TITLE: Amplicon multiplex PCR sequencing of Rift Valley fever virus (RVFV) on Illumina MiSeq 
AUTHORS: John Juma, Samuel O. Oyola, Samson Konongoi, Isidore Nsengimana [Rwanda Inspectorate, Competition and Consumer Protection Authority], r.k.mwangi, James Akoko, Richard Nyamota, c.muli, e.kiritu, p.dobi, s.osiany, Amos Onwong'a, rwanja8, Rosemary Sang, Alan Christoffels, Kristina Roesel, Bernard Bett, Samuel O. Oyola

[DESCRIPTION]
Amplicon sequencing protocol for Rift Valley fever virus (RVFV)

[STEPS]
SECTION: RNA extraction
1. Extract viral RNA from serum or cell-culture supernatants using QIAamp Viral RNA kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. Begin with a volume of 140 µL 

SECTION: RT-qPCR
2. Determine cycle threshold (Ct) values on RNA samples using probe-based reverse transcription quantitative real-time PCR against the highly conserved domain on the L-segment of the virus (using 5' Fam reporter dye and 3' BHQ1 quencher dye).

 
     RVFV segment      Primer name      Sequence 5’-3’        L      RVFL-2912fwdGG      TGAAAATTCCTGAGACACATGG        L      RVFL-2981revAC      ACTTCCTTGCATCATCTGATG        L      RVFL-probe-2950      CAATGTAAGGGGCCTGTGTGGACTTGTG   
 

Mix the following components in PCR strip-tubes/plate

 


 
Incubate the reaction on a Applied Biosystems machine as follows:

50 °C for 10 min 
95 °C for 2 min 
95 °C for 3 s for 40 cycles
60 °C for 30 s 

SECTION: cDNA synthesis
3. Prepare RNA samples and include a negative control (nuclease-free water) per library. If previously frozen, mix by vortexing briefly and quick spin to collect the liquid. At all times, keep the samples on ice.
Mix the following components in PCR strip-tubes/plate. Gently mix by pipetting and performing quick spin to collect the liquid.

 


 


3. Incubate the reaction as follows:

25 °C for 2 min 
55 °C for 10 min 
95 °C for 1 min 
Hold at 4 °C 

SECTION: Primer pool preparation
4. If making up primer pools from individual oligos fully resuspend lyophilised oligos in 1xTE to a concentration of 100 micromolar (µM) , vortex thoroughly and spin down.


SECTION: Primer pool preparation
4.1. Sort all odd regions primers into one or more tube racks. Add 5 µL of each odd region primer to a 1.5 mL Eppendorf tube labelled "Pool 1 (100 micromolar (µM))". Repeat the process for all even region primers for Pool 2. These are your 100 micromolar (µM) stocks of each primer pool.


 

SECTION: Primer pool preparation
4.2. Dilute 100 micromolar (µM) pools 1:10 in molecular grade water, to generate 10 micromolar (µM) primer stocks.


 

 


SECTION: Multiplex PCR
5. 
SECTION: Multiplex PCR
5.1. Set up the two PCR reactions per sample as follows in strip-tubes or plates. Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.


 

 

SECTION: Multiplex PCR
5.2. Add 4.5 µL cDNA to each of the PCR reactions, gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.


 

SECTION: Multiplex PCR
5.3. Set-up the following program on the thermal cycler:

Step                                Temperature                        Time                            Cycles

Heat activation            98 °C                     30 s                  1
Denaturation                95 °C                     15 s                 35
Annealing                     63 °C                     5 min                 35
Hold                              4 °C                         Indefinite                         1

SECTION: Amplicon clean-up
6. Combine the two pools of amplicons:
Add 12.5 µL of each primer pool (Pool 1 and Pool 2, total of 25 µL) in new PCR strip-tubes/plate. 

Perform  NEBNext Sample Purification Beads/AMPure XP bead cleanup as follows:
SECTION: Amplicon clean-up
6.1. Add 20 µL  (0.8X) of AMPure XP beads (thoroughly vortexed and at Room temperature) to the combined amplicons plate. Cover the plate with seal, gently mix on a plate mixer and pulse spin to bring down the components at the bottom of the tube. Incubate at Room temperature for 5 min (5 minutes).

SECTION: Amplicon clean-up
6.2. Place the tube/plate on a magnetic stand for 5 min or until the beads have pelleted and the supernatant is completely clear.

SECTION: Amplicon clean-up
6.3.  Remove and discard the liquid from each well with a multichannel pippette, being careful not to touch the bead pellet. 


 

SECTION: Amplicon clean-up
6.4. Add 200 µL of freshly prepared, Room temperature 80% ethanol to each well/tube, incubate for 30 s at Room temperature and then carefully remove and discard the supernatant.


 

SECTION: Amplicon clean-up
6.5. Repeat ethanol wash (step 6.3 and 6.4).
Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
SECTION: Amplicon clean-up
6.6. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.


 

SECTION: Amplicon clean-up
6.7. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 28 µL  0.1X TE or Elution Buffer (EB).
SECTION: Amplicon clean-up
6.8. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min (2 minutes) at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

SECTION: Amplicon clean-up
6.9. Place the tube/plate on the magnetic stand. After 5 min (5 minutes) (or when the solution is clear).

SECTION: Amplicon clean-up
6.10. Transfer 25 µL to a new PCR tube, ensuring no beads are transferred. 

