TITLE: RCA-NGS for RNA viruses with ONT V14 chemistry
AUTHORS: Masayasu Misu, Tomoki Yoshikawa, Satoko Sugimoto, Yuki Takamatsu, Takeshi Kurosu, Yukiteru Ouji, Masahide Yoshikawa, Masayuki Shimojima, Hideki Ebihara, Masayuki Saijo

[DESCRIPTION]
Note that this version of the protocl was adopted to V14 chemistry of ONT.

This RCA-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 an isothermal DNA amplification technique, such as rolling circle amplification (RCA). 

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 genomic 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-PBK004/ PCS109).
 




SECTION: cSP6-polyA Linker DNA ligation
8. The linker-ligated viral RNA is purified using 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 the RNA in 10 µL 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
9. The viral RNA is reverse transcribed using   .
5′ phosphorylated SP6 primer is used for reverse transcription.
 


SECTION: The viral genomic 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 genomic 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 genomic 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 genomic 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 genomic 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 genomic RNA extraction
4.6. Add 50 µL  Elution Buffer.
 13000 x g, 1 min

 

SECTION: Remove unwanted DNA
6.1. Add an 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.
Incubate  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
9.1. Set up pre-mixture

10 µL  RNA (~ 50ng)
1 µL  50 μM SP6 primer
1 µL  nuclease-free H2O
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
9.2. Total 20 μl reaction

13 µL  pre-mixture sample
4 µL  5X SSIV Buffer
1 µL  100mM DTT
1 µL  RNase OUT -   
1 µL  SuperScript IV Reverse Transcriptase

Mix gently by flicking the tube, and spin down.
 55 °C 10 min 
 80 °C 10 min 
 


SECTION: RNase H treatment
10.  Add 1 µL    .
37 °C  20 min 

SECTION: (Optional step) Short cDNA fragment removal
12. Short cDNA fragment is removed from the viral RNA sample using   
Prepare AMpure XP beads for use; resuspend by vortexing.
Transfer amplified DNA sample to 1.5ml low binding tube.

 

SECTION: (Optional step) Short cDNA fragment removal
12.1. Add 8 µL of TE to the 12 µL of elute to adjust 20 µL .

SECTION: (Optional step) Short cDNA fragment removal
13. Size selection of the cDNA sample is performed using   .
X0.8 volume of AMPure beads recovers more than 200 bp of nucleic acids.
SECTION: (Optional step) Short cDNA fragment removal
13.1. Add 16 µL AMPure beads and mix by pipetting.
Incubate on rotor mixer.
 5 min  Room temperature 
SECTION: (Optional step) Short cDNA fragment removal
13.2. Spin down and pellet on a magnet. 
Wait for 1 min and pipette off the supernatant.
SECTION: (Optional step) Short cDNA fragment removal
13.3. Wash twice by 100 µL  70 % ethanol and remove the ethanol using a pipette and discard.
SECTION: (Optional step) Short cDNA fragment removal
13.4. Spin down and pipette off any residual ethanol.
SECTION: (Optional step) Short cDNA fragment removal
13.5. Resuspend pellet in 12 µL  nuclease-free water. 
Incubate on a rotor mixer.
 5 min Room temperature 

SECTION: (Optional step) Short cDNA fragment removal
13.6. Spin down and pellet the beads on the magnet until the elute is clear and colourless.
SECTION: (Optional step) Short cDNA fragment removal
13.7. Remove retain 12 µL  elute into a new tube.
SECTION: Circularization of cDNA
14. The cDNA is circularized using CircLigase II ssDNA Ligase - Biosearch Technologies Catalog #CL9021K.
SECTION: Circularization of cDNA
14.1. Total 20 μl reaction

12 µL  cDNA
2 µL  10X reaction buffer
1 µL  50 mM MnCl2
4 µL 5M Betaine
1 µL CircLigase II
 
Mix by pipetting and spin down.
60 °C 60 min 
80 °C 10 min 

SECTION: Circularization of cDNA
15. cDNA is purified using  
  (Add 36 µL AMPure beads)
Elute the pellet in 10 µL  nuclease-free H2O.
SECTION: Amplification of cDNA by rolling circle amplification (RCA)
16. cDNA is amplified by Rolling circle amplification (RCA) using  GenomiPhi V3 Ready-To-Go DNA Amplification Kit - Cytiva Catalog #25-6601-24. 

SECTION: T7 endonuclease treatment
19. The amplified cDNA by RCA is digested using   to remove branching.

The following protocol is modified based on the Native barcoding amplicons (with EXP-NBD104, EXPNBD114,and SQK-LSK109) protocol (NBA_9093_v109_revA_12Nov2019) provided by Oxford Nanopore Technologies website. 
SECTION: Amplification of cDNA by rolling circle amplification (RCA)
16.1. Total 20 μl reaction

10 µL  cDNA
10 µL 2X denaturation buffer

Mix by pipetting and spin down.
95 °C 3 min 
4 °C 
SECTION: Amplification of cDNA by rolling circle amplification (RCA)
16.2. Add 20 μl denatured sample to Ready to go GenomiPhi cake.

30 °C 240 min
65 °C 10 min 

SECTION: Amplification of cDNA by rolling circle amplification (RCA)
17. The cDNA is purified using   
  (Add 36 µL AMPure beads)
Elute the pellet in 40 µL  nuclease-free H2O.
SECTION: T7 endonuclease treatment
20. The cDNA is purified using   .
  (Add 30 µL AMPure beads)

Resuspend pellet in 13 µL  nuclease-free H2O. 
SECTION: DNA repair and end-prep
21. The purified cDNA is end-prepped using 
  and
 
SECTION: DNA repair and end-prep
22. The cDNA is purified using   .
  (Add 15 µL AMPure beads)

Resuspend pellet in 10 µL  nuclease-free H2O. 
SECTION: Native barcode ligation
24. The end-prepped cDNA is ligated with native barcode using Native Barcoding Kit V14 - Oxford Nanopore Technologies Catalog #SQK-NBD114.24 and   .
SECTION: Native barcode ligation
25. Add 20 µL TE(pH8.0).
SECTION: Adaptor ligation
28. Pool each barcoded sample into a  0.2ml PCR tube (Total 100–200 fmol).

 


SECTION: T7 endonuclease treatment
19.1. Total 30 μl reaction

x µL (1.0 μg) DNA
3 µL NEBuffer 2
1.5 µL T7 endonuclease I
25-x µL nuclease-free H2O

Mix by pipetting and spin down.
37 °C 30 min 
SECTION: DNA repair and end-prep
21.1. Total 15 μl reaction

12 µL DNA
0.875 µL NEB Next FFPE DNA repair buffer
0.5 µL NEB Next FFPE DNA repair Mix
0.875 µL Ultra II end-prep reaction buffer
0.75 µL Ultra II end-prep reaction Mix

Mix by pipetting and spin down.
20 °C 30 min 
65 °C 5 min 
SECTION: Native barcode ligation
24.1. Total 20 μl reaction

x µL DNA(about 400ng)
1.5 µL native barcode
10 µL Blunt/TA ligase master mix
8.5-x µL nuclease-free H2O

Mix by pipetting and spin down.
25 °C 20 min 




SECTION: Adaptor ligation
29. Adaptor Ligation with pooled samples is performed using
Ligation Sequencing Kit - Oxford Nanopore Technologies Catalog #SQK-NBD114.24 and 
  .

SECTION: Adaptor ligation
29.1. Total 20 μl reaction

x µL DNA (100-200 fmol)
2 µL Native Adapter (NA)
4 µL NEB Next Quick Ligation Reaction Buffer(5X)
2 µL Quick T4 DNA ligase
12-x µL nuclease-free H2O

mix gently and incubate. 
25 °C 20 min 
SECTION: Adaptor ligation
30. The adaptor-ligated cDNA is purified using   .
Prepare AMpure XP beads for use; resuspend by vortexing.
Transfer amplified DNA sample to 1.5ml low binding tube.

SECTION: Adaptor ligation
30.1. Add 10 µL AMPure XP reagent and mix by pipetting.
Incubate on a rotor mixer.
 5 min  Room temperature 
SECTION: Adaptor ligation
30.2. Spin down and pellet on a magnet. Wait for 1 min and pipette off the supernatant.
SECTION: Adaptor ligation
30.3. Wash twice by 100 µL Short Fragment Buffer(SFB) and remove the SFB using a pipette and discard.
SECTION: Adaptor ligation
30.4. Spin down and pipette off any residual SFB.
SECTION: Adaptor ligation
30.5. Resuspend pellet in 13 µL  Elution Buffer (EB) 
5 min Room temperature  and tapping occasionally.

SECTION: Adaptor ligation
30.6. Spin down and pellet the beads on the magnet until the elute is clear and colourless.
SECTION: Adaptor ligation
30.7. Remove retain 13 µL  elute into a new tube.
SECTION: DNA repair and end-prep
23. 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: Amplification of cDNA by rolling circle amplification (RCA)
18. 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: Native barcode ligation
27. 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.
Convert nanogram(ng) into femtomole(fmol) by a calculator. 
 

SECTION: Adaptor ligation
31. 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: Sequencing by MinION
32. Sequencing according to the manufacturer's instructions.
SECTION: cDNA purification using AMPure XP
11. cDNA is purified using   
Prepare AMpure XP beads for use; resuspend by vortexing.
Transfer amplified DNA sample to 1.5ml low binding tube.
SECTION: cDNA purification using AMPure XP
11.1. Add 36 µL AMPure XP reagent and mix by pipetting.
Incubate on rotor mixer.
 5 min  Room temperature 
SECTION: cDNA purification using AMPure XP
11.2. Spin down and pellet on a magnet. 
Wait for 1 min and pipette off the supernatant.
SECTION: cDNA purification using AMPure XP
11.3. Wash twice by 100 µL 70 % ethanol and remove the ethanol using a pipette and discard.
SECTION: cDNA purification using AMPure XP
11.4. Spin down and pipette off any residual ethanol.
SECTION: cDNA purification using AMPure XP
11.5. Resuspend pellet in 12 µL  TE(pH 8.0). 
Incubate on a rotor mixer.
 5 min  Room temperature 
SECTION: cDNA purification using AMPure XP
11.6. Spin down and pellet the beads on the magnet until the elute is clear and colourless.
SECTION: cDNA purification using AMPure XP
11.7. Remove and retain 12 µL  elute into a new tube.
SECTION: Native barcode ligation
26. The cDNA is purified using   .
  (Add 20 µL AMPure beads)

Resuspend pellet in 12 µL  nuclease-free H2O.