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. Preparação do material Fúngica <ol><li> Crescer Oídio <ol><li> Cultivar plantas em câmaras de crescimento com as seguintes condições: 22 ° C dia temperatura, 20 ° C Temperatura de noite, 80% de humidade relativa, 14 h duração do dia e uma intensidade de luz de 125 μE / m 2 / s (fornecida pela iluminação fluorescente ). </li><li> Infectar plantas de pepino (Cucumis sativa variedade de Bush Champion) com Golovinomyces cichoracearum UCSC1 estirpe como descrito 18, 29. </li></ol></li><li> Colheita Oídio conídios <ol><li> Colheita conídios de plantas infectadas em 7 - 10 dias após a inoculação com um pequeno vácuo com um filtro em linha (11? M) na qual os conídios acumular. Aplicando uma pressão suave, executar a tubeira de vácuo ao longo da superfície da folha de recolher os conídios. NOTA: O rendimento médio de G. cichoracearum é de 20 mg de conídios de um fortemente INFECTED folha de pepino de cerca de 130 cm2 de área. </li><li> Escova de cerca de 150 mg do conídios (aproximadamente uL de volume 375, ou conídios de 7-8 folhas de pepino infectadas) a partir do filtro sobre uma folha de papel limpa. A partir daqui, transferir conídios em 2 mL criotubos com diâmetro dois 5/32 polegadas pré-arrefecida (em azoto líquido) bolas de moagem aço. voltar imediatamente para tubos de azoto líquido. tubos de armazenar a -80 ° C. </li></ol></li><li> Breaking Open conídios por moagem <ol><li> Flash congelar placas de alumínio de moinho de esferas em azoto líquido. Carga criotubos em rack de moinho de bolas. Agitar vigorosamente os criotubos com bolas de aço e conídios em moinho de bolas durante 30 s a 30 ciclos / s (à temperatura ambiente). recongelar imediatamente criotubos em azoto líquido. NOTA: Inicialmente, acesso a uma amostra de conídios por microscopia uma ampliação de 100X para a eficiência de ruptura da parede celular durante a moagem. Se necessário, para moer um adicional de 1 - 2 ciclos de 30 s durante 30 ciclos / s, but manter o número de rodadas a um mínimo. A avaliação de rotina de quebra de conídios por microscopia nesta fase é desnecessária uma vez que foram estabelecidas condições ideais. </li></ol></li></ol> 2. Purificao de ADN genómico <ol><li> conídios de lise <ol><li> Trabalhar rapidamente para evitar o descongelamento conídios, remover as bolas de aço de criotubos com um ímã. Adicionar 700 uL de 65 ° C, tampão de lise (Tabela 1) e vórtice 5 - 10 s até uma suspensão é formada. </li><li> Adicionar 300? L pré-aquecido (65 ° C) 5% (v / v) de sarcosil e inverter cuidadosamente para misturar (uma inversão por 3 s) cinco vezes. Incubar os tubos durante 30 min a 65 ° C; invertido suavemente 3 vezes em 10, 20 e 30 min. Utilizando uma ponta de pipeta de amplo calibre, transferir a solução inteira para novo tubo de microcentrifugação de 2 ml. Em todos os passos subsequentes, misturar por inversão suave, lento e transferir as soluções contendo ADN com pontas de pipeta de grande calibre. </li></ol></li><li> Extração de DNA I <ol><li> Adicionar um volume de cloroforma: álcool isoamílico (24: 1 v / v) para a soluo de lise e inverter cuidadosamente para misturar cinco vezes. Incubar durante 10 minutos à temperatura ambiente, invertendo suavemente para misturar cinco vezes no ponto a meio caminho e novamente no final da incubação. Centrifuga-se a temperatura ambiente durante 15 min a 14.000 x g. Usando ampla-furo ponta de pipeta, transferir cuidadosamente a fase aquosa para um novo tubo de microcentrifugação de 2 ml; evitar a inclusão do material da interface. </li></ol></li><li> DNA Precipitation I <ol><li> Adicionar um volume de temperatura ambiente a 100% de isopropanol (cerca de 750 mL) e inverter cuidadosamente para misturar seis vezes. Centrifuga-se a temperatura ambiente durante 15 min a 14.000 x g. Remova cuidadosamente o sobrenadante e descartar. </li><li> Adicionar 450 mL de pré-refrigerada% de etanol (-20 ° C) 70. Centrifugar durante 5 min à temperatura ambiente a 14000 x g. Remova cuidadosamente o sobrenadante e descartar. Centrifugar a 1.000 xg durante 3 s, remover cuidadosamente o sobrenadante com uma pipeta de ponta fina e descartar. Secar o sedimento durante 15 minutos, umatemperatura ambiente t. Não secar por mais de 15 min. </li><li> Ressuspender o sedimento em 300 ul de TE (10 mM Tris-Cl, ácido etilenodiaminotetra-acético 1 mM, pH 8,0). Incubar durante a noite a 4 ° C. Agite suavemente para voltar a suspender qualquer material residual