SECTION: Gel electrophoresis or Tapestation
7. Use remaining volumes from Pool 1 and Pool 2 to confirm amplification (step 5.3). 
SECTION: Gel electrophoresis or Tapestation
7.1. Make 1% agarose gels with enough wells for all samples. 
SECTION: Gel electrophoresis or Tapestation
7.2. Load 2 µL of the 100 bp ladder into gel on either side of each row of wells.

SECTION: Gel electrophoresis or Tapestation
7.3. Dispense 2 µL of 6X loading dye into each sample with a multichannel pipette, mix and load 2 µL 2of this mix into the gel. 

SECTION: Gel electrophoresis or Tapestation
7.4. Run at 240V for 20 min. Visualize PCR products, confirm bands of approximately 400bp size.

SECTION: Amplicon quantification
8. Quantify amplicons using Qubit dsDNA High Sensitivity kit and plate reader according to directions.
SECTION: Library preparation
9. Prepare sequencing libraries with NEBNext Ultra II RNA Library Prep kit at half volume, as follows.
SECTION: Library preparation
9.1. End-Prep

Add the following components to a sterile nuclease-free tube:


 

Set a 100 µL or 200 µL  pipette to 25 µL and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.

In a thermal cycler with lid heated to 75 °C, run the following program:

Temperature                            Time
20 °C                              30 min 
65 °C                              30 min 
4 °C                                 Indefinite

SECTION: Library preparation
9.2. Adaptor-ligation


Add the following components directly to the End Prep Reaction Mixture


 



 


SECTION: Library preparation
9.3. Set a 100 µL or 200 µL 2pipette to 40 µL and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.


 

SECTION: Library preparation
9.4. Incubate at 20 °C for 15 min (15 minutes) in a thermal cycler with the heated lid off.

SECTION: Library preparation
9.5. Add 1.5 µL of USER® Enzyme to the ligation mixture from Step 9.4.


 

SECTION: Library preparation
9.6. Mix well and incubate at 37 °C for  15 min (15 minutes) with the heated lid set to ≥ 47 °C.


 

SECTION: PCR Enrichment of Adaptor-ligated DNA
10. Follow Section 10.1. if you are using the following oligos: Use option A for any NEBNext Oligo kit where index primers are supplied in tubes. These kits have the forward and reverse primers supplied in separate tubes. Primers are supplied at 10 micromolar (µM). 

Follow Section 10.2. if you are using the following oligos: Use Option B for any NEBNext Oligo kit where index primers are supplied in a 96-well plate format. These kits have the forward and reverse (i7 and i5) primers combined. Primers are supplied at 10 micromolar (µM).



SECTION: PCR Enrichment of Adaptor-ligated DNA
10.1. Add the following components to a sterile strip tube:

Separate Forward and Reverse Primers


 


SECTION: PCR Enrichment of Adaptor-ligated DNA
10.2. Add the following components to a sterile strip tube:

Premixed Forward and Reverse Primers


 


SECTION: PCR Enrichment of Adaptor-ligated DNA
10.3. Set a 100 µL pipette to  20 µL and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.

SECTION: PCR Enrichment of Adaptor-ligated DNA
10.4. Run the PCR program to amplify the libraries:

Step                                        Temperature                                    Time                                Cycles
Initial Denaturation            98 °C                             30 s                     1
Denaturation                      98 °C                             10 s                     7
Annealing                           65 °C                             75 s                      7
Extension                           65 °C                             5 min                     1
Hold                                    4 °C                                 Indefinite



SECTION: Library Clean-up
11. Clean Up Libraries
Repeat the same clean up process as step 6 using 20 µL of AMPure beads or NEBNext Sample Purification Beads and 28 µL of Elution Buffer (EB)/ 0.1X TE.

SECTION: Library quantification and normalization
12. 
SECTION: Library quantification and normalization
12.1. Analyze 2 µL library using a Qubit dsDNA HS Assay kit

SECTION: Library quantification and normalization
12.2. Calculate the molarity value using the following formula. Use the band size from gel electrophoresis or Tapestation readings (step 7).

Library concentration (  ) / (660 g/mol   * average library size (bp)) * 10^6 



SECTION: Gel electrophoresis or Tapestation
7.5. Run pooled cDNA amplicons on a TapeStation® without cleanup. To run on a TapeStation, dilute an aliquot of the pooled amplicons 10-fold with 0.1X TE Buffer and run 2 µL on a DNA High Sensitivity ScreenTape.

SECTION: Library quantification and normalization
12.3. Normalize each library by dilution with nuclease free water. 
SECTION: Library quantification and normalization
12.4. Pool equal volume (e.g. 5 µL ) from each of the normalized libraries into a single 1.5 mL Eppendorf tube.

SECTION: Sequencing
13. Denature and load pooled libraries as follows:
SECTION: Sequencing
13.1. Denature the pooled libraries by mixing 5 µL of pooled libraries and 5 µL of 0.2N NaOH solution.

SECTION: Sequencing
13.2. Incubate for 5 min (5 minutes)

SECTION: Sequencing
13.3. Add 990 µL of HT1 buffer and mix well with denatured pooled library by pipetting up and down 10 times with P1000. 

SECTION: Sequencing
13.4. Load 600 µL of the denatured, diluted pooled library into the loading position of the Illumina reagent cartridge (V2, 300 cycle kit). Load reagent cartridge, flow cell, and PR2 buffer into Miseq instrument, confirm the metrics and start the run.