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

Vi takker Taylor AuBuchon-eldre, Jordan Teisher, og Kristina Zudock for hjelp i prøvetaking herbarium prøver og Missouri botaniske hagen for tilgang til herbarium prøver for destruktive prøvetaking. Dette arbeidet var støtte av et stipend fra National Science Foundation (DEB-1457748).

1. før å starte
<ol><li>Gjør frisk cetyl trimethylammonium bromide (CTAB) buffer17 ved å legge til 20 g CTAB, 10 g polyvinylpyrrolidone (PVP) 40, 100,0 mL 1 M Tris pH 8.0, 40 mL 0,5 M ethylenediaminetetraacetic syre (EDTA) pH 8.0, 280.0 mL 5 M NaCl og 400.0 mL reagens vann sammen, og bringe det totale volumet til 1 L bruker reagens-grade vann. Justere pH til 8.0. Merk: Flere reagenser kan legges til CTAB avhengig av sekundær forbindelser i individuelle taxa. Se Allen et al. <sup...

<ol>
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Protokollen presenteres her er en omfattende og robust metode for DNA isolasjon og sekvensering biblioteket forberedelse fra tørkede planter prøver. Konsistensen av metoden og minimalt behov for å endre det basert på prøven kvaliteten gjør det skaleres for store herbarium-baserte sekvensering prosjekter. Inkludering av et valgfritt reamplification trinn for lav yield biblioteker tillater inkludering av lav kvalitet, lav mengde, sjeldne, eller historisk viktige eksempler som ellers ikke ville være egnet for sekvens...

Herbarium samlinger er en potensielt verdifulle arter og genomisk mangfold for studier inkludert phylogenetics1,2,3, Populasjonsgenetikk4,5, konservering biologi6, invasiv Art biologi7og trekk utviklingen8. Muligheten til å få et rikt mangfold av arter, bestander, geografiske steder og tidspunkt høydepunkter "skattekiste"9 som herbarium. Historisk har degradert natur herbarium-avledet DNA hindret PCR-baserte prosjekter, ofte relegating forskere til å bruke bare indikatorer i høy kopien, for eksempel områder chloroplast genomet eller interne transkribert distansestykke (ITS) i ribosomal RNA. Kvaliteten på prøvene og DNA variere mye på metoder for bevaring9,10, med dobbel-strandet pauser og fragmentering av varme i tørking prosessen blir de vanligste formene for skader, skaper den såkalte 90% DNA Lås opp som har encumbered PCR-baserte studier11. Bortsett fra fragmentering er nest vanligste problemet i herbarium genomics forurensning, slik som avledet fra endophytic sopp13 eller sopp ervervet postmortem etter samling, men før montering i den herbarium12, skjønt Dette problemet kan bli løst bioinformatically gitt rett fungal databasen (se nedenfor). En tredje, og mindre vanlig, problemet er sekvensen modifisering gjennom cytosine deamination (C/G→T/A)14, selv om det anslås for å være lav (~ 0,03%) i herbarium prøver11. Med bruk av høy gjennomstrømming sekvensering (HTS), kan spørsmålet om fragmentering overvinnes med kort leser og sekvensering dybde12,15, la genomisk nivå datainnsamling fra mange prøver med lav kvalitet DNA, og noen ganger tillater hele genomet sekvensering15.
Herbarium prøver stadig oftere brukes og er en større del av Fylogenetiske prosjekter16. En gjeldende utfordring å bruke herbarium prøver for HTS er konsekvent få tilstrekkelig double-strandet DNA, en nødvendig forutsetning for sekvensering protokoller, fra mange arter i tide, uten å optimalisere metoder for enkelte prøver. I denne utredningen er en protokoll for DNA utvinning og biblioteket forberedelse av herbarium demonstrert som utnytter eksisterende metoder og modifiserer dem til å tillate rask og repliserbar. Denne metoden gir mulighet for full behandling fra prøven til et bibliotek av 24 prøver i 13 h, med 8 h hands-on tid, eller 16 h, 9 h hands-on tid, når det valgfrie reamplification trinnet kreves. Samtidig behandling av flere prøver er oppnåelig, men den begrensende faktoren er sentrifuge kapasitet og tekniske ferdigheter. Protokollen er utformet krever bare vanlig laboratoriumutstyr (thermocycler, sentrifuger og magnetiske står) i stedet for spesialisert utstyr, som en forstøveren eller sonicator, for shearing DNA.
DNA kvalitet, fragment størrelse og antall er begrensende faktorer for bruk av herbarium høy gjennomstrømming sekvensering eksperimenter. Andre metoder for å isolere herbarium DNA og lage høy gjennomstrømming sekvensering biblioteker har vist verktøyet å bruke så lite som 10 ng DNA16; men de krever eksperimentelt bestemme optimal antall PCR sykluser kreves for biblioteket forberedelse. Dette blir upraktisk når håndtere meget små mengder levedyktig dobbel strandet DNA (dsDNA), som noen herbarium eksemplarer produserer bare nok DNA for en enkelt bibliotek forberedelse. Metoden som presenteres her bruker en enkelt antall sykluser uavhengig av eksempel kvaliteten, så ingen DNA er tapt i biblioteket optimering skritt. I stedet startes et reamplification skritt når biblioteker ikke oppfyller du minimumsbeløpene som kreves for sekvenser. Mange herbarium prøver sjeldne og svært lite stoff vanskelig å rettferdiggjøre destruktive prøvetaking i mange tilfeller. Mot dette presentert protokollen tillater dsDNA inngang størrelser mindre enn 1,25 ng i biblioteket forberedelsesprosessen, utvide omfanget av levedyktig prøver for høy gjennomstrømming sekvensering og minimere behovet for destruktive prøvetaking av prøver.
Følgende protokollen er optimalisert for gress og testet på hundrevis av forskjellige arter fra herbarium prøver, men vi forventer at protokollen kan brukes til mange andre plante grupper. Det inkluderer et valgfritt utvinning skritt som kan brukes til å lagre lav kvalitet og/eller sjeldne eksemplarer. Basert på over to hundre herbarium prøvene testet, fungerer denne protokollen på prøver med lav vev input og kvalitet, slik at for bevaring av sjeldne eksemplarer gjennom minimal destruktive prøvetaking. Her er det vist at denne protokollen kan gi høy kvalitet biblioteker som kan være sekvensert for phylogenomics-baserte prosjekter.

