TITLE: Rabies Virus Sequencing using Illumina- MiSeq
AUTHORS: Chakrakodi N Varun, Dhanya K, Ashwini M Ananda, Reeta Mani

[DESCRIPTION]
This Rabies whole genome sequencing protocol has been derived and modified from the Illumina COVIDSeq RUO sequencing pipeline. The protocol has been modified and optimised for sequencing Rabies virus (RABV). The methodology uses RABV-specific primers that have been designed in-house. In brief, the RNA is extracted from samples and converted to cDNA. RABV sequencing library is generated and sequenced using MiSeq.

[BEFORE_START]
All the processes should be performed in Biosafety cabinets. Ensure you have separate Biosafety cabinets for RNA extraction, Reagent preparation, and Template addition.



[GUIDELINES]
Ensure all Biosafety guidelines are followed as per the working policy.

Please ensure you keep a log of sample processing and use a template to locate sample wells.

The protocol is based on sequencing a total of 96 samples, which includes a known positive and negative control. At least one negative control is mandatory to assess possible contamination. Positive control with a known sequence is mandatory to estimate the quality of the run.

The complete library preparation process takes approximately two days at our laboratory.  It is advisable to use "Safe Stops" as needed.



[STEPS]
SECTION: Samples and Extraction
1. Samples:
The following samples (human or animal sources) may be used for viral RNA extraction for Rabies lyssavirus.
1. Brain Tissue or Nuchal skin: Homogenise the tissue by crushing a small piece in a sterile environment. Transfer the contents to a vial and vortex and spin down the sample. Retrieve the supernatant.
2. Saliva
SECTION: Samples and Extraction
2. RNA Extraction:
The RNA is extracted using theQIAmp Viral RNA Mini Kit, as per the procedure outlined in the QIAamp Viral RNA Mini Handbook (https://www.qiagen.com/us/resources/download.aspx?id=c80685c0-4103-49ea-aa72-8989420e3018&lang=en).
SECTION: Samples and Extraction
2.1. Add 140 µL of the supernatant or saliva sample to the Buffer AVL–carrier RNA in the microcentrifuge tube. Mix by pulse-vortexing for 15 s.

SECTION: Samples and Extraction
2.2. Incubate at Room temperature for 20 min.

SECTION: Samples and Extraction
2.3. Briefly centrifuge the tube to remove drops from the inside of the lid.
SECTION: Samples and Extraction
2.4. Add 560 µL ethanol (96–100%) to the sample, and mix by pulse-vortexing for 15 s. After mixing, briefly centrifuge the tube to remove drops from inside the lid.

SECTION: Samples and Extraction
2.5. Carefully apply 630 µL of the solution from step 2.4 to the QIAamp Mini column (in a 2 mL collection tube) without wetting the rim. Close the cap, and centrifuge at 8000 rpm for 1 min. Place the QIAamp Mini column into a clean 2 mL collection tube, and discard the tube containing the filtrate.
SECTION: Samples and Extraction
2.6. Carefully open the QIAamp Mini column, and repeat the step.
 


SECTION: Samples and Extraction
2.7. Carefully open the QIAamp Mini column, and add 500 µL Buffer AW1. Close the cap, and centrifuge at 8000 rpm for 1 min. Place the QIAamp Mini column in a clean 2 mL collection tube (provided), and discard the tube containing the filtrate.

SECTION: Samples and Extraction
2.8. Carefully open the QIAamp Mini column, and add 500 µL Buffer AW2. Close the cap and centrifuge at full speed 14.000 rpm for 3 min. 
SECTION: Samples and Extraction
2.9. Place the QIAamp Mini column in a clean 1.5 mL microcentrifuge tube. Discard the old collection tube containing the filtrate. Carefully open the QIAamp Mini column and add 60 µL of Buffer AVE. Close the cap and incubate at Room temperature for 1 min.

 

SECTION: Samples and Extraction
2.10. Centrifuge at 8000 rpm for 1 min. The eluted RNA can be used immediately or stored at ≤ -80 °C . 


