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.

1. Preparazione del materiale Fungal <ol><li> Crescere Oidio <ol><li> Crescere le piante in camere di crescita con le seguenti condizioni: 22 ° C Temperatura di giorno, temperatura 20 ° C notte, 80% di umidità relativa, 14-h giornata intera e un&#39;intensità luminosa di 125 μE / m 2 / s (fornito da lampade fluorescenti ). </li><li> Infettare piante di cetriolo (Cucumis sativa varietà Bush Champion) con Golovinomyces cichoracearum ceppo UCSC1 come descritto 18, 29. </li></ol></li><li> Fare la raccolta Oidio Conidi <ol><li> Raccolto conidi da piante infette a 7 - 10 giorni dopo l&#39;inoculazione con un piccolo vuoto con un filtro in linea (11 micron) su cui i conidi accumulano. Applicando una leggera pressione, eseguire l&#39;ugello di vuoto lungo la superficie della foglia per raccogliere il conidi. NOTA: La resa media di G. cichoracearum è di 20 mg di conidi da un pesantemente infeCTED foglia di cetriolo di circa 130 cm 2 nella zona. </li><li> Spazzolare circa 150 mg di conidi (circa 375 volumi mL o conidi di 7-8 foglie cetriolo infetti) dal filtro su un foglio di carta pulita. Da qui, trasferire conidi in 2 mL cryovials con diametro due 5/32-inch pre-raffreddata (in azoto liquido) palle di macinazione in acciaio. ritornare immediatamente tubi di azoto liquido. conservare le provette a -80 ° C. </li></ol></li><li> Rottura aperta Conidi da Ball Milling <ol><li> Flash congelare piatti di alluminio di mulino a palle in azoto liquido. Carico cryovials in cremagliera mulini a palle. Agitare vigorosamente la cryovials con conidi e acciaio palline in mulino a sfere per 30 s a 30 cicli / s (a temperatura ambiente). ricongelare immediatamente cryovials in azoto liquido. NOTA: Inizialmente, accedere ad un campione di conidi mediante microscopia a ingrandimento 100X per l&#39;efficienza di rottura della parete cellulare durante la macinazione. Se necessario, macinare per altri 1 - 2 cicli di 30 s per 30 cicli / s, but mantenere il numero di giri al minimo. La valutazione di routine di rottura conidica al microscopio in questa fase non è necessaria una volta stabilite le condizioni ottimali. </li></ol></li></ol> 2. Il DNA genomico Purificazione <ol><li> conidi Lysis <ol><li> Lavorando in fretta per evitare lo scongelamento conidi, rimuovere le sfere di acciaio da cryovials con un magnete. Aggiungere 700 microlitri 65 ° C tampone di lisi (Tabella 1) e vortex 5 - 10 s fino a formare un impasto liquido. </li><li> Aggiungere 300 microlitri pre-riscaldato (65 ° C) 5% (v / v) sarcosilL e capovolgere delicatamente per miscelare (1 inversione per 3 s) cinque volte. Incubare le provette per 30 min a 65 ° C; invertire delicatamente 3 volte a 10, 20 e 30 min. Usando una pipetta ampio tunnel, trasferire l&#39;intera soluzione di nuovo 2 ml provetta. In tutte le fasi successive, mescolare delicatamente, lenta inversione e trasferire le soluzioni di DNA contenenti con punte di pipette ampio tunnel. </li></ol></li><li> DNA Extraction I <ol><li> Aggiungere 1 volume di cloroforma: alcool isoamilico (24: 1 v / v) alla soluzione di lisi e capovolgendo delicatamente per miscelare cinque volte. Incubare per 10 minuti a temperatura ambiente, delicatamente per miscelare invertendo cinque volte a metà strada e di nuovo alla fine dell&#39;incubazione. Centrifugare a temperatura ambiente per 15 minuti a 14.000 x g. Utilizzando ampio tunnel punta della pipetta, trasferire accuratamente lo strato acquoso al nuovo 2 ml provetta; evitare di includere materiale dall&#39;interfaccia. </li></ol></li><li> DNA Precipitazioni I <ol><li> Aggiungere 1 volume temperatura ambiente 100% isopropanolo (circa 750 mL) e capovolgere delicatamente per miscelare sei volte. Centrifugare a temperatura ambiente per 15 minuti a 14.000 x g. rimuovere con attenzione il surnatante e gettarlo. </li><li> Aggiungere 450 microlitri pre-raffreddata (-20 ° C) 70% di etanolo. Centrifugare per 5 minuti a temperatura ambiente a 14.000 x g. rimuovere con attenzione il surnatante e gettarlo. Centrifugare a 1000 xg per 3 s, rimuovere con attenzione il surnatante con punta fine della pipetta e scartare. Far asciugare il pellet per 15 minuti unat temperatura ambiente. Non asciugare per più di 15 min. </li><li> Risospendere il pellet in 300 microlitri TE (10 mM Tris-Cl, 1 mM di acido etilendiammina tetra-acetico, pH 8,0). Incubare per una notte a 4 ° C. Delicatamente flick per sospendere di nuovo il materiale residuo che non è