Name: optimization and comparative analysis of plant organellar dna enrichment methods suitable for next-generation sequencing
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Nos gustaría agradecer el financiamiento del Departamento de Agricultura de Estados Unidos - Servicio de Investigación Agrícola y de la National Science Foundation (IOS 1025881 e IOS 1361554). Damos las gracias a R. Caspers por el mantenimiento de invernaderos y el cuidado de las plantas. También damos las gracias al Centro de Genómica de la Universidad de Minnesota, donde se realizaron los preparativos de la biblioteca de Illumina y la secuenciación. También estamos agradecidos por los comentarios de los editores de la revista y cuatro revisores anónimos que fortalecieron aún más nuestro manuscrito. También agradecemos a la OCDE por una beca a SK para integrar estos protocolos para proyectos colaborativos con colegas en Japón.

1. Generación de materiales vegetales para el aislamiento orgánico y la extracción de ADN <ol><li> Crecimiento estándar de plántulas de trigo <ol><li> Plante las semillas en vermiculita en pequeñas ollas cuadradas con 4 a 6 semillas por esquina. Traslado a un invernadero o cámara de crecimiento con un ciclo de luz de 16 h, 23 ºC día / 18 ºC noche. </li><li> Regar las plantas cada día. Fertilice las plantas con ¼ de cucharadita de fertilizante granular 20-20-20 NPK durante la germinación y...

