Name: optimization and comparative analysis of plant organellar dna enrichment methods suitable for next-generation sequencing
Uploaded: 2017-07-28T12:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing at JoVE.com

我们要承认美国农业部农业研究服务处和国家科学基金会（IOS 1025881和IOS 1361554）的资助。感谢R. Caspers进行温室维护和植物保养。我们还感谢明尼苏达大学基因组学中心，其中进行了Illumina图书馆的制备和测序。我们也感谢期刊编辑和四位匿名审稿人的意见，进一步加强了我们的稿件。我们也感谢经合组织与SK的合作，将这些协议项目与日本的同事整合起来。

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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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+of+wild+and+domesticated+rices+(Oryza+AA+genome+species)+based+upon+whole+chloroplast+genome+sequences.">Relationships of wild and domesticated rices (Oryza AA genome species) based upon whole chloroplast genome sequences.</a> Sci Rep. 5 (13957), 1-9 (2015).</li><li>Iorizzo, M., 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=De+novo+assembly+of+the+carrot+mitochondrial+genome+using+next+generation+sequencing+of+whole+genomic+DNA+provides+first+evidence+of+DNA+transfer+into+an+angiosperm+plastid+genome.">De novo assembly of the carrot mitochondrial genome using next generation sequencing of whole genomic DNA provides first evidence of DNA transfer into an angiosperm plastid genome.</a> BMC Plant Biol. 12 (61), 1-17 (2012).</li><li>Park, 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=Complete+sequences+of+organelle+genomes+from+the+medicinal+plant+Rhazya+stricta+(Apocynaceae)+and+contrasting+patterns+of+mitochondrial+genome+evolution+across+asterids.">Complete sequences of organelle genomes from the medicinal plant Rhazya stricta (Apocynaceae) and contrasting patterns of mitochondrial genome evolution across asterids.</a> BMC Genomics. 15 (405), 1-18 (2014).</li><li>Skippington, E., Barkman, T. 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. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+with+a+wider+applicability+to+isolate+plant+mitochondria+for+mtDNA+extraction.">An improved method with a wider applicability to isolate plant mitochondria for mtDNA extraction.</a> Plant Methods. 11 (56), 1-11 (2015).</li><li>Ejaz, M., 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+small+scale+methods+for+the+rapid+and+efficient+extraction+of+mitochondrial+DNA+from+wheat+crop+suitable+for+down-stream+processes.">Comparison of small scale methods for the rapid and efficient extraction of mitochondrial DNA from wheat crop suitable for down-stream processes.</a> Genet Mol Res. 13 (4), 10320-10331 (2014).</li><li>Eubel, H., Heazlewood, J. L., Millar, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subfractionation+of+plant+mitochondria+for+proteomic+analysis.">Isolation and subfractionation of plant mitochondria for proteomic analysis.</a> Methods Mol Biol. 355, 49-62 (2007).</li><li>Hao, W., Fan, S., Hua, W., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+extraction+and+assembly+methods+for+simultaneously+obtaining+plastid+and+mitochondrial+genomes.">Effective extraction and assembly methods for simultaneously obtaining plastid and mitochondrial genomes.</a> PLoS One. 9 (9), e108291 (2014).</li><li>Pomeroy, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+respiratory+properties+of+mitochondria+isolated+from+developing+winter+wheat+seedlings.">Studies on the respiratory properties of mitochondria isolated from developing winter wheat seedlings.</a> Plant Physiol. 53 (4), 653-657 (1974).</li><li>Taylor, N. L., Stroher, E., Millar, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+organelle+isolation+and+characterization.">Arabidopsis organelle isolation and characterization.</a> Methods Mol Biol. 1062, 551-572 (2014).</li><li>Triboush, S. O., Danilenko, N. G., Davydenko, O. 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>. QIAamp DNA Mini and Blood Mini Handbook Available from: <a target="_blank" href="https://www.qiagen.com/ch/resources/">https://www.qiagen.com/ch/resources/</a> (2016)</li><li>. User developed protocol: Isolation of genomic DNA from plants and filamentous fungi using the QIAGEN Genomic-tip - (EN) Available from: <a target="_blank" href="https://www.qiagen.com/ch/resources/">https://www.qiagen.com/ch/resources/</a> (2001)</li><li>. QIAGEN Genomic DNA Handbook Available from: <a target="_blank" href="https://www.qiagen.com/ch/resources/">https://www.qiagen.com/ch/resources/</a> (2012)</li><li>Bolger, A. 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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+gapped-read+alignment+with+Bowtie+2.">Fast gapped-read alignment with Bowtie 2.</a> Nat Methods. 9 (4), 357-359 (2012).</li><li>Bendich, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+chloroplasts+and+mitochondria+contain+so+many+copies+of+their+genome?.">Why do chloroplasts and mitochondria contain so many copies of their genome?.</a> Bioessays. 6 (6), 279-282 (1987).</li><li>Kumar, R. A., Oldenburg, D. J., Bendich, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+DNA+damage,+molecular+integrity,+and+copy+number+for+plastid+DNA+and+mitochondrial+DNA+during+maize+development.">Changes in DNA damage, molecular integrity, and copy number for plastid DNA and mitochondrial DNA during maize development.</a> J Exp Bot. 65 (22), 6425-6439 (2014).</li><li>Ma, J., Li, X. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organellar+genome+copy+number+variation+and+integrity+during+moderate+maturation+of+roots+and+leaves+of+maize+seedlings.">Organellar genome copy number variation and integrity during moderate maturation of roots and leaves of maize seedlings.</a> Curr Genet. 61 (4), 591-600 (2015).</li><li>Lang, E. 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=Simultaneous+isolation+of+pure+and+intact+chloroplasts+and+mitochondria+from+moss+as+the+basis+for+sub-cellular+proteomics.">Simultaneous isolation of pure and intact chloroplasts and mitochondria from moss as the basis for sub-cellular proteomics.</a> Plant Cell Rep. 30 (2), 205-215 (2011).</li><li>Tobin, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+fractionation+of+plant+tissues.+Isolation+of+chloroplasts+and+mitochondria+from+leaves.">Subcellular fractionation of plant tissues. Isolation of chloroplasts and mitochondria from leaves.</a> Methods Mol Biol. 59, 57-68 (1996).</li></ol>

