Evaluation of Viral RNA Recovery Methods in Vectors by Metagenomic Sequencing

Identification and characterization of viral genomes in vectors including ticks and mosquitoes positive for pathogens of great public health concern using metagenomic next generation sequencing (mNGS) has challenges. One such challenge is the ability to efficiently recover viral RNA which is typically dependent on sample processing. We evaluated the quantitative effect of six different extraction methods in recovering viral RNA in vectors using negative tick homogenates spiked with serial dilutions of tick-borne encephalitis virus (TBEV) and surrogate Langat virus (LGTV). Evaluation was performed using qPCR and mNGS. Sensitivity and proof of concept of optimal method was tested using naturally positive TBEV tick homogenates and positive dengue, chikungunya, and Zika virus mosquito homogenates. The amount of observed viral genome copies, percentage of mapped reads, and genome coverage varied among different extractions methods. The developed Method 5 gave a 120.8-, 46-, 2.5-, 22.4-, and 9.9-fold increase in the number of viral reads mapping to the expected pathogen in comparison to Method 1, 2, 3, 4, and 6, respectively. Our developed Method 5 termed ROVIV (Recovery of Viruses in Vectors) greatly improved viral RNA recovery and identification in vectors using mNGS. Therefore, it may be a more sensitive method for use in arbovirus surveillance.

i. Using the pipette, measure the volume of the PCR product and pipette into a 1.5mL Eppendorf LoBind tube. ii.
Add 5 times the volume of PB buffer to the measured PCR product volume and mix well iii.
Transfer the violet column to a 2 ml collection vessel iv.
Pipette the entire contents of the tube into the violet column v.
Centrifuge the column at 17`900 x g for 60 sec vi.
Discard liquid in the 2 ml collecting vessel vii.
Place the column in the same collecting vessel viii.
Pipette 750 μL PE buffer into the column ix.
Centrifuge the column at 17`900 x g for 60 sec x.
Discard liquid in the 2 ml collecting vessel xi.
Place the column in the same collecting vessel xii.
Centrifuge the column at 17`900 x g for 60 sec xiii.
Transfer the column to a 1.5ml LoBind tube xiv.
Pipette 30μL-50μL TE buffer directly onto the membrane without touching the membrane xv.
Incubate for 1 min at room temperature xvi.
Centrifuge the column at 17`900 x g for 60 sec xvii.
In the 1.5ml LoBind tube is the purified DNA *Pause point. DNA can be further processed or stored at 4 ° C / -20 ° C 75. Determine the purified DNA's concentration by using Qubit (step 40 -56). The DNA yield at this point will vary depending on the quality of starting DNA, but is usually 1-5 μg.

Primer removal
Note: A reaction input of 2.1 μg is recommended to yield sufficient product for subsequent deep sequencing workflows (>1 μg). Determine the purified DNA's concentration by using Qubit (step 40 -56). The DNA yield at this point will vary depending on the quality of starting DNA, but is usually 1-5 μg.
76. Combine the following component on ice: buffer and Agencourt AMPure XP beads to room temperature. 81. Freeze thaw the adapter and / or barcodes on ice, vortex and centrifuge briefly. 82. Insert the "Ion Plus and Ion Xpress Plus" program card into the Library Builder. CRITICAL Only when the Library Builder is switched off. Activate the library builder.

Note:
The library is ligated with adapters A and P1 so that the sequencing can take place. Furthermore, you have the option to load your library with a barcode (Adapter Barcode and P1 Adapter) to load several libraries on one chip. Use the tables below to select the volume of adapters or barcodes.
83. Mix the diluted adapters (50 μl) with the LdN buffer by pipetting up and down then centrifuge the tube briefly 84. When the cartridges have completely thawed, make sure that all the solutions are at the bottom of the wells by gently tapping on the bench top 85. Loading and programming the Library Builder: set up following the manufacturer's instructions.
86. Select the program on the Library Builder as follows:  No size selection  Pre-sheared 87. Once the programme is complete, remove the library from the machine and transfer it to a new clean 1.5 ml LoBind tube. AMPure XP beads into the library, and mix by flicking the tube 91. Incubate the mixture for 3 mins at room temperature on shaker at 300 rpm 92. Transfer the tube mixture on the magnetic rack and incubate for 3 mins at room temperature to separate beads from the supernatant. 93. Carefully pipette the clear solution (supernatant) from the tube mixture without disturbing the beads into a new LoBind tube. Note: This is the size selected library. 94. Calculating amount of Agencourt AMPure XP beads for clean up. Measure the volume of the size selected library, and multiple this volume by 1.8. this will give the volume of AMPure beads required for clean up. 95. Add volume of AMPure beads required for clean up to the tube containing the size selected library 96. Incubate for 5 min at room temperature on a shaker at 300 rpm 97. Place the sample tube on the magnetic rack and incubate for 3 min at room temperature (until a clear supernatant solution forms and beads have concentrated on the wall of the tube). 98. Whilst the tube is still on the magnetic rack, carefully pipette and discard the supernatant. 99. Add 200 μl of 70% ethanol into the sample tube without disturbing the bead pellet and incubate for 30 sec at room temperature. 100. Carefully pipette the ethanol from the sample tube and discard. 101. Repeat step 105 -106 making sure that the sample tube is still on the magnetic rack 102. Allow the pellet to air dry for 5 mins at room temperature. Make sure not to over dry the pellet. 103. Remove the sample tube from the magnetic rack and resuspend the pellet with 30 μl of TE buffer (pipette at least 10 times with the pipette tip up and down) 104. Incubate for 2 min at room temperature 105. Place the sample tube on the magnetic rack, incubate for 2 min at room temperature and pipette the clear supernatant (eluate = DNA library) into a new LoBind tube.
*Pause point. Store the DNA library at 4° C for up to 1 week or freeze in a freezer at -20° C for longer storage or continue directly with the quality check.
H. Quantification and quality check of the DNA library. * Timing approximately 2 hours and 30 min including set up and running time 106. Measure the concentration of the DNA library using Qubit (step 40 -56) and dilute the library to be analyzed by the bioanalyzer to 1-10 ng / μl in nuclease free water. 107. Check for the quality of the DNA library using an Agilent Bioanalyzer High Sensitivity DNA assay as described in the manufacture's manual. Note: this is to ensure that the fragment length of the DNA library is within the expected range (200 -400bp) for sequencing on the Ion torrent S5 platform. 108. Quantification of the DNA library. Calculate the molar concentration in pM of the library by creating an excel table of the Ion Torrent "Library Conversion Table (pg / uL to pM)" using the formula below; Molar concentration in pM = 1 (660 X Peak size of library) X (Stock library in pg/ μl) X 1000000 Example:  Concentration of sample determined by Qubit = 9.12ng/μl  Stock library in pg/μl = 9.12ng/ul x 1000 = 9120pg/μl  Peak size (bp) of the library determined by the Bioanalyzer = 300 Therefore, Molar concentration = 1 (660 X 300) X (9120) X 1000000 Molar concentration = 46060.6pM I. Deep sequencing. * Timing approximately 23 hours including pre setup and running time. 109. Calculating the estimated dilution factor for the DNA library. Divide the calculated pM concentration from the library conversion excel table by 3 (e.g. 38071 / 3 = 12690.3pM), then further divide this value by 80 (e.g 12690.3 / 80 = 158.6). This gives the dilution factor of 1: 159 for the DNA library. Note: we recommend diluting the DNA library in E1 or TE buffer. Inhouse laboratory optimization (data not shown) indicated that estimating the dilution factor of the DNA library using this formula yields enough amount of library required for loading on the Ion torrent chip for sequencing on the Ion S5 platform. 110. Automated loading of library on the chip using Ion Chef system. As described in the manufacture's manual and protocols, 111. Sequencing with Ion torrent S5 platform. As described in the manufacture's manual and protocols.