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	<li>Liberatore, K. L., Dukowic-Schulze, S., Miller, M. E., Chen, C., Kianian, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mitochondria+in+plant+development+and+stress+tolerance.">The role of mitochondria in plant development and stress tolerance.</a> Free Radic Biol Med. 100, 238-256 (2016).</li><li>Samaniego Castruita, J. A., Zepeda Mendoza, M. L., Barnett, R., Wales, N., Gilbert, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odintifier--A+computational+method+for+identifying+insertions+of+organellar+origin+from+modern+and+ancient+high-throughput+sequencing+data+based+on+haplotype+phasing.">Odintifier--A computational method for identifying insertions of organellar origin from modern and ancient high-throughput sequencing data based on haplotype phasing.</a> BMC Bioinformatics. 16 (232), 1-13 (2015).</li><li>Zhang, T., Zhang, X., Hu, S., Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+procedure+for+plant+organellar+genome+assembly,+based+on+whole+genome+data+from+the+454+GS+FLX+sequencing+platform.">An efficient procedure for plant organellar genome assembly, based on whole genome data from the 454 GS FLX sequencing platform.</a> Plant Methods. 7 (38), 1-8 (2011).</li><li>Wambugu, P. W., Brozynska, M., Furtado, A., Waters, D. L., Henry, R. 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J., Rice, D. W., Palmer, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturized+mitogenome+of+the+parasitic+plant+Viscum+scurruloideum+is+extremely+divergent+and+dynamic+and+has+lost+all+nad+genes.">Miniaturized mitogenome of the parasitic plant Viscum scurruloideum is extremely divergent and dynamic and has lost all nad genes.</a> Proc Natl Acad Sci U S A. 112 (27), E3515-E3524 (2015).</li><li>Wicke, S., Schneeweiss, G. M., H&#246;randl, E., Appelhans, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+1.">Chapter 1.</a> Next Generation Sequencing in Plant Systematics. , (2015).</li><li>Sloan, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+ring+to+rule+them+all?+Genome+sequencing+provides+new+insights+into+the+'master+circle'+model+of+plant+mitochondrial+DNA+structure.">One ring to rule them all? Genome sequencing provides new insights into the 'master circle' model of plant mitochondrial DNA structure.</a> New Phytol. 200 (4), 978-985 (2013).</li><li>Woloszynska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heteroplasmy+and+stoichiometric+complexity+of+plant+mitochondrial+genomes--though+this+be+madness,+yet+there's+method+in't.">Heteroplasmy and stoichiometric complexity of plant mitochondrial genomes--though this be madness, yet there's method in't.</a> J Exp Bot. 61 (3), 657-671 (2010).</li><li>Ahmed, Z., Fu, Y. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+isolation+of+chloroplast+DNA+and+mitochondrial+DNA+from+Sunflower.">A method for isolation of chloroplast DNA and mitochondrial DNA from Sunflower.</a> Plant Mol Biol Rep. 16 (2), 183-189 (1998).</li><li>Pinard, R., 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=Assessment+of+whole+genome+amplification-induced+bias+through+high-throughput,+massively+parallel+whole+genome+sequencing.">Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing.</a> BMC Genomics. 7 (216), 1-21 (2006).</li><li>Lamble, 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=Improved+workflows+for+high+throughput+library+preparation+using+the+transposome-based+Nextera+system.">Improved workflows for high throughput library preparation using the transposome-based Nextera system.</a> BMC Biotechnol. 13 (104), 1-10 (2013).</li><li>Raley, C., 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=Preparation+of+next-generation+DNA+sequencing+libraries+from+ultra-low+amounts+of+input+DNA:+Application+to+single-molecule,+real-time+(SMRT)+sequencing+on+the+Pacific+Biosciences+RS+II.">Preparation of next-generation DNA sequencing libraries from ultra-low amounts of input DNA: Application to single-molecule, real-time (SMRT) sequencing on the Pacific Biosciences RS II.</a> bioRxiv. , (2014).</li><li>Tsai, Y. C., 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=Resolving+the+Complexity+of+Human+Skin+Metagenomes+Using+Single-Molecule+Sequencing.">Resolving the Complexity of Human Skin Metagenomes Using Single-Molecule Sequencing.</a> MBio. 7 (1), e01948 (2016).</li><li>Feehery, G. R., 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+method+for+selectively+enriching+microbial+DNA+from+contaminating+vertebrate+host+DNA.">A method for selectively enriching microbial DNA from contaminating vertebrate host DNA.</a> PLoS One. 8 (10), e76096 (2013).</li><li>Yigit, E., Hernandez, D. I., Trujillo, J. T., Dimalanta, E., Bailey, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+and+metagenome+sequencing:+Using+the+human+methyl-binding+domain+to+partition+genomic+DNA+derived+from+plant+tissues.">Genome and metagenome sequencing: Using the human methyl-binding domain to partition genomic DNA derived from plant tissues.</a> Appl Plant Sci. 2 (11), 1-6 (2014).</li><li>Noyszewski, A. K., 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=Accelerated+evolution+of+the+mitochondrial+genome+in+an+alloplasmic+line+of+durum+wheat.">Accelerated evolution of the mitochondrial genome in an alloplasmic line of durum wheat.</a> BMC Genomics. 15 (67), 1-16 (2014).</li><li>. 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M., Lohse, M., Usadel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimmomatic:+a+flexible+trimmer+for+Illumina+sequence+data.">Trimmomatic: a flexible trimmer for Illumina sequence data.</a> Bioinformatics. 30 (15), 2114-2120 (2014).</li><li>Ogihara, Y., 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=Structural+dynamics+of+cereal+mitochondrial+genomes+as+revealed+by+complete+nucleotide+sequencing+of+the+wheat+mitochondrial+genome.">Structural dynamics of cereal mitochondrial genomes as revealed by complete nucleotide sequencing of the wheat mitochondrial genome.</a> Nucleic Acids Res. 33 (19), 6235-6250 (2005).</li><li>Ogihara, Y., 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=Structural+features+of+a+wheat+plastome+as+revealed+by+complete+sequencing+of+chloroplast+DNA.">Structural features of a wheat plastome as revealed by complete sequencing of chloroplast DNA.</a> Mol Genet Genomics. 266 (5), 740-746 (2002).</li><li>International Wheat Genome Sequencing Consortium (IWGSC). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chromosome-based+draft+sequence+of+the+hexaploid+bread+wheat+(Triticum+aestivum)+genome.">A chromosome-based draft sequence of the hexaploid bread wheat (Triticum aestivum) genome.</a> Science. 345 (6194), (2014).</li><li>Langmead, B., Salzberg, S. 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Hasta la fecha, la mayoría de los estudios de secuenciación organelar se centran en los métodos tradicionales de CC para enriquecer el ADN específico. Métodos para aislar orgánulos de diversas plantas se han descrito, incluyendo el musgo 40 ; Monocotiledóneas tales como trigo 15 y avena 11 ; Y dicotiledóneas tales como arabidopsis 11 , girasol 17 , y colza 14 . La mayor?...

