TITLE: PCR-NGS for RNA viruses
AUTHORS: Masayasu Misu, Tomoki Yoshikawa, Satoko Sugimoto, Yuki Takamatsu, Takeshi Kurosu, Yukiteru Ouji, Masahide Yoshikawa, Masayuki Shimojima, Hideki Ebihara, Masayuki Saijo

[DESCRIPTION]
This PCR-NGS were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. 

These methods enable whole RNA viral genome sequencing by combining the following techniques:
 1) Removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment.
 2) The terminal of viral genome sequence determination by barcoded linkers ligation.
 3) Amplification of the viral genomic cDNA using ligated linker sequences-specific PCR. 

This method can be exploited to determine any whole RNA viral genomes (i.e., single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes).



[BEFORE_START]
This method was only confirmed to work with the working stocks that contain isolated RNA viruses at least 3.0 × 105  TCID50 per ml.
It is recommended to check no bacterial contamination(e.g., Mycoplasma spp.).

[STEPS]
SECTION: Preparation for virus supernatant
1. Centrifuge the working stock virus to remove debris.
 6000 x g, 10 min 

SECTION: Preparation for virus supernatant
3. Unwanted DNA and RNA mainly originating from the virus-infected cells are digested using . 
SECTION: The viral RNA extraction
4. The viral genomic RNA extraction is performed using   .
SECTION: Remove unwanted DNA
5. Unwanted DNA mainly from the virus-infected cells in the RNA sample is digested using a   .
SECTION: cSP6-polyA Linker DNA ligation
7. 
The viral RNA is ligated with cSP6-polyA Linker DNA using  .
The RNA is ligated to the 3' end with the barcoded(complementary sequence of SP6 (cSP6)) polyA linker DNA. It is able to identify the 3’ terminal viral genome sequence. The PolyA sequence is required for reverse transcription for ONT kit (SQK-SQK-PBK004/PCS109).
 




SECTION: cSP6-polyA Linker DNA ligation
8. The viral RNA purification by NucleoSpin RNA Clean-up XS - Takara, Catalog #740903.10.
  

Fill the sample to 100 μl with 80 μl TE (pH 8.0) and add 100 μl (equal volume) of Buffer RCU.

Eluted by 10 μl of RNase-free H2O and transfer the sample to a 0.2 ml PCR tube.
SECTION: Remove unwanted DNA
6. The viral RNA is purified using NucleoSpin RNA Clean-up XS - Takara, Catalog #740903.10.
SECTION: Reverse transcription with strand-switching,  SQK-PBK004/ PCS109
9. The viral RNA is reverse transcribed using Maxima H Minus Reverse Transcriptase - Life Technologies, Catalog #EP0752, PCR barcoding kit - Oxford Nanopore Technologies Catalog #SQK-PBK004, cDNA-PCR Sequencing kit - Oxford Nanopore Technologies Catalog #SQK-PCS109.

The following protocol is modified based on the cDNA-PCR Sequencing protocol (PCSB_9086_v109_revK_14Aug2019) provided by Oxford Nanopore Technologies website. 

 


SECTION: PCR with barcoding
10. PCR enzyme; 
KOD One PCR Master Mix - TOYOBO Catlog #KMM-101
               or
  


SECTION: PCR with barcoding
11. Add 1 µL  .

37 °C 15 min 
80 °C 15 min 
 
SECTION: PCR with barcoding
12. The PCR product is purified using  .
Prepare AMpure XP reagent for use; resuspend by vortexing.
Transfer amplified DNA sample to 1.5ml low binding tube.

SECTION: PCR with barcoding
13. DNA concentration is measured using a Qubit 4 Fluorometer with  .

199 µL 1X working solution
1 µL  DNA

Mix by vortexing.

Incubate 2 min Room temperature and measure.

 

SECTION: The viral RNA extraction
4.1. Add 400 µL of binding buffer (with 4 µL  PolyA carrier RNA).

Mix gently by ~5 times pipetting and flicking thoroughly the tube, and spin down.
Room temperature 10 min 
SECTION: The viral RNA extraction
4.2. Transfer the sample to a High Pure Filter Tube.
 8000 x g, 1 min 

Discard the flow-through liquid and Collection Tube, and insert the Filter Tube into a new Collection Tube. 
SECTION: The viral RNA extraction
4.3. Add 500 µL  of  inhibitor removal bo transfer the sample to a High Pure Filter Tube.
 8000 x g, 1 min 

Discard the flow-through liquid and Collection Tube, and insert the Filter Tube into a new Collection Tube. 
SECTION: The viral RNA extraction
4.4. Add 450 µL  of  wash buffer.
 8000 x g, 1 min 

Discard the flow-through liquid and Collection Tube, and insert the Filter Tube into a new Collection Tube. 
SECTION: The viral RNA extraction
4.5. Add 450 µL  of  wash buffer.
 13000 x g, 1 min and discard the flow-through liquid.

Discard the Collection Tube and insert the Filter Tube into a 1.5 ml tube(  ).  
SECTION: The viral RNA extraction
4.6. Add 50 µL  Elution Buffer.
 13000 x g, 1 min

 

SECTION: Remove unwanted DNA
6.1. Add equal volume 56 µL  of Buffer RCU and mix gently.
SECTION: Remove unwanted DNA
6.4. Wash the column by 200 µL Buffer RA3.
 11000 x g, 2 min

Discard the flow-through liquid and Collection Tube, and insert the NucleoSpin RNA XS Column into a Nuclease-free Collection Tube(1.5 ml).
SECTION: Remove unwanted DNA
6.2. Transfer the sample to a NucleoSpin RNA XS Column.
 11000 x g, 1 min 
SECTION: Remove unwanted DNA
6.3. Wash the column by 400 µL Buffer RA3.
 11000 x g, 1 min

Discard the flow-through liquid and Collection Tube, and insert the NucleoSpin RNA XS Column into a new Collection Tube. 
SECTION: Remove unwanted DNA
6.5. Add 10 µL  RNase-free H2O.
 11000 x g, 1 min
Transfer the sample to a 0.2 ml PCR tube -  .
SECTION: cSP6-polyA Linker DNA ligation
7.1. Total 20 μl reaction

10 µL Purified RNA
1 µL 10 μM the cSP6-polyA linker DNA
2 µL 10X T4 RNA Ligase Reaction Buffer
6 µL 50% PEG8000 solution
1 µL T4 RNA Ligase 2, truncated KQ

Mix gently by pipetting and spin down.
Incubation  25 °C 15 min 

SECTION: Preparation for virus supernatant
2. Transfer 180 µL virus supernatant to a 1.5ml screw cap tube.
SECTION: Preparation for virus supernatant
3.1. Total 201 μl reaction

180 µL virus supernatant
20 µL 10X Micrococcal Nuclease Reaction Buffer
1 µL  Micrococcal nuclease

Mix by pipetting and spin down.
37 °C 60 min 


SECTION: Remove unwanted DNA
5.1. Total 56 μl reaction

50 µL the eluted RNA
5 µL  10X reaction buffer
1 µL  DNase I

Mix gently by pipetting and spin down.
37 °C 30 min 
SECTION: Reverse transcription with strand-switching,  SQK-PBK004/ PCS109
9.1. Set up pre-mixture 1

9 µL  RNA (~ 50ng)
1 µL  VN primer (VNP)
1 µL  10mM dNTP -   

Mix gently by flicking the tube, and spin down.
65 °C 5 min  and 4 °C 1 min 


SECTION: Reverse transcription with strand-switching,  SQK-PBK004/ PCS109
9.3. Add 1 µL  Maxima H Minus Reverse Transcriptase and mix gently by flicking the tube, and spin down. (Total 20 μl reaction).

42 °C 90 min 
85 °C 5 min 


SECTION: Reverse transcription with strand-switching,  SQK-PBK004/ PCS109
9.2. Set up pre-mixture 2

11 µL pre-mixture 1
4 µL  5X RT buffer
1 µL  nuclease-free H2O
1 µL  RNase OUT -   
2 µL  Strand-Switching Primer(SSP)

Mix gently by flicking the tube, and spin down.
 42 °C 2 min 


SECTION: PCR with barcoding
10.1. PCR reaction is as follows:

5 µL  cDNA
3 µL  LWB (barcoding primer)
42 µL  nuclease-free water
50 µL  PCR enzyme (KOD One / Q5)
The reaction mix should be aliquoted in appropriate portions in accordance with the PCR machine used.

<KOD One PCR Master Mix>
Step                                                 Temperature               Time                       
Heat Activation               98 °C        15 s               

30 cycles of 3 steps
Denaturation                   98 °C        10 s                  
Annealing                        62 °C        5 s     
Extension                        68 °C         35sec or 5 sec/kb           
                                         68 °C        2 min   

 
                                        


<Q5 Hot Start High-Fidelity 2X Master Mix>
Step                                                 Temperature               Time                       
Heat Activation               98 °C        30 s               

30 cycles of 3 steps
Denaturation                   98 °C        10 s              
Annealing                        72 °C        10 s        
Extension                        72 °C         40 sec/kb          
                                         72 °C        2 min      
                                        


SECTION: Adaptor Ligation
14. Add 1 µL  of Rapid Adaptor (RAP)(SQK-PBK004, SQK-PCS109) to 11 µL  library DNA(total approximately 100 fmol).

Mix gently and incubate 25 Room temperature 5 min.

SECTION: PCR with barcoding
12.1. Add 80 µL AMPure XP reagent and mix by pipetting.
Incubate on rotor mixer.
 5 min  Room temperature 

SECTION: PCR with barcoding
12.4. Spin down and pipette off any residual ethanol.
SECTION: PCR with barcoding
12.2. Spin down and pellet on a magnet. Wait for 1 min and pipette off the supernatant.
SECTION: PCR with barcoding
12.6. Spin down and pellet the beads on the magnet until the elute is clear and colourless.
SECTION: PCR with barcoding
12.3. Wash three times by 200 µL  70 % ethanol and remove the ethanol using a pipette and discard.
SECTION: PCR with barcoding
12.5. Resuspend pellet in 12 µL  Elution Buffer (EB). 
37 °C 3 min  and tapping occasionally.
Incubate on rotor mixer.
 7 min
SECTION: PCR with barcoding
12.7. Remove retain 12 µL  elute into a new tube.

SECTION: Sequencing by MinION
15. Sequencing according to the manufacturer's instructions.