Jan 11, 2023
MinION Sequencing protocol for Rabies virus of Arctic lineage
- pramadaprasad1,
- Harsha P.K.1,
- Chitra Pattabiraman1
- 1National Institute of Mental Health and Neuro Sciences
Protocol Citation: pramadaprasad, Harsha P.K., Chitra Pattabiraman 2023. MinION Sequencing protocol for Rabies virus of Arctic lineage. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4jzb8lo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 23, 2022
Last Modified: January 11, 2023
Protocol Integer ID: 74417
Keywords: MinION, Sequencing, Rabies
Abstract
An ONT MinION-based sequencing protocol to retrieve whole genomes of Rabies virus of the Artic lineage. Primers were selected using PrimalScheme (https://primalscheme.com/) and theprotocol was modified from the Zika and SARS-CoV-2 sequencing protocols using the same tool.
Protocol materials
NEBNext Ultra II End Prep Enzyme MixNew England BiolabsCatalog #E7646
Step 11
Native Barcoding Expansion 1-12 (PCR-free)Catalog #EXP-NBD104
Step 12
NEB Blunt/TA Ligase Master Mix Catalog #M0367
Step 12
Flow Cell Priming Kit Oxford Nanopore TechnologiesCatalog #EXP-FLP002
Step 15.2
LunaScript RT SuperMix Kit - 100 rxnsNew England BiolabsCatalog #E3010L
Step 2.1
QIAamp Viral RNA Mini KitQiagenCatalog #52904
Step 1
Q5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L
Step 6
AMPure XP beadsBeckman Coulter
Step 10
Qubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
In 4 steps
NEBNext Ultra II End Prep Reaction BufferNew England BiolabsCatalog #E7647
Step 11
Native Barcoding Expansion 13-24 (PCR-free)Oxford Nanopore TechnologiesCatalog #EXP-NBD114
Step 12
Ligation sequencing kit 1DOxford Nanopore TechnologiesCatalog #SQK-LSK109
Step 15.2
ONT MinION Flow Cell R9.4.1Oxford Nanopore TechnologiesCatalog #FLO-MIN106D
Step 15.2
RNA Extraction
RNA Extraction
Extract and purify RNA from Brain tissue homogenate using QIAamp Viral RNA Mini KitQiagenCatalog #52904 .
Note
Removal of genomic DNA by on column DNase treatment (optional)
Genomic DNA can be removed by on column DNase I (RNase-free) - 5,000 unitsNew England BiolabsCatalog #M0303L treatment for 30 min at room temperature before the wash steps.
cDNA Preparation
cDNA Preparation
20m
20m
Note
Extracted RNA samples are mixed thoroughly by brief vortex and spin down to collect all liquid at the bottom.
20m
Prepare the master mix for cDNA Prep as follows in a PCR plate/ tubes for 10μL reaction.
| A | B | |
| Component | Volume | |
| Lunascrpit RT Supermix (5X) | 2µl | |
| Template | 8µl | |
| Total | 10 µl |
LunaScript RT SuperMix Kit - 100 rxnsNew England BiolabsCatalog #E3010L
Note
Prepare all reagents in a biosafety cabinet designated for clean work.
Incubate the reaction mix following the conditions mentioned below in a thermo cycler:
25 °C for 00:02:00
55 °C for 00:10:00
95 °C for 00:01:00
Hold at 4 °C
Amplicon PCR
Amplicon PCR
5h
5h
Primers are designed using Primal Scheme. Resuspend the lyophilized oligos fully in Nuclease free water / 1X TE to a concentration of 100µM, vortex thoroughly,Nuclease-free, and spin down.
RABV_Arctic_primers.csv 5h
Separate odd and even primer sets. Add 5µl of each odd primer to a 1.5mL LoBind tube labelled Pool 1. Repeat the same for all even primers for Pool 2. These are the 100µM stocks of each primer pool.
Dilute 100µM stock pools (1:10) in Nuclease free water, to generate 10µM primer stock. A final concentration of 15 nM per primer is used. Here we use a total of 39 primers in Pool 1 and 39 primers in Pool 2. Here 0.75µl of per primer pool (10µM) is required for a 25µl reaction.
Set up two PCR reactions per sample (for Pool 1 and 2) as follows in tubes or plates. Mix by gentle pipetting and pulse spin to collect liquid at the bottom.
| A | B | C | |
| Component | Reaction Mix 1 | Reaction Mix 2 | |
| 5X Q5 Reaction Buffer | 5µL | 5µL | |
| 10 mM dNTPs | 0.5µL | 0.5µL | |
| Q5 Hot Start DNA Polymerase | 0.25µL | 0.25µL | |
| Pool 1 (10µM) | 0.75µL | 0µL | |
| Pool 2 (10µM) | 0µL | 0.75µL | |
| Nuclease-free water | 18.5µL | 18.5µL |
Q5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L
Add 2.5µl cDNA to each of the PCR reactions, mix by gentle pipetting and spin the tube to collect all liquid at the bottom of the tube.
