITS primerler mantar ile amplifiye edilmelidir külleme temsil eden bir grup. bu amplikonun ve bakteriyel veya bitki primerleri ile üretilen herhangi bir amplikon DNA kirlenme kaynağı belirlemek üzere dizilenmiştir edilmelidir. Sadece külleme ITS dizileri mantar amplikonuna bulunmalıdır. </li></ol></li></ol>

<ol>
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The authors have nothing to disclose.

Zorunlu biotrophic külleme mantar saf, yüksek moleküler ağırlıklı genomik DNA elde etmek için, daha önce tarif edilen yöntemlerin değiştirilmiş bir versiyonu 30 geliştirilmiştir. Bu optimize edilmiş bir protokol kullanılarak ortalama verim 150 mg conidia başına 7 mg DNA, bir önceki protokol ile elde edilenin iki katıdır. Ayrıca, ortalama boyutu 48.5 kbp boyunca yaklaşık 20 kbp yükselmiştir. Bu protokol cucumber- G. cichoacearum sisteminde optimize edildi. biotr...

<html> Pudra küfleri zorunlu biotrophic mantar bitkinin bir grup birlikte alındığında, bitki hastalık dünya çapında 1 büyük nedenidir, bu patojenler bulunmaktadır. Taksonomik ailesi Erisyphaceae 2 içinde beş kabileler halinde gruplandırılmış olan külleme 900&#39;ün üzerinde açıklanan türler vardır. ekonomik önemleri ve onların ana geliştirmek samimi bir ilişki için her iki dolayı, külleme hastalıkları&gt; 100 yıldır araştırma konusu olmuştur. enfeksiyon üzerine, toz küfler bu patojenin yararlanmak için, hücreli yapının, metabolizma ve ana moleküler biyolojide önemli değişiklikler ortaya çıkarır. Bununla birlikte, toz küfü çalışma nedeniyle bunların zorunlu yaşam tarzı ve saf kültürde mantar büyümesi yapılmamış 5, 4, 3 tarif edilmemiştir için özellikle zor olduğu,&quot;xref&quot;&gt; 6, 7, 8. Geçici transformasyon bazı türler 9, 10, rapor edilmiştir, ancak toz küfü güvenilir ve sabit bir genetik transformasyon ayrıca henüz gerçekleştirilmiş değildir. külleme genomunun sıralama ve montaj nedeniyle genomun kendisinin bir dizi özellik zor olduğu kanıtlanmıştır. Diğer mantar genomların - (180 Mbp 120) göreli ve 60 oluşur - Toz halinde küf genomları büyüktür% 90 eşit olarak dağıtılmış tekrar eden kısımlar 11. Bu elemanlar, uzun terminal tekrarı, hem de edilmemiş tekrarlayan elementleri içerir. Tek külleme türleri, Blumeria graminis f iki formae SPECIALES. sp. hordei ve f. sp. tritici (BGH sırasıyla BGT) hem de üzüm külleme Erysiphe necator, arın sıralandı ve birkaç diğerleri için taslak genomları 12, 13, 14 tamamlanmıştır. Genomların tekrarlayan doğası düzeneği zorlaştırmıştır ve tamamlanan BGH genom 2 arasında bir L50 Mb 12 ile 6,989 supercontigs içine monte edilmiştir. Büyük genomesi rağmen, toz küfler sırasıyla BGH&#39;nin ve Bgt tahmin 5.845 ve 6,540 genleri ile, protein kodlama genlerinin az sayıda sahip gibi görünmektedir. Birbirini izleyen toz küfler de hayatta kalma 11, 12, 13, 14 ev sahibi bitki üzerinde mantar bağımlılığı ile tutarlıdır başka mantarlar içerisinde gerekli olan en az 99 çekirdek genlerden yoksun görünmektedir. telomerlerin, sentromer, ribozomal RN yakın Tekrarlanan dizilerBir gen dizileri ve transpoze edilebilir elemanları zengin bolgelerın zayıf kısa okuma dizileme stratejilerinden monte edilir ve yeterince temsil genom tertibatları 15 genellikle edilir. Bu bölgelerin genom dizileri meydana boşlukların çok sorumlu olduğu düşünülen ve bu tekrar eden kısımlar 16 kendi geniş genişleme ile toz küfü için geçerli olan. Yüksek plastik genom bölümlerinin genellikle tekrar eden bölgeler 3 bulunmaktadır. Bunlar kromozom yeniden düzenlemelerinin bir alanı olarak hizmet ve genellikle bu efektör proteinlerini kodlayan genler ve ikincil metabolizma enzimleri kodlayan genler gibi güçlü selektif basınç altındaki genleri kodlayan. Tek moleküllü uzun okuma sekans teknolojilerini gelişmeler genomları 15 tekrarlamalı bölgelerde dizileme için potansiyel bir çözüm sağlar. Örneğin, Faino ve diğ. (2015) da dahil olmak üzere, uzun-dizi teknolojisi ve optik m okuma bulunduapping gen açıklamaları gen ek açıklama sayısını artırarak (ve kısmen sayısını azaltarak ya da eksik, genom içinde tekrar eden DNA dizilerinin oranını üçe bunları fungal bitki patojen Verticillium Dahlia iki soy için bir boşluk daha az genom dizisi üretmek için izin ) ve ortaya genom 17 yeniden düzenlemelerinin. en az boyutları&gt; 20 kbp bu uzun okuma dizileme teknolojileri, yüksek kaliteli, genomik DNA, yüksek konsantrasyonlarda kullanılması için, ihtiyaç vardır. Burada conidia gelen konidial toplanması, yüksek molekül ağırlıklı DNA &#39;nın arıtılması için yöntemleri tarif eder ve külleme türün Golovinomyces cichoracearum kullanarak kalite kontrol değerlendirmeleri salatalık 18 üzerinde büyümüş. Bu protokol B. Keller laboratuvar grubunda (Zürich Üniversitesi, Zürih İsviçre) 13, 19 geliştirilen bir protokol dayanmaktadırve artmış DNA verimlerine yol açan çok sayıda modifikasyonları ve büyüklüğü, DNA&gt; 48.5 kbp daha yüksek bir oranını içermektedir. Protokol ayrıca Enstitüsü 20, 21, 22 Enerji Ortak Genom United States Department of tavsiyelerine göre kalite kontrol adımlarını içerir. Liziz tamponu içerisinde yer alan her bir reaktif, ve her bir saflaştırma aşaması için mantık fonksiyonu olarak (2008) 23 Henry tarif edilmiştir. Bitki ve mikrobiyal dokularından, yüksek moleküler ağırlıklı genomik DNA&#39;nın izolasyonu için uygun yayınlanmış protokoller bu protokol 24, 25, 26, 27, 28 tasarımında başvurulmuştur.