作者宣称他们没有竞争的利益。 本出版物中提及的商品名称或商业产品仅用于提供具体信息，并不意味着美国农业部的建议或认可。 USDA是一个平等的机会提供者和雇主。

迄今为止，大多数细胞器测序研究集中于传统的DC方法，以丰富特定的DNA。已经描述了从不同植物中分离细胞器的方法，包括苔藓40 ;单子叶植物如小麦15和燕麦11 ;和双子叶植物如拟南芥11 ，向日葵17和油菜籽14 。大多数协议集中在叶组织<s...

<html>基因组测序是解析重要植物性状的潜在遗传基础的有力工具。大多数基因组测序研究集中于核基因组内容，因为大多数基因位于细胞核中。然而，细胞器基因组，包括线粒体（跨真核生物）和质（植物;在特殊形式，叶绿体，工作在光合作用）促进生物发育，应激反应和整体健康至关重要的1显著的遗传信息。细胞器基因组通常包括在用于核基因组测序的总DNA的提取，虽然方法，以减少前，DNA提取的细胞器号码也采用2。许多研究已经使用测序结果从总的gDNA提取组装细胞器基因组3，4，5，外部参照"&gt; 6,7。然而，当研究的目标是把重点放在细胞器基因组，使用总的gDNA增加了测序成本，因为很多的读取，但‘丢失’的核的DNA序列，特别是在植物具有大核基因组此外，由于重复和传输细胞器序列的进核基因组和细胞器之间，解决测序的正确的映射位置读取到适当的基因组是生物信息学有挑战性2,8。细胞器基因组的从核基因组的纯化是一个减少这些问题的策略，进一步的生物信息学策略可用于分离映射到线粒体和叶绿体之间同源区域的读数。 虽然来自许多植物物种的细胞器基因组已被测序，但是对于细胞器基因组多样性的广度几乎不了解可用于野生种群或栽培育种池。还知道细胞器基因组是由于重复序列9之间的重组而经历显着的结构重排的动态分子。此外，每个细胞器中含有多个拷贝的细胞器基因组，并且每个细胞内都含有多个细胞器。并非所有这些基因组的拷贝都是相同的，这被称为异质性。与"主圈"的规范图相比，现在越来越多的证据表明，细胞基因组结构更复杂，包括亚基因组圆，线性染色体，线性连接子和支链结构10 。植物细胞器基因组的组装由于其相对较大的尺寸和大量的反向和直接重复而进一步复杂化。 用于细胞器分离，DNA纯化和随后基因组的传统方案<html> È测序往往笨重且需要大体积的组织输入的，具有几克到几百个向上必要为起点11，12，13，14，15，16，17克幼叶组织的组成。当组织受限时，这使得细胞器基因组测序不可及。在某些情况下，种子数量有限，例如当需要在代际基础上进行序列或在必须通过杂交维持的雄性不育系中时。在这些情况下，可以纯化细胞器DNA，然后进行全基因组扩增。然而，全基因组扩增可以引入显着的测序偏差，这在评估结构变异，亚基因组结构和异质性水平时是一个特别的问题&gt; 18。用于短读序列技术的图书馆准备工作的最新进展克服了低输入障碍，以避免全基因组扩增。例如，Illumina Nextera XT库制备试剂盒允许使用少至1ng的DNA作为输入19 。然而，用于长读取测序应用（如PacBio或Oxford Nanopore测序技术）的标准库制备仍然需要相对较高量的输入DNA，这可能对组织基因组测序构成挑战。最近，新的用户制作，长读测序的协议已被开发，以减少输入量和帮助促进在获得的DNA微克，批量样品的基因组测序是困难的20，21。然而，获得高分子量纯的细胞器级分以进入这些文库制剂仍是一个挑战。 我们寻求o比较和优化不需要全基因组扩增的适用于NGS的细胞器DNA富集和分离方法。具体来说，我们的目标是确定从有限起始材料（如叶子子样品）中丰富高分子量细胞器DNA的最佳实践。这项工作提供了丰富细胞DNA的方法的比较分析:（1）修改的传统差速离心方案与（2）基于使用市售DNA CpG-甲基结合结构域蛋白质下拉法的DNA分级方案22施用于植物组织23 。我们建议从小麦叶组织中分离细胞叶DNA的最佳实践，这可能容易地扩展到其他植物和组织类型。