Set up the following PCR conditions in the thermal cycler.
Stage 0 °C 00:00:00 Cycles
Heat Activation 98 °C 00:00:30 1
Denaturation 98 °C 00:00:15 25-35
Annealing 65 °C 00:05:00 25-35
Hold 4 °C Indefinite 1
CITATION
PCR clean-up
PCR clean-up
Label the tubes/plates and pool the PCR reactions for each samples into a single LoBind tube .
| A | B | |
| PCR pool 1 | 25µL | |
| PCR pool 2 | 25µL | |
| Nuclease Free water | 0µL | |
| Total | 50µL |
Note
When higher viral loads are anticipated based on Ct values < 20 then reduce the amount of amplicons to 15-20µL each and make up the volume to 50µl with Nuclease free water.
Clean-up the amplicons using AMPure XP beadsBeckman Coulter .
Thaw the AMPure beads to room temperature and vortex thoroughly to ensure that the beads are well suspended/ until the solution is uniformly brown in colour.
Add 0.4x volume (0.4:1) of AMPure beads to the sample tube and mix gently by either flicking or pipetting. For example add 20,µl AMPure beads to a 50 µl reaction.
Spin the tubes to collect entire liquid at the bottom and incubate the tubes at room temperature for 00.05.00.
Place the tubes on magnetic stand and incubate for 00:02:00 or until the beads have settled and the supernatant is completely colourless.
Carefully remove and discard the supernatant, without disturbing the bead pellet.
Add 200µl of 70% ethanol or enough to cover the pellet and wait for 30s.Carefully remove and discard ethanol delicately without disrupting the bead pellet.
and repeat Spin to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible.
Incubate the tube with open lid for 00:00:30 or until the pellet is dried (if the pellet dries completely it will crack and become difficult to resuspend)
Resuspend pellet in 30µl Elution Buffer (EB), mix gently by either flicking or pipetting and incubate for 00:02:00.Make sure that the pellet is completely resuspended.
Place on magnetic stand and transfer sample to a clean 1.5mL LoBind tube ensuring no beads are transferred into this tube.
Quantify 1µl product using the Qubit Fluorometer using the Qubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854 .
Qubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
End-Prep Reaction
End-Prep Reaction
40m
40m
In a new PCR plate/ tubes set up the following reaction for each sample. Prepare Master mix and aliquote it into tubes . If the Qubit reading is > 15ng/ul ,
| A | B | |
| Component | Volume | |
| PCR dilution from previous step | 3.3µL | |
| Ultra II End Prep Reaction Buffer | 1.2µL | |
| Ultra II End Prep Enzyme Mix | 0.5µL | |
| Nuclease-free water | 5µL | |
| Total | 10µL |
If the Qubit reading is 5ng/ul -15ng/ul
| A | B | |
| Component | Volume | |
| PCR dilution from previous step | 5µL | |
| Ultra II End Prep Reaction Buffer | 1.2µL | |
| Ultra II End Prep Enzyme Mix | 0.5µL | |
| Nuclease-free water | 3.3µL | |
| Total | 10µL |
If the Qubit reading is <5ng/ul,
| A | B | |
| Component | Volume | |
| PCR dilution from previous step | 8.3µL | |
| Ultra II End Prep Reaction Buffer | 1.2µL | |
| Ultra II End Prep Enzyme Mix | 0.5µL | |
| Nuclease-free water | 0µL | |
| Total | 10µL |
NEBNext Ultra II End Prep Reaction BufferNew England BiolabsCatalog #E7647 NEBNext Ultra II End Prep Enzyme MixNew England BiolabsCatalog #E7646
Reaction conditions for End Prep:
Incubate
25 °C for 00:15:00
65 °C for 00:15:00
On ice for 00:01:00
Native Barcoding
Native Barcoding
In a new labelled tube/plate prepare the following components.
| A | B | |
| Component | Volume | |
| End-preparation reaction mixture | 0.75µL | |
| NBXX barcode | 1.25µL | |
| Blunt/TA Ligase Master Mix | 5µL | |
| Nuclease-free water | 3µL | |
| Total | 10µL |
Native Barcoding Expansion 1-12 (PCR-free)Catalog #EXP-NBD104 Native Barcoding Expansion 13-24 (PCR-free)Oxford Nanopore TechnologiesCatalog #EXP-NBD114
NEB Blunt/TA Ligase Master Mix Catalog #M0367
Incubate at
25 °C for 00:20:00
65 °C for 00:10:00
On ice for 00:01:00
In a new 1.5mL LoBind tube pool all barcoded reactions together.
