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.

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>

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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><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

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

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 ...

Ricerca

Genetica

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.

Sequenziamento del DNA nanoporo per analisi Metagenomica del suolo

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 ...

Ambiente

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.

Scoperta di romanzo sequenza genomica sottrattiva

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.

Analisi a livello di genoma della distribuzione delle modificazioni isoniche utilizzando il metodo di sequenziamento dell'immunoprecipitazione della cromatina in Magnaporthe oryzae

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.

Estrazione di DNA ad alta peso molecolare genomico da terreni e sedimenti

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

Biologia

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.

Una piattaforma di Microfluidic ad altissima velocità di trasmissione per il sequenziamento del genoma di cella singola

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

Bioingegneria

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.

Sequenziamento di lettura ultra-lungo per analisi di DNA Genomic intero

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.

Estrazione di DNA ad alta peso molecolare da Mats microbica

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.

Purificare l&#39;Impure: sequenziamento metagenomi e Metatranscriptomes da campioni complessi animali associate

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.

Purificazione genoma a livello di extrachromosomal circolare DNA dalle cellule eucariotiche

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