<table><tbody><tr><td>2-mercaptoethanol (beta-mercaptoethanol; BME)</td><td>Sigma Aldrich</td><td>M3148-100ml</td><td></td></tr><tr><td>2-propanol (Isopropyl alcohol/isopropanol), bioreagent</td><td>Sigma Aldrich</td><td>I9516</td><td></td></tr><tr><td>agarose, Bio-Rad Cetified Megabase agarose</td><td>Bio-Rad</td><td>1613108</td><td></td></tr><tr><td>analytical balance</td><td>Mettler Toledo</td><td>AB54-S</td><td></td></tr><tr><td>balance</td><td>Mettler Toledo</td><td>PB1502-S</td><td></td></tr><tr><td>bovine serum albumin (BSA)</td><td>Sigma Aldrich</td><td>B4287-25G</td><td></td></tr><tr><td>Ceramic grinding cylinders, 3/8in x 7/8in</td><td>SPEX SamplePrep</td><td>2183</td><td></td></tr><tr><td>Cryogenic Blocks compatible with tissue homogenizer for holding 50 mL tubes</td><td>SPEX SamplePrep</td><td>2664</td><td></td></tr><tr><td>DNaseI</td><td>Sigma</td><td>DN25</td><td></td></tr><tr><td>ethanol, absolute</td><td>Decon Laboratories</td><td>2716</td><td></td></tr><tr><td>Ethylenediamine Tetraacetic Acid (EDTA), 0.5 M Solution, pH 8.0</td><td>Fisher</td><td>BP2482-500</td><td></td></tr><tr><td>gel imaging system</td><td></td><td></td><td></td></tr><tr><td>gel stain</td><td></td><td></td><td>Such as GelRed or Ethidium Bromide</td></tr><tr><td>grinding pestle, wide tip for 2 mL conical tubes</td><td></td><td></td><td></td></tr><tr><td>Guanidine-HCl, 8 M solution</td><td>ThermoFisher</td><td>24115</td><td></td></tr><tr><td>LightCycler 480 SYBR Green I Master</td><td>Roche</td><td>4707516001</td><td></td></tr><tr><td>liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>Lysing enzymes from Trichoderma harzianum</td><td>Sigma</td><td>L1412</td><td></td></tr><tr><td>Magnesium Chloride</td><td>G Bioscience</td><td>24115</td><td></td></tr><tr><td>magnetic rack</td><td>ThermoFisher</td><td>A13346</td><td></td></tr><tr><td>microcentrifuge tubes, LoBind 1.5 mL&amp;nbsp;</td><td>Eppendorf</td><td>22431021</td><td></td></tr><tr><td>microcentrifuge tubes, standard nuclease-free 1.5 mL</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>microcentrifuge, refrigerated</td><td>Sorvall&amp;nbsp;</td><td>Legend X1R</td><td>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><td>microcentrifuge, room temperature</td><td>Eppendorf</td><td>5424</td><td>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><td>Microcon DNA Fast Flow Centrifugal Filter Units</td><td>EMD Millipore</td><td>MRCFOR100</td><td></td></tr><tr><td>Miracloth, 1 square per sample cut to fit funnel</td><td>EMD Millipore</td><td>475855</td><td></td></tr><tr><td>NEBNext Microbiome DNA Enrichment Kit</td><td>New England Biolabs</td><td>E2612L</td><td></td></tr><tr><td>parafilm</td><td>Parafilm M</td><td>PM992</td><td></td></tr><tr><td>plastic pots and trays</td><td></td><td></td><td></td></tr><tr><td>polyvinylpyrrolidone (PVP)</td><td>Fisher</td><td>BP431-100</td><td></td></tr><tr><td>Proteinase K</td><td>Qiagen</td><td>19131</td><td></td></tr><tr><td>Pulsed-Field Gel Electrophoresis rig (e.g. CHEF DR III)</td><td>Bio-Rad</td><td>1703697</td><td></td></tr><tr><td>purification beads, Agencourt AMpureXP beads</td><td>Beckman Coulter</td><td>A63881</td><td></td></tr><tr><td>QIAamp DNA Mini Kit</td><td>Qiagen</td><td>51304</td><td></td></tr><tr><td>Qiagen 20/g Genomic Tip DNA Extraction Kit</td><td>Qiagen</td><td>10223</td><td></td></tr><tr><td>Qiagen Buffer EB (elution buffer)</td><td>Qiagen</td><td>19086</td><td></td></tr><tr><td>Qiagen DNA Extraction Buffer Set</td><td>Qiagen</td><td>19060</td><td></td></tr><tr><td>QiaRack</td><td>Qiagen</td><td>19015</td><td></td></tr><tr><td>qPCR machine (e.g. Roche Light Cycler 480)</td><td>Roche</td><td></td><td></td></tr><tr><td>qPCR plate sealing film</td><td>Roche</td><td>4729757001</td><td></td></tr><tr><td>qPCR plate, 96 well plate</td><td>Roche</td><td>4729692001</td><td></td></tr><tr><td>Qubit&amp;nbsp;assay tubes</td><td>Life Technologies</td><td>Q32856</td><td></td></tr><tr><td>Qubit Broad Spectrum assay kit</td><td>Life Technologies</td><td>Q32850</td><td></td></tr><tr><td>Qubit High Sensitivity assay kit</td><td>Life Technologies</td><td>Q32851</td><td></td></tr><tr><td>RNaseA</td><td>Qiagen</td><td>19101</td><td></td></tr><tr><td>Serological pipettes (20 mL) and pipet-aid</td><td>Fisher</td><td>13-678-11</td><td></td></tr><tr><td>Small funnels, 1 per sample</td><td></td><td></td><td></td></tr><tr><td>Sodium Chloride</td><td>Ambion</td><td>AM9759</td><td></td></tr><tr><td>Soft paintbrush, 2 per sample</td><td></td><td></td><td></td></tr><tr><td>SPEX SamplePrep 2010 Geno/Grinder or another type of tissue homogenizer</td><td>SPEX SamplePrep</td><td></td><td>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><td>Sucrose</td><td>Omnipure</td><td>8550</td><td></td></tr><tr><td>TBE</td><td></td><td></td><td></td></tr><tr><td>thermomixer</td><td></td><td></td><td></td></tr><tr><td>Tris</td><td>Sigma</td><td>T2819-100ml</td><td></td></tr><tr><td>Triton X-100</td><td>Promega</td><td>H5142</td><td></td></tr><tr><td>tube rotater</td><td></td><td></td><td></td></tr><tr><td>tubes, 50 mL conical polypropylene</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>tubes, 50 mL high-speed polypropylene&amp;nbsp;</td><td>ThermoScientific/Nalgene</td><td>3119-0050</td><td>e.g. Nalgene Oakridge tubes or equivalent</td></tr><tr><td>vermiculite</td><td></td><td></td><td></td></tr><tr><td>water bath</td><td></td><td></td><td></td></tr><tr><td>water, sterile and certified Nuclease-free&amp;nbsp;</td><td>Fisher</td><td>1481</td><td></td></tr><tr><td>water, sterile milliQ</td><td></td><td></td><td></td></tr></tbody></table>

