TITLE: MinION Sequencing protocol for Rabies virus of Arctic lineage
AUTHORS: pramadaprasad, Harsha P.K., Chitra Pattabiraman

[DESCRIPTION]
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. 





[STEPS]
SECTION: RNA Extraction
1. Extract and purify RNA from Brain tissue homogenate using  .
 
 

SECTION: Amplicon PCR
3. 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.

  

SECTION: Amplicon PCR
4. 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.
SECTION: Amplicon PCR
5. 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.
SECTION: Amplicon PCR
6. 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. 


 
  


SECTION: Amplicon PCR
7. 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.
SECTION: Amplicon PCR
8. Set up the following PCR conditions in the thermal cycler.

 Stage                              °C                                       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

 


SECTION: PCR clean-up
9. Label the tubes/plates and pool the PCR reactions for each samples into a single LoBind tube . 

 

 

SECTION: PCR clean-up
10. Clean-up the amplicons using  .

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.


SECTION: PCR clean-up
10.1. 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.
SECTION: PCR clean-up
10.2. Spin the tubes to collect entire liquid at the bottom and incubate the tubes at room temperature for 00.05.00.
SECTION: PCR clean-up
10.3. Place the tubes on magnetic stand and incubate for 00:02:00 or until the beads have settled and the supernatant is completely colourless.
SECTION: PCR clean-up
10.4. Carefully remove and discard the supernatant, without disturbing the bead pellet.
SECTION: PCR clean-up
10.5. 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.
SECTION: PCR clean-up
10.6.   and repeat  Spin to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible.

SECTION: PCR clean-up
10.7. 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)
SECTION: PCR clean-up
10.8. 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.
SECTION: PCR clean-up
10.9. Place on magnetic stand and transfer sample to a clean 1.5mL LoBind tube ensuring no beads are transferred into this tube.
SECTION: PCR clean-up
10.10. Quantify 1µl product using the Qubit Fluorometer using the   .


  

SECTION: End-Prep Reaction
11. 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 ,

 
If the Qubit reading is 5ng/ul -15ng/ul

 
If the Qubit reading is <5ng/ul,

 
     

SECTION: End-Prep Reaction
11.1. Reaction conditions for End Prep: 
Incubate 
25 °C for  00:15:00
65 °C for 00:15:00
on ice for 00:01:00

SECTION: Native Barcoding
12. In a new labelled tube/plate prepare the following components.
 

    
  

SECTION: Native Barcoding
12.1. Incubate at 
25 °C  for 00:20:00
65 °C  for 00:10:00
on ice for 00:01:00

SECTION: Native Barcoding
12.2. In a new 1.5mL LoBind tube pool all  barcoded reactions together.
SECTION: Native Barcoding
12.3. 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.
SECTION: Native Barcoding
12.4. Briefly vortex and mix the reactions and incubate at room temperature for 00.05.00. 
SECTION: Native Barcoding
12.5. 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.
SECTION: Native Barcoding
12.6. 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.
SECTION: Native Barcoding
12.7. Add 200µl of 70% ethanol to wash the pellet. Carefully remove and discard ethanol, being careful not to touch the bead pellet.
SECTION: Native Barcoding
12.8. Spin to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible.
SECTION: Native Barcoding
12.9. 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).
SECTION: Native Barcoding
12.10. Resuspend pellet in 30µl Elution Buffer(EB), mix gently by either flicking or pipetting and incubate for 00:02:00.
SECTION: Native Barcoding
12.11. Place on magnet stand and transfer sample to a clean 1.5mL Eppendorf tube ensuring no beads are transferred into this tube.
SECTION: Native Barcoding
12.12. Quantify 1µl of the barcoded amplicons using the Qubit Flurometer.
 
  

SECTION: Adapter Ligation
13. Prepare the following reaction for AMII Adapter ligation in a PCR tube.

 

SECTION: Adapter Ligation
13.1. Incubate at 25 °C  for 00:20:00, possibly in a thermo cycler.

SECTION: Post adapter ligation Clean-up
14. 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.

SECTION: Post adapter ligation Clean-up
14.1. Incubate for 00:05:00 at room temperature.
SECTION: Post adapter ligation Clean-up
14.2. 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.





SECTION: Post adapter ligation Clean-up
14.3. 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.
SECTION: Post adapter ligation Clean-up
14.4. Repeat step 14.3 to perform a second SFB wash.
SECTION: Post adapter ligation Clean-up
14.5. Brief spin and remove any residual SFB. Add 15µl EB (ONT) and resuspend beads by gentle flicking or pipette mixing.
SECTION: Post adapter ligation Clean-up
14.6. 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
SECTION: Post adapter ligation Clean-up
14.7. Quantify 1µl of the barcoded amplicons using the Qubit Fluorometer.
 
  

SECTION: Flow cell Priming and Sequencing
15. Thaw the following reagents.

Sequencing buffer (SQB)
Loading beads (LB)
Flush buffer (FB)
Flush tether (FLT)
SECTION: Flow cell Priming and Sequencing
15.1. Add 30µl FLT to the FB tube and mix well by vortex and spin down.
SECTION: Flow cell Priming and Sequencing
15.2. Prepare the following library dilution for sequencing in a LoBind tube:

 
      


SECTION: cDNA Preparation
2. 
 

SECTION: Flow cell Priming and Sequencing
15.3. Flowcell Priming, loading of the library and sequencing are performed based on the following protocol:
dx.doi.org/10.17504/protocols.io.bbmuik6w
SECTION: cDNA Preparation
2.1. Prepare the master mix for cDNA Prep as follows in a PCR plate/ tubes for 10μL reaction.

 
  

 


SECTION: cDNA Preparation
2.2. 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