Name: robust dna isolation and high-throughput sequencing library construction for herbarium specimens
Uploaded: 2018-03-08T14:30:00
Description: Watch this Scientific Journal Video about Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens at JoVE.com

破壊的なサンプリングの標本へのアクセスのためのミズーリの植物園とサンプリング標本、援助のクリスティーナ Zudock ヨルダン Teisher、テイラー AuBuchon-高齢者を感謝いたします。この作品は全米科学財団 (DEB-1457748) からの助成金による支援です。

1. 開始前に
<ol><li>新鮮なセチル トリメチル アンモニウム臭化 (CTAB) バッファー17 CTAB、ポリビニルピロリドン (PVP)、40 100.0 mL 1 M トリス pH 8.0、0.5 M エチレンジアミン四酸 (EDTA) pH 8.0、280.0 mL 40 mL 5 の 10 g の 20 グラムを追加することにより作る M NaCl および 400.0 mL の試薬水一緒に、水 1 L 試薬グレードを使用する合計ボリュームをもたらす。8.0 pH を調整します。 注: 追加試?...

<ol>
	<li>Savolainen, V., Cu&#233;noud, P., Spichiger, R., Martinez, M. D. P., Cr&#232;vecoeur, M., Manen, J. 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+use+of+herbarium+specimens+in+DNA+phylogenetics:+Evaluation+and+improvement.">The use of herbarium specimens in DNA phylogenetics: Evaluation and improvement.</a> Plant Syst Evo. 197 (1-4), 87-98 (1995).</li><li>Zedane, L., Hong-Wa, C., Murienne, J., Jeziorski, C., Baldwin, B. G., Besnard, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Museomics+illuminate+the+history+of+an+extinct,+paleoendemic+plant+lineage+(Hesperelaea,+Oleaceae)+known+from+an+1875+collection+from+Guadalupe+Island,+Mexico.">Museomics illuminate the history of an extinct, paleoendemic plant lineage (Hesperelaea, Oleaceae) known from an 1875 collection from Guadalupe Island, Mexico.</a> Bio J Linn Soc. 117 (1), 44-57 (2016).</li><li>Teisher, J. K., McKain, M. R., Schaal, B. A., Kellogg, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyphyly+of+Arundinoideae+(Poaceae)+and+Evolution+of+the+Twisted+Geniculate+Lemma+Awn.">Polyphyly of Arundinoideae (Poaceae) and Evolution of the Twisted Geniculate Lemma Awn.</a> Ann Bot. , (2017).</li><li>Cozzolino, S., Cafasso, D., Pellegrino, G., Musacchio, A., Widmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variation+in+time+and+space:+the+use+of+herbarium+specimens+to+reconstruct+patterns+of+genetic+variation+in+the+endangered+orchid+Anacamptis+palustris.">Genetic variation in time and space: the use of herbarium specimens to reconstruct patterns of genetic variation in the endangered orchid Anacamptis palustris.</a> Conserv Gen. 8 (3), 629-639 (2007).</li><li>Wandeler, P., Hoeck, P. E. A., Keller, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Back+to+the+future:+museum+specimens+in+population+genetics.">Back to the future: museum specimens in population genetics.</a> Tre Eco &amp; Evo. 22 (12), 634-642 (2007).</li><li>Rivers, M. C., Taylor, L., Brummitt, N. A., Meagher, T. R., Roberts, D. L., Lughadha, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+many+herbarium+specimens+are+needed+to+detect+threatened+species?.">How many herbarium specimens are needed to detect threatened species?.</a> Bio Conserv. 144 (10), 2541-2547 (2011).</li><li>Saltonstall, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptic+invasion+by+a+non-native+genotype+of+the+common+reed,+Phragmites+australis,+into+North+America.">Cryptic invasion by a non-native genotype of the common reed, Phragmites australis, into North America.</a> PNAS USA. 99 (4), 2445-2449 (2002).</li><li>Besnard, 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=From+museums+to+genomics:+old+herbarium+specimens+shed+light+on+a+C3+to+C4+transition.">From museums to genomics: old herbarium specimens shed light on a C3 to C4 transition.</a> J Exp Bot. 65 (22), 6711-6721 (2014).</li><li>S&#228;rkinen, T., Staats, M., Richardson, J. E., Cowan, R. S., Bakker, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+open+the+treasure+chest?+Optimising+DNA+extraction+from+herbarium+specimens.">How to open the treasure chest? Optimising DNA extraction from herbarium specimens.</a> PLoS ONE. 7 (8), e43808 (2012).</li><li>Harris, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+analysis+of+tropical+plant+species:+An+assessment+of+different+drying+methods.">DNA analysis of tropical plant species: An assessment of different drying methods.</a> Plant Syst Evo. 188 (1-2), 57-64 (1994).</li><li>Staats, 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=DNA+damage+in+plant+herbarium+tissue.">DNA damage in plant herbarium tissue.</a> PLoS ONE. 6 (12), e28448 (2011).</li><li>Bakker, F. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbarium+genomics:+plastome+sequence+assembly+from+a+range+of+herbarium+specimens+using+an+Iterative+Organelle+Genome+Assembly+pipeline.">Herbarium genomics: plastome sequence assembly from a range of herbarium specimens using an Iterative Organelle Genome Assembly pipeline.</a> Bio J of the Linn Soc. 117 (1), 33-43 (2016).</li><li>Camacho, F. J., Gernandt, D. S., Liston, A., Stone, J. K., Klein, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endophytic+fungal+DNA,+the+source+of+contamination+in+spruce+needle+DNA.">Endophytic fungal DNA, the source of contamination in spruce needle DNA.</a> Mol Eco. 6 (10), 983-987 (1997).</li><li>Hofreiter, M., Jaenicke, V., Serre, D., Von Haeseler, A., P&#228;&#228;bo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequences+from+multiple+amplifications+reveal+artifacts+induced+by+cytosine+deamination+in+ancient+DNA.">DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA.</a> Nucl Acids Res. 29 (23), 4793-4799 (2001).</li><li>Staats, 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=Genomic+treasure+troves:+Complete+genome+sequencing+of+herbarium+and+insect+museum+specimens.">Genomic treasure troves: Complete genome sequencing of herbarium and insect museum specimens.</a> PLoS ONE. 8 (7), e69189 (2013).</li><li>Bakker, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbarium+genomics:+skimming+and+plastomics+from+archival+specimens.">Herbarium genomics: skimming and plastomics from archival specimens.</a> Webbia. 72 (1), 35-45 (2017).</li><li>Doyle, J. J., Doyle, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+DNA+isolation+procedure+for+small+quantities+of+fresh+leaf+tissue.">A rapid DNA isolation procedure for small quantities of fresh leaf tissue.</a> Phytochem Bul. 19, 11-15 (1987).</li><li>Allen, G. C., Flores-Vergara, M. A., Krasynanski, S., Kumar, S., Thompson, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+protocol+for+rapid+DNA+isolation+from+plant+tissue+using+cetryltrimethylammonium+bromide.">A modified protocol for rapid DNA isolation from plant tissue using cetryltrimethylammonium bromide.</a> Nat Prot. 1, 2320-2325 (2006).</li><li>Twyford, A. D., Ness, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+complete+plastid+genome+seqeuncing.">Strategies for complete plastid genome seqeuncing.</a> Mol Eco Resour. , (2016).</li><li>Aird, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+and+minimizing+PCR+amplification+bias+in+Illumina+sequencing+libraries.">Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries.</a> Genome Bio. 12 (2), R18 (2011).</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> Bioinf. 30, 2114-2120 (2014).</li><li>Grigoriev, I. V., 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=MycoCosm+portal:+gearing+up+for+1000+fungal+genomes.">MycoCosm portal: gearing up for 1000 fungal genomes.</a> Nucl Acids Res. 42 (1), D699-D704 (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 Meth. 9 (4), 357-359 (2012).</li><li>Herbarium Genomics. Available from: <a target="_blank" href="https://github.com/mrmckain/">https://github.com/mrmckain/</a> (2017)</li><li>. Fast-Plast: Rapid de novo assembly and finishing for whole chloroplast genomes Available from: <a target="_blank" href="https://github.com/mrmckain/">https://github.com/mrmckain/</a> (2017)</li><li>McKain, M. R., McNeal, J. R., Kellar, P. R., Eguiarte, L. E., Pires, J. C., Leebens-Mack, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+of+rapid+diversification+and+convergent+origins+of+active+pollination+within+Agavoideae+(Asparagaceae).">Timing of rapid diversification and convergent origins of active pollination within Agavoideae (Asparagaceae).</a> Am J Bot. 103 (10), 1717-1729 (2016).</li><li>McKain, M. R., Hartsock, R. H., Wohl, M. M., Kellogg, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Verdant:+automated+annotation,+alignment,+and+phylogenetic+analysis+of+whole+chloroplast+genomes.">Verdant: automated annotation, alignment, and phylogenetic analysis of whole chloroplast genomes.</a> Bioinf. , (2016).</li><li>Staton, S. E., Burke, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposome:+A+toolkit+for+annotation+of+transposable+element+families+from+unassembled+sequence+reads.">Transposome: A toolkit for annotation of transposable element families from unassembled sequence reads.</a> Bioinf. 31 (11), 1827-1829 (2015).</li><li>Bao, W., Kojima, K. K., Kohany, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repbase+Update,+a+database+of+repetitive+elements+in+eukaryotic+genomes.">Repbase Update, a database of repetitive elements in eukaryotic genomes.</a> Mobile DNA. 6 (1), 11 (2015).</li><li>. Transposons Available from: <a target="_blank" href="https://github.com/mrmckain/">https://github.com/mrmckain/</a> (2017)</li><li>Wei&#223;, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+patterns+of+damage+and+decay+kinetics+of+DNA+retrieved+from+plant+herbarium+specimens.">Temporal patterns of damage and decay kinetics of DNA retrieved from plant herbarium specimens.</a> Royal Soc Open Sci. 3 (6), 160239 (2016).</li><li>Sawyer, S., Krause, J., Guschanski, K., Savolainen, V., P&#228;&#228;bo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+patterns+of+nucleotide+misincorporations+and+DNA+fragmentation+in+ancient+DNA.">Temporal patterns of nucleotide misincorporations and DNA fragmentation in ancient DNA.</a> PLoS ONE. 7 (3), e34131 (2012).</li><li>Head, S. 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=Library+construction+for+next-generation+sequencing:+overviews+and+challenges.">Library construction for next-generation sequencing: overviews and challenges.</a> BioTechniques. 56 (2), 61-64 (2014).</li><li>Grover, C. E., Salmon, A., Wendel, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+sequence+capture+as+a+powerful+tool+for+evolutionary+analysis.">Targeted sequence capture as a powerful tool for evolutionary analysis.</a> Am J Bot. 99, 312-319 (2012).</li></ol>