<html> La secuenciación del genoma es una poderosa herramienta para disecar la base genética subyacente de rasgos importantes de las plantas. La mayoría de los estudios de secuenciación del genoma se centran en el contenido del genoma nuclear, ya que la mayoría de los genes se encuentran en el núcleo. Sin embargo, los genomas organelos, incluyendo las mitocondrias (a través de eucariotas) y plastidios (en las plantas, la forma especializada, el cloroplasto, trabaja en fotosíntesis) aportan información genética significativa esencial para el desarrollo de organismos, la respuesta al estrés y la aptitud general 1 . Genomas organelas se incluyen normalmente en el total de extracciones de ADN destinados a la secuenciación del genoma nuclear, aunque los métodos para reducir el número de organelos antes de la extracción de ADN también se emplean [ 2] . Muchos estudios han utilizado resultados de secuenciación de las extracciones totales de gDNA para ensamblar genomas organelos 3 , 4 , 5 ,Xref &quot;&gt; 6 , 7. Sin embargo, cuando el objetivo del estudio es centrarse en los genomas organelares, el uso del gDNA total incrementa los costos de secuenciación debido a que muchas lecturas están&quot; perdidas &quot;en las secuencias de ADN nuclear, particularmente en plantas con genomas nucleares grandes Además, debido a la duplicación y transferencia de secuencias organelares en el genoma nuclear y entre orgánulos, la resolución de la posición correcta de la cartografía de secuenciación lee al genoma adecuado es bioinformatically desafiante 2 , 8. La purificación de los genomas organelares del genoma nuclear es uno Estrategia para reducir estos problemas.Más estrategias bioinformáticas se pueden utilizar para separar las lecturas que mapa a las regiones de homología entre las mitocondrias y los cloroplastos. Mientras que los genomas organelares de muchas especies de plantas han sido secuenciados, poco se sabe acerca de la amplitud de la diversidad de genomas organelosDisponible en poblaciones silvestres o en piscinas de cría cultivadas. También se sabe que los genomas organelares son moléculas dinámicas que experimentan un reordenamiento estructural significativo debido a la recombinación entre secuencias repetidas 9 . Además, múltiples copias del genoma organelo están contenidas dentro de cada orgánulo, y múltiples organelos están contenidos dentro de cada célula. No todas las copias de estos genomas son idénticos, que se conoce como heteroplasmia. En contraste con el cuadro canónico de &quot;círculos maestros&quot;, ahora hay evidencia creciente de una imagen más compleja de las estructuras del genoma organelar, incluidos los círculos sub-genómica, cromosomas lineales, concatamers lineales y estructuras ramificadas [ 10] . El ensamblaje de los genomas organelares de las plantas se complica aún más por sus tamaños relativamente grandes y sus sustanciales repeticiones invertidas y directas. Los protocolos tradicionales para el aislamiento organelar, la purificación del ADN y el subsiguiente genoma<html> La secuenciación a menudo es engorrosa y requiere grandes volúmenes de entrada de tejido, con varios gramos a más de cientos de gramos de tejido de hoja joven necesarios como punto de partida 11 , 12 , 13 , 14 , 15 , 16 , 17 . Esto hace inaccesible la secuenciación del genoma organelo cuando el tejido es limitado. En algunas situaciones, las cantidades de semillas son limitadas, como cuando es necesario secuenciar sobre una base generacional o en líneas masculinas estériles que tienen que ser mantenidas por cruce. En estas situaciones, el ADN organelar puede purificarse y luego someterse a amplificación de todo el genoma. Sin embargo, la amplificación del genoma completo puede introducir sesgo de secuenciación significativo, que es un problema particular al evaluar la variación estructural, las estructuras sub-genómicas y los niveles de heteroplasmia&gt; 18. Los avances recientes en la preparación de bibliotecas para tecnologías de secuenciación de lectura corta han superado barreras de entrada baja para evitar la amplificación del genoma completo. Por ejemplo, el kit de preparación de biblioteca de Illumina Nextera XT permite que se utilice tan poco como 1 ng de ADN como entrada 19 . Sin embargo, las preparaciones de bibliotecas estándar para aplicaciones de secuenciación de larga lectura, como las tecnologías de secuenciación PacBio o Oxford Nanopore, todavía requieren una cantidad relativamente alta de ADN de entrada, lo que puede representar un reto para la secuenciación del genoma organelo. Recientemente, se han desarrollado nuevos protocolos de secuenciación de larga lectura para reducir las cantidades de entrada y ayudar a facilitar la secuenciación del genoma en muestras en las que es difícil obtener cantidades de microgramos de ADN 20 , 21 . Sin embargo, la obtención de fracciones orgánicas puras de alto peso molecular para alimentar estas preparaciones de biblioteca sigue siendo un desafío. BuscamosO comparar y optimizar los métodos de enriquecimiento y aislamiento de ADN organelar adecuados para NGS sin necesidad de amplificación de todo el genoma. Específicamente, nuestro objetivo era determinar las mejores prácticas para enriquecer el ADN organelar de alto peso molecular a partir de materiales de partida limitados, como una submuestra de una hoja. Este trabajo presenta un análisis comparativo de los métodos para enriquecer el ADN organelar: (1) un protocolo de centrifugación diferencial tradicional modificado versus (2) un protocolo de fraccionamiento de ADN basado en el uso de un ADN comercialmente disponible CpG-metil-binding domain protein pulldown approach 22 aplicado al tejido vegetal 23 . Recomendamos las mejores prácticas para el aislamiento de ADN organelar de tejido de hoja de trigo, que puede ser fácilmente extendido a otras plantas y tipos de tejidos.

