TITLE: Nucleic Acid Extraction, Amplification and Library Construction for Viral Metagenomic Sequencing.
AUTHORS: Jing-Zhe (Ginger) Jiang, Hong-Ying Wei

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span> This protocol is a continuation of the previous protocol (</span><a href="https://dx.doi.org/10.17504/protocols.io.m4yc8xw" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">dx.doi.org/10.17504/protocols.io.m4yc8xw</span></a><span>) for viral metagenomic researches with the tissues of Molluscs, such as abalone, oyster, clam,  scallop and so on. This maybe also applicable for other animals, such as shrimp and fish, based on the principle of the protocol. But we havn't verified this idea yet.</span></div></div>

[STEPS]
?. [Viral DNA/RNA Extraction]
Now, the supernatant from step 13 of below protocol is ready, We will use HP Viral DNA/RNA Kit (Omega Bio-Tek, Norcross, GA, USA) to extract both the viral DNA and RNA sitimutaniously.
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♠ The kit HP Viral DNA/RNA Kit is not indispensable. You can replace it with any brand of kits, such as QIAamp cador Pathogen Mini Kit (Qiagen), as long as it suitable for extraction both viral DNA and RNA from liquid samples.♣ The entire procedure MUST BE RNase free
?. [Viral DNA/RNA Extraction]
Follow the kit protocol, then you will acquire 15-50 μl of viral DNA/RNA solution.
DO NOT add carrier RNA, as carrier RNA will account  a high percentage in the dataset from high throughput seqencing.
?. [Viral DNA/RNA Extraction]
Use Qubit™ (Thermo Fisher) and Qubit™ dsDNA HS Assay Kit (Thermo Fisher) to specifically quantify the concentration of dsDNA. If the amount is high that means previous extraction is successful.
If you like, you can also use Qubit™ RNA HS Assay Kit to specifically quantify RNA. But we don't recommand Qubit™ ssDNA Assay Kit, as it is not specific.
?. [Choosing Amplification Strategy]
Now, we are ready to use REPLI-g® Cell WGA & WTA Kit (Qiagen) to amplify both viral DNA RNA, or only viral RNA.
♠ We havn't fully assessed the performances of three amplification strategies. ♣ However, we found that the production of WTA is usually lower than WGA and RT-WGA protocol. Sometime, the barren production of WTA will even not be qualified to the further library construction.♥ And the differences in terms of viral diversity with three strategies are still under assessment.♦ We just use half of the required volume of kit protocol to save some money.◊ DO READ THROUGH the whole REPLI-g Cell WGA & WTA Handbook
?. [for viral RNA amplification (WTA)]
Place 3 μl nucleic acid, add 2 μl Lysis Buffer. Mix by vortexing and centrifuge briefly.
?. [for viral RNA amplification (WTA)]
Incubate at 24°C for 5 min.
24 °C
?. [for viral RNA amplification (WTA)]
Add 1 μl gDNA Wipeout Buffer the lysed sample. Mix by vortexing and centrifuge briefly.
?. [for viral RNA amplification (WTA)]
Incubate at 42°C for 10 min.
[Wiping out DNA]
?. [for viral RNA amplification (WTA)]
Prepare Quantiscript RT mix (Table 1).Table 1. Quantiscript RT mix  AB1Component
Volume/reaction
2RT/Polymerase Buffer
2&nbsp;μl
3H2O sc
0.5&nbsp;μl
4Random Primer
0.5 μl
5Oligo dT Primer
0.5&nbsp;μl
6Quantiscript RT Enzyme Mix
0.5 μl
7Total volume
4&nbsp;μl
AB1Component
Volume/reaction
2RT/Polymerase Buffer
2&nbsp;μl
3H2O sc
0.5&nbsp;μl
4Random Primer
0.5 μl
5Oligo dT Primer
0.5&nbsp;μl
6Quantiscript RT Enzyme Mix
0.5 μl
7Total volume
4&nbsp;μl
♠ gDNA Wipeout buffer will be deactivate in this Mix. So the DNA is kept during RT reaction.♣ The using of both random and oligo dT Primers will improve the proformance of RT reaction.♥ NOTE: The Quantiscript RT mix must be prepared fresh.
?. [for viral RNA amplification (WTA)]
Add 4 μl RT mix to the aliquot (6 μl) in step 8. Mix by vortexing and centrifuge briefly.
?. [for viral RNA amplification (WTA)]
Incubate at 42°C for 60 min
[Reverse Transcripting]
?. [for viral RNA amplification (WTA)]
Stop the reaction by incubating at 95ºC for 3 min, then cool on ice.
[Stopping RT]
[Cooling]
?. [for viral RNA amplification (WTA)]
Prepare the ligation mix (Table 2).Table 2 Ligation mix  AB1Component
Volume/reaction
2Ligase Buffer
4 μl
3Ligase Mix
1 μl
4Total volume
5 μl
AB1Component
Volume/reaction
2Ligase Buffer
4 μl
3Ligase Mix
1 μl
4Total volume
5 μl
IMPORTANT: When preparing the ligation mix, add the components in the order shown in Table 2. NOTE: The ligation mix must be prepared fresh.
?. [for viral RNA amplification (WTA)]
Add 5 μl ligation mix to the WTA ready reaction (10 μl) from step 12. Mix by vortexing and centrifuge briefly.
?. [for viral RNA amplification (WTA)]
Incubate at 24°C for 30 min.
[Ligating]
?. [for viral RNA amplification (WTA)]
Stop the reaction by incubating at 95°C for 5 min.
[Stopping ligating]
?. [for viral RNA amplification (WTA)]
Prepare REPLI-g SensiPhi amplification mix (Table 3).Table 3 Preparing REPLI-g Sensiphi amplification mix  AB1Component
Volume/reaction
2REPLI-g sc Reaction Buffer
14.5 μl
3REPLI-g Sensiphi DNA Polymerase
0.5 μl
4Total volume
15 μl
AB1Component
Volume/reaction
2REPLI-g sc Reaction Buffer
14.5 μl
3REPLI-g Sensiphi DNA Polymerase
0.5 μl
4Total volume
15 μl
NOTE: The REPLI-g SensiPhi amplification mix must be prepared fresh.
?. [for viral RNA amplification (WTA)]
Add 15 μl REPLI-g SensiPhi amplification mix to the ligation reaction (15 μl) from step 16. Mix by vortexing and centrifuge briefly.
?. [for viral RNA amplification (WTA)]
Incubate at 30°C for 2h.
[Amplifying]
?. [for viral RNA amplification (WTA)]
Stop the reaction by incubating at 65°C for 5 min.
[Stopping Amplifying]
♠ If not being used directly, store the amplified DNA at –15 ºC to –30ºC until required for downstream applications.♣ We recommend storage of the amplified DNA at a concentration of at least 100 ng/μl.
?. [Cleaning with AMPure Beads]
Adjust the volume to 100 μl by adding the appropriate volume of TE buffer. Then equilibrate to room temperature (15–25°C) for no longer than 10 min.
[RT equilibrating]
?. [Cleaning with AMPure Beads]
Add Fully mixed 0.6 × volume of AMPure Beads (Beckman Coulter) to the solution from above step. Mix by gently pipetting entire volume up and down 10 times
♠ Take out AMPure Beads from refrigerator to sit in RT for at least 15 min.♣ Make sure AMPure Beads is full mixed before use, this will ensure the purification effect.
?. [Cleaning with AMPure Beads]
Incubate at RT for 15 min.
?. [Cleaning with AMPure Beads]
Place the tube on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant.
Be careful not to disturb the beads that contain DNA targets (Caution: do not discard beads).
?. [Cleaning with AMPure Beads]
Add 200-600 μl of freshly prepared 80% ethanol to the tube while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
?. [Cleaning with AMPure Beads]
Repeat above Step to do the ethanol clean again.
?. [Cleaning with AMPure Beads]
Put the tube back in the magnetic stand,completely remove the residual ethanol, and air dry beads for 10 minutes while the tube is on the magnetic stand with lid open.
?. [Cleaning with AMPure Beads]
Elute the DNA target from the beads with 15 μl nuclease-free water. Mix well on a vortex mixer or by pipetting up and down, and put the tube in the magnetic stand until the solution is clear.
?. [Cleaning with AMPure Beads]
Transfer the supernatant to a clean PCR tube.
?. [Cleaning with AMPure Beads]
We recommend using the transposase base library construction strategy. These kit convert the complex steps of DNA fragment, end repair and adaptor connection into a one-step enzymatic reaction, which significantly reduce the demanded amount of the initial DNA and shorten the time of library construction.
?. [for both viral DNA and RNA amplification (RT-WGA)]
Place 3 μl nucleic acid, add 2 μl Lysis Buffer. Mix by vortexing and centrifuge briefly.
?. [for both viral DNA and RNA amplification (RT-WGA)]
Incubate at 24°C for 5 min
[Lysising]
?. [for both viral DNA and RNA amplification (RT-WGA)]
Preparing Quantiscript RT mix (Table 1): Table 1. Quantiscript RT mix  AB1Component
Volume/reaction
2RT/Polymerase Buffer
2&nbsp;μl
3gDNA Wipeout Buffer
1&nbsp;μl
4H2O sc
0.5 μl
5Random Primer
0.5 μl
6Oligo dT Primer
0.5 μl
7Quantiscript RT Enzyme Mix
0.5 μl
8Total volume
5&nbsp;μl
AB1Component
Volume/reaction
2RT/Polymerase Buffer
2&nbsp;μl
3gDNA Wipeout Buffer
1&nbsp;μl
4H2O sc
0.5 μl
5Random Primer
0.5 μl
6Oligo dT Primer
0.5 μl
7Quantiscript RT Enzyme Mix
0.5 μl
8Total volume
5&nbsp;μl
♠ gDNA Wipeout buffer will be deactivate in this Mix. So the DNA is kept during RT reaction.♣ The using of both random and oligo dT Primers will improve the proformance of RT reaction.♥ NOTE: The Quantiscript RT mix must be prepared fresh.
?. [for both viral DNA and RNA amplification (RT-WGA)]
Add 5 μl RT mix to the aliquot (5 μl) in step 6. Mix by vortexing and centrifuge briefly.
?. [for both viral DNA and RNA amplification (RT-WGA)]
Incubate at 42°C for 60 min
[Reverse Transcripting]
?. [for both viral DNA and RNA amplification (RT-WGA)]
Stop the reaction by incubating at 95°C for 3 min, then cool on ice.
[Stopping RT]
?. [for both viral DNA and RNA amplification (RT-WGA)]
Prepare the ligation mix (Table 2).Table 2 Ligation mix  AB1Component
Volume/reaction
2Ligase Buffer4 μl
3Ligase Mix1 μl
4Total volume
5 μl
AB1Component
Volume/reaction
2Ligase Buffer4 μl
3Ligase Mix1 μl
4Total volume
5 μl
IMPORTANT: When preparing the ligation mix, add the components in the order shown in Table 2. NOTE: The ligation mix must be prepared fresh.
?. [for both viral DNA and RNA amplification (RT-WGA)]
Add 5 μl ligation mix to the WTA ready reaction (10 μl) from step 10. Mix by vortexing and centrifuge briefly.
?. [for both viral DNA and RNA amplification (RT-WGA)]
Incubate at 24°C for 30 min.
[Ligation]
?. [for both viral DNA and RNA amplification (RT-WGA)]
Stop the reaction by incubating at 95°C for 5 min,then cool on ice.
[Stopping Ligation]
?. [for both viral DNA and RNA amplification (RT-WGA)]
Prepare REPLI-g SensiPhi amplification mix (Table 3).Table 3 Preparing REPLI-g Sensiphi amplification mix  AB1Component
Volume/reaction
2REPLI-g sc Reaction Buffer
14.5 μl
3REPLI-g Sensiphi DNA Polymerase
0.5 μl
4Total volume
15 μl
AB1Component
Volume/reaction
2REPLI-g sc Reaction Buffer
14.5 μl
3REPLI-g Sensiphi DNA Polymerase
0.5 μl
4Total volume
15 μl
NOTE: The REPLI-g SensiPhi amplification mix must be prepared fresh.
?. [for both viral DNA and RNA amplification (RT-WGA)]
Add 15 μl REPLI-g SensiPhi amplification mix to the ligation reaction (15 μl) from step 14. Mix by vortexing and centrifuge briefly.
?. [for both viral DNA and RNA amplification (RT-WGA)]
Incubate at 30°C for 2h to amplify DNA and cDNA
[Amplifying]
?. [for both viral DNA and RNA amplification (RT-WGA)]
Stop the reaction by incubating at 65°C for 5 min.
[Stopping Amplifying]
♠ If not being used directly, store the amplified DNA at –15 ºC to –30ºC until required for downstream applications.♣ We recommend storage of the amplified DNA at a concentration of at least 100 ng/μl.
?. [Cleaning with AMPure Beads]
Adjust the volume to 100 μl by adding the appropriate volume of TE buffer. Then equilibrate to room temperature (15–25°C) for no longer than 10 min.
[RT equilibratign]
?. [Cleaning with AMPure Beads]
Add Fully mixed 0.6 × volume of AMPure Beads (Beckman Coulter) to the solution from above step. Mix by gently pipetting entire volume up and down 10 times
♠ Take out AMPure Beads from refrigerator to sit in RT for at least 15 min.♣ Make sure AMPure Beads is full mixed before use, this will ensure the purification effect.
?. [Cleaning with AMPure Beads]
Incubate at RT for 15 min.
[Incubating]
?. [Cleaning with AMPure Beads]
Place the tube on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant.
Be careful not to disturb the beads that contain DNA targets (Caution: do not discard beads).
[Incubating]
?. [Cleaning with AMPure Beads]
Add 200-600 μl of freshly prepared 80% ethanol to the tube while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
[Incubating]
?. [Cleaning with AMPure Beads]
Repeat above Step to do the ethanol clean again.
