Name: optimization and comparative analysis of plant organellar dna enrichment methods suitable for next-generation sequencing
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We would like to acknowledge funding from the United States Department of Agriculture-Agricultural Research Service and from the National Science Foundation (IOS 1025881 and IOS 1361554). We thank R. Caspers for greenhouse maintenance and plant care. We also thank the University of Minnesota Genomics Center, where the Illumina library preparations and sequencing were performed. We are also grateful for the comments from the journal editors and four anonymous reviewers that further strengthened our manuscript. We also thank OECD for a fellowship to SK to integrate these protocols for collaborative projects with colleagues in Japan.

1. Generation of Plant Materials for Organellar Isolation and DNA Extraction
<ol>
	<li>Standard growth of wheat seedlings
	<ol>
		<li>Plant seeds in vermiculite in small, square pots with 4 - 6 seeds per corner. Transfer to a greenhouse or growth chamber with a 16 h light cycle, 23 &#186;C day/18 &#186;C night.</li>
		<li>Water the plants each day. Fertilize the plants with &#188; teaspoon of granular 20-20-20 N-P-K fertilizer upon germination and at 7 days post-germination.</li>
	</ol>
	</li>
	<li><st...

<ol>
	<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>

To date, most organellar sequencing studies center on traditional DC methods to enrich for specific DNA. Methods to isolate organelles from diverse plants have been described, including moss40; monocots such as wheat15 and oats11; and dicots such as arabidopsis11, sunflower17, and rapeseed14. Most protocols focus on leaf tissue13,<sup class="xre...

Genome sequencing is a powerful tool to dissect the underlying genetic basis of important plant traits. Most genome-sequencing studies focus on the nuclear genome content, as the majority of genes are located in the nucleus. However, organellar genomes, including the mitochondria (across eukaryotes) and plastids (in plants; the specialized form, the chloroplast, works in photosynthesis) contribute significant genetic information essential to organismal development, stress response, and overall fitness1. Organellar genomes are typically included in total DNA extractions intended for nuclear genome sequencing, although methods to reduce organelle numbers prior to DNA extraction are also employed2. Many studies have used sequencing results from total gDNA extractions to assemble organellar genomes3,4,5,6,7. However, when the target of the study is to focus on organellar genomes, using the total gDNA increases the sequencing costs because many reads are &#34;lost&#34; to the nuclear DNA sequences, particularly in plants with large nuclear genomes. Moreover, due to the duplication and transfer of organellar sequences into the nuclear genome and between organelles, resolving the correct mapping position of sequencing reads to the proper genome is bioinformatically challenging2,8. The purification of organellar genomes from the nuclear genome is one strategy to reduce these problems. Further bioinformatics strategies may be used to separate reads that map to regions of homology between the mitochondria and chloroplasts.
While the organellar genomes from many plant species have been sequenced, little is known about the breadth of organellar genome diversity available in wild populations or in cultivated breeding pools. Organellar genomes are also known to be dynamic molecules that undergo significant structural rearrangement due to recombination between repeat sequences9. Moreover, multiple copies of the organellar genome are contained within each organelle, and multiple organelles are contained within each cell. Not all copies of these genomes are identical, which is known as heteroplasmy. In contrast to the canonical picture of &#34;master circles,&#34; there is now growing evidence for a more complex picture of organellar genome structures, including sub-genomic circles, linear chromosomes, linear concatamers, and branched structures10. The assembly of plant organellar genomes is further complicated by their relatively large sizes and substantial inverted and direct repeats.
Traditional protocols for organellar isolation, DNA purification, and subsequent genome sequencing are often cumbersome and require large volumes of tissue input, with several grams to upwards of hundreds of grams of young leaf tissue necessary as a starting point11,12,13,14,15,16,17. This makes organellar genome sequencing inaccessible when tissue is limited. In some situations, seed amounts are limited, such as when it is necessary to sequence on a generational basis or in male sterile lines that have to be maintained via crossing. In these situations, organellar DNA can be purified and then subjected to whole-genome amplification. However, whole-genome amplification can introduce significant sequencing bias, which is a particular problem when assessing structural variation, sub-genomic structures, and heteroplasmy levels18. Recent advances in library preparation for short-read sequencing technologies have overcome low-input barriers to avoid whole-genome amplification. For example, the Illumina Nextera XT library preparation kit allows for as little as 1 ng of DNA to be used as input19. However, standard library preparations for long-read sequencing applications, such as PacBio or Oxford Nanopore sequencing technologies, still require a relatively high amount of input DNA, which can pose a challenge for organellar genome sequencing. Recently, new user-made, long-read sequencing protocols have been developed to reduce the input amounts and to help facilitate genome sequencing in samples where obtaining microgram-quantities of DNA is difficult20,21. However, obtaining high-molecular weight, pure organellar fractions to feed into these library preparations remains a challenge.
We sought to compare and optimize organellar DNA enrichment and isolation methods suitable for NGS without the need of whole-genome amplification. Specifically, our goal was to determine best practices to enrich for high-molecular weight organellar DNA from limited starting materials, such as a subsample of a leaf. This work presents a comparative analysis of methods to enrich for organellar DNA: (1) a modified, traditional differential centrifugation protocol versus (2) a DNA fractionation protocol based on the use of a commercially available DNA CpG-methyl-binding domain protein pulldown approach22 applied to plant tissue23. We recommend best practices for the isolation of organellar DNA from wheat leaf tissue, which may be readily extended to other plants and tissue types.

<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

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 protocols presented in this manuscript describe two distinct methods to enrich for organellar DNA from plant tissue. The conditions presented here reflect optimization for wheat tissue. A comparison of key steps in the protocols, required tissue input, and DNA output are described in Figure 1. The steps of the DC protocol we tested follow similar conditions to those described previously (Figure 1A). Harvested tissue must be p...

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

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.

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.

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