ここで提示されたプロトコルは、DNA の隔離および植物の乾燥標本からのライブラリの準備をシーケンス処理の包括的かつ堅牢な方法です。メソッドとそれを変更する必要は最小限の一貫性は、それは大規模な標本に基づくシーケンス プロジェクトのスケーラブルな標本の品質確認に基づいています。低降伏点ライブラリのオプション reamplification ステップを含めることは、低品質、低量、珍...

標本コレクションは、種と系統1,2,3, 集団遺伝学4,5, 保全を含む研究のためのゲノムの多様性の両方の可能性がある貴重な情報源生物学6、外来種生物学7、および形質進化8。種、人口、地理的な場所、タイム ポイントの豊かな多様性を取得する機能は、標本は、「宝箱」9を強調表示します。歴史的に、標本由来 DNA の劣化した自然が多い領域の葉緑体ゲノムや、リボソームの内部転写スペーサー (ITS) などの高いコピー内で見つかったマーカーのみを使用する研究者を追いやって、PCR ベースのプロジェクトを妨げています。RNA。標本と DNA の品質によって異なります広く保全9,10二本鎖切断と損傷の最も一般的な形であること、作成の乾燥工程で使用される熱から断片化の手法、いわゆる 90 %dna ロックアップ PCR ベースの研究11を妨害しています。断片化、別標本のゲノムの第 2 最も一般的な問題は汚染、生菌13から派生する菌類コレクション後、標本12にもマウントする前に死後を取得したようにこの問題は、右真菌データベース (下記参照) を与えられた解決の bioinformatically をすることができます。11押し葉標本の低い (〜 0.03%) と推定されている、3 番目、およびより少なく共通の問題はシトシン脱アミノ化 (C/G→T/A)14日シーケンス変更です。短いリードとシーケンス深さ12,15、低品質の多数の標本から遺伝子レベルのデータ集録が可能高スループット シーケンス (HTS) の出現により、断片化の問題を克服できます。DNA と時々 全ゲノム シーケンス15を許可します。
標本サンプルはより頻繁に使用されるなる、系統のプロジェクト16の大きなコンポーネントです。標本を用いた高温超伝導体の現在の課題が個々 のための方法を最適化することがなく、タイムリーに多くの種から十分な二本鎖 DNA、シーケンス プロトコルは、必要な前提条件を一貫して取得します。標本。本稿では、既存の手段を活用して、高速かつ複製可能な結果を可能にするそれらを変更に DNA の抽出と標本のライブラリの準備のためのプロトコルが示されています。この方法は 13 h、8 h のハンズオン タイム、または 16 h、9 h、ハンズオン時間、オプション reamplification ステップが必要な場合 24 サンプルのライブラリに試験片から処理が完了すること。多くのサンプルの同時処理は、制限要因は遠心力と技術的なスキルが達成可能で。プロトコルは DNA をせん断してだけ典型的な実験装置 (たち、遠心とマグネット スタンド) ネブライザー、超音波発生装置などの特殊な機器ではなくを必要とする設計されています。
DNA の品質、フラグメント サイズと数量は、高スループット シーケンス実験の標本の使用のための要因を制限しています。標本の DNA を分離し、高スループット シーケンス ライブラリを作成する他の方法は 10 として少し使用の有用性を実証している DNA16; ngただし実験的 PCR の最適な数を決定する必要なライブラリの準備のために必要なサイクルです。これは、いくつか標本が単一のライブラリの準備のため十分な DNA のみを生成するよう実行可能なダブルの非常に小さな金額を扱う鎖 DNA (dsDNA)、非現実的ななります。ライブラリの最適化の手順で DNA は失われませんので、ここで紹介した方法は単一サンプルの質に関係なくサイクル数を使用します。代わりに、ライブラリは、シーケンスに必要な最低限の量を満たしていない場合に、reamplification ステップが呼び出されます。多くの植物標本サンプルはまれであり、多くの場合有害なサンプリングを正当化するが難しく少し材料を所有しています。これを対抗するため提案するプロトコルにより、dsDNA 入力サイズを 1.25 未満 ng のライブラリの準備プロセス、高スループット シーケンスと供試体の破壊のサンプリングのための必要性を最小限に抑えるのための実行可能なサンプルの範囲を拡大します。
次のプロトコルを草用に最適化されており、我々 はプロトコルことができます他の多くの植物のグループに適用されることを期待するものの、標本サンプルから別の種の何百ものテストします。低品質または珍しい標本を保存するために使用できる省略可能な回復手順が含まれています。テスト 200 以上の標本を基に、このプロトコルは、最小限の破壊的なサンプリングによって珍しい標本の保存を可能にする低組織入力と品質、標本で動作します。ここでこのプロトコルがゲノミクス ベース プロジェクトのシーケンスことができます高品質ライブラリを提供できることを示す.