andato in soluzione. </li></ol></li><li> Per eliminare la contaminazione di RNA, aggiungere 10 ml RNasi A (10 mg / mL). Delicatamente capovolgere per mescolare tre volte. Centrifugare a 1000 xg per 3 s. Incubare a 37 ° C per 2 h. </li><li> DNA Extraction II <ol><li> Aggiungere 300 microlitri fenolo: cloroformio: alcool isoamilico (25: 24: 1 v / v) e capovolgere delicatamente per miscelare cinque volte. Incubare 10 minuti a temperatura ambiente, delicatamente invertendo mescolare cinque volte al giro di boa e di nuovo alla fine dell&#39;incubazione. Centrifugare 15 min a temperatura ambiente a 14.000 x g. Usando un puntale ampio tunnel, trasferire il surnatante in nuova 1,5 ml provetta. </li></ol></li><li> DNA Precipitazioni II <ol><li> Aggiungere 0,01 volume di 3 M acetato di sodio pH 5,2 (approximately 3 ml), capovolgere delicatamente per miscelare cinque volte. Aggiungere 2,5 volumi pre-raffreddata (-20 ° C) 100% di etanolo (circa 750 mL). Delicatamente capovolgere per mescolare cinque volte. Incubare per una notte a -20 ° C. </li><li> Centrifugare 30 min a 4 ° C a 14.000 x g. rimuovere con attenzione il surnatante e gettarlo. </li><li> Aggiungere 450 microlitri pre-raffreddata (-20 ° C) 70% di etanolo. Centrifugare per 5 minuti a 4 ° C a 14.000 x g. rimuovere con attenzione il surnatante e gettarlo. Centrifugare a 1000 xg per 3 s. rimuovere con attenzione il surnatante con punta fine pipetta e degli scarti. </li><li> Far asciugare il pellet per 30-60 minuti a temperatura ambiente. Risospendere il pellet in 27,5 microlitri TE. Incubare per una notte a 4 ° C. Delicatamente flick per sospendere di nuovo il materiale residuo che non è andato in soluzione. </li><li> Aliquotare 2,5 ml in 22,5 microlitri TE (volume finale 25 ml) per prove di controllo di qualità (vedi sotto). campioni di DNA Conservare a 4 ° C fino alla presentazione per il sequenziamento. Per lo stoccaggio, conservare i campioni a lungo terminea -80 ° C e ridurre al minimo i cicli di gelo-disgelo per evitare taglio. NOTA: Fare attenzione durante la dispensazione in questa fase, come l&#39;elevato peso molecolare DNA genomico possono essere difficili da pipettare correttamente, ed è importante per garantire che &quot;QC aliquota&quot; è una rappresentazione accurata del campione di DNA genomico. </li></ol></li></ol> 3. Controllo Qualità <ol><li> Purezza del campione <ol><li> Utilizzando un TE vuoto, valutare la purezza del campione di DNA caricando 1 ml di QC Aliquotare su uno spettrofotometro piccolo volume. Record A260 / A280 e A260 / A230 letture. DNA puro avrà un rapporto A260 / A280 del rapporto di circa 1,8 e A260 / A230 tra 1,8 e 2,2. </li></ol></li><li> La quantificazione del DNA genomico <ol><li> Eseguire la quantificazione di QC aliquota utilizzando un kit di analisi quantificazione del DNA a doppio filamento commerciale secondo le istruzioni del produttore. </li></ol></li><li> DNA valutazione della qualità I <ol><li> Carico di circa 60 ng di DNA genomico eladder DNA su un gel di agarosio 0,7% in TAE 1x (40 mM Tris, 20 mM acido acetico, e 1 mM EDTA, pH 8,0). Apparecchiatura gel funzionare a 70 V per 40 - 45 min fino bande di DNA genomico sono circa 2 cm dal pozzo e banda separazione nella scala è evidente. Queste condizioni possono essere necessario regolare basato su apparato gel elettroforesi disponibili. </li></ol></li><li> DNA valutazione della qualità II <ol><li> Carico di circa 60 ng di DNA genomico e molecolare elevato peso DNA ladder (adatto per pulsato-field gel electrophoresis) su un gel di agarosio 1% in 0,5x TBE (44.5 mM Tris, 44,5 mM di acido borico, 1 mM EDTA, pH 8,3) e eseguire in 0.5x TBE tampone di corsa con un 5 - campo pulsato protocollo elettroforesi su gel 80 kbp forma d&#39;onda per 16 h. </li></ol></li><li> La valutazione di batteri, funghi e contaminanti vegetali <ol><li> Eseguire la reazione a catena della polimerasi (PCR) per amplificare le opportune distanziatori interni trascritti (ITS) regioni dei geni ribosomali utilizzando primer e condizioni di amplificazione listacato nella tabella 2. Carico 10 microlitri della reazione risultante su un gel. NOTA: Una band che rappresenta l&#39;oidio regione ITS deve essere amplificato con il primer fungine ITS. DNA da questo amplicone e ogni amplicone generata con i primer o vegetali batteriche dovrebbe essere sequenziato per determinare l&#39;origine della contaminazione. Solo oidio sequenze ITS dovrebbe essere trovato nel amplicone fungine. </li></ol></li></ol>