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

植物细胞器基因组包含大的重复元件，其可能经历配对或重组以形成复合结构和/或亚基因组片段。细胞器基因组也存在于给定细胞或组织类型（异质性）内的混合物中，并且丰富的亚型可能在整个发育过程中或当处于应力（亚化学计量转移）时）发生变化。需要下一代测序（NGS）技术来更深入地了解细胞器基因组结构和功能。传统的测序研究使用几种方法来获得细胞器DNA:（1）如果使用大量的起始组织，将其均质化并进行差速离心和/或梯度纯化。 （2）如果使用较少量的组织（ 即如果种子，材料或空间有限），则按照（1）所述进行相同的处理，然后进行全基因组扩增以获得足够的DNA。 （3）生物信息学分析可用于分析使用总基因组DNA并解析出细胞器读数。所有这些方法都有固有的挑战和权衡。在（1）中，可能难以获得如此大量的起始组织;在（2）中，全基因组扩增可能引入测序偏倚;在（3）中，核和细胞器基因组之间的同源性可能会干扰组装和分析。在具有大核基因组的植物中，富集细菌DNA以降低生物信息学分析的测序成本和序列复杂度是有利的。在这里，我们将传统的差速离心法与第四种方法（适应性CpG-甲基下拉法）进行比较，将总基因组DNA分离成核和细胞器级分。两种方法为NGS产生足够的DNA，对于细胞器序列高度富集的DNA，尽管在线粒体和叶绿体中具有不同的比例。我们介绍了这些方法对小麦叶组织的优化，并讨论了主要优点和d在样本输入，协议易用性和下游应用的上下文中的每种方法的优点。

本手册中提出的方案描述了从植物组织中丰富细胞器DNA的两种不同的方法。这里提出的条件反映了小麦组织的优化。 图1中描述了协议，组织输入和DNA输出中的关键步骤的比较。我们测试的DC协议的步骤遵循与先前描述的相似的条件（ 图1A ）。收获的组织必须新鲜处理，并进行差速离心和/或梯度以分离完整的细胞器。在细胞器?...

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

提出了两种植物细菌DNA富集方法的比较和优化:传统差速离心和基于甲基化状态的总gDNA分馏。我们评估产生的DNA数量和质量，证明在短读下一代测序中的性能，并讨论长读单分子测序中使用的潜力。

适用于下一代测序的植物细菌DNA富集方法的优化与比较分析

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

optimization-comparative-analysis-plant-organellar-dna-enrichment

Research

JoVE Journal

Genetics

12.8K 次观看.  United States Department of Agriculture-Agricultural Research Service. 提出了两种植物细菌DNA富集方法的比较和优化:传统差速离心和基于甲基化状态的总gDNA分馏。我们评估产生的DNA数量和质量，证明在短读下一代测序中的性能，并讨论长读单分子测序中使用的潜力。 

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

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for 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 of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 &#181;g DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., &#62; 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.

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.

超高分子量基因组DNA的分离纯化白粉病对长读测序

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics ...

科研

遗传学

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.

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

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

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.

