TITLE: Universal, amplicon-based sequencing method for Canine Distemper Virus (CDV)
AUTHORS: Gábor  Tóth, Zsófia Lanszki, Gabor  Kemenesi

[DESCRIPTION]
Canine distemper virus is a multihost pathogen wich mostly affects family Caniade (dog, fox, coyote, wolf) but it is also occur in other carnivorous families like Mustilidae (ferret, skunk, badger, mink, weasel, otter), Procyonidae (racoon, lesser panda, kinkajou), Hyanidae (hyenas) or Ursidae (bear).

Increasing surveillance needed to identify new CDV variants and to understand the dynamics of CDV epidemiology . There are available  vaccines against the virus, but it is important to asses the efficacy of the vaccine against these novel strains. To maintain these follow up efforts we developed an universal amplicon based sequencing method which is capable to generate data from the previously described CDV strains. 

The protocol was forked and designed by the original work of Josh Quick (https://www.protocols.io/view/ncov-2019-sequencing-protocol-v2-bdp7i5rn?version_warning=no). 

Now this method is in experimental phase therefore it should be widely tested to evaluate it's efficiency.


Some literature of CDV:

-Martella, V., Elia, G. and Buonavoglia, C. (2008) ‘Canine Distemper Virus’, Veterinary Clinics of North America: Small Animal Practice, 38(4), pp. 787–797. doi: 10.1016/J.CVSM.2008.02.007.
-Kapil, S. and Yeary, T. J. (2011) ‘Canine Distemper Spillover in Domestic Dogs from Urban Wildlife’, Veterinary Clinics of North America: Small Animal Practice, 41(6), pp. 1069–1086. doi: 10.1016/J.CVSM.2011.08.005.
-Day, M. J. et al. (2020) ‘Aetiology of Canine Infectious Respiratory Disease Complex and Prevalence of its Pathogens in Europe’, Journal of Comparative Pathology, 176, pp. 86–108. doi: 10.1016/J.JCPA.2020.02.005.
-Takeda, M. et al. (2020) ‘Animal morbilliviruses and their cross-species transmission potential’, Current Opinion in Virology, 41, pp. 38–45. doi: 10.1016/J.COVIRO.2020.03.005.
-Decaro, N., Buonavoglia, C. and Barrs, V. R. (2020) ‘Canine parvovirus vaccination and immunisation failures: Are we far from disease eradication?’, Veterinary Microbiology, 247, p. 108760. doi: 10.1016/J.VETMIC.2020.108760.
-Koç, B. T., Akkutay-Yoldar, Z. and Oğuzoğlu, T. Ç. (2021) ‘New members to Arctic-like lineage of canine distemper virus from Turkey’, Comparative Immunology, Microbiology and Infectious Diseases, 78, p. 101678. doi: 10.1016/J.CIMID.2021.101678.
-Chang, Z. et al. (2021) ‘Spatiotemporal dynamics for an impulsive eco-epidemiological system driven by canine distemper virus’, Applied Mathematics and Computation, 402, p. 126135. doi: 10.1016/J.AMC.2021.126135.

Literature of the amplicon-based sequencing:

-Quick, Joshua et al. “Real-time, portable genome sequencing for Ebola surveillance.”Naturevol. 530,7589 (2016): 228-232. doi:10.1038/nature16996
-Quick, Joshua et al. “Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples.”Nature protocolsvol. 12,6 (2017): 1261-1276. doi:10.1038/nprot.2017.066

[STEPS]
SECTION: cDNA preparation
1. Mix the following components in an 0.2mL 8-strip tube;

Component                                        Volume

50µM random hexamers                 1 µL   
10mM dNTPs mix (10mM each)    1 µL
Template RNA                                   11 µL
Total                                                    13 µL 
 

 

SECTION: cDNA preparation
2. Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
SECTION: cDNA preparation
3. Incubate the reaction as follows:

 65 °C for 5 min
Place on ice for 1 min 
SECTION: cDNA preparation
4. Add the following to the annealed template RNA: 

Component	                                Volume

 SSIV Buffer	                                4 µL 
 100mM DTT                                 1 µL 
 RNaseOUT RNase Inhibitor 	1 µL 
 SSIV Reverse Transcriptase	1 µL 
 Total	                                        20 µL 


 

SECTION: cDNA preparation
5. Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
SECTION: cDNA preparation
6. Incubate the reaction as follows:

50 °C 50 min 
70 °C 10 min
Hold at 5 °C 

SECTION: Primer pool preparation
7. If required resuspend lyophilised primers at a concentration of 100µM each


 


Universal CDV primers for 1000 basepair long amplicon set: 
  

Universal CDV primers for 2000 basepair long amplicon set: 
  

SECTION: Primer pool preparation
8. Generate primer pool stocks by adding 5 µL of each odd region primer to a 1.5 mL Eppendorf labelled “Pool 1 (100µM)” and each even region primer to a 1.5 mL Eppendorf labelled  “Pool 2 (100µM)”. These are your 100µM stocks of each primer pool.