que não entrar em solução. </li></ol></li><li> Para eliminar a contaminação de ARN, adicionam-se 10 ul de ARNase A (10 mg / mL). Inverta suavemente para misturar três vezes. Centrifugar a 1.000 xg durante 3 s. Incubar a 37 ° C durante 2 h. </li><li> Extracção de DNA II <ol><li> Adicionar 300 ul de fenol: clorofórmio: álcool isoamílico (25: 24: 1 v / v) e inverter cuidadosamente para misturar cinco vezes. Incubar 10 min a temperatura ambiente, invertendo suavemente para misturar cinco vezes a meio caminho e novamente no final da incubação. Centrifugar 15 min a temperatura ambiente a 14000 x g. Utilizando uma ponta de pipeta de amplo calibre, transferir o sobrenadante para um novo tubo de microcentrifugação de 1,5 mL. </li></ol></li><li> ADN Precipitation II <ol><li> Adicionar 0,01 volume de 3 M de acetato de sódio pH 5,2 (approximately 3 mL), inverter cuidadosamente para misturar cinco vezes. Adicionar 2,5 volumes de pré-arrefecida (-20 ° C) de 100% de etanol (cerca de 750 mL). Inverta suavemente para misturar cinco vezes. Incubar durante a noite a -20 ° C. </li><li> Centrifugar 30 min a 4 ° C a 14000 x g. Remova cuidadosamente o sobrenadante e descartar. </li><li> Adicionar 450 mL de pré-refrigerada% de etanol (-20 ° C) 70. Centrifugar durante 5 minutos a 4 ° C a 14000 x g. Remova cuidadosamente o sobrenadante e descartar. Centrifugar a 1.000 xg durante 3 s. Remova cuidadosamente o sobrenadante com ponteira fina e descarte. </li><li> Secar o sedimento durante 30-60 min à temperatura ambiente. Ressuspender o pellet em 27,5 ul de TE. Incubar durante a noite a 4 ° C. Agite suavemente para voltar a suspender qualquer material residual que não entrar em solução. </li><li> Alíquota de 2,5 mL em 22,5 ul de TE (volume final 25 mL) para ensaios de controlo de qualidade (ver abaixo). As amostras de ADN armazenar a 4 ° C até à apresentação para a sequenciação. Para o armazenamento, armazenar amostras a longo prazoa -80 ° C e minimizar a ciclos de congelação-descongelação para evitar distorção. NOTA: Tome cuidado ao pipetar nesta etapa como o ADN genómico de elevado peso molecular podem ser difíceis de pipetar adequadamente, e é importante para assegurar que &quot;QC Alíquota&quot; é uma representação precisa da amostra de DNA genómico. </li></ol></li></ol> 3. Controle de Qualidade <ol><li> Pureza amostra <ol><li> Usando um TE em branco, avaliar a pureza da amostra de ADN por carregamento de 1 mL da alíquota QC em um espectrofotômetro de pequeno volume. Registro A260 / A280 e A260 / A230 leituras. ADN puro terá uma razão A260 / A280 de cerca de 1,8 e A260 / A230 razão entre 1,8 e 2,2. </li></ol></li><li> Quantificação do ADN genómico <ol><li> Realizar a quantificação da alíquota QC usando um kit comercial de cadeia dupla do ADN de ensaio de quantificação de acordo com as instruções do fabricante. </li></ol></li><li> DNA Quality Assessment I <ol><li> Carga de aproximadamente 60 ng de ADN genómico e umaescada de DNA em um gel de agarose a 0,7% em 1x TAE (Tris 40 mM, ácido acético 20 mM, e EDTA 1 mM, pH 8,0). aparelho de gel executar a 70 V durante 40 - 45 minutos até as bandas de ADN genómico são aproximadamente 2 cm da separação bem e banda na escada é aparente. Estas condições podem necessitar de ser ajustada com base no aparelho de electroforese em gel disponível. </li></ol></li><li> DNA Quality Assessment II <ol><li> Carga de aproximadamente 60 ng de ADN genómico e uma escada de DNA de peso molecular elevado (adequado para electroforese em gel de campo pulsado) sobre um gel de agarose a 1% em TBE 0,5x (Tris 44,5 mM, ido bico 44,5 mM, EDTA 1 mM, pH 8,3) e executado em tampão de corrida 0,5x TBE usando um 5 - de campo pulsado protocolo de electroforese em gel de 80 kpb da forma de onda durante 16 h. </li></ol></li><li> Avaliação de bactérias, fungos e Contaminantes de plantas <ol><li> Realizar a reacção em cadeia da polimerase (PCR) para amplificar os espaçadores internos transcritos (ITS) apropriados regiões dos genes ribossomais, utilizando os iniciadores e as condições de amplificação listaed na Tabela 2. Carga de 10 pL da reacção resultante num gel. NOTA: Uma banda que representava o oídio região ITS deve ser amplificado com o fúngica SEUS iniciadores. ADN a partir deste amplicão e qualquer amplic gerado com os iniciadores, ou de plantas bacterianas deve ser sequenciado para determinar a origem da contaminação. Apenas oídio sequências ITS deve ser encontrada no amplicon fúngica. </li></ol></li></ol>