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		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Veriti Thermal Cycler</td>
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			<td style="width:104px;">4452300</td>
			<td style="width:147px;">96 well&#160;</td>
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			<td height="21" style="height:21px;width:303px;">Gel Imaging System</td>
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			<td style="width:104px;">c300</td>
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			<td height="21" style="height:21px;width:303px;">Microfuge 20 Series</td>
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			<td style="width:104px;">B30137</td>
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			<td height="21" style="height:21px;width:303px;">Digital Dry Bath</td>
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			<td height="21" style="height:21px;width:303px;">Electrophoresis System</td>
			<td style="width:180px;">EasyCast</td>
			<td style="width:104px;">B2</td>
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			<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>
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			<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>
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			<td height="21" style="height:21px;width:303px;">Mini centrifuge</td>
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			<td height="21" style="height:21px;width:303px;">Vortex-Genie 2</td>
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			<td style="width:104px;">12-812</td>
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			<td height="21" style="height:21px;width:303px;">Mortar</td>
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			<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>
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			<td height="21" style="height:21px;width:303px;">Centrifuge tubes</td>
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			<td style="width:104px;">21-403-161</td>
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			<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

Herbaria er en uvurderlig kilde til plantemateriale som kan brukes i en rekke biologiske studier. Bruk av herbarium er forbundet med en rekke utfordringer som eksempel bevaring kvalitet, degradert DNA og ødeleggende utvalg av sjeldne eksemplarer. Mer effektivt bruke herbarium materiale i store sekvensering prosjekter, er pålitelig og skalerbar metode DNA isolasjon og biblioteket forberedelser nødvendig. Dette dokumentet demonstrerer en robust, begynnelsen-til-ende protokoll for DNA isolasjon og høy gjennomstrømming biblioteket bygging fra herbarium prøver som ikke krever endringer for individuelle prøver. Denne protokollen er skreddersydd for lav kvalitet tørket plante materiale og tar fordel av eksisterende metoder ved å optimalisere vev sliping, endre biblioteket størrelse utvalg og innføre et valgfritt reamplification trinn for lav yield biblioteker. Reamplification lav yield DNA biblioteker kan redde prøver fra uerstattelige og potensielt verdifulle herbarium prøver, benektende behovet for ytterligere destruktive prøvetaking og uten introdusere merkbar sekvensering bias for vanlig Fylogenetiske programmer. Protokollen er testet på hundrevis av grasarter, men forventes å bli anvendelig for bruk i andre plante linjene etter bekreftelse. Denne protokollen kan begrenses av ekstremt degradert DNA, der fragmentene ikke finnes i området ønsket størrelse, og sekundære metabolitter i noen plantemateriale som hemmer ren DNA isolasjon. Samlet introduserer denne protokollen en rask og omfattende metode som tillater DNA isolasjon og biblioteket utarbeidelse av 24 prøvene i mindre enn 13 h, med bare 8 timer aktiv hands-on tid med minimale endringer.

DNA isolasjon og endelige biblioteket avkastning I denne studien ble effekten av protokollen for isolering av herbarium DNA og utvinning av høy kvalitet sekvensering biblioteker demonstrert med 50 forskjellige prøver med den eldste 1920 og den yngste fra 2012 (tabell 2). For hver prøve, var ca 10 mg av blad vev brukes for DNA isolasjon. Grønnere blad vev ble foretrukket hvis tilgjengelig, og ingen vev med åpenbare fungal smitte ble valgt. Vellykke...

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

Robust DNA isolasjon og høy gjennomstrømming sekvensering biblioteket konstruksjon for Herbarium prøver

Denne artikkelen viser en detaljert protokoll for DNA isolasjon og høy gjennomstrømming sekvensering biblioteket bygging fra herbarium materiale inkludert redning av svært dårlig kvalitet 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 ...

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.

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