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>
	<li>Agrios, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Pathology. , (1969).</li><li>Braun, U., Cook, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Taxonomic manual of the Erysiphales (Powdery Mildews). , (2012).</li><li>Both, M., Csukai, M., Stumpf, M. P. H., Spanu, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+of+Blumeria+graminis+indicate+dynamic+changes+to+primary+metabolism+during+development+of+an+obligate+biotrophic+pathogen.">Gene expression profiles of Blumeria graminis indicate dynamic changes to primary metabolism during development of an obligate biotrophic pathogen.</a> Plant Cell. 17 (7), 2107-2122 (2005).</li><li>Glawe, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+powdery+mildews:+A+review+of+the+world's+most+familiar+(yet+poorly+known)+plant+pathogens.">The powdery mildews: A review of the world's most familiar (yet poorly known) plant pathogens.</a> Annu Rev Phytopathol. 46, 27-51 (2008).</li><li>Fabro, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+expression+profiling+Arabidopsis+at+the+stage+of+Golovinomyces+cichoracearum+haustorium+formation.">Genome-wide expression profiling Arabidopsis at the stage of Golovinomyces cichoracearum haustorium formation.</a> Plant Physiol. 146 (3), 1421-1439 (2008).</li><li>H&#252;ckelhoven, R., Panstruga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology+of+the+plant-powdery+mildew+interaction.">Cell biology of the plant-powdery mildew interaction.</a> Curr Opin Plant Biol. 14 (6), 738-746 (2011).</li><li>Micali, C. O., Neumann, U., Grunewald, D., Panstruga, R., O'Connell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+of+a+specialized+plant-fungal+interface+during+host+cell+internalization+of+Golovinomyces+orontii+haustoria.">Biogenesis of a specialized plant-fungal interface during host cell internalization of Golovinomyces orontii haustoria.</a> Cell Microbiol. 13 (2), 210-226 (2011).</li><li>Chandran, D., Inada, N., Hather, G., Kleindt, C. K., Wildermuth, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microdissection+of+Arabidopsis+cells+at+the+powdery+mildew+infection+site+reveals+site-specific+processes+and+regulators.">Laser microdissection of Arabidopsis cells at the powdery mildew infection site reveals site-specific processes and regulators.</a> Proc Natl Acad Sci. 107 (1), 460-465 (2010).</li><li>Spanu, P. D., Panstruga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Powdery+mildew+genomes+in+the+crosshairs.">Powdery mildew genomes in the crosshairs.</a> New Phytol. 195 (1), 20-22 (2012).</li><li>Vela-Corc&#237;a, D., Romero, D., Tor&#233;s, J. A., De Vicente, A., P&#233;rez-Garc&#237;a, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+transformation+of+Podosphaera+xanthii+by+electroporation+of+conidia.">Transient transformation of Podosphaera xanthii by electroporation of conidia.</a> BMC Microbiol. 15 (20), (2015).</li><li>Hacquard, S., Francis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in Botanical Research. 70, 109-142 (2014).</li><li>Spanu, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+expansion+and+gene+loss+in+powdery+mildew+fungi+reveal+tradeoffs+in+extreme+parasitism.">Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism.</a> Science. 330 (6010), 1543-1546 (2010).</li><li>Wicker, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+wheat+powdery+mildew+genome+shows+the+unique+evolution+of+an+obligate+biotroph.">The wheat powdery mildew genome shows the unique evolution of an obligate biotroph.</a> Nat Genet. 45 (9), 1092 (2013).</li><li>Jones, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+genomic+structural+variation+in+the+grape+powdery+mildew+pathogen,+Erysiphe+necator.">Adaptive genomic structural variation in the grape powdery mildew pathogen, Erysiphe necator.</a> BMC Genomics. 15, 1081 (2014).</li><li>Thomma, B. P. H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mind+the+gap;+seven+reasons+to+close+fragmented+genome+assemblies.">Mind the gap; seven reasons to close fragmented genome assemblies.</a> Fungal Genet Biol. 90, 24-30 (2016).</li><li>Parlange, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+major+invasion+of+transposable+elements+accounts+for+the+large+size+of+the+Blumeria+graminis+f.sp.+tritici+genome.">A major invasion of transposable elements accounts for the large size of the Blumeria graminis f.sp. tritici genome.</a> Funct Integr Genomics. 11, 671-677 (2011).</li><li>Faino, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+real-time+sequencing+combined+with+optical+mapping+yields+completely+finished+fungal+genome.">Single-molecule real-time sequencing combined with optical mapping yields completely finished fungal genome.