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.
