RCA-NGS for RNA viruses with ONT V14 chemistry
Tomoki Yoshikawa, Masayasu Misu, Satoko Sugimoto, Yuki Takamatsu, Takeshi Kurosu, Yukiteru Ouji, Masahide Yoshikawa, Masayuki Shimojima, Hideki Ebihara, Masayuki Saijo
Abstract
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:
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removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment
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the terminal of viral genome sequence determination by barcoded linkers ligation
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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
Preparation for virus supernatant
Centrifuge the working stock virus to remove debris.
6000x g
Transfer 180µL virus supernatant to a 1.5ml screw cap tube.
Unwanted DNA and RNA mainly originating from the virus-infected cells are digested using
Total 201 μl reaction
180µLvirus supernatant20µL10X Micrococcal Nuclease Reaction Buffer1µLMicrococcal nuclease
Mix by pipetting and spin down.
37°C 1h 0m 0s
The viral genomic RNA extraction
The viral genomic RNA extraction is performed using
Add 400µL of binding buffer (with 4µL PolyA carrier RNA).
Mix gently by ~5 times pipetting and flicking thoroughly the tube, and spin down.
Room temperature 0h 10m 0s
Transfer the sample to a High Pure Filter Tube.
8000x g
Discard the flow-through liquid and Collection Tube, and insert the Filter Tube into a new Collection Tube.
Add 500µL of inhibitor removal bo transfer the sample to a High Pure Filter Tube.
8000x g
Discard the flow-through liquid and Collection Tube, and insert the Filter Tube into a new Collection Tube.
Add 450µL of wash buffer.
8000x g
Discard the flow-through liquid and Collection Tube, and insert the Filter Tube into a new Collection Tube.
Add 450µL of wash buffer.
13000x g and discard the flow-through liquid.
Discard the Collection Tube and insert the Filter Tube into a 1.5 ml tube -
Add 50µL Elution Buffer.
13000x g
Remove unwanted DNA
Unwanted DNA mainly from the virus-infected cells in the RNA sample is digested using a
Total 56 μl reaction
50µLthe eluted RNA5µL10X reaction buffer1µLDNase I
Mix gently by pipetting and spin down.
37°C 0h 30m 0s
The viral RNA is purified using NucleoSpin RNA Clean-up XS - Takara, Catalog #740903.10.
Add an equal volume 56µL of Buffer RCU and mix gently.
Transfer the sample to a NucleoSpin RNA XS Column.
11000x g
Wash the column by 400µL Buffer RA3.
11000x g
Discard the flow-through liquid and Collection Tube, and insert the NucleoSpin RNA XS Column into a new Collection Tube.
Wash the column by 200µL Buffer RA3.
11000x g
Discard the flow-through liquid and Collection Tube, and insert the NucleoSpin RNA XS Column into a Nuclease-free Collection Tube(1.5 ml).
Add 10µL RNase-free H2O.
11000x g
Transfer the sample to a 0.2 ml PCR tube -
cSP6-polyA Linker DNA ligation
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).
Total 20 μl reaction
10µLPurified RNA1µL10 μM the cSP6-polyA linker DNA2µL10X T4 RNA Ligase Reaction Buffer6µL50% PEG8000 solution1µLT4 RNA Ligase 2, truncated KQ
Mix gently by pipetting and spin down.
Incubate 25°C 0h 15m 0s
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 10µL RNase-free H2O and transfer the sample to a 0.2 ml PCR tube.
Reverse transcription
The viral RNA is reverse transcribed using
5′ phosphorylated SP6 primer is used for reverse transcription.
Set up pre-mixture
10µLRNA (~ 50ng)1µL50 μM SP6 primer1µLnuclease-free H2O1µL10mM dNTP -
Mix gently by flicking the tube, and spin down.
65°C 0h 5m 0s and 4°C 0h 1m 0s
Total 20 μl reaction
13µLpre-mixture sample4µL5X SSIV Buffer1µL100mM DTT1µLRNase OUT -1µLSuperScript IV Reverse Transcriptase
Mix gently by flicking the tube, and spin down.
55°C 0h 10m 0s
80°C 0h 10m 0s
RNase H treatment
Add 1µL 37°C 0h 20m 0s
cDNA purification using AMPure XP
cDNA is purified using
Prepare AMpure XP beads for use; resuspend by vortexing.
Transfer amplified DNA sample to 1.5ml low binding tube.
Add 36µL AMPure XP reagent and mix by pipetting.
Incubate on rotor mixer.
0h 5m 0s Room temperature
Spin down and pellet on a magnet.
Wait for 0h 1m 0s and pipette off the supernatant.
Wash twice by 100µL 70 % ethanol and remove the ethanol using a pipette and discard.
Spin down and pipette off any residual ethanol.
Resuspend pellet in 12µL TE(pH 8.0).
Incubate on a rotor mixer.
`0h 5m 0s` `Room temperature`
Spin down and pellet the beads on the magnet until the elute is clear and colourless.
Remove and retain 12µL elute into a new tube.
(Optional step) Short cDNA fragment removal
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.
Add 8µL of TE to the 12µL of elute to adjust 20µL .
Size selection of the cDNA sample is performed using
X0.8 volume of AMPure beads recovers more than 200 bp of nucleic acids.
Add 16µL AMPure beads and mix by pipetting.