<table border="0" cellpadding="0" cellspacing="0" width="733">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Veriti Thermal Cycler</td>
			<td style="width:180px;">Applied Biosystems</td>
			<td style="width:104px;">4452300</td>
			<td style="width:147px;">96 well&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Gel Imaging System</td>
			<td style="width:180px;">Azure Biosystems</td>
			<td style="width:104px;">c300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microfuge 20 Series</td>
			<td style="width:180px;">Beckman Coulter</td>
			<td style="width:104px;">B30137</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Digital Dry Bath</td>
			<td style="width:180px;">Benchmark Scientific</td>
			<td style="width:104px;">BSH1001</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Electrophoresis System</td>
			<td style="width:180px;">EasyCast</td>
			<td style="width:104px;">B2</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">PURELAB flex 2 (Ultra pure water)</td>
			<td style="width:180px;">ELGA&#160;</td>
			<td style="width:104px;">89204-092</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">DNA LoBind Tube&#160;</td>
			<td style="width:180px;">Eppendorf</td>
			<td>30108078</td>
			<td style="width:147px;">2 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Mini centrifuge</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:104px;">12-006-901</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Vortex-Genie 2</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:104px;">12-812</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Mortar</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:104px;">S02591</td>
			<td style="width:147px;">porcelain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pestle</td>
			<td style="width:180px;">fisher Scientific</td>
			<td style="width:104px;">S02595</td>
			<td style="width:147px;">porcelain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Centrifuge tubes</td>
			<td style="width:180px;">fisher Scientific</td>
			<td style="width:104px;">21-403-161</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microwave</td>
			<td style="width:180px;">Kenmore</td>
			<td style="width:104px;">405.7309231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Qubit Assay Tubes</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:104px;">Q32856</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">0.2 ml Strip tube and Cap for PCR</td>
			<td style="width:180px;">VWR</td>
			<td style="width:104px;">20170-004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Qubit 2.0 Fluorometer</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:104px;">Q32866</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Balance</td>
			<td style="width:180px;">Mettler Toledo</td>
			<td style="width:104px;">PM2000</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Liquid Nitrogen Short-term Storage</td>
			<td style="width:180px;">Nalgene</td>
			<td style="width:104px;">F9401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Magnetic-Ring Stand&#160;</td>
			<td style="width:180px;">ThermoFisher Scientific&#160;</td>
			<td style="width:104px;">AM10050</td>
			<td style="width:147px;">96 well&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Water Bath</td>
			<td style="width:180px;">VWR</td>
			<td style="width:104px;">89032-210</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Hot Plate Stirrers</td>
			<td style="width:180px;">VWR</td>
			<td style="width:104px;">97042-754</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Liquid Nitrogen</td>
			<td style="width:180px;">Airgas</td>
			<td style="width:104px;">UN1977</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">1 X TE Buffer</td>
			<td style="width:180px;">Ambion</td>
			<td style="width:104px;">AM9849</td>
			<td style="width:147px;">pH 8.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">CTAB</td>
			<td style="width:180px;">AMRESCO</td>
			<td style="width:104px;">0833-500G</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">2-MERCAPTOETHANOL</td>
			<td style="width:180px;">AMRESCO</td>
			<td style="width:104px;">0482-200ML</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Ribonuclease A</td>
			<td style="width:180px;">AMRESCO</td>
			<td style="width:104px;">E866-5ML</td>
			<td style="width:147px;">10 mg/ml solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Agencourt AMPure XP</td>
			<td style="width:180px;">Beckman Coulter</td>
			<td style="width:104px;">A63882</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Sodium Chloride</td>
			<td style="width:180px;">bio WORLD</td>
			<td style="width:104px;">705744</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Isopropyl Alcohol</td>
			<td style="width:180px;">bio WORLD</td>
			<td style="width:104px;">40970004-1</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Nuclease Free water</td>
			<td style="width:180px;">bio WORLD</td>
			<td style="width:104px;">42300012-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Isoamyl Alcohol</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:104px;">A393-500</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Sodium Acetate Trihydrate</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:104px;">s608-500</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">LE Agarose</td>
			<td style="width:180px;">GeneMate</td>
			<td style="width:104px;">E-3120-500</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">100bp PLUS DNA Ladder</td>
			<td style="width:180px;">Gold Biotechnology</td>
			<td style="width:104px;">D003-500</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">EDTA, Disodium Salt</td>
			<td style="width:180px;">IBI Scientific</td>
			<td style="width:104px;">IB70182</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Qubit dsDNA HS Assay Kit</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:104px;">Q32854</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">TRIS</td>
			<td style="width:180px;">MP Biomedicals</td>
			<td style="width:104px;">103133</td>
			<td style="width:147px;">ultra pure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Gel Loading Dye Purple (6 X)</td>
			<td style="width:180px;">New England BioLabs</td>
			<td style="width:104px;">B7024S</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">NEBNext dsDNA Fragmentase</td>
			<td style="width:180px;">New England BioLabs</td>
			<td style="width:104px;">M0348L</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">NEBNext Ultra II DNA Library Prep Kit for Illumina&#160;</td>
			<td style="width:180px;">New England BioLabs</td>
			<td style="width:104px;">E7645L</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">NEBNext Multiplex Oligos for Illumina</td>
			<td style="width:180px;">New England BioLabs</td>
			<td style="width:104px;">E7600S</td>
			<td style="width:147px;">Dual Index Primers Set 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">NEBNext Q5 Hot Start HiFi PCR Master Mix</td>
			<td style="width:180px;">New England BioLabs</td>
			<td style="width:104px;">M0543L</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Mag-Bind RXNPure Plus</td>
			<td style="width:180px;">Omega bio-tek</td>
			<td style="width:104px;">M1386-02</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">GelRed 10000 X</td>
			<td style="width:180px;">Pheonix Research</td>
			<td style="width:104px;">41003-1</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Phenol solution</td>
			<td style="width:180px;">SIGMA Life Science</td>
			<td style="width:104px;">P4557-400ml</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">PVP40</td>
			<td style="width:180px;">SIGMA-Aldrich</td>
			<td style="width:104px;">PVP40-50G</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Chloroform</td>
			<td style="width:180px;">VWR</td>
			<td style="width:104px;">EM8.22265.2500</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Ethanol</td>
			<td style="width:180px;">Koptec</td>
			<td style="width:104px;">V1016</td>
			<td style="width:147px;">200 Proof</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Silica sand</td>
			<td style="width:180px;">VWR</td>
			<td style="width:104px;">14808-60-7</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reamplification primers</td>
			<td colspan="2">Integrated DNA Technologies</td>
			<td>see text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Sequencher v.5.0.1</td>
			<td style="width:180px;">GeneCodes</td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