有针对性的下一代测序和生物信息学管道评价宪政疾病的遗传决定因素

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

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples

Gene expression analysis by RNA sequencing (RNA-seq) enables unique insights into clinical samples that can potentially lead to mechanistic understanding of the basis of various diseases as well as resistance and/or susceptibility mechanisms. However, FFPE tissues, which represent the most common method for preserving tissue morphology in clinical specimens, are not the best sources for gene expression profiling analysis. The RNA obtained from such samples is often degraded, fragmented, and chemically modified, which leads to suboptimal sequencing libraries. In turn, these generate poor quality sequence data that may not be reliable for gene expression analysis and mutation discovery. In order to make the most of FFPE samples and obtain the best possible data from low quality samples, it is important to take certain precautions while planning experimental design, preparing sequencing libraries, and during data analysis. This includes the use of appropriate metrics for precise sample quality control (QC), identifying the best methods for various steps during the sequencing library generation, and careful library QC. In addition, applying correct software tools and parameters for sequence data analysis is critical in order to identify artifacts in RNA-seq data, filter out contamination and low quality reads, assess uniformity of gene coverage, and measure the reproducibility of gene expression profiles among biological replicates. These steps can ensure high accuracy and reproducibility for profiling of very heterogeneous RNA samples. Here we describe the various steps for sample QC, library preparation and QC, sequencing, and data analysis that can help to increase the amount of useful data obtained from low quality RNA, such as that obtained from FFPE-RNA tissues.

This method describes the steps to improve the quality and quantity of sequence data that can be obtained from formalin-fixed paraffin-embedded (FFPE) RNA samples. We describe the methodology to more accurately assess the quality of FFPE-RNA samples, prepare sequencing libraries, and analyze the data from FFPE-RNA samples.

降解FFPE-RNA样品测序与分析优化

Gene expression analysis by RNA sequencing (RNA-seq) enables unique insights into clinical samples that can potentially lead to mechanistic understanding ...

<meta charset="utf8"></head><body>药用植物的 DNA 条形码：鉴定和系统发育分析

Application of DNA Barcoding to Identify Medicinal Plants

Medicinal plants are valuable resources globally and are used worldwide to maintain health and treat disease; however, the presence of adulteration obstructs their development. DNA barcoding, a technique for species identification by standard DNA regions, facilitates prompt and accurate identification of traditional medicinal plants. The process of DNA barcoding entails six basic steps: 1) processing the medicinal plants, 2) extracting high-quality total DNA from the medicinal plants using centrifugal column method, 3) amplifying target DNA region internal transcribed spacer 2 (ITS2) with universal primers of plants and performing Sanger sequencing, 4) splicing and aligning sequence to obtain the target sequence, 5) matching the barcode sequence against the barcode library for identification, 6) aligning sequence, comparing intraspecific and interspecific variation, constructing phylogenetic neighbor-joining tree. As shown in the results, the universal primer can amplify the target region. Basic Local Alignment Search Tool (BLAST) demonstrates the percentage identified was 100%, and the neighbor-joining tree demonstrates that the splicing sequences were clustered with the A. sinensis OR879715.1 clade, and the clade support value is 100. This protocol provides a reference for applying DNA barcoding technology as an effective method to identify medicinal plants and adulterants.

<meta charset="utf8"></head><body>作者聚焦：利用 DNA 条形码技术提高药用植物鉴定的效率

The present protocol describes the use of DNA barcoding technology to authenticate plant-based medicinal materials, and the medicinal plant Angelica sinensis (Oliv.) Diels was identified as an example.

DNA 条形码在药用植物鉴定中的应用

Medicinal plants are valuable resources globally and are used worldwide to maintain health and treat disease; however, the presence of adulteration ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

收集和唾液DNA提取用于新一代测序

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

医学

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

低质量和低数量肿瘤活检丰枯台过程的集成优化了有针对性的下一代测序

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

适用于下一代测序的植物细菌DNA富集方法的优化与比较分析

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DNA 条形码在药用植物鉴定中的应用

DNA 条形码在药用植物鉴定中的应用

DNA 条形码在药用植物鉴定中的应用

收集和唾液DNA提取用于新一代测序

低质量和低数量肿瘤活检丰枯台过程的集成优化了有针对性的下一代测序