                                                Number of primers:                                        Final volume:
CDV_1000_pool_1:                            63                                                         315 µL 
CDV_1000_pool_2:                            50                                                         250 µL 

CDV_2000_pool_1:                            32                                                         160 µL 
CDV_2000_pool_2:                            27                                                         135 µL 

 

SECTION: Primer pool preparation
9. Dilute this primer pool 1:10 in molecular grade water, to generate 10µM primer stocks. It is recommend that multiple aliquots of each primer pool are made to in case of degradation or contamination. Our recommendation is that yous should use 50 µL from the stock solution and add 450 µL nuclease-free water to generate the working solution of the primers.
 
 

SECTION: Multiplex PCR
10. In the mastermix hood set up the multiplex PCR reactions as follows in 0.2mL 8-strip PCR tubes:


Reactions with the 1000 bp amplicon set:

Component                                             Pool 1                Pool 2 

5X Q5 Reaction Buffer                           5 µL           5 µL
10 mM dNTPs                                         0.5 µL       0.5 µL 
Q5 Hot Start DNA Polymerase             0.25 µL     0.25 µL 
V3 Primer Pool 1 or 2 (10µM)               2.5 µL          2.0 µL
Nuclease-free water                              14.25 µL  14.75 µL
Total                                                         22.5 µL     22.5 µL


Reactions with the 2000 bp amplicon set:

Component                                             Pool 1                Pool 2 

5X Q5 Reaction Buffer                           5 µL           5 µL
10 mM dNTPs                                         0.5 µL       0.5 µL 
Q5 Hot Start DNA Polymerase             0.25 µL     0.25 µL 
V3 Primer Pool 1 or 2 (10µM)               2.0 µL          2.0 µL
Nuclease-free water                              14.75 µL  14.75 µL
Total                                                         22.5 µL     22.5 µL


 

SECTION: Multiplex PCR
11. In the extraction and sample addition cabinet add 2.5 µL cDNA to each tube and mix well by pipetting.
 

SECTION: Multiplex PCR
12. Pulse centrifuge the tubes to collect the contents at the bottom of the tube.
SECTION: Multiplex PCR
13. Set-up the following program on the thermal cycler:  

Step                        Temperature     Time                    Cycles

Heat Activation     98 °C        30 s    1
Denaturation         98 °C        15 s     25-35 
Annealing              64 °C        5 min     25-35
Hold                       4 °C            Indefinite              1
 

SECTION: Quantification and normalisation
15. Label another 1.5 mL Eppendorf tube for each sample.
 

You should measure the concentration and dilute the sample to appropriate concentration for the end-prep and barcoding reactions.

1000bp_set:        25 ng / 1 µL 

2000bp_set:        50 ng / 1 µL 


 

SECTION: Native barocoding
16. Barcode the amplicon pools using the one-pot native barcoding approach.
 


SECTION: Native barocoding
17. Quantify 1 µL of the barcoded amplicon pool using the Quantus Fluorometer using the ONE dsDNA assay. 
 

SECTION: Native barocoding
18. Set up the following AMII adapter ligation and clean-up with SFB.
 

SECTION: Native barocoding
19. Quantify 1 µL of the final library using the Quantus Fluorometer using the ONE dsDNA assay. 
 

 


SECTION: MinION sequencing
36. Prime the flowcell and load 50 ng sequencing library onto the flowcell if you used only the CDV_1000 bp set.
Prime the flowcell and load 100 ng sequencing library onto the flowcell if you used only the CDV_2000 bp set.

If you combined the two set you should load between 50 ng  and 100 ng .





36. Start the sequencing run using MinKNOW.





SECTION: Clean up
14. Label a  1.5 mLEppendorf tube for each sample and assemble the following PCR dilution for each sample:

Component                                        Volume

Pool 1 PCR reaction                        25 µL   
Pool 2 PCR reaction                        25 µL
SPRI Beads                                       25 µL
70% EtOH                                           2X100 µL 
Nuclease-free water                        25 µL 

SECTION: Clean up
14.1. In a new 1.5 ml Eppendorf tube pool all 25 µl PCR product from the same primer set (1000 pool 1-2 or 2000 pool 1-2)reactions together.

SECTION: Clean up
14.2. Add 0.5x volume of SPRI beads to the sample tube and mix gently by either flicking or pipetting. If you were performed 25 µL  reactions it should be also 25 µL .

SECTION: Clean up
14.3. Pulse centrifuge to collect all liquid at the bottom of the tube.
SECTION: Clean up
14.4. Incubate for 5 min  at room temperature.

SECTION: Clean up
14.5. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
SECTION: Clean up
14.6. Add 100 µL   70% EtOH and resuspend beads completely by pipette mixing.

SECTION: Clean up
14.7. Pulse centrifuge to collect all liquid at the bottom of the tube.
SECTION: Clean up
14.8. Remove supernatant and discard.
SECTION: Clean up
14.9. Repeat steps 14.6-14.9 to perform a second EtOH wash.
SECTION: Clean up
14.10. Pulse centrifuge to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible using a P10 pipette.
SECTION: Clean up
14.11. With the tube lid open incubate for 2 min  or until the pellet loses it's shine (if the pellet dries completely it will crack and become difficult to resuspend).

SECTION: Clean up
14.12. Resuspend pellet in 25 µL  nuclease-free water, mix gently by either flicking or pipetting and incubate for 2 min .

SECTION: Clean up
14.13. Place on magnet and transfer sample to a clean 1.5mL Eppendorf tube ensuring no beads are transferred into this tube.