<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

külleme mantar ekonomik açıdan önemli fungal bitki patojenlerinin bir gruptur. Nispeten küçük nedeniyle iyi gelişmiş genetik ve genomik kaynak eksikliğinden kısmen, moleküler biyoloji ve bu patojenlerin genetik az şey bilinmektedir. Bu organizmalar, genom sekanslama ve montaj engelleyici zorlaştırmıştır büyük, tekrarlayan genomlara sahiptir. Burada, tek bir toz halinde küf türleri, Golovinomyces cichoracearum yüksek moleküler ağırlıklı genomik DNA toplanması, ekstraksiyon, saflaştırma ve kalite kontrol değerlendirilmesi için yöntemleri tarif eder. Açıklanan protokol, optimize edilmiş bir fenol / kloroform genomik DNA izolasyonu, ardından sporlar mekanik bozulmasını içerir. Tipik verim 150 mg konidia başına 7 ug DNA kullanılmıştır. Bu prosedür kullanılarak izole edilmektedir genomik DNA, uzun okuma sekanslama (yani&gt; 48.5 kbp) için uygundur. Kalite kontrol önlemleri, genomik DNA&#39;nın boyutu ile, verim ve saflığı sağlamak için de vardırBu metot dahilinde tarif. Burada tarif edilen kalite genomik DNA dizilenmesi düzeneği ve bu da daha iyi bir anlaşılmasına yol, birden çok külleme genomlarının, karşılaştırılması için izin verir ve bu tarımsal patojen kontrolü gelişmiş olur.

G. saflaştırılan genomik DNA 60 ng temsili bir örneği, jel elektroforezi kullanılarak bir agaroz jel üzerinde yürütülmüştür cichoracearum ve palslı-alan jel elektroforez kullanılarak sırasıyla Şekil 1 ve 2 de gösterilmiştir. Marjinal geçmek ve kalite kontrolü başarısız geçiren Genomik DNA preparasyonu temsil edilmektedir. tamamen kalite kontrolü yapılan genomik DNA preparatları uzun okuma ge...

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.

Uzun Okuma Dizi Yapmak İçin Küllemesi Yüksek Moleküler Ağırlık Genomik DNA saflaştırılması

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

Research

JoVE Journal

Genetics

11.6K Görüntüleme.  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.

Video: 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.

Yeni nesil Sıralama için Uygun Bitki Organelleri DNA Zenginleştirme Yöntemlerinin Optimizasyonu ve Karşılaştırmalı Analizi

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

Genetik

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.

Nanopore DNA sıralama Metagenomic toprak analizleri için

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 ...

Çevre

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.

Eksiltici genomik tarafından roman sıra bulma

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.

Magnaporthe oryzae'de Kromatin İmmün Önkapipitasyon Dizileme Yöntemi Kullanılarak Histone Modifikasyonlarının Genom Çapında Analizi

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.

Topraklar ve tortulları Yüksek Moleküler Ağırlık Genomik DNA çıkarımı

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

Biyoloji

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.

Tek hücreli genom sıralama için bir üretilen iş Ultrahigh mikrosıvısal Platform

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

Biyomühendislik

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.

Bütün genomik DNA analizi için olağanüstü uzun okunur sıralama

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.

Mikrobiyal Paspas Yüksek Moleküler Ağırlıklı DNA çıkarımı

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.

Saf olmayan Arındırıcı: Sıralama Metagenomes ve Metatranscriptomes Kompleksi Hayvan ilişkili Örneklerinden

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.

Ökaryotik Hücrelerde gelen ekstrakromozomal dairesel DNA genom Arıtma

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