robust dna isolation and high-throughput sequencing library construction for herbarium specimens

標本は、さまざまな生物学的研究で使用できる植物材料の非常に貴重な源です。標本の使用はサンプル保存品質、劣化 DNA、珍しい標本の破壊的なサンプリングを含む課題の番号に関連付けられます。効果的に大規模なプロジェクトの標本材料を使用、するために DNA の分離およびライブラリの準備の信頼性と拡張性の高い方法が必要です。本稿は、DNA 分離、高速ライブラリ構築に標本から個々 のサンプルの変更を必要としない堅牢で、始めから終わりまでプロトコルを示します。このプロトコルは、組織研削、ライブラリ サイズ選択を変更して低収量ライブラリのオプション reamplification ステップを導入することを最適化することにより従来の低品質の乾燥植物素材とを利用に合わせて調整します。低収量 DNA ライブラリの reamplification は、かけがえのない、潜在的に貴重な標本、追加破壊サンプリングし、共通認識できるシーケンス バイアスを導入することがなく、必要性を否定することから派生したサンプルを救うことができます。系統のアプリケーション。プロトコル数百草種でテストされていますが確認後他の植物の系統での使用に適応します。このプロトコルは、フラグメントは目的のサイズの範囲では存在しない、非常に低下した DNA ときれいな DNA 分離を抑制する二次代謝産物いくつかの植物材料の存在によって制限することができます。全体的に、このプロトコルは DNA の隔離と最小限の変更でアクティブなハンズオン タイムの 8 時間だけで 13 h 未満の 24 サンプル調製されたライブラリでは、高速かつ包括的な方法を紹介します。

DNA の隔離および最終的なライブラリ収量 本研究では 1920 年からの最も古いと 2012 年 (表 2) から最年少 50 の異なるサンプルを使用して標本の DNA の分離および高品質シーケンス ライブラリの回復用のプロトコルの有効性を示した.各サンプルは、葉の組織の約 10 mg は DNA の隔離に使われました。場合は、環境に優しい葉組織が支持され、明?...

Watch this Scientific Journal Video about Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens at JoVE.com

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

堅牢な DNA の隔離および標本の高スループット シーケンス ライブラリーの構築

この記事では、DNA の隔離及び非常に質の悪い DNA の救助を含む標本材料から高スループット シーケンス ライブラリ構築のための詳しいプロトコルを示します。

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 ...

