Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

1. General Virus Purification by Differential Centrifugation Using Standard Method by Covey et al. ²⁵

1. First, cut 80-100 g of leaves from diseased plants and grind in a waring blender at 4 °C using 200 mL grinding buffer (0.5 M NaH₂PO₄, 0.5 M Na₂HPO₄ (pH 7.2). and 0.5% (w/v) Na₂SO₃). Wear a laboratory coat and gloves for all steps of this procedure.
2. Then, transfer the homogenate (300 mL) to a 1.0 L beaker. Add 18 g of urea and 25 mL of 10% nonionic detergent (t-Oct-C₆H₄-(OCH₂CH₂)₉OH) to the homogenate inside a chemical hood.
   NOTE: For this step it is best to wear safety goggles and a simple breathing mask for personal protection.
3. Stir with a magnetic stirrer briefly in the hood and cover the beaker with the foil. Then transfer the foil covered beaker to a cold room and stir with a magnetic stirrer overnight at 4 °C.
4. Transfer the homogenate to centrifuge rotor bottles (250 mL containers) and centrifuge in a fixed angle rotor at 4,000 x g for 10 min at 4 °C. In a chemical fume hood, recover the supernatant and filter through 4 layers of cheesecloth.
5. Divide the homogenate among 38.5 mL polypropylene centrifuge tubes and centrifuge for 2.5 h at 40,000 x g at 4 °C. Typically, check for the presence of a green pellet at the bottom of the tube and a white pellet along the length of the tube. Pour off the supernatant and retain both pellets; place samples on ice.
   NOTE: The green pellet contains chloroplasts, starch, and other organelles.
6. Working in a chemical hood, use a rubber policeman to separate the pellets. Resuspend the white pellet in each rotor bottle in 1 mL of ddH₂O over the course of 1-2 h while maintaining the suspensions overnight at 4 °C to allow the materials to fully dissolve into solution. Centrifuge the suspension at 6,000 x g and at 4 °C for 10 min to remove the remaining debris.
7. Centrifuge the concentrated suspension at 136,000 x g for 2 h at 4 °C to pellet virions. Resuspend the pellets in 1 mL of buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl₂).
   NOTE: An optional step is to treat virions with DNAse I (10 µg/mL) for 10 min at 37 °C to remove non-encapsidated DNA, i.e., contaminating chloroplast and mitochondrial DNA. Then, inactivate the DNAse I by adding EDTA to 1 mM.
8. Disrupt virions with 40 µL of 2 µg/µL proteinase K at 37 °C for 15 min.
9. Work inside a chemical hood to recover virion DNA by organic extraction. Wear a face shield, gloves, and a laboratory coat during the extraction for protection against potential acute health effects. Add 1 volume of phenol-chloroform-isoamyl alcohol (49:50:1) to the sample and shake by hand for 20 s. Centrifuge at room temperature for 5 min at 16,000 x g. Remove the upper aqueous phase and transfer to a new tube. Repeat this extraction two or more times. Dispose of the organic phase by placing it in a glass waste bottle for proper institutional chemical disposal²⁶.
10. Concentrate the DNA using ethanol precipitation. Use 0.3 M final concentration of sodium acetate (pH 5.2) and 2.5 volumes of 95% ethanol. Place samples at -20 °C for 30-60 min and centrifuge at 13,000 x g for 10-20 min to pellet the DNA²⁶.
11. Working at a laboratory bench, resuspend the DNA pellet in 1 mL of 0.1 mM TE buffer (pH 8.0). Filer the suspension through a commercial gel filtration column (normally used for polymerase chain reaction (PCR) clean up) to eliminate salts and low molecular weight material that might impede NGS.
12. Analyze the samples by 1% agarose gel electrophoresis using ethidium bromide staining to view the quality of the preparations. Assess the quality of DNA using a nanodrop spectrophotometer.
    NOTE: A ratio of sample absorbance at 260 λ and 280 λ between 1.85 and 2.0 typically indicates that the preparation is "clean" of impurities and is of the desired quality.
13. Analyze the quality of DNA (use 5 pg to 10 ng) using a chip based capillary electrophoresis instrument.
    NOTE: Quality output shows clean peaks, representing DNA fragments distributed by size along an X-axis. Peak height indicates abundance of the fragment. Jagged peaks indicate partially degraded fragments or chemical contaminants. Round curves represent a smear of DNA indicating poor quality

2. Library Preparation Using DNA and Emulsion-based Clonal Amplification (emPCR Amplification)

Note: The library is typically prepared by a NGS facility which carries out customer-oriented work.

1. Shear a solution of DNA (> 200 ng) using a nebulizer which converts the DNA to fragments. Ligate the commercial adapters according to the manual's instructions²⁷.
2. Carry out emPCR amplification of the DNA sample according to the manufacturer's instructions^28,29,30. Repeat the wash step three times, and after each wash, pellet the beads in a minicentrifuge for 10 s. Discard the supernatant after each wash.
   NOTE: The procedure begins with preparation of the capture beads by washing in the commercial wash buffer provided with the kit. emPCR is commonly used for template amplification for NGS.
3. Heat denature the DNA or RNA at 95 °C for 2 min and then 4 °C until ready to use. Use 200-million molecules of DNA/RNA to 5 million capture beads in a final volume of 30 µL. Prepare a mock sample alongside the DNA/RNA sample and carry out the following steps with the nucleic acid sample as well as the mock sample.
4. Perform emulsification by vortexing the tube of emulsion oil for 10 s at maximum speed, then pour the entire content (4 mL) into a plastic stirring tube that is compatible with a platform homogenizer. Place the stirring tube on the platform to mix the emulsion at 2,000 rpm for 5 min.
5. Dispense 100 µL aliquots of emulsion into 8-strip cap tubes or into a 96-well plate. Cap the tubes or seal the plate and carry out emPCR using the manufacturer's recommended program²⁸.
   NOTE: After the PCR is complete, check the wells to see if the emulsion is intact and then proceed. Discard the entire well if the emulsion is broken.
6. Wear a lab coat and work in a chemical hood to collect the amplified DNA beads (ADB). Vacuum aspirate the emulsion from the wells and collect the beads in a 50 mL tube. Rinse the wells twice with 100 µL of isopropanol and aspirate the rinse to the same 50 mL tube.
7. Vortex the collected emulsions and resuspend the ADB with isopropanol to a final volume of 35 mL. Pellet the ADB at 930 x g for 5 min. Remove the supernatant and add 10 mL of enhancing buffer. Vortex the ADB and then wash by adding isopropanol to 40 mL final volume Centrifuge and discard the supernatant after each wash and repeat the wash step twice.
8. Carry out a final wash using ethanol in place of isopropanol. Add enhancing buffer to 35 mL final volume, vortex, and pellet the beads at 930 x g for 5 min. Remove the supernatant but leave 2 mL of enhancing buffer.
9. Transfer the suspension to a microcentrifuge tube and briefly centrifuge to pellet the ADB. After discarding the supernatant, rinse the ADB pellet twice with 1 mL of enhancing buffer. Centrifuge and discard the supernatant after each wash.
10. To prepare for the DNA library bead enrichment, add 1 mL of 1 N NaOH to the beads. Vortex the ADB and then incubate for 2 min at room temperature. Centrifuge and discard the supernatant. Repeat this wash step once.
11. Add 1 mL of annealing buffer, then vortex the ADB and incubate for 2 min at room temperature. Briefly centrifuge and discard the supernatant. Repeat this step again using 100 µL of annealing buffer.
12. To anneal sequencing primers to the DNA, add 15 µL of Seq Primer A and 15 µL of Seq Primer B provided in the kit. Briefly mix by vortexing and place the microcentrifuge tube in a heat block at 65 °C for 5 min. Transfer to ice for 2 min.
13. Wash three times with 1.0 mL of annealing buffer. Vortex for 5 s and discard the supernatant each time.
14. Before sequencing, measure the number of beads using a commercial bead counter. There should be at least 500,000 enriched beads.
    NOTE: The bead counter is a special device that measures the beads in a supplied microcentrifuge tube.

3. General mRNA Isolation and dsDNA Synthesis Starting with Infected Canna Leaves that Test by RT-PCR for CaYMV Using Reported Diagnostic Primers

1. Wear a laboratory coat and latex gloves for personal protection in all subsequent steps. Working at a laboratory bench, collect 12 samples from the leaves and plunge the samples into liquid nitrogen. Use a bead mill for homogenization. Use a commercial kit that provides a standard column-based method for total plant RNA isolation. Add the guanidine-isothiocyanate lysis buffer provided by the kit to the ground sample and shake for 20 s.
2. Add ethanol and mix thoroughly, according to kit instructions. Add each homogenate to a spin column that binds RNA to the membrane. Wash three times and elute the RNA into a recovery tube²⁴.
3. Quantify the RNA using a spectrophotometer to measure the ratio of absorbance at 260 λ and 280 λ. Verify the RNA integrity using 1% agarose gel electrophoresis stained with ethidium bromide.
   NOTE: An absorbance ratio between 1.85 and 2.0 indicates that the preparation is of the desired quality. Treat RNA with DNase I (10 µg/mL) for 10 min at 37 °C. Use a commercial spin column to concentrate RNA in RNase-free water³¹. Pool RNA samples before proceeding.
4. Use an rRNA removal kit to remove plant ribosomal RNA. Aliquot magnetic beads to the microcentrifuge tube and wash twice with RNase-free water. Vortex the tube aliquot to resuspend, place the tube on a magnetic stand and wait for liquid to clear. Discard the supernatant and replace with the magnetic bead resuspension solution. Vortex to resuspend and add 1 µL of RNase Inhibitor.
   NOTE: Such kits use oligo-dT bound to magnetic beads which hybridize to mRNA. The method uses standard magnet bead separation technology to recover transcripts²⁴.
5. Combine 500 ng to 1.25 µg of RNA, RNase-free water, and reaction buffers provided by the kit. Place the mixture for 10 min at 50 °C. Remove from heat and add washed magnetic beads in RNAse free water. Vortex briefly and set at room temperature for 5 min.
6. Place on a magnetic stand and wait for the liquid to clear. Transfer the supernatant to a fresh microcentrifuge tube. Set on ice.
7. Use a solution-based capture method for enrichment of exosomes and 200 ng of RNA to prepare the cDNA library.
   NOTE: The double strand cDNA library is typically prepared by a NGS facility which carries out customer-oriented work.
8. Fragment the RNA using a commercial RNA fragmentation solution (0.136 g ZnCl₂ and 100 mM Tris-HCl pH 7.0). Add 2 µL of solution to 18 µL of RNA (200 ng total). Spin tubes briefly in the microcentrifuge, place the samples at 70 °C for 30 s, and transfer to ice. Stop the reaction using 2 µL of 0.5 M EDTA pH 8.0 and 28 µL of 10 mM Tris-HCl pH 7.5.
9. Bind RNA to magnetic beads by mixing at room temperature for 10 min. Use a magnetic concentrator to collect the beads and discard the supernatant. Wash the beads three times with 200 µL of 70% ethanol. Discard each wash and then air dry the pelleted beads at room temperature for 3 min. Resuspend in 19 µL of 10 mM Tris-HCl pH 7.5.
10. Anneal random primers to fragmented RNA by heating to 70 °C for 10 min and then place the tube on ice for 2 min. Prepare the first strand and second strand cDNA using a standard commercial cDNA synthesis kit.
11. Purify the double-strand cDNA using a magnetic bead concentrator. Wash with 800 µL of 70% ethanol three times. Discard each wash and air dry the pellets at room temperature for 3 min. Resuspend in 16 µL of 10 mM Tris-HCl pH 7.5. Use the magnetic bead concentrator to separate the beads from the double-stranded cDNA, which is now in solution. Remove the cDNA by pipetting into a new 200 µL PCR tube.
12. Carry out fragment end repair using Taq polymerase and a mixture of deoxyribonucleotides provided by a commercial library preparation kit. The commercial kit provides pre-diluted adapters to add to each end of the double-strand cDNA using commercial ligase at 25 °C for 10 min.

4. NGS of DNA Library Prepared from Crude Virus Preparation and dsDNA Library Prepared from mRNA

1. Use a standard high throughput pyrosequencing instrument and follow all recommended manufacturers' protocols to generate direct readouts of DNA sequences. Use commercial sequencing reagents, including fluorescently labeled nucleotides.
   NOTE: For details refer manufacturer's instructions provided with the instrument.
2. Carry out post-sequencing analysis using genome assembly software which automatically assembles reads to produce the first set of contigs with an average length of < 700 bp. Use the FastQC software on the iPlant/CyVerse website which performs quality control checks on the raw sequence data³². Select sequences with Phred scores ≥ 30 to continue to reconstruct longer sequences from smaller sequence reads²⁴ using mapping and amplicon software.
   NOTE: For details refer manufacturer's instruction.
3. Submit these assembled contigs to NCBI-BLASTn analysis using MEGABLAST default module as well as Viridplantae (TaxID: 33090) and Viruses (TaxID: 10239) as the limiting organismal names³³. Gather the subpopulation of contigs that show high similarity to reported Badnavirus genomes into a report.
4. Verify that the joined scaffolds that represent one or more candidate full-length virus genomes, correctly produce in-frame sequences that have the same organization as the standard badnavirus genome. To do this, input the candidate full length virus genome into a plasmid drawing software. Then confirm the first 15 nucleotides consists of a tRNA^met (TGGTATCAGAGCGAG) which is highly conserved among badnaviruses. Locate the potential polyadenylation signal near the 3' end of the genome. Annotate the complete genome to identify the presence of two small ORFs and one large ORF encoding a polyprotein. Then use the ExPASy portal translate tool to identify the badnavirus ORF1, ORF2, and ORF3 translation products³⁴.
   NOTE: This scientific software is free and will generate circular DNA, identify all open reading frames, and provides an immediate output to verify that the sequence represents the full length circular DNA genome.
5. Use open source multiple sequence comparison tools, MUSCLE and CLUSTALW, to compare the virus genomes obtained from DNA and RNA analyses^35,36.
6. Search the NCBI nucleotide database to obtain the full genome sequences of 30 badnavirus species and export them as a document in .fasta format. Upload sequences to a software that conducts evolutionary genetic analysis of sequences along with the virus genome sequences obtained by NGS. Generate multiple sequence alignments and Maximum likelihood trees using MUSCLE³⁷.