Incubate on rotor mixer.
0h 5m 0s Room temperature
Spin down and pellet on a magnet.
Wait for 0h 1m 0s and pipette off the supernatant.
Wash twice by 100µL 70 % ethanol and remove the ethanol using a pipette and discard.
Spin down and pipette off any residual ethanol.
Resuspend pellet in 12µL nuclease-free water.
Incubate on a rotor mixer.
`0h 5m 0s` `Room temperature`
Spin down and pellet the beads on the magnet until the elute is clear and colourless.
Remove retain 12µL elute into a new tube.
Circularization of cDNA
The cDNA is circularized using CircLigase II ssDNA Ligase - Biosearch Technologies Catalog #CL9021K.
Total 20 μl reaction
12µLcDNA2µL10X reaction buffer1µL50 mM MnCl24µL5M Betaine1µLCircLigase II Mix by pipetting and spin down.
60°C 1h 0m 0s
80°C 0h 10m 0s
cDNA is purified using
(Add 36µL AMPure beads)
Elute the pellet in 10µL nuclease-free H2O.
Amplification of cDNA by rolling circle amplification (RCA)
cDNA is amplified by Rolling circle amplification (RCA) using GenomiPhi V3 Ready-To-Go DNA Amplification Kit - Cytiva Catalog #25-6601-24.
Total 20 μl reaction* 10µL cDNA
10µL2X denaturation buffer Mix by pipetting and spin down.95°C0h 3m 0s4°C
Add 20 μl denatured sample to Ready to go GenomiPhi cake.30°C 4h 0m 0s``65°C 0h 10m 0s
The cDNA is purified using
(Add 36µL AMPure beads)
Elute the pellet in 40µL nuclease-free H2O.
DNA concentration is measured using a Qubit 4 Fluorometer with
199µL1X working solution1µLDNA
Mix by vortexing.
Incubate 0h 2m 0s Room temperature and measure.
T7 endonuclease treatment
The amplified cDNA by RCA is digested using
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.
Total 30 μl reaction* xµL (1.0 μg) DNA
3µLNEBuffer 21.5µLT7 endonuclease I25-xµLnuclease-free H2O Mix by pipetting and spin down.37°C0h 30m 0s
The cDNA is purified using
(Add 30µL AMPure beads)
Resuspend pellet in 13µL nuclease-free H2O.
DNA repair and end-prep
The purified cDNA is end-prepped using
Total 15 μl reaction
12µLDNA0.875µLNEB Next FFPE DNA repair buffer0.5µLNEB Next FFPE DNA repair Mix0.875µLUltra II end-prep reaction buffer0.75µLUltra II end-prep reaction Mix
Mix by pipetting and spin down.
20°C 0h 30m 0s
65°C 0h 5m 0s
The cDNA is purified using
(Add 15µL AMPure beads)
Resuspend pellet in 10µL nuclease-free H2O.
DNA concentration is measured using a Qubit 4 Fluorometer with
199µL1X working solution1µLDNA
Mix by vortexing.
Incubate 0h 2m 0s Room temperature and measure.
Native barcode ligation
The end-prepped cDNA is ligated with native barcode using Native Barcoding Kit V14 - Oxford Nanopore Technologies Catalog #SQK-NBD114.24 and
Total 20 μl reaction
xµLDNA(about 400ng)1.5µLnative barcode10µLBlunt/TA ligase master mix8.5-xµLnuclease-free H2O Mix by pipetting and spin down.25°C0h 20m 0s
Add 20µL TE(pH8.0).
The cDNA is purified using
(Add 20µL AMPure beads)
Resuspend pellet in 12µL nuclease-free H2O.
DNA concentration is measured using a Qubit 4 Fluorometer with
199µL1X working solution1µLDNA
Mix by vortexing.
Incubate 0h 2m 0s Room temperature and measure.
Convert nanogram(ng) into femtomole(fmol) by a calculator.
Adaptor ligation
Pool each barcoded sample into a 0.2ml PCR tube (Total 100–200 fmol).
Adaptor Ligation with pooled samples is performed using Ligation Sequencing Kit - Oxford Nanopore Technologies Catalog #SQK-NBD114.24 and
Total 20 μl reaction* xµL DNA (100-200 fmol)
2µLNative Adapter (NA)4µLNEB Next Quick Ligation Reaction Buffer(5X)2µLQuick T4 DNA ligase12-xµLnuclease-free H2O mix gently and incubate.25°C0h 20m 0s
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.
Add 10µL AMPure XP reagent and mix by pipetting.
Incubate on a rotor mixer.
0h 5m 0s Room temperature
Spin down and pellet on a magnet. Wait for 0h 1m 0s and pipette off the supernatant.
Wash twice by 100µL Short Fragment Buffer(SFB) and remove the SFB using a pipette and discard.
Spin down and pipette off any residual SFB.
Resuspend pellet in 13µL Elution Buffer (EB) 0h 5m 0s Room temperature and tapping occasionally.
Spin down and pellet the beads on the magnet until the elute is clear and colourless.
Remove retain 13µL elute into a new tube.
DNA concentration is measured using a Qubit 4 Fluorometer with
199µL1X working solution1µLDNA
Mix by vortexing.
Incubate 0h 2m 0s Room temperature and measure.
Sequencing by MinION
Sequencing according to the manufacturer's instructions.