?. [Cleaning with AMPure Beads]
Put the tube back in the magnetic stand,completely remove the residual ethanol, and air dry beads for 10 minutes while the tube is on the magnetic stand with lid open.
[Air Drying]
?. [Cleaning with AMPure Beads]
Elute the DNA target from the beads with 15 μl nuclease-free water. Mix well on a vortex mixer or by pipetting up and down, and put the tube in the magnetic stand until the solution is clear.
?. [Cleaning with AMPure Beads]
Transfer the supernatant to a clean PCR tube.
?. [Library Construction]
We recommend using the transposase base library construction strategy. These kit convert the complex steps of DNA fragment, end repair and adaptor connection into a one-step enzymatic reaction, which significantly reduce the demanded amount of the initial DNA and shorten the time of library construction.
?. [for viral DNA amplification (WGA)]
Place 3 μl nucleic acid, add 2 μl Lysis Buffer. Mix by vortexing and centrifuge briefly.
?. [for viral DNA amplification (WGA)]
Preparing Quantiscript RT mix (Table 1): Table 1. Quantiscript RT mixComponent
Volume/reaction
RT/Polymerase Buffer
2&nbsp;μl
gDNA Wipeout Buffer
1&nbsp;μl
H2O sc
0.5 μl
Random Primer
0.5 μl
Oligo dT Primer
0.5 μl
Quantiscript RT Enzyme Mix
0.5 μl
Total volume
5&nbsp;μl
Component
Volume/reaction
RT/Polymerase Buffer
2&nbsp;μl
gDNA Wipeout Buffer
1&nbsp;μl
H2O sc
0.5 μl
Random Primer
0.5 μl
Oligo dT Primer
0.5 μl
Quantiscript RT Enzyme Mix
0.5 μl
Total volume
5&nbsp;μl
♠ gDNA Wipeout buffer will be deactivate in this Mix. So the DNA is kept during RT reaction.♥ NOTE: The Quantiscript RT mix must be prepared fresh.
?. [for viral DNA amplification (WGA)]
Add 5 μl RT mix to the aliquot (5 μl) in step 6. Mix by vortexing and centrifuge briefly.
?. [for viral DNA amplification (WGA)]
Incubate at 24°C for 5 min
[Lysising]
?. [for viral DNA amplification (WGA)]
Stop the reaction by incubating at 95°C for 3 min, then cool on ice.
[Stopping RT]
♥ NOTE: Comparing to the RT-WGA case, the step of "incubating 60min at 42 °C" is skipped here to avoid reverse transcription and subsequent amplification of RNA .
?. [for viral DNA amplification (WGA)]
Prepare the ligation mix (Table 2).Table 2 Ligation mixComponent
Volume/reaction
Ligase Buffer4 μl
Ligase Mix1 μl
Total volume
5 μl
Component
Volume/reaction
Ligase Buffer4 μl
Ligase Mix1 μl
Total volume
5 μl
IMPORTANT: When preparing the ligation mix, add the components in the order shown in Table 2. NOTE: The ligation mix must be prepared fresh.
?. [for viral DNA amplification (WGA)]
Add 5 μl ligation mix to the WTA ready reaction (10 μl) from step 10. Mix by vortexing and centrifuge briefly.
?. [for viral DNA amplification (WGA)]
Incubate at 24°C for 30 min.
[Ligation]
?. [for viral DNA amplification (WGA)]
Stop the reaction by incubating at 95°C for 5 min,then cool on ice.
[Stopping Ligation]
?. [for viral DNA amplification (WGA)]
Prepare REPLI-g SensiPhi amplification mix (Table 3).Table 3 Preparing REPLI-g Sensiphi amplification mixComponent
Volume/reaction
REPLI-g sc Reaction Buffer
14.5 μl
REPLI-g Sensiphi DNA Polymerase
0.5 μl
Total volume
15 μl
Component
Volume/reaction
REPLI-g sc Reaction Buffer
14.5 μl
REPLI-g Sensiphi DNA Polymerase
0.5 μl
Total volume
15 μl
NOTE: The REPLI-g SensiPhi amplification mix must be prepared fresh.
?. [for viral DNA amplification (WGA)]
Add 15 μl REPLI-g SensiPhi amplification mix to the ligation reaction (15 μl) from step 14. Mix by vortexing and centrifuge briefly.
?. [for viral DNA amplification (WGA)]
Incubate at 30°C for 2h to amplify DNA and cDNA
[Amplifying]
?. [for viral DNA amplification (WGA)]
Stop the reaction by incubating at 65°C for 5 min.
[Stopping Amplifying]
♠ If not being used directly, store the amplified DNA at –15 ºC to –30ºC until required for downstream applications.♣ We recommend storage of the amplified DNA at a concentration of at least 100 ng/μl.