5. Quality Assessment of De Novo Sequencing by PCR Amplification of Virus Genomes from Infected Plants

1. Input the newly identified full-length badnavirus genome sequences (.fasta format) into the free online Primer3 tool to derive PCR primers³⁸. Identify primer sets that will produce staggered products of 1,000-1,500 bp along the entire length of the virus genome(s). Send the sequences to a service facility that will synthesize and deliver PCR primers.
   NOTE: The output identifies acceptable primer pairs with common and acceptable melting temperatures and precise primer locations along the introduced sequences.
2. Working at a laboratory bench and wearing a lab coat and gloves, isolate 5 µg of DNA from the virus-infected and healthy control leaves using an automated method that involves standard paramagnetic cellulose particles to isolate DNA from plant material³⁹ . Freeze leaf material (20-40 mg) in liquid nitrogen in a microcentrifuge tube and grind using a bead mill. Combine the sample with lysis buffer in a microcentrifuge tube and add RNase A to each sample. Vortex the sample for 10-20 s and briefly spin the sample to remove solid particles.
   NOTE: Paramagnetic cellulose particles have high DNA-binding capacity and isolate high yields of pure DNA. The standard commercial silica column methods for DNA isolation do not efficiently extract DNA from a wide variety of plant species. Consequently, dozens of methods exist that are modifications of these procedures to improve the efficiency for individual plant species. The automated paramagnetic cellulose particle method was chosen because it yields more and higher quality DNA from more than 25 herbaceous angiosperm species⁴⁰.
3. Use commercial reagent cartridges for automated paramagnetic DNA isolation. Add 300 µL of nuclease free water to each commercial reagent cartridge and transfer plant lysate to the same cartridge. Place the cartridge in the cartridge rack, place a plunger in the well closest to the elution tube, and place elution buffer into the elution tube. Load cartridges into the automated nucleic acid isolation machine and run the plant DNA isolation protocol^41,42.
4. Carry out PCR to derive a set of overlapping PCR products. Use 5 µM of each forward and reverse primer with 35 cycles of PCR amplification. Use the following cycling conditions: denaturation at 95 °C for 60 s, annealing at 50 °C for 45 s, and extension at 72 °C for 1-2 min with a final extension at 72 °C for 7-10 min. Use a prepackaged gel filtration column to eliminate salts and low molecular weight material as in step 1.2³¹.
5. Calculate a 3:1 molar ratio of PCR product to vector to determine the amount of PCR product to ligate to 50 ng of linearized pGEM plasmid⁴³. Use a control insert DNA to determine whether the ligations work efficiently. Perform the ligation overnight using T4 DNA ligase (3 U/µL) at 4 °C. Then transform commercially prepared JM109 competent Escherichia coli cells. Use control 100 pg of uncut plasmid DNA as a positive control for efficient transformation. Plate 100 µL of transformed cells onto LB-agar plates with antibiotic and blue/white selection to recover ligated plasmids²⁶. Incubate plates for 16-24 h at 37 °C.
   NOTE: The pGEM vector has a lacZ gene which encodes β-galactosidase. Transformed bacteria grown on a plate containing 100 µg/mL ampicillin, 0.5 mM IPTG, 80 µg/mL 5-bromo-4-chloro-3-indoyl-β-D-galactopyranosidase (X-gal) will turn blue because of β-galactosidase activity. The pGEM plasmid is linearized in a manner that disrupts the lacZ gene. Colonies that contain the PCR product inserts disrupt the lacZ gene and do not metabolize X-gal. These colonies are white. Thus colonies with an insert can be differentiated from those without an insert by the color of the colony (white versus blue)²⁶.
6. Isolate DNA from three colonies using a standard column-based plasmid isolation kit³⁹. Sequence three plasmids of transformation product. Compare each DNA sequence with the de novo assembled virus genomes produced by NGS. Use CLUSTALW to align the sequences and to ensure that they are appropriately ordered.