<ol>
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The authors have nothing to disclose.

Per ottenere puro, alto peso molecolare DNA genomico dal obbligato biotrofico funghi oidio, una versione modificata di metodi precedentemente descritti è stato sviluppato 30. La resa media usando questo protocollo ottimizzato è 7 ug di DNA a 150 mg conidi, un raddoppio della resa ottenuta con un protocollo precedente. Inoltre, la dimensione media è aumentata da circa 20 a oltre 48,5 kbp kbp. Questo protocollo è stato ottimizzato nel cucumber- sistema cichoracearum G.. Per ottenere la...

<html> Mal bianco sono un gruppo di piante obbligato fungina biotrofico agenti patogeni che, nel loro insieme, sono la principale causa di malattie delle piante in tutto il mondo 1. Ci sono oltre 900 specie descritte di oidio, che sono stati tassonomico raggruppati in cinque tribù all&#39;interno della famiglia Erisyphaceae 2. Sia per la loro importanza economica e l&#39;intimo rapporto si sviluppano con i loro ospiti, malattie oidio sono stati oggetto di ricerca per&gt; 100 anni. Al momento l&#39;infezione, mal bianco suscitano drastici cambiamenti nella struttura cellulare, il metabolismo e la biologia molecolare dei loro ospiti, a beneficio di questo patogeno. Tuttavia, lo studio di mal bianco è particolarmente difficile a causa della loro vita obbligato, e crescita del fungo in coltura pura non è ancora stato descritto 3, 4, 5,&quot;xref&quot;&gt; 6, 7, 8. Affidabile, stabile trasformazione genetica di mal bianco ha inoltre non è ancora stata compiuta, anche se la trasformazione transitoria è stata riportata in alcune specie 9, 10. Il sequenziamento e l&#39;assemblaggio di polverulenti genomi muffa ha dimostrato difficile a causa di una serie di caratteristiche del genoma stesso. Genomi oidio sono grandi (120 - 180 Mbp) rispetto ad altri genomi fungini, e consistono 60 - 90% elementi ripetitivi uniformemente distribuiti 11. Questi elementi includono ripetizioni terminali non-lungo, così come gli elementi ripetitivi non categorizzati. Due speciales formae di una singola specie oidio, Blumeria graminis f. sp. hordei e f. sp. tritici (BGH e Bgt, rispettivamente), così come l&#39;uva oidio Erysiphe necator, been sequenziato, e un progetto di genomi per molti altri sono stati completati 12, 13, 14. La ripetitività dei genomi ha reso difficile il montaggio, e il genoma BGH pronto è stato assemblato in 6.989 supercontigs con L50 di 2 Mb 12. Nonostante il grande genoma, i mal bianco sembrano avere un piccolo numero di geni che codificano proteine, con 5.845 e 6.540 geni predetti in BGH e Bgt, rispettivamente. I oidi sequenziate appaiono anche mancare almeno 99 geni essenziali che sono essenziali in altri funghi, che è coerente con la dipendenza dei funghi sulla loro pianta ospite per la sopravvivenza 11, 12, 13, 14. sequenze ripetitive vicino telomeri, centromeri, RN ribosomialeUn array e regioni arricchite in elementi trasponibili gene sono scarsamente assemblati dalle strategie di sequenziamento short-letto e sono spesso sottorappresentati negli assiemi genoma 15. Tali regioni sono ritenuti responsabili di molte delle lacune che si verificano in sequenze genomiche, e questo vale per i mal bianco con la loro vasta espansione elementi ripetitivi 16. Altamente regioni del genoma di plastica si trovano spesso in tali regioni ripetitive 3. Essi servono come sito di riarrangiamenti e spesso codificano geni sotto forte pressione selettiva, come i geni che codificano proteine ​​effettrici e geni codificanti enzimi del metabolismo secondario. I progressi nelle tecnologie di sequenziamento di lunga lettura singola molecola di fornire una possibile soluzione per il sequenziamento tra le regioni ripetitive di genomi 15. Ad esempio, Faino et al. (2015) hanno trovato che l&#39;inclusione di lunga sequenza leggi le tecnologie e m otticoANALITICI permesso loro di produrre una sequenza genomica gap-meno due ceppi della pianta fungina patogeni Verticillium dalia, triplicando