<table border="0" cellpadding="0" cellspacing="0" width="685">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2-mercaptoethanol (beta-mercaptoethanol; BME)</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:119px;">M3148-100ml</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2-propanol (Isopropyl alcohol/isopropanol), bioreagent</td>
			<td>Sigma Aldrich</td>
			<td>I9516</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">agarose, Bio-Rad Cetified Megabase agarose</td>
			<td>Bio-Rad</td>
			<td>1613108</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">analytical balance</td>
			<td>Mettler Toledo</td>
			<td>AB54-S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">balance</td>
			<td>Mettler Toledo</td>
			<td>PB1502-S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bovine serum albumin (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>B4287-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ceramic grinding cylinders, 3/8in x 7/8in</td>
			<td>SPEX SamplePrep</td>
			<td>2183</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cryogenic Blocks compatible with tissue homogenizer for holding 50 ml tubes</td>
			<td>SPEX SamplePrep</td>
			<td>2664</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNaseI</td>
			<td>Sigma</td>
			<td>DN25</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ethanol, absolute</td>
			<td>Decon Laboratories</td>
			<td>2716</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethylenediamine Tetraacetic Acid (EDTA), 0.5M Solution, pH8.0</td>
			<td>Fisher</td>
			<td>BP2482-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gel imaging system</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">gel stain</td>
			<td></td>
			<td></td>
			<td style="width:167px;">Such as GelRed or Ethidium Bromide</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">grinding pestle, wide tip for 2 ml conical tubes</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Guanidine-HCl, 8M solution</td>
			<td>ThermoFisher</td>
			<td>24115</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LightCycler 480 SYBR Green I Master</td>
			<td>Roche</td>
			<td>4707516001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">liquid nitrogen</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lysing enzymes from Trichoderma harzianum</td>
			<td>Sigma</td>
			<td>L1412</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride</td>
			<td>G Bioscience</td>
			<td>24115</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">magnetic rack</td>
			<td>ThermoFisher</td>
			<td>A13346</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">microcentrifuge tubes, LoBind 1.5 ml&#160;</td>
			<td>Eppendorf</td>
			<td>22431021</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">microcentrifuge tubes, standard nuclease-free 1.5 ml</td>
			<td>Eppendorf</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:216px;">microcentrifuge, refrigerated</td>
			<td>Sorvall&#160;</td>
			<td>Legend X1R</td>
			<td style="width:167px;">Or equivalent product, must be capable of reaching at least 18,000 x g with rotors for 50 ml tubes, Oak Ridge tubes, and 1.5 ml tubes</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:216px;">microcentrifuge, room temperature</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td style="width:167px;">Or equivalent product, must be capable of reaching at least 18,000 x g with rotor for 1.5 ml and 2 ml microcentrifuge tubes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microcon DNA Fast Flow Centrifugal Filter Units</td>
			<td>EMD Millipore</td>
			<td>MRCFOR100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Miracloth, 1 square per sample cut to fit funnel</td>
			<td>EMD Millipore</td>
			<td>475855</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NEBNext Microbiome DNA Enrichment Kit</td>
			<td>New England Biolabs</td>
			<td>E2612L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">parafilm</td>
			<td>Parafilm M</td>
			<td>PM992</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">plastic pots and trays</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polyvinylpyrrolidone (PVP)</td>
			<td>Fisher</td>
			<td>BP431-100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proteinase K</td>
			<td>Qiagen</td>
			<td>19131</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pulsed-Field Gel Electrophoresis rig (e.g. CHEF DR III)</td>
			<td>Bio-Rad</td>
			<td>1703697</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">purification beads, Agencourt AMpureXP beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAamp DNA Mini Kit</td>
			<td>Qiagen</td>
			<td>51304</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Qiagen 20/g Genomic Tip DNA Extraction Kit</td>
			<td>Qiagen</td>
			<td>10223</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Qiagen Buffer EB (elution buffer)</td>
			<td>Qiagen</td>
			<td>19086</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Qiagen DNA Extraction Buffer Set</td>
			<td>Qiagen</td>
			<td>19060</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QiaRack</td>
			<td>Qiagen</td>
			<td>19015</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">qPCR machine (e.g. Roche Light Cycler 480)</td>
			<td>Roche</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">qPCR plate sealing film</td>
			<td>Roche</td>
			<td>4729757001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">qPCR plate, 96 well plate</td>
			<td>Roche</td>
			<td>4729692001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Qubit&#160;assay tubes</td>
			<td>Life Technologies</td>
			<td>Q32856</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Qubit Broad Spectrum assay kit</td>
			<td>Life Technologies</td>
			<td>Q32850</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Qubit High Sensitivity assay kit</td>
			<td>Life Technologies</td>
			<td>Q32851</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNaseA</td>
			<td>Qiagen</td>
			<td>19101</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Serological pipettes (20 ml) and pipet-aid</td>
			<td>Fisher</td>
			<td>13-678-11</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Small funnels, 1 per sample</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Chloride</td>
			<td>Ambion</td>
			<td>AM9759</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Soft paintbrush, 2 per sample</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="260">
			<td height="260" style="height:260px;width:216px;">SPEX SamplePrep 2010 Geno/Grinder or another type of tissue homogenizer</td>
			<td>SPEX SamplePrep</td>
			<td></td>
			<td style="width:167px;">Or another comparable tissue homogenizer. If you do not have access to a tissue homogenizer, then grinding in a pre-chilled mortar and pestle will suffice (see protocol for details). However, a homogenizer will give more consistent results and total homogenization time is reduced.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td>Omnipure</td>
			<td>8550</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TBE</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">thermomixer</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td>Sigma</td>
			<td>T2819-100ml</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td>Promega</td>
			<td>H5142</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tube rotater</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">tubes, 50 mL conical polypropylene</td>
			<td>Corning</td>
			<td>352070</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">tubes, 50 ml high-speed polypropylene&#160;</td>
			<td>ThermoScientific/Nalgene</td>
			<td>3119-0050</td>
			<td style="width:167px;">e.g. Nalgene Oakridge tubes or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">vermiculite</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">water bath</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">water, sterile and certified Nuclease-free&#160;</td>
			<td>Fisher</td>
			<td>1481</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">water, sterile milliQ</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