 


SECTION: cDNA Conversion
3. The extracted RNA is annealed using random hexamers to prepare for cDNA synthesis during this process.

Thaw the EPH3 (Elution Prime Fragment 3HC Mix) at Room temperature.
 

Label a new PCR plate as CDNA.

Add 8.5 µl EPH3 to each well.
Add 8.5 µl eluted sample to each well.
Seal and shake at 1600 rpm for 1 min.
Centrifuge at 1000 rpm for 1 min.

Set up a PCR (RABV Anneal program) as follows:

65 °C for 3 min
Hold at 4 °C

SECTION: cDNA Conversion
4. This step reverse transcribes the RNA fragments primed with random hexamers into first-strand cDNA using reverse transcriptase.

Thaw the FSM (First Strand Mix) and RVT (Reverse Transcriptase) reagents inRoom temperature 

For 96 samples, prepare a master mix in a 1.7 ml tube as follows.

FSM- 720 µL
RVT- 80 µL 

Add 8 µl master mix to each well of the CDNA plate.
Seal and shake at 1600 rpm for 1 min.
Centrifuge at 1000 rpm for 1 min.

Set up a PCR (RABV FSM program) as follows:

Choose the preheat lid option
Set the reaction volume to 25 µL
25 °C  5 min
50 °C 10 min
80 °C 5 min
Hold at 4 °C

SECTION: Targeted Amplification of cDNA
5. Preparation of RABV Primer Pools
SECTION: Targeted Amplification of cDNA
5.1. Prepare the primer pool mix as provided in the RABV_PrimerPool sheets. Store the Rabies Primer Pools (Odd and Even Primer mix) at -20 °C

  


 

SECTION: Targeted Amplification of cDNA
5.2. Prepare two master mixes as follows.

1. Odd Rabies PrimerPool Master Mix

IPM (Illumina PCR Mix):1260 µL
Odd PrimerPool Mix:361.20 µL
Nuclease Free Water:394.8 µL

2. Even Rabies PrimerPool Master Mix

IPM (Illumina PCR Mix):1260 µL
Odd PrimerPool Mix:361.20 µL
Nuclease Free Water:394.8 µL


 

SECTION: Targeted Amplification of cDNA
5.3. Amplification PCR

Label two PCR plates as
(i) RABV_Odd plate
(ii) RABV_Even Plate


Add 20 µL of Odd Rabies PrimerPool Master Mix to each well of the RABV_Odd plate 
Add 5 µL of cDNA synthesised in the previous step to the corresponding well of the  RABV_Odd plate.
Add 20 µL of Even Rabies PrimerPool Master Mix to each well of the RABV_Even plate 
Add 5 µL of cDNA synthesised in the previous step to the corresponding well of the  RABV_even plate.
Seal and shake at 1600 rpm for 1 min.
Centrifuge at 1000 rpm for 1 min.

Set up two PCR's (RABV amplification program) as follows:

Choose the preheat lid option

Set the reaction volume to 25 µL
98 °C for 3 min
35 cycles of:
         98 °C for 15 s
         63 °C for 5 min
Hold at 4 °C


 

SECTION: Tagment of PCR Amplicons
6. This step uses EBLTS (Enrichment Bead-Linked Transposomes) to tagment PCR amplicons, which is a process that fragments and tags the PCR amplicons with adapter sequences.
SECTION: Tagment of PCR Amplicons
6.1. Thaw EBLTS and TB1 Buffer at Room temperature
 

Label a new PCR plate as TAG.

Transfer 10 µLfrom each well of the RABV_Odd plate to the corresponding well of the TAG plate.
Transfer 10 µLfrom each well of the RABV_Even Plate to the corresponding well of the TAG plate
SECTION: Tagment of PCR Amplicons
6.2. Prepare Tagmentation Master Mix in a 15 ml tube as follows.

