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

We thank Taylor AuBuchon-Elder, Jordan Teisher, and Kristina Zudock for assistance in sampling herbarium specimens, and the Missouri Botanical Garden for access to herbarium specimens for destructive sampling. This work was support by a grant from the National Science Foundation (DEB-1457748).

1. Prior to Start
<ol>
	<li>Make fresh cetyl trimethylammonium bromide (CTAB) buffer17 by adding 20 g of CTAB, 10 g of polyvinylpyrrolidone (PVP) 40, 100.0 mL 1 M Tris pH 8.0, 40 mL of 0.5 M ethylenediaminetetraacetic acid (EDTA) pH 8.0, 280.0 mL of 5 M NaCl, and 400.0 mL of reagent water together, and bring the total volume to 1 L using reagent-grade water. Adjust the pH to 8.0. 
	NOTE: Additional reagents can be added to CTAB depending on secondary compounds in individual taxa. See Allen ...

<ol>
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The protocol presented here is a comprehensive and robust method for DNA isolation and sequencing library preparation from dried plant specimens. The consistency of the method and minimal need to alter it based on specimen quality make it scalable for large herbarium-based sequencing projects. The inclusion of an optional reamplification step for low yield libraries allows the inclusion of low quality, low quantity, rare, or historically important samples that would otherwise not be suitable for sequencing.
<p class=...

Herbarium collections are a potentially valuable source of both species and genomic diversity for studies including phylogenetics1,2,3, population genetics4,5, conservation biology6, invasive species biology7, and trait evolution8. The ability to obtain a rich diversity of species, populations, geographical locations, and time points highlights the &#34;treasure chest&#34;9 that is the herbarium. Historically, the degraded nature of herbarium-derived DNA has hindered PCR-based projects, often relegating researchers to using only markers found in high copy, such as regions of the chloroplast genome or the internal transcribed spacer (ITS) of the ribosomal RNA. Quality of specimens and DNA vary extensively based on methods of preservation9,10, with double-stranded breaks and fragmentation from heat used in the drying process being the most common forms of damage, creating the so-called 90% DNA lock-up that has encumbered PCR-based studies11. Aside from fragmentation, the second most prevalent issue in herbarium genomics is contamination, such as that derived from endophytic fungi13 or fungi acquired postmortem after collection but before mounting in the herbarium12, though this problem can be solved bioinformatically given the right fungal database (see below). A third, and less common, problem is sequence modification through cytosine deamination (C/G&#8594;T/A)14, although it is estimated to be low (~0.03%) in herbarium specimens11. With the advent of high-throughput sequencing (HTS), the issue of fragmentation can be overcome with short reads and sequencing depth12,15, allowing genomic-level data acquisition from numerous specimens with low quality DNA, and even sometimes permitting whole genome sequencing15.
Herbarium samples are becoming more frequently used and are a larger component of phylogenetic projects16. A current challenge of using herbarium specimens for HTS is consistently obtaining sufficient double stranded DNA, a necessary prerequisite for sequencing protocols, from numerous species in a timely manner, without needing to optimize methods for individual specimens. In this paper, a protocol for DNA extraction and library preparation of herbarium specimens is demonstrated that takes advantage of existing methods and modifies them to allow for fast and replicable results. This method allows for complete processing from specimen to a library of 24 samples in 13 h, with 8 h hands-on time, or 16 h, with 9 h hands-on time, when the optional reamplification step is required. Simultaneous processing of more samples is achievable, though the limiting factor is centrifuge capacity and technical skill. The protocol is designed to require only typical laboratory equipment (thermocycler, centrifuge, and magnetic stands) instead of specialized equipment, such as a nebulizer or sonicator, for shearing DNA.
DNA quality, fragment size, and quantity are limiting factors for the use of herbarium specimens in high-throughput sequencing experiments. Other methods for isolating herbarium DNA and creating high-throughput sequencing libraries have demonstrated the utility of using as little as 10 ng of DNA16; however they require experimentally determining the optimum number of PCR cycles required for library preparation. This becomes impractical when dealing with exceedingly small amounts of viable double stranded DNA (dsDNA), as some herbarium specimens produce only enough DNA for a single library preparation. The method presented here uses a single number of cycles regardless of sample quality, so no DNA is lost in library optimization steps. Instead, a reamplification step is invoked when libraries do not meet the minimum amounts needed for sequencing. Many herbarium samples are rare and possess little material making it difficult to justify destructive sampling in many cases. To counter this, the presented protocol allows dsDNA input sizes less than 1.25 ng into the library preparation process, expanding the scope of viable samples for high-throughput sequencing and minimizing the need for destructive sampling of specimens.
The following protocol has been optimized for grasses and tested on hundreds of different species from herbarium samples, although we expect that the protocol can be applied to many other plant groups. It includes an optional recovery step that can be used to save low quality and/or rare specimens. Based on over two hundred herbarium specimens tested, this protocol works on specimens with low tissue input and quality, allowing for the preservation of rare specimens through minimal destructive sampling. Here it is shown that this protocol can provide high quality libraries that can be sequenced for phylogenomics-based projects.

<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

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

DNA Isolation and Final Library Yield 
In this study, the efficacy of the protocol for the isolation of herbarium DNA and the recovery of high quality sequencing libraries was demonstrated using fifty different samples with the oldest from 1920 and the youngest from 2012 (Table 2). For each sample, approximately 10 mg of leaf tissue was used for DNA isolation. Greener leaf tissue was favored if available, and no tissue with obvious fungal contaminati...

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

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

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.

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

Research

JoVE Journal

Genetics

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.

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

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.

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

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.

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.

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.

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.

&#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.

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.

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.

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)

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

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

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

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