optimization and comparative analysis of plant organellar dna enrichment methods suitable for next-generation sequencing

Los genomas organelares de las plantas contienen grandes elementos repetitivos que pueden someterse a apareamiento o recombinación para formar estructuras complejas y / o fragmentos sub-genómicos. Los genomas organelas también existen en mezclas dentro de una célula o tipo de tejido dado (heteroplasmia), y una abundancia de subtipos puede cambiar durante el desarrollo o cuando está bajo tensión (desplazamiento subestequiométrico). Se requieren tecnologías de secuenciación de próxima generación (NGS) para obtener una comprensión más profunda de la estructura y la función del genoma organelar. Los estudios de secuenciación tradicionales utilizan varios métodos para obtener ADN organelar: (1) Si se utiliza una gran cantidad de tejido de partida, se homogeneiza y se somete a centrifugación diferencial y / o gradiente de purificación. (2) Si se utiliza una cantidad menor de tejido ( es decir, si las semillas, el material o el espacio son limitados), se realiza el mismo proceso que en (1), seguido por amplificación del genoma completo para obtener suficiente ADN. (3) El análisis bioinformático puede ser usado para buscarEl ADN genómico total y analizar las lecturas organelas. Todos estos métodos tienen desafíos inherentes y compensaciones. En (1), puede ser difícil obtener una cantidad tan grande de tejido de partida; En (2), la amplificación de todo el genoma podría introducir un sesgo de secuenciación; Y en (3), la homología entre los genomas nucleares y organelas podría interferir con el montaje y el análisis. En plantas con genomas nucleares grandes, es ventajoso enriquecer el ADN organelar para reducir los costos de secuenciación y la complejidad de la secuencia para los análisis bioinformáticos. Aquí, comparamos un método de centrifugación diferencial tradicional con un cuarto método, un enfoque adaptado CpG-metil pulldown, para separar el ADN genómico total en fracciones nucleares y organelares. Ambos métodos producen suficiente ADN para NGS, el ADN que está altamente enriquecido para las secuencias organelares, aunque en diferentes proporciones en las mitocondrias y los cloroplastos. Se presenta la optimización de estos métodos para el tejido foliar de trigo y se discuten las principales ventajas ydLas ventajas de cada enfoque en el contexto de la entrada de la muestra, la facilidad del protocolo, y la aplicación descendente.

Los protocolos presentados en este manuscrito describen dos métodos distintos para enriquecer el ADN organelar del tejido vegetal. Las condiciones aquí presentadas reflejan la optimización para el tejido de trigo. En la Figura 1 se describe una comparación de los pasos clave en los protocolos, la entrada de tejido requerida y la salida de ADN. Los pasos del protocolo DC que probamos siguen condiciones similares a las descritas anteriormente ( <strong cla...

Watch this Scientific Journal Video about Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing at JoVE.com

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Se presentan la comparación y optimización de dos métodos de enriquecimiento de ADN organelar de plantas: centrifugación diferencial tradicional y fraccionamiento del gDNA total basado en el estado de metilación. Evaluamos la cantidad y calidad de ADN resultante, demostramos el rendimiento en secuenciación de próxima generación de lectura corta y discutimos el potencial de uso en la secuenciación de una molécula de larga lectura.

Optimización y Análisis Comparativo de Métodos de Enriquecimiento de ADN de Organelos Vegetales Adecuados para Secuenciación de Próxima Generación