</a> mBio. 6, (2015).</li><li>Adam, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Erysiphe+cichoracearum+and+E.+cruciferarum+and+a+survey+of+360+Arabidopsis+thaliana+accessions+for+resistance+to+these+two+powdery+mildew+pathogens.">Comparison of Erysiphe cichoracearum and E. cruciferarum and a survey of 360 Arabidopsis thaliana accessions for resistance to these two powdery mildew pathogens.</a> Mol Plant Microbe In. 12 (12), 1031-1043 (1999).</li><li>Bourras, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+avirulence+loci+and+allele-specific+effector+recognition+control+the+PM3+race-specific+resistance+of+wheat+to+powdery+mildew.">Multiple avirulence loci and allele-specific effector recognition control the PM3 race-specific resistance of wheat to powdery mildew.</a> Plant Cell. 27 (10), 2991-3012 (2015).</li><li>Henry, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant genotyping II: SNP technology. , (2008).</li><li>Healey, A., Furtado, A., Cooper, T., Henry, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol:+a+simple+method+for+extracting+next-generation+sequencing+quality+genomic+DNA+from+recalcitrant+plant+species.">Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species.</a> Plant Methods. 10 (21), (2014).</li><li>Fang, G., Hammar, S., Grumet, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quick+and+inexpensive+method+for+removing+polysaccharides+from+plant+genomic+DNA.">A quick and inexpensive method for removing polysaccharides from plant genomic DNA.</a> Biotechniques. 13 (1), 52-54 (1992).</li><li>Jordon-Thaden, I. E., Chanderbali, A. S., Gitzendanner, M. A., Soltis, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+CTAB+and+TRIzol+protocols+improve+RNA+extraction+from+chemically+complex+Embryophyta.">Modified CTAB and TRIzol protocols improve RNA extraction from chemically complex Embryophyta.</a> Appl Plant Sci. 3 (5), 1400105 (2015).</li><li>Murray, M. G., Thompson, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+high+molecular+weight+plant+DNA.">Rapid isolation of high molecular weight plant DNA.</a> Nucleic Acids Res. 8 (19), 4321-4325 (1980).</li><li>Greco, M., S&#225;ez, C. A., Brown, M. T., Bitonti, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+effective+method+for+high+quality+co-extraction+of+genomic+DNA+and+total+RNA+from+low+biomass+Ectocarpus+siliculosus,+the+model+brown+alga.">A simple and effective method for high quality co-extraction of genomic DNA and total RNA from low biomass Ectocarpus siliculosus, the model brown alga.</a> PLoS One. 9 (5), e96470 (2014).</li><li>Wilson, I. W., Schiff, C. L., Hughes, D. E., Somerville, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+trait+loci+analysis+of+powdery+mildew+disease+resistance+in+the+Arabidopsis+thaliana+accession+Kashmir-1.">Quantitative trait loci analysis of powdery mildew disease resistance in the Arabidopsis thaliana accession Kashmir-1.</a> Genetics. 158 (3), 1301-1309 (2001).</li></ol>

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 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

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

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.

Sequenciamento de captura de DNA de ligação de metilo em tecidos de pacientes

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

Genética

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 ...

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.

Geração de imunoprecipitação da cromatina nativa sequenciamento de bibliotecas para análise da densidade nucleossoma

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: Uma alta taxa de transferência baseada no sequenciamento técnica para identificar a tarde replicando regiões do 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 do DNA robusto e construção de biblioteca de sequenciamento de alto rendimento para espécimes de herbário

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.

Deteção de evento raro usando o DNA de correção de erro e a sequenciação do ARN

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.

Isolando, sequenciamento e análise MicroRNAs extracelular das células-tronco mesenquimais humanas

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.

Sequenciamento de mRNA de sangue total usando sequenciamento de nanoporos

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.

Identificação de RNAs circulares usando sequenciamento de 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.

Geração de modificações genómicas definidas usando CRISPR-CAS9 em células-tronco pluripotentes humanas

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