The overall goal of this protocol is to increase the quantity of isolated double-stranded DNA and subsequent high-throughput sequencing libraries from herbarium specimens, maximizing their utility in phylogenomic studies. This method unlocks the potential of herbarium specimens that would otherwise not be useful for sequencing projects as genomic sequences because of extensive sample degradation. The main advantage of this technique is that it is robust enough that rare and historically significant herbarium specimens can be used without the need for extensive destructive sampling.This technique extends the depth of taxon sampling in systematic spaced projects. Species acquisition is a limiting step, but with this protocol, historical collections can be easily used into these studies. Add liquid nitrogen and 30 to 50 milligrams of sterilized sand to grind one centimeter square of preselected herbarium tissue to fine powder in pre-chilled mortar and pestle.Adequate grinding is essential for successful DNA isolation. And care should be taken that the sample is completely pulverized. Next, add the frozen powder to two two-milliliter tubes, filling no more than half of the tube volume.Then add 600 microliters of warm CTAB solution to each tube. Thoroughly mix the powder by inverting and vortexing. Then incubate the samples in a water bath maintained at 65 degrees Celsius for one to 1.5 hours.In the meantime, vortex every 15 minutes. Then centrifuge the samples at 10, 000 g for five minutes at room temperature. After centrifugation, transfer approximately 500 microliters of the supernatant to a new, fresh set of labeled tubes.To the supernatant, add four microliters of 10 milligrams per milliliter of RNase A to each tune. Then mix the contents of the tube by inverting or pipetting. Incubate the samples in a heat block or water bath maintained at 37 degrees Celsius for 15 minutes.Once the tubes have obtained room temperature, add approximately 500 microliters of 25:24:1 phenol, chloroform, isoamyl alcohol mixture to the samples. Then mix the contents of the tubes by inverting or repeated pipetting. Then centrifuge the samples at 12, 000 g for 15 minutes at room temperature.Transfer the upper aqueous layer, which is approximately 400 microliters, to a fresh set of labeled tubes. To the aqueous layer in each tube, add approximately 400 microliters of 24:1 chloroform, isoamyl alcohol mixture. Mix the sample thoroughly by repeated pipetting or inversion.Then centrifuge the tubes at 12, 000 g for five minutes and transfer approximately 300 microliters of upper aqueous layer to pre-chilled, labeled tubes. Next, add 300 microliters of pre-chilled isopropanol and 12 microliters of 2.5 molar sodium acetate. Then incubate the tubes at negative 20 degrees Celsius for 30 to 60 minutes.After incubation, remove the samples from the freezer. Next, centrifuge the samples at 12, 000 g for 15 minutes at room temperature. After centrifugation, wash the pellet with approximately 300 to 500 microliters of 70%ethanol.Again, centrifuge the tubes at 12, 000 g for 10 minutes. Then without disturbing the pellet, carefully remove the supernatant. Next, air-dry the pellets and dissolve the DNA in 50 microliters of 1X Tris-EDTA buffer.Add one to 16 microliters of isolated DNA and two microliters of fragmentation reaction buffer in a 0.2-milliliter polymerase chain reaction tube. Adjust the final volume to 18 microliters with nuclease-free water. Next, add two microliters of double-stranded DNA fragmentation enzyme and vortex the mixture briefly for tree seconds.Then incubate the samples at 37 degrees Celsius for 8.5 minutes in the thermocycler. Post incubation, add five microliters of 0.5 molar EDTA to the tubes. Add 25 microliters of nuclease-free water to adjust the total volume of the sheared DNA to 50 microliters.Then add 45 microliters of SPRI beads maintained at room temperature to 50 microliters of sheared DNA and mix the two thoroughly by pipetting it up and down. Leave the sample containing the tubes for five minutes. Then transfer the tubes on a magnetic plate and wait for another five minutes.Carefully expel the supernatant. Next, add 200 microliters of fresh 80%ethanol to the tubes on the magnetic stand and wait for another 30 seconds at room temperature. After 30 seconds, carefully remove the supernatant.Leave the lid of the tube open to air-dry the beads for five minutes on