TITLE: TELEvir Field Protocol 
AUTHORS: Anna S Fomsgaard

[DESCRIPTION]
The TELE-Vir project key aim is to develop a fast point-of-incidence (poi) toolbox for identification and characterisation of emerging virus threats for humans and/or domestic and wildlife animals. 
Presented here is a protocol including sample pretreatment, NA extraction and random amplification for metagenomic virus detection using the MinION device (Oxford Nanopore Technologies)

[STEPS]
SECTION: Pretreatment
1. 
  

 

 
400 µL 
50 µL  
50 µL 
15 min 
 

SECTION: Pretreatment
2. Add 500 µL (or equivalent to 1 ml sample material in total)

SECTION: Pretreatment
3. Poor the diluted sample into the lid of the Syringe Filter (Minsart). The filter can be placed on a sterile surface meanwhile e.g. the Syringe Filter paper lid (inner side).
SECTION: Pretreatment
4. Suck in air corresponding to app 1 ml in a 5 ml syringe
SECTION: Pretreatment
5. Extract the 1 ml sample material from the petri dish to the 5 ml syringe
SECTION: Pretreatment
6. Attach the 0,22 µM Minisart syringe filter to the 5 ml syringe with Luer-Lok
SECTION: Pretreatment
7. Filter the sample and air directly into a 4.5 ml cryotube containing 1 ml MPLB-buffer. Put lid on, mix by turning tube.

  

SECTION: Extraction
9. Kit used for hand held NA extraction:
  

 

 

Cylinder magnet
A rubber band to attach the magnet to a tube.
A 50 ml Nunc tube or similar to pour excess solutions into for disposing. 

Following the protocol by:
 

SECTION: Extraction
10. Per X number of samples, aliquot in cryotubes or Eppendorf tubes
 960 µLMagnetic Glass Particles (MGPs) in an Eppendorf tube
4 mLWash buffer I in a 4.5 ml cryotube
1.5 mL Wash buffer II in an Eppendorf tube
 3 mLWash buffer III in a 3.6 ml cryotube
 100 µL Elution buffer in an Eppendorf tube
Prepare one sample at the time. 
Approximate time  10 min  
SECTION: Extraction
11. Add 960 µl MGPs to 2 ml solution of sample and MPLB-buffer
SECTION: Extraction
12. Put lid on and turn tube in hand gently until well mixed
SECTION: Extraction
13. Attach magnet and trap beads.
SECTION: Extraction
14. Pour solution into trash tube. The MGPs should stay behind trapped on the side of the tube.
SECTION: Extraction
15. Pour Wash buffer I in the sample tube
SECTION: Extraction
16. Put a lid on, remove magnet and turn the tube in hand gently until well mixed
SECTION: Extraction
17. Attach magnet and trap beads.
SECTION: Extraction
18. Pour solution into trash tube. The MGPs should stay behind trapped on the side of the tube.
SECTION: Extraction
19. Pour Wash buffer II in the sample tube
SECTION: Extraction
20. Put a lid on, remove magnet and turn the tube in hand gently until well mixed
SECTION: Extraction
21. Attach magnet and trap beads.
SECTION: Extraction
22. Pour solution into trash tube. The MGPs should stay behind trapped on the side of the tube.
SECTION: Extraction
23. Pour Wash buffer III in the sample tube
SECTION: Extraction
24. Put a lid on, remove magnet and turn the tube in hand gently until well mixed
SECTION: Extraction
25. Attach magnet and trap beads.
SECTION: Extraction
26. Pour solution into trash tube. The MGPs should stay behind trapped on the side of the tube.
SECTION: Extraction
27. Pour Elution buffer in the sample tube
SECTION: Extraction
28. Put a lid on, remove magnet and turn the gently until beads are eluted.
28. Transfer 10 µL to a 0.2 ml PCR tube.

SECTION: WTA &amp; WGA
29. 
SECTION: Extraction
29. Transfer 10 µL to 2 x 0.2 ml PCR tube 

SECTION: Whole Transcriptome and Genome Amplification
30. Equipment to enable portability
- A MiniPCR to run isothermal incubations
- A powerbank that can provide 20V. Use one to power the miniPCR and another to power the MinION for approximately 5 hours. Use brand of choice.
- A salad swing as a hand driven centrifuge. Use brand of choice.
- A coolbox for reagents and cooling samples. Use brand of choice.
 

 

 
Kit used for WTA and WGA
  
Quantiscript RT Enzyme Mix, Ligase Mix, and REPLI-g SensiPhi DNA Polymerase should be thawed on ice.
All other components can be thawed at room temperature (15–25°C)
SECTION: Whole Transcriptome and Genome Amplification
32. Cleanup for WTA 
Add 2 µL to PCR tube with 10 µl eluted sample material
10 min 42 °C  on MiniPCR

SECTION: Whole Transcriptome and Genome Amplification
33. Repair (WGA) and Reverse Transcription (WTA)

SECTION: Whole Transcriptome and Genome Amplification
31. The two aliquots of same sample enables uniform whole genome amplification (WGA) and whole transcriptome amplification (WTA) in parallel reactions.
SECTION: Whole Transcriptome and Genome Amplification
33.1. For WGA, mix following and add 10 µL to sample
 
        RT/Polymerase   Buffer  4   µl    gDNA Wipeout   buffer  2 µl    H2O sc  1 µl    Random primer  1 µl    Random Hex-P   primer (20µM)  1 µl    WGA Ready   Enzym  1 µl  
 
 

SECTION: Whole Transcriptome and Genome Amplification
33.2. For WTA, mix following and add 8 µL to sample
 
        RT/Polymerase   Buffer  4   µl    H2O sc  1 µl    Random primer  1 µl    Random Hex-P   primer (20µM)  1 µl    Quantiscript   RT Enzym Mix  1 µl  
 
SECTION: Whole Transcriptome and Genome Amplification
33.3. Incubate 
60 min 42 °C  On MiniPCR
Afterwards immediatly 
3 min 95 °C on MiniPCR

SECTION: Whole Transcriptome and Genome Amplification
34. Ligation
Mix following and add 10 µL to each tube 
 
     Ligase   buffer      8 µl        Ligase Mix      2 µl   
 Incubate
30 min 24 °C 
5 min 95 °C 
Hold at 4 °C 

SECTION: Whole Transcriptome and Genome Amplification
35. Amplification
Mix following and add 30 µL to each tube
 
        Repli-g   sc reasction buffer  29   µl    Repli-g   SensiPhi DNA polymerase  1 µl  
 Incubate
120 min 30 °C 
5 min 65 °C 
Hold at 4 °C 

SECTION: Inactivation
8. Incubate 
20 min for viral inactivation

 


 

SECTION: Library Preparation
37. 
Multiple rapid library preparation kits are available through Oxford Nanopore Technology. 
For Workshop we use. 
  
 



SECTION: Whole Transcriptome and Genome Amplification
36. For library preparation use amplified material without MGPs.