la proporzione di sequenze di DNA ripetute nel genoma, aumentando il numero di annotazioni geniche (e riducendo il numero di parziale o mancanti annotazioni geniche ) e genoma rivelatrice riarrangiamenti 17. Impiegare queste tecnologie di sequenziamento lunghe lettura, elevate concentrazioni di DNA genomico di alta qualità, con minime dimensioni&gt; 20 kbp, sono necessari. Qui si descrivono i nostri metodi per la raccolta dei conidi, purificazione del DNA ad alto peso molecolare da conidi e le nostre valutazioni controllo qualità utilizzando le specie oidio Golovinomyces cichoracearum cresciuti su cetriolo 18. Questo protocollo si basa su un protocollo sviluppato nel gruppo laboratorio B. Keller (Università di Zurigo, Zurigo Svizzera) 13, 19e include diverse modifiche che hanno portato a un incremento delle rese di DNA e una maggiore percentuale di DNA&gt; 48,5 kbp dimensioni. Il protocollo include anche misure di controllo di qualità sulla base delle raccomandazioni da parte del Dipartimento dell&#39;energia degli Stati Uniti Joint Genome Institute 20, 21, 22. La funzione di ciascun reagente inclusa nel tampone di lisi, e la logica per ogni fase di purificazione, sono come descritto in Henry (2008) 23. Protocolli pubblicati disponibili per l&#39;isolamento di alto peso molecolare DNA genomico da tessuti vegetali e microbici sono stati consultati anche durante la progettazione di questo protocollo 24, 25, 26, 27, 28.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MM400 Ball Mill</td>
			<td>Retsch</td>
			<td>20.745.0001</td>
			<td>Or equivalent ball mill</td>
		</tr>
		<tr>
			<td>2mL tubes for ball milling</td>
			<td>Sarstedt&#160;</td>
			<td>72.694.007</td>
			<td></td>
		</tr>
		<tr>
			<td>5/32&#34; 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&#176;C&#160;</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&#176;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&#160;</td>
			<td>38-022621807</td>
			<td>Or equivalent microcentrifuge</td>
		</tr>
		<tr>
			<td>Chlorform:IAA (24:1 v/v)</td>
			<td>Sigma-Aldrich&#160;</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 (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&#160;</td>
			<td>W292907</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>34923</td>
			<td>Chill to -20&#176;C prior use</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase, Dnase-free (10mg/ml)</td>
			<td>Thermo Fisher Scientific</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium metabisulfite</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P2522</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Lauryl Sacroinate</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>L9150</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>10708976001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>EDS</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Cetyltrimethyl ammonium bromide (CTAB)</td>
			<td>Sigma-Aldrich&#160;</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 10mg/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>&#160;Agarose Ultrapure Bioreagent</td>
			<td>VWR International LLC</td>
			<td>JT4063</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneRuler 1kb Plus DNA Ladder</td>
			<td>Thermo Fisher Scientific</td>
			<td>FERSM1332</td>
			<td></td>
		</tr>
		<tr>
			<td>8-48kb CHEF DNA size standards</td>
			<td>Bio-Rad</td>
			<td>170-3707</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippin Prep&#160;&#160;</td>
			<td>Life Technologies Corporation</td>
			<td>4472172</td>
			<td>Or equivalent pulse-field gel aparatus</td>
		</tr>
		<tr>
			<td>Whatman&#160;qualitative filter paper, Grade 1</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>WHA1001042</td>
			<td>Or equivalent growth chamber</td>
		</tr>
		<tr>
			<td>Percival&#160; 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&#160;</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 &#176;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