TB1 1008 µL
EBLTS 336 µL
Nuclease Free Water 1680 µL

 

Add 30 μl master mix to each well in the TAG plate.
Seal and shake at 1600 rpm for 1 min
Centrifuge at 1000 rpm for 1 min
SECTION: Tagment of PCR Amplicons
6.3. Set up PCR (TAG program) as follows:

Choose the preheat lid option
Set the reaction volume to 50 μl

55 °C for 5 min
Hold at 10 °C
SECTION: Post Tagmentation Clean Up
7. This step washes the adapter-tagged amplicons before PCR amplification.
SECTION: Post Tagmentation Clean Up
7.1. Vortex ST2 (Stop Tagment Buffer 2) and TWB (Tagmentation Wash Buffer) before use.

Centrifuge the TAG plate at 500 x g for 1 min.
Add 10 µL ST2 to each well of the TAG plate.
Seal and shake at 1600 rpm for 1 minute.
Incubate at Room temperature for 5 min.
Centrifuge at   for 1 min.
Place on the magnetic stand and wait until the liquid is clear (3 min).


 

SECTION: Post Tagmentation Clean Up
7.2. Wash the beads as follows:

Remove from the magnetic stand.
Add 100 µL TWB to each well.
Seal and shake at 1600 rpm for 1 min.
Centrifuge   for 1 min.
Place on the magnetic stand and wait until the liquid is clear (~3 minutes).

 

Remove and discard all supernatant from each well.

Wash beads a second time.
Leave supernatant in theplate for the second wash to prevent beads from overdrying.
SECTION: Amplify Tagmented Amplicons and Indexing
8. This step amplifies the tagmented amplicons using a PCR program. The PCR step adds prepaired 10 base pair Index 1 (i7) adapters, Index 2 (i5) adapters, and sequences required for sequencing
SECTION: Amplify Tagmented Amplicons and Indexing
8.1. Prepare the Master mix as follows, in a 15 ml tube.

EPM (Enhanced PCR Mix): 2016 µL
Nuclease-free water: 2016 µL


 

SECTION: Amplify Tagmented Amplicons and Indexing
8.2. Add 40 µL PCR Master Mix to each well.
Add 10 µL index adapters to each well of the PCR plate.
Seal and shake at 1600 rpm for 1 min
If the liquid is visible on the seal, centrifuge at 500 x gfor 1 minute.
Inspect to make sure beads are resuspended.

Set up a PCR (Amplification and Indexing PCR) as follows:

Choose the preheat lid option and set to 100 °C
Set the reaction volume to 50 µL

72 °C for 3 min
98 °C for 3 min
7 cycles of:
     98 °C for 20 s
     60 °C for 30 s
    72 °C for 1 min
72 °C for 3 min
Hold at 10 °C

SECTION: Pool and Clean Up Libraries
9. This step combines libraries from each 96-well sample plate into one 1.7 ml tube. Libraries of optimal size are then bound to magnetic beads, and fragments that are too small or large are washed away.
SECTION: Pool and Clean Up Libraries
9.1. Centrifuge the TAG plate at 500 x g for 1 min.
Place on the magnetic stand and wait until the liquid is clear (~3 minutes).
Transfer 5 µL library from each well of the TAG plate into a 1.7 ml tube.
Vortex the tubes to mix, and then centrifuge briefly.
Add 0.9x of IPB.


 

Vortex to mix.
Incubate at Room temperaturefor 5 min.
Centrifuge briefly.

Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
Remove and discard all supernatant.
SECTION: Pool and Clean Up Libraries
9.2. Wash beads as follows.

Keep on the magnetic stand and add 1000 µLfresh 80% Ethanol.
Incubate at Room temperature 30 s.
Remove and discard all supernatant.

Wash beads a second time.
Centrifuge briefly.
Use a 20 µl pipette to remove all residual EtOH.


 

SECTION: Pool and Clean Up Libraries
9.3. Add 55 µL Resuspension Buffer (RSB)
Vortex to mix, and then centrifuge briefly.
Incubate at room temperature for 2 min.
Place on the magnetic stand and wait until the liquid is clear (~2 minutes).
Transfer 50 µL supernatant to a fresh new microcentrifuge tube.