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.

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

Research

JoVE Journal

Genetics

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

This workflow allows novice researchers to leverage advanced computational resources such as cloud computing to carry out pairwise comparative transcriptomics. It also serves as a primer for biologists to develop data scientist computational skills, e.g. executing bash commands, visualization and management of large data sets. All command line code and further explanations of each command or step can be found on the wiki (<a href="https://wiki.cyverse.org/wiki/x/dgGtAQ">https://wiki.cyverse.org/wiki/x/dgGtAQ</a>). The Discovery Environment and Atmosphere platforms are connected together through the CyVerse Data Store. As such, once the initial raw sequencing data has been uploaded there is no more need to transfer large data files over an Internet connection, minimizing the amount of time needed to conduct analyses. This protocol is designed to analyze only two experimental treatments or conditions. Differential gene expression analysis is conducted through pairwise comparisons, and will not be suitable to test multiple factors. This workflow is also designed to be manual rather than automated. Each step must be executed and investigated by the user, yielding a better understanding of data and analytical outputs, and therefore better results for the user. Once complete, this protocol will yield de novo assembled transcriptome(s) for underserved (non-model) organisms without the need to map to previously assembled reference genomes (which are usually not available in underserved organism). These de novo transcriptomes are further used in pairwise differential gene expression analysis to investigate genes differing between two experimental conditions. Differentially expressed genes are then functionally annotated to understand the genetic response organisms have to experimental conditions. In total, the data derived from this protocol is used to test hypotheses about biological responses of underserved organisms.

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

This protocol outlines a comparative de novo transcriptome assembly and annotation workflow for novice bioinformaticians. The workflow is available for free entirely through CyVerse and connected by the Data Store. Command line and graphical user interfaces are used, but all code needed is available to copy and paste.

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

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species.
Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family,&#160;it could be easily used to other prokaryote and eukaryote species

Here, we present a protocol for tracing genomic DNA (gDNA) contamination in RNA samples. The presented method utilizes primers specific for the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) genes. The method is suited for reliable and sensitive detection of DNA contamination in most eukaryotes and prokaryotes.

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

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.

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

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.

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.

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

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

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

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

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)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

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

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

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

Long INterspersed Elements-1 (LINEs/L1s) are repetitive elements that can copy and randomly insert in the genome resulting in genomic instability and mutagenesis. Understanding the expression patterns of L1 loci at the individual level will lend to the understanding of the biology of this mutagenic element. This autonomous element makes up a significant portion of the human genome with over 500,000 copies, though 99% are truncated and defective. However, their abundance and dominant number of defective copies make it challenging to identify authentically expressed L1s from L1-related sequences expressed as part of other genes. It is also challenging to identify which specific L1 locus is expressed due to the repetitive nature of the elements. Overcoming these challenges, we present an RNA-Seq bioinformatic approach to identify L1 expression at the locus specific level. In summary, we collect cytoplasmic RNA, select for polyadenylated transcripts, and utilize strand-specific RNA-Seq analyses to uniquely map reads to L1 loci in the human reference genome. We visually curate each L1 locus with uniquely mapped reads to confirm transcription from its own promoter and adjust mapped transcript reads to account for mappability of each individual L1 locus. This approach was applied to a prostate tumor cell line, DU145, to demonstrate the ability of this protocol to detect expression from a small number of the full-length L1 elements.

Here, we present a bioinformatic approach and analyses to identify LINE-1 expression at the locus specific level.

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

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule F&#246;rster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.

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

Presented here is a protocol for performing single-molecule F&#246;rster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.