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

The overall goal of this experiment is to compare two methods used to isolate high quality organellar DNA, both plastid and mitochondria DNA from plant leaf tissue that is suitable for next-generation sequencing. These methods can help answer key questions in plant biology such as the extent of organellar diversity across taxa and how changes in organellar genome sequence or structure affect development and stress response. We will compare two techniques, the differential centrifugation method that enriches for one specific organellar type and the meta fractionation technique that recovers total organellar DNA from smaller frozen tissue samples.The standard procedure for growing wheat seedlings, is to plant seeds in vermiculite, in square pots with four to six seeds per corner. Transfer the pots to a greenhouse with a 16 hour light cycle. Water the plants each day.Fertilize the plants with one quarter teaspoon of granular 20-20-20 MPK fertilizer upon germination, and again at seven days post germination. For adulation of wheat seedlings, plant seeds and care for plants as in the standard procedure. But place the pots in a dark growth chamber.An alternative to growing these seedlings at a dark growth chamber is to cover the plants in the greenhouse but with proper ventilation. The first step in this procedure is to isolate the organelles. Harvest five grams of fresh tissue and rinse it in cold, sterile water in a chilled beaker on ice.Using scissors, cut the leaf tissue into approximately one centimeter pieces directly into a pre-chilled 50 millimeter tube containing two ceramic grinding cylinders. Keep the samples on ice throughout the procedure. To avoid cross-contamination, it is important to change the scissors between samples.Take the samples to the fume hood and add 20 milliliters of STE buffer to each 50 milliliter tube. Place the samples into pre-chilled cryogenic grinding blocks in a tissue grinding apparatus and grind the samples for 30 seconds at 1, 750 rotations per minute. Place the samples on ice for about one minute.Rotate the sample positions and grind for another 30 seconds at 1, 750 rotations per minute. Return the samples to the ice bucket. For each sample, insert a funnel into a clean 50 milliliter tube placed in ice and place one layer of filtration cloth into the funnel.Pre-wet the filtration cloth with five milliliters of STE buffer and save the flow through. Pour the homogenized tissue into the funnel. Rinse the grinding tube with 15 milliliters of STE buffer, recap and invert the tube to rinse walls and lid and pour its contents into the funnel.Carefully remove the ceramic stones and then squeeze and wring out the filtration cloth into the funnel. To avoid cross-contamination, change gloves between samples. Wrap the tube caps with paraffin film to avoid spillage.Centrifuge at 2, 000 x G, at four degree celsius for 10 minutes. Next, use a serological pipette to carefully aspirate the supernatant without disturbing the pellet and place it in a 50 milliliter high speed centrifuge tube with a tight sealing gasket. Place a small beaker of ice on a scale, tear the scale and weigh each supernatant.Use STE buffer to balance the tubes to within 0.1 grams and then centrifuge at 18, 000 x G in four degree celsius for 20 minutes. When the centrifugation is complete, return samples to the fume hood and discard the supernatant. Add one milliliter of ST buffer to the pellet and resuspend gently using a soft paintbrush.Add 24 milliliters of ST buffer to get a final volume of 25 milliliters. Gently swirl the paintbrush around in a suspension to mix, and then press the paintbrush on the side of a tube to remove all liquid before removing the paintbrush. After balancing the tubes, centrifuge at 18, 000 x G in four degree celsius for 20 minutes.During this centrifugation, prepare DNA's 1 solution as described in the text protocol and aliquot 200 microliters to a 1.5 milliliter tube per sample. When the centrifugation is complete, discard the supernatant and blot the tube. To each high speed centrifuge tube, add 300 microliters of STE buffer and resuspend the pellet using a soft paint brush.Place the paint brush in the previously prepared 1.5 milliliter tube containing 200 microliters of DNA's 1 solution and swirl the paint brush to remove any residual pellet stuck in the brush. Pipette the DNA's 1 solution into the high speed centrifuge tube and gently swirl to mix. Wrap paraffin