<ol>
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The authors have nothing to disclose.

A fim de se obter alta ADN genómico puro, de peso molecular a partir do obrigatório biotrófica fungos de oídio, uma versão modificada dos métodos anteriormente descritos foi desenvolvido 30. O rendimento médio usando este protocolo optimizado é de 7 g de ADN por 150 mg de conídios, uma duplicação do rendimento obtido com um protocolo anterior. Além disso, o tamanho médio aumentou de cerca de 20 kpb a mais de 48,5 kpb. Este protocolo foi optimizado no sistema cichoracearum G....

<html> Oídios são um grupo de obrigatório planta fúngico biotrófica agentes patogénicos que, quando tomados em conjunto, são a maior causa de doenças de plantas em todo o mundo 1. Existem mais de 900 espécies descritas de oídio, que foram taxonomicamente agrupadas em cinco tribos dentro da família Erisyphaceae 2. Devido tanto à sua importância econômica e a íntima relação que desenvolvem com seus anfitriões, doenças de oídio foram objecto de investigação por&gt; 100 anos. Após a infecção, oídio provocar mudanças drásticas na estrutura celular, metabolismo e biologia molecular de seus hospedeiros, para beneficiar deste patógeno. No entanto, o estudo de oídios é particularmente difícil devido ao seu estilo de vida obrigatória, e o crescimento do fungo em cultura pura ainda não foi descrito 3, 4, 5,&quot;xref&quot;&gt; 6, 7, 8. Reliable transformação, estável genética de oídios ainda não foi também conseguida, embora a expressão transiente tem sido relatada em algumas espécies 9, 10. O sequenciamento e montagem de genomas oídio tem sido difícil devido a uma série de características do próprio genoma. Genomas oídio são grandes (120-180 MBP) em relação a outros genomas de fungos, e consistem em 60 - 90% de elementos repetitivos uniformemente distribuídas 11. Estes elementos incluem repeties terminais longas não, bem como elementos repetitivos não classificadas. Dois speciales formae de uma única espécie de oídio, Blumeria graminis f. sp. hordei e f. sp. tritici (BGH e Bgt, respectivamente), bem como o míldio pulverulento da uva Erysiphe necator, têm abelhan sequenciado, e os projectos de genomas de vários outros foram concluídos 12, 13, 14. A natureza repetitiva dos genomas fez montagem difícil, e o genoma Bgh completada foi montado em 6.989 supercontigs com um L50 de 2 Mb 12. Apesar do grande genoma, os míldios pulverulentos parecem ter um pequeno número de genes que codificam proteínas, com 5.845 e 6.540 genes previstos em Bgh e Bgt, respectivamente. Os oídios sequenciados também parecem carecer de pelo menos 99 genes principais que são essenciais em outros fungos, o que é consistente com a dependência dos fungos na sua planta hospedeira para a sobrevivência de 11, 12, 13, 14. seqüências repetitivas perto telômeros, centrômeros, RN ribossomalA matrizes e as regiões enriquecidas em elementos transponíveis de genes são mal montada a partir de estratégias de sequenciação de curto-ler e são frequentemente sub-representados em conjuntos de 15 genoma. Tais regiões são pensados para ser responsável por muitas das lacunas que ocorrem em sequências do genoma, e isto aplica-se aos míldios pulverulentos, com o seu grande expansão de elementos repetitivos 16. Altamente regiões do genoma de plástico são freqüentemente encontrados nessas regiões repetitivas 3. Eles servem como um local de rearranjos de cromossomas e muitas vezes codificam genes sob forte pressão selectiva, tais como os genes que codificam para proteínas efectoras e os genes que codificam as enzimas do metabolismo secundário. Advances in única molécula de tecnologias de sequenciação de leitura longa fornecer uma solução potencial para a sequenciação através das regiões repetitivas de genomas 15. Por exemplo, Faino et al. (2015) verificaram que a inclusão de sequência de leitura longa e tecnologias m ópticoapping lhes permitiu produzir uma sequência de genoma lacuna-menos para duas estirpes de planta fúngico patógeno dália Verticillium, triplicando a proporção de sequências repetitivas de ADN no genoma, aumentando o número de notas de genes (e a redução do número de parcial ou ausente anotações gene ) e revelador genoma rearranjos 17. Para empregar estas tecnologias de sequenciamento de leitura longa, altas concentrações de DNA genômico de alta qualidade, com o mínimo de tamanhos&gt; 20 kbp, são necessários. Descrevemos aqui os nossos métodos para a recolha de conídios, purificação de DNA de alto peso molecular a partir de conídios e nossas avaliações de controlo de qualidade utilizando as espécies de oídio Golovinomyces cichoracearum cultivadas em pepino 18. Este protocolo baseia-se num protocolo desenvolvido no laboratório grupo B. Keller (Universidade de Zurique, Zurique Suíça) 13, 19e inclui várias modificações que levaram ao aumento da produção de ADN e uma proporção mais elevada de ADN de&gt; 48,5 kpb em tamanho. O protocolo também inclui etapas de controle de qualidade com base nas recomendações do Departamento de Estados Unidos da Joint Genome Institute Energia 20, 21, 22. A função de cada um dos reagentes incluídos no tampão de lise, e a razão para cada passo de purificação, são como descrito em Henry (2008) 23. Disponíveis protocolos publicados para o isolamento de DNA de alto peso molecular genómico a partir de tecidos de plantas e microbianas foram também consultados durante a concepção deste protocolo 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