I funghi oidio sono un gruppo di economicamente importanti patogeni delle piante fungine. Relativamente poco si sa sulla biologia molecolare e genetica di questi agenti patogeni, in parte a causa della mancanza di risorse genetiche e genomiche ben sviluppati. Questi organismi hanno grandi, genomi ripetitivi, che hanno reso il sequenziamento del genoma e l&#39;assemblaggio proibitivo difficile. Qui, descriviamo i metodi per la valutazione raccolta, l&#39;estrazione, la purificazione e controllo di qualità di alto peso molecolare DNA genomico da specie muffa uno polverulente, Golovinomyces cichoracearum. Il protocollo descritto comprende distruzione meccanica delle spore seguiti da un ottimizzato fenolo / cloroformio estrazione di DNA genomico. Una resa tipica era 7 ug di DNA a 150 mg conidi. Il DNA genomico che viene isolato utilizzando questa procedura è adatto per il sequenziamento lungo lettura (cioè,&gt; 48,5 kbp). misure di controllo qualità per garantire la dimensione, la resa e la purezza del DNA genomico sono anchedescritto in questo metodo. Sequenziamento del DNA genomico della qualità descritta qui consentirà per l&#39;assemblaggio e il confronto dei genomi multipli oidio, che a sua volta portare ad una migliore comprensione e un migliore controllo di questo patogeno agricolo.

Un esempio rappresentativo di 60ng purificato DNA genomico da G. cichoracearum eseguito su un gel di agarosio usando elettroforesi su gel e utilizzando campo pulsato elettroforesi su gel sono mostrati nelle Figure 1 e 2 rispettivamente,. preparazioni di DNA genomico che passano, marginalmente accettazione e di rifiuto controllo qualità sono rappresentati. preparazioni di DNA genomico che passano completamente il controllo della...

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.

Purificazione di alto peso molecolare genomico da oidio per lungo-Read Sequencing

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 Views.  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

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.

Metil-binding cattura sequenziamento del DNA per tessuti del paziente

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

Ricerca

Genetica

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.

Ottimizzazione e analisi comparativa dei metodi di arricchimento del DNA organologico vegetale adatti per la sequenziazione di nuova generazione

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

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

Generazione di immunoprecipitazione della cromatina nativo sequenziamento librerie per analisi di densità del nucleosoma

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

G2-seq: Un'elevato Throughput sequenziamento-base tecnica per identificare replica ritardata regioni del genoma

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

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

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

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

Isolamento del DNA robusto e costruzione della libreria High-throughput sequenziamento per campioni di erbario

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Rilevamento di eventi rari utilizzando la sequenza di RNA e DNA di correzione d'errore

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

Isolare, sequenziamento e analisi dei microRNA extracellulari dalle cellule staminali mesenchimali umane

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

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

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

Sequenziamento dell'mRNA da sangue intero utilizzando il sequenziamento di NanoPore

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

Identification of Circular RNAs using RNA Sequencing

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein interactions, and regulation of parental gene transcription. In classical next generation RNA sequencing (RNA-seq), circRNAs are typically overlooked as a result of poly-A selection during construction of mRNA libraries, or are found at very low abundance, and are therefore difficult to isolate and detect. Here, a circRNA library construction protocol was optimized by comparing library preparation kits, pre-treatment options and various total RNA input amounts. Two commercially available whole transcriptome library preparation kits, with and without RNase R pre-treatment, and using variable amounts of total RNA input (1 to 4 &#181;g), were tested. Lastly, multiple tissue types; including liver, lung, lymph node, and pancreas; as well as multiple brain regions; including the cerebellum, inferior parietal lobe, middle temporal gyrus, occipital cortex, and superior frontal gyrus; were compared to evaluate circRNA abundance across tissue types. Analysis of the generated RNA-seq data using six different circRNA detection tools (find_circ, CIRI, Mapsplice, KNIFE, DCC, and CIRCexplorer) revealed that a stranded total RNA library preparation kit with RNase R pre-treatment and 4 &#181;g RNA input is the optimal method for identifying the highest relative number of circRNAs. Consistent with previous findings, the highest enrichment of circRNAs was observed in brain tissues compared to other tissue types.

Circular RNAs (circRNAs) are non-coding RNAs that may have roles in transcriptional regulation and mediating interactions between proteins. Following assessment of different parameters for construction of circRNA sequencing libraries, a protocol was compiled utilizing stranded total RNA library preparation with RNase R pre-treatment and is presented here.

Identificazione di RNA circolari mediante il sequenziamento dell'RNA

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

Generazione di modifiche genomiche definite utilizzando CRISPR-CAS9 nelle cellule staminali pluripotenti umane

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...