film around the top of each tube and incubate at 37 degrees celsius in a water bath for 30 minutes.During the incubation, gently mix by swirling. Using a pipette tip with a wide orifice, gently pipette the pellet mixture out of the tube and place it in a 1.5 milliliter low binding tube. Add 500 microliters of 400 millimolar EDTA, pH 8.0 to the high speed centrifuge tube.Gently pipette to completely remove the residual pellet and transfer the EDTA to the low binding tube containing the pellet mixture. Gently mix by inversion. Centrifuge at 18, 000 x G, at four degree celsius for 20 minutes.Subsequently, discard the supernatant, blot each tube and use the pellet immediately for DNA isolation. In this method, organellar DNA is enriched from total genomic DNA that was extracted using commercial DNA extraction columns. Prepare the required amount of MBD2 FC protein bound magnetic beads, per the manufacturer's instructions and scale the reactions to use between one and two micrograms of total input DNA.In this demonstration, 160 microliters of beads is required for one microgram of total input DNA. To capture methylated nuclear DNA, add one microgram of input DNA to a tube containing 160 microliters of MBD2 bound magnetic beads for each individual sample and gently pipette up and down with a wide bore tip. Add the appropriate volume of 5x bind wash buffer for a final concentration of 1x and pipette the sample up and down with a wide bore tip, a few times to mix.Rotate the tubes at room temperature for 15 minutes. For the success of the pull down, it is critical to thoroughly mix the beads during the incubation, particularly, if there is significant bead clumping. To prevent bead clumping and to ensure pull down of the methylated DNA, gently pipette the samples with a wide bore pipette tip and flick the samples two to three times throughout the incubation.Briefly spin the tubes containing the DNA and magnetic bead mixture. Place the tubes on a magnetic rack for at least five minutes to collect the beads to the side of the tubes. The solution should appear clear, it contains un-methylated organellar enriched DNA.Using wide bore tips, carefully remove the cleared supernatant without disturbing the beads and transfer the supernatant to a clean, low binding two milliliter microcentrifuge tube. Store the samples at 20 or 80 degrees celsius. Acute PCR assay was employed to assess the relative abundances of three organelle specific implicons in total genomic DNA and the organellar DNA fraction obtained from both methods.Overall, the data indicate that the differential centrifugation method, preferentially enriches for mitochondria. The unmethylated fraction of the methylfractionated total genomic DNA, shows substantial enrichment of both organellar implicons. Percentages of reads mapped to the mitochondria or chloroplast chinese spring and chris cultivated wheat reference genomes are consistent with the QPCR results.Differential centrifugation yield DNA that is more enriched in mitochondria DNA and methylfractionation yields DNA that likely reflects the native abundance of the two organellar genomes. Sample labels including E, designate idyllated samples and NE designate non-idyllated samples. Interestingly, idyllation did not change organellar abundances.In both methods, the theoretical coverage of the mitochondria or chloroplast referenced genomes, exceeds 100x coverage, even when 12 libraries atremultiplexed. The total genomic DNA migrates as a diffused smear in post field gel electrophoresis and exceeds 50 kilobases. The nuclear fraction after methylfractionation decreases in size, but remains centered around 50 kilobases, suggesting that methylfractioanted DNA is most suitable for long read sequencing.Once mastered, both organellar DNA extraction techniques can be done in one to two days, from sample collection to assessment of final product, if performed properly. The methylfractionation technique has the potential to greatly expand the range of organellar DNA studies to rare and difficult samples, since tissue inputs are extremely low. However, differential centrifugation may still be preferable when solely mitochondria DNA is required for downstream studies.Likewise, centrifugation steps maybe adjusted to preferentially extract plastid DNA if desired. Both organellar DNA extraction methods yield DNA that is suitable for next generation sequencing but methylfractionation yields high molecular rate DNA that is ideal for long read sequencing.