Os fungos de oídio são um grupo de economicamente importantes agentes patogénicos fúngicos de plantas. Relativamente pouco se sabe sobre a biologia molecular e genética de estes agentes patogénicos, em parte devido à falta de recursos genéticos e genómicos bem desenvolvidos. Estes organismos possuem grandes genomas, repetitivas, que fizeram a sequenciação e montagem do genoma proibitivamente difícil. Aqui, nós descrevemos métodos para a avaliação de recolha, a extracção, purificação e controle de qualidade de ADN genómico de elevado peso molecular a partir de espécies de míldio em pó, um Golovinomyces cichoracearum. O protocolo descrito inclui ruptura mecânica de esporos seguido por uma extracção de ADN genómico optimizado fenol / clorofórmio. Um rendimento típico foi de 7 g de ADN por 150 mg de conídios. O ADN genómico que é isolado utilizando este procedimento é adequado para a sequenciação de leitura longa (isto é,&gt; 48,5 kpb). medidas de controlo de qualidade para assegurar o tamanho, rendimento e a pureza do ADN genómico são igualmentedescrito neste método. A sequenciação do ADN genómico da qualidade descrito aqui vai permitir a montagem e a comparação dos múltiplos genomas de oídio, que por sua vez irá conduzir a uma melhor compreensão e uma melhor controle deste patogénio agrícola.

Um exemplo representativo de 60 ng de ADN genómico purificado de G. cichoracearum passado em gel de agarose, utilizando electroforese em gel e usando electroforese em gel de campo pulsado são mostrados nas Figuras 1 e 2, respectivamente. preparações de ADN genómico que passam, marginalmente passam e falham o controlo de qualidade são representados. preparações de ADN genómico que passam completamente o controlo de quali...

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.

Purificação de DNA de alto peso molecular genômico de oídio de longo 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 visualizações.  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.

Vídeo: 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.

Otimização e Análise Comparativa de Métodos de Enriquecimento de DNA de Plantas Organizadas Adequado para Seqüenciamento de Próxima Geração

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

Genética

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.

Sequenciamento de DNA Nanopore para análise de solo Metagenomic

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.

Sequência de romance descoberta pela genômica subtrativa

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.

Análise em todo o genoma da distribuição de modificações de histona usando o Método de Sequenciamento de Imunoprecipitação de Chromatina em 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.

Extração de DNA genômico de alto peso molecular de solos e sedimentos

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.

Uma plataforma Microfluidic de ultra alta taxa de transferência para o sequenciamento do genoma de célula única

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

Bioengenharia

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.

Sequenciamento de leitura ultralonga para análise de DNA genômico completo

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.

Extração de DNA de alto peso molecular de Esteiras Microbianas

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.

Purificando o Impure: Sequenciação metagenomes e metatranscriptomas do Complexo amostras associadas a animais

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.

A purificação de todo o genoma de DNA circular Extracromossômica de células eucarióticas

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