Rabies Virus Sequencing using Illumina- MiSeq

Chakrakodi N Varun, Dhanya K, Ashwini M Ananda, Reeta Mani

Published: 2024-01-28 DOI: 10.17504/protocols.io.ewov1qbrogr2/v1

Abstract

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.

Steps

Samples and Extraction

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

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).

2.1.

Add 140µL of the supernatant or saliva sample to the Buffer AVL–carrier RNA in the microcentrifuge tube. Mix by pulse-vortexing for 0h 0m 15s.

2.10.

Centrifuge at 8000rpm,0h 0m 0s for 0h 1m 0s. The eluted RNA can be used immediately or stored at ≤ -80°C .

Note

It is advised to check the quality of extracted RNA using Nanodