Ringraziamo Taylor AuBuchon-Elder, Jordan Teisher e Kristina Zudock per assistenza negli esemplari di erbario di campionamento e il giardino botanico del Missouri per l'accesso agli esemplari di erbario per campionamento distruttivo. Questo lavoro è stato supporto da una sovvenzione della National Science Foundation (DEB-1457748).

<ol>
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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. 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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. 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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. 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Gli autori dichiarano che non hanno nessun interessi concorrenti.

Il protocollo presentato qui è un metodo completo e robusto per isolamento del DNA e sequenziamento preparazione libreria dagli esemplari di piante essiccate. La consistenza del metodo e minima necessità di modificarla basata su esemplare qualità rendono esso scalabile per progetti di grande erbario di sequenziamento. L'inclusione di un passo opzionale reamplification per librerie di basso rendimento permette l'inclusione di bassa qualità, quantità di basso, rara o storicamente importanti campioni che altrimenti non...

Erbario collezioni sono una fonte potenzialmente importante di entrambe le specie e diversità genomica per studi compreso phylogenetics1,2,3, genetica delle popolazioni4,5, conservazione biologia6, specie invasive biologia7e tratto evolution8. La capacità di ottenere una ricca diversità di specie, popolazioni, luoghi geografici e punti di tempo mette in evidenza il "forziere"9 che è l'erbario. Storicamente, la natura degradata del DNA erbario-derivato ha ostacolato progetti basati su PCR, relegando spesso i ricercatori a usando solo gli indicatori trovati in alta copia, quali le regioni del genoma del cloroplasto o il distanziatore interno trascritto (ITS) della ribosomiale RNA. Qualità dei campioni e DNA variano ampiamente basata su metodi di conservazione9,10, con rotture a doppio filamento e la frammentazione da calore utilizzato nel processo di essiccazione, essendo le forme più comuni di danno, creando il cosiddetto 90% DNA di lock-up che ha gravato studi basati su PCR11. A parte la frammentazione, il problema secondo più prevalente in erbario genomica è contaminazione, come quello derivato da funghi endofiti13 o funghi acquisito post mortem dopo la raccolta, ma prima di montare nell'erbario12, però Questo problema può essere risolto bioinformatically dato il database proprio fungo (Vedi sotto). Un terzo problema, meno comune, è modifica di sequenza attraverso citosina deaminazione (C/G→T/A)14, anche se si stima essere bassa (~ 0,03%) in campioni di erbario11. Con l'avvento di sequenziatori di alto-rendimento (HTS), il problema di frammentazione può essere superato con brevi letture e sequenziamento profondità12,15, permettendo l'acquisizione di dati a livello genomico dalle numerosi esemplari con bassa qualità DNA e a volte anche permettendo di sequenziamento del genoma intero15.
I campioni di erbario stanno diventando sempre più frequentemente utilizzati e una maggiore componente di filogenetica progetti16. Una sfida attuale della utilizzando campioni di erbario per HTS è costantemente ottenere sufficiente DNA a doppia elica, un prerequisito indispensabile per protocolli di sequenziamento, da numerose specie in modo tempestivo, senza la necessità di ottimizzare i metodi per individuo esemplari. In questa carta, un protocollo per l'estrazione di DNA e preparazione di libreria di campioni di erbario è dimostrato che si avvale dei metodi esistenti e li modifica per consentire risultati veloci e replicabili. Questo metodo consente completo elaborazione da esemplare a una libreria di 24 campioni in 13 h, con tempo di preparazione manuale h 8, o 16 h, con hands-on tempo h 9, quando è richiesto il passaggio opzionale reamplification. Elaborazione simultanea di più campioni è realizzabile, anche se il fattore limitante è la capacità della centrifuga e abilità tecnica. Il protocollo è progettato per richiedere solo attrezzature tipici del laboratorio (termociclatore, centrifuga e supporti magnetici) invece di attrezzature specializzate, come un nebulizzatore o un sonicatore, per la tosatura del DNA.
Qualità del DNA, la dimensione del frammento e la quantità sono fattori per l'utilizzo di campioni di erbario a esperimenti di sequenziamento ad alta velocità limitanti. Altri metodi per isolare il DNA di erbario e creazione di librerie di sequenziamento ad alte prestazioni hanno dimostrato l'utilità dell'utilizzo di appena 10 ng di DNA16; Tuttavia hanno bisogno di determinare sperimentalmente il numero ottimale di PCR cicli necessari per la preparazione di biblioteca. Questo diventa impraticabile quando trattare con estremamente piccole quantità di vitali double stranded DNA (dsDNA), come alcuni esemplari di erbario producono solo abbastanza DNA per una preparazione unica libreria. Il metodo presentato qui utilizza un singolo numero di cicli indipendentemente dalla qualità del campione, quindi nessun DNA si perde nelle fasi di ottimizzazione biblioteca. Invece, un passo di reamplification viene richiamato quando le librerie non soddisfano gli importi minimi necessari per la sequenza. Molti campioni di erbario sono rari e possiedono poco materiale che lo rende difficile da giustificare campionamento distruttivo in molti casi. Per contrastare ciò, il protocollo presentato permette di dsDNA ingresso taglie meno di 1,25 ng nel processo di preparazione di biblioteca, ampliando l'ambito dei campioni praticabili per high throughput sequenziamento e riducendo al minimo la necessità di campionamento distruttivo degli esemplari.
Il seguente protocollo è stato ottimizzato per erbe e testato su centinaia di specie diverse dai campioni di erbario, anche se ci aspettiamo che il protocollo può essere applicato a molti altri gruppi di piante. Esso comprende un passaggio di ripristino opzionale che può essere utilizzato per salvare bassa qualità e/o rari esemplari. Questo protocollo basato su oltre duecento esemplari di erbario testati, funziona sugli esemplari con tessuto basso input e qualità, consentendo la conservazione di esemplari rari tramite campionamento distruttivo minimo. Qui è indicato che questo protocollo può fornire librerie di alta qualità che possono essere ordinati in sequenza per progetti basati su phylogenomics.

<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

Gli erbari sono una preziosa fonte di materiale vegetale che può essere utilizzato in una varietà di studi biologici. L'uso di campioni di erbario è associato con una serie di sfide tra cui qualità di conservazione del campione, DNA degradato e campionamento distruttivo di rari esemplari. Per poter utilizzare in modo più efficace materiale di erbario in progetti di sequenziamento di grandi dimensioni, è necessario un metodo affidabile e scalabile di preparazione di isolamento e libreria di DNA. Questa carta dimostra un protocollo robusto, inizio-to-end per DNA alto-rendimento e isolamento costruzione della libreria da esemplari che non richiedono la modifica di singoli campioni. Questo protocollo è su misura per la bassa qualità secchi pianta materiale e approfitta dei metodi esistenti ottimizzando tessuto rettifica, modifica selezione dimensione biblioteca ed introducendo un passo opzionale reamplification per librerie di basso rendimento. Reamplification di librerie di basso rendimento del DNA può salvare i campioni provenienti da campioni di erbario potenzialmente preziosi e insostituibili, negando la necessità per ulteriore campionamento distruttivo e senza introdurre bias di sequenziamento discernibile per comune applicazioni filogenetiche. Il protocollo è stato testato su centinaia di specie di erba, ma dovrebbe essere adatto per l'impiego in altri lignaggi pianta dopo la verifica. Questo protocollo può essere limitato dal DNA estremamente degradato, dove non esistono frammenti nel campo dimensione desiderata, e di metaboliti secondari presenti in alcuni materiale vegetale che inibiscono pulito isolamento del DNA. Nel complesso, questo protocollo introduce un metodo veloce e completo che permette di isolamento del DNA e preparazione della biblioteca di 24 campioni in meno di 13 ore, con solo 8 h di tempo attivo hands-on con modifiche minime.

Isolamento del DNA e la resa finale biblioteca In questo studio, l'efficacia del protocollo per l'isolamento del DNA di erbario e al recupero delle librerie di sequenziamento di alta qualità è stata dimostrata utilizzando cinquanta diversi campioni con la più antica dal 1920 e il più giovane dal 2012 (tabella 2). Per ogni campione, circa 10 mg di tessuto fogliare è stato usato per isolamento del DNA. Più verde tessuto fogliare è stato favorito s...

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

Isolamento del DNA robusto e costruzione della libreria High-throughput sequenziamento per campioni di erbario

Questo articolo viene illustrato un protocollo dettagliato per isolamento di DNA e la costruzione della libreria sequenziamento ad alte prestazioni da materiale di erbario tra cui salvataggio di eccezionalmente scarsa qualità del 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 ...

robust-dna-isolation-high-throughput-sequencing-library-construction

Research

JoVE Journal

Genetics

10.5K Views.  Donald Danforth Plant Science Center. Questo articolo viene illustrato un protocollo dettagliato per isolamento di DNA e la costruzione della libreria sequenziamento ad alte prestazioni da materiale di erbario tra cui salvataggio di eccezionalmente scarsa qualità del DNA.

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

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

Metil-binding cattura sequenziamento del DNA per tessuti del paziente

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

Ricerca

Genetica

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

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 transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Una piattaforma robotica per high-throughput Protoplast Isolamento e Trasformazione

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 ...

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.

Purificazione di alto peso molecolare genomico da oidio per lungo-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 ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

Generazione di immunoprecipitazione della cromatina nativo sequenziamento librerie per analisi di densità del nucleosoma

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

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

Un protocollo universale per gRNA su larga scala produzione di libreria da qualsiasi fonte di DNA

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

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 been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

G2-seq: Un'elevato Throughput sequenziamento-base tecnica per identificare replica ritardata regioni del genoma

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

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

Sequenziamento di MicroRNA retrospettiva: Protocollo di preparazione libreria DNA complementare utilizzando campioni di RNA di paraffina-incastonato formalina-fisso

&#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

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.

Rilevamento di eventi rari utilizzando la sequenza di RNA e DNA di correzione d'errore

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

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state when water is supplied. The genomic sequencing of microscopic animals such as tardigrades risks bacterial contamination that sometimes leads to erroneous interpretations, for example, regarding the extent of horizontal gene transfer in these animals. Here, we provide an ultralow input method to sequence the genome of the tardigrade, Hypsibius dujardini, from a single specimen. By employing rigorous washing and contaminant exclusion along with an efficient extraction of the 50 ~ 200 pg genomic DNA from a single individual, we constructed a library sequenced with a DNA sequencing instrument. These libraries were highly reproducible and unbiased, and an informatics analysis of the sequenced reads with other H. dujardini genomes showed a minimal amount of contamination. This method can be applied to unculturable tardigrades that could not be sequenced using previous methods.

Contamination during the genomic sequencing of microscopic organisms remains a large problem. Here, we show a method to sequence the genome of a tardigrade from a single specimen with as little as 50 pg of genomic DNA without whole genome amplification to minimize the risk of contamination.

Bassissimo ingresso il sequenziamento del genoma libreria preparazione da un singolo esemplare di Tardigrade

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

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

Estrazione del DNA high-throughput e genotipizzazione delle larve di Zebrafish 3dpf di pinna Clipping

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