the magnetic stand. Then remove the tubes from the magnet stand and elute the DNA from the beads in 55 microliters of 0.1X Tris-EDTA buffer. Mix the DNA thoroughly by repeated pipetting.Then incubate the sample at room temperature for five minutes. Again, place the tube on the magnetic stand and wait for the solution to clear in approximately two minutes. Remove 52 microliters of the supernatant and then perform DNA quantification analysis as outlined in the text protocol.To 50 microliters of cleaned and sheared DNA, add three microliters of endonuclease, phosphate tailing enzymes, and seven microliters of reaction buffer. Mix the DNA and the other components thoroughly by pipetting up and down. Then spin the tubes to remove the bubbles.After spinning, transfer the samples to a thermocycler and set the program. Dilute the adaptor 25 to 50-fold to achieve a working concentration of 0.6 to 0.3 micromolar. For high-throughput short-read sequencing, add 30 microliters of ligation master mix, one microliter of ligation enhancer, and 2.5 microliters of adaptor to the tubes.Mix the contents of the tubes thoroughly by pipetting up and down. Again, spin the tubes to remove any bubbles. Then incubate the tube at 20 degrees Celsius for 15 minutes.Add three microliters of commercial mixture of uracil-DNA glycosylase and DNA glycosylase lyase endonuclease VIII to the tubes. Adjust the total volume to 96.5 microliters. Mix the components thoroughly and incubate at 37 degrees Celsius for 15 minutes in the thermocycler.In the tube, prepare the polymerase chain reaction mix by adding all the reagents to cleaned, adaptor-ligated DNA. Then mix the reaction well by vortexing. Once the vortex is over, put the samples in the thermocycler and start the thermocycler amplification setting.Add about 25 microliters of magnetic beads maintained at room temperature to the sample and mix by repeated pipetting. Incubate the sample for five minutes. Then transfer the tubes on a magnetic stand and wait for another five minutes.After five minutes, remove the supernatant to a new set of labeled tubes. Next, to the supernatant, add six microliters of SPRI beads maintained at room temperature and mix thoroughly. After incubating the samples for five minutes, transfer the tubes on the magnetic plate and wait another five minutes.Remove and discard the supernatant. Then add 200 microliters of fresh 80%ethanol to the tubes on the magnetic stand. Next, incubate the tubes at room temperature for 30 seconds.After 30 seconds, carefully remove the supernatant and air-dry the beads for five minutes with the lid of the tube open while still on the magnetic stand. Now remove the tubes from the magnet. Elute the target DNA from the beads into 33 microliters of 0.1X Tris-EDTA buffer and subject to thorough mixing.After proper mixing, incubate at room temperature for five minutes. Then transfer the tubes onto the magnetic stand and wait for about two minutes until the solution turns clear. Pipette 30 microliters of the supernatant to two-milliliter tubes for storage.To show the isolation of herbarium DNA, an agarose gel was run with double-stranded DNA isolated from different herbarium specimens. Using 10 milligrams of leaf tissue, almost 3.56 to 2, 610 nanograms of double-stranded DNA isolated was loaded onto the agarose gel. The gel shows degraded herbarium DNA in a smear form in all the lanes.Next, in order to show recovery of high-quality sequencing library from DNA isolated from 10 herbarium specimens, an agarose gel was run. Before loading on the agarose gel, approximately 1.26 to 464 nanograms of the isolated herbarium DNA was subjected to enzymatic shearing and clean up with 90%bead volume. The agarose gel shows recovery of high-quality sequencing library from the DNA of 10 herbarium specimens.Final sequencing libraries show a primary band between 300 to 500 base pairs. Then to show the shotgun sequence of herbarium-derived DNA, a circular plot of TK 686 chloroplast genome was obtained. The sequence shows the total length of the DNA, inverted repeat region, and presence of large and small single-copy regions.Once mastered, this technique can be performed on 24 samples in less than 13 hours with only about eight hours of active hands-on time. After this procedure, other methods, such as sequence capture, can be performed on the resulting libraries to generate nuclear data for phylogenetic studies.

Research

JoVE Journal

Genetics

Methyl-binding DNA capture Sequencing for Patient Tissues

患者の組織のためのメチル結合DNA捕捉シーケンシング

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

遺伝学

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

ハイスループットプロトプラスト単離と変革のためのロボットプラットフォーム

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

ネイティブのクロマチン免疫沈降のヌクレオソーム密度解析ライブラリをシーケンスの生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

大規模な gRNA ライブラリ生産 DNA の任意のソースからの普遍的なプロトコル

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

G2-seq: 高スループット シーケンス ベースを識別する方法後半ゲノムの領域を複製します。

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

ホルマリン固定パラフィン包埋 RNA 試料を用いた回顧マイクロ Rna シーケンス: 相補的な DNA ライブラリの準備のプロトコル

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

エラー修正 DNA ・ RNA シーケンスを用いた珍しいイベント検出

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

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

超低入力ヒトゲノム単一 Tardigrade の標本からのライブラリの準備

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

ハイスループット DNA の抽出およびフィン クリッピングが 3dpf ゼブラフィッシュ仔のジェノタイピング

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

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

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

円の染色体構造の次世代シーケンシングによる遺伝子制御配列の高スループット識別キャプチャ (4 C seq)

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

堅牢な DNA の隔離および標本の高スループット シーケンス ライブラリーの構築

堅牢な DNA の隔離および標本の高スループットシーケンスライブラリーの構築