Research

JoVE Journal

Genetics

Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved (Non-model) Organisms

Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved Non-model Organisms

Aprovechando los recursos de CyVerse para<em&gt; De Novo</em&gt; Transcriptómica Comparativa de los Organismos de Discapacidad (no Modelos)

This workflow allows novice researchers to leverage advanced computational resources such as cloud computing to carry out pairwise comparative ...

Investigación

Genética

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

Evaluación de la contaminación del ADN en las muestras de RNA basadas en ADN Ribosomal

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

Purificación de ADN de alto peso molecular genómico de oídio para Long-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 ...

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

Robusto de ADN y la secuenciación de alto rendimiento biblioteca construcción de especímenes de herbario

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 ...

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Dirigida tubería de Bioinformática y secuenciación de próxima generación para evaluar determinantes genéticos de la enfermedad constitucional

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Detección de eventos raros usando ADN Error corregido y secuenciación de ARN

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

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

Amplificación de cerca integrales provirus VIH-1 para secuenciación de próxima generación

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

Captura de alto rendimiento identificación de secuencias reguladoras de genes utilizando secuenciación de próxima generación de conformación Circular cromosoma (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

RNA Next-Generation Sequencing and a Bioinformatics Pipeline to Identify Expressed LINE-1s at the Locus-Specific Level

Secuenciación de próxima generación de ARN y una tubería bioinformática para identificar los LINE-1s expresados en el nivel específico de locus

Long INterspersed Elements-1 (LINEs/L1s) are repetitive elements that can copy and randomly insert in the genome resulting in genomic instability and ...

Single-Molecule F&ouml;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Single-Molecule F&#246;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Métodos de transferencia de energía por resonancia de una sola molécula para la investigación en tiempo real de la resolución de Holliday Junction por GEN1