Add 0.4x volume of AMPure beads to the sample tube and mix by either flicking or gentle pipetting. For example add 96µl AMPure beads to 240µl pooled barcoding reactions.
Briefly vortex and mix the reactions and incubate at room temperature for 00.05.00.
Place on magnetic stand and incubate for 00:02:00 or until the beads have pelleted and the supernatant is completely clear. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
Remove the tube from the magnetic stand and add 200µl SFB and resuspend beads completely using pipette. Spin down to collect liquid at the bottom of the tube and place on the magnetic stand. Remove supernatant and discard.
Add 200µl of 70% ethanol to wash the pellet. Carefully remove and discard ethanol, being careful not to touch the bead pellet.
Spin to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible.
With the tube lid open incubate for 00:00:30 or until the pellet is dried (if the pellet dries completely it will crack and become difficult to resuspend).
Resuspend pellet in 30µl Elution Buffer(EB), mix gently by either flicking or pipetting and incubate for 00:02:00.
Place on magnet stand and transfer sample to a clean 1.5mL Eppendorf tube ensuring no beads are transferred into this tube.
Quantify 1µl of the barcoded amplicons using the Qubit Flurometer.
Equipment
Qubit
NAME
Flurometer
TYPE
Invitrogen
BRAND
Q33228
SKU
LINK
Qubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
Adapter Ligation
Adapter Ligation
25m
25m
Prepare the following reaction for AMII Adapter ligation in a PCR tube.
| A | B | |
| Component | Volume | |
| Barcoded amplicon pool | 30µL | |
| NEBNext Quick Ligation Reaction Buffer (2X) | 40µL | |
| Adapter Mix (AMII) | 5µL | |
| Quick T4 DNA Ligase | 5µL | |
| Total | 80µL |
Incubate at 25 °C for 00:20:00, possibly in a thermo cycler.
Post adapter ligation Clean-up
Post adapter ligation Clean-up
Add 80µl (1:1) of AMPure beads to the sample tube and mix gently by either flicking or pipetting. Spin to collect all liquid at the bottom of the tube.
Incubate for 00:05:00 at room temperature.
Place on magnetic rack and incubate for 00:02:00 or until the beads have pelleted and the supernatant is completely clear. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
Add 200µl SFB and resuspend beads completely by pipette mixing. Brief spin to collect all liquid at the bottom of the tube. Remove supernatant and discard.
Repeat step 14.3 to perform a second SFB wash.
Brief spin and remove any residual SFB. Add 15µl EB (ONT) and resuspend beads by gentle flicking or pipette mixing.
Incubate at room temperature for 00:02:00 and place on magnetic stand until clear. Transfer final library to a new 1.5mL LoBind tube
Quantify 1µl of the barcoded amplicons using the Qubit Fluorometer.
Equipment
Qubit
NAME
Flurometer
TYPE
Invitrogen
BRAND
Q33228
SKU
LINK
Qubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
Flow cell Priming and Sequencing
Flow cell Priming and Sequencing
Thaw the following reagents.
Sequencing buffer (SQB)
Loading beads (LB)
Flush buffer (FB)
Flush tether (FLT)
Add 30µl FLT to the FB tube and mix well by vortex and spin down.
Prepare the following library dilution for sequencing in a LoBind tube:
| A | B | |
| SQB | 37.5µL | |
| LB | 25.5µL | |
| Library | 12-14µL | |
| Total | 75-80µL |
Ligation sequencing kit 1DOxford Nanopore TechnologiesCatalog #SQK-LSK109 Flow Cell Priming Kit Oxford Nanopore TechnologiesCatalog #EXP-FLP002 ONT MinION Flow Cell R9.4.1Oxford Nanopore TechnologiesCatalog #FLO-MIN106D
Flowcell Priming, loading of the library and sequencing are performed based on the following protocol:
Citations
Step 8
Quick J, Grubaugh ND, Pullan ST, Claro IM, Smith AD, Gangavarapu K, Oliveira G, Robles-Sikisaka R, Rogers TF, Beutler NA, Burton DR, Lewis-Ximenez LL, de Jesus JG, Giovanetti M, Hill SC, Black A, Bedford T, Carroll MW, Nunes M, Alcantara LC Jr, Sabino EC, Baylis SA, Faria NR, Loose M, Simpson JT, Pybus OG, Andersen KG, Loman NJ. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples.
https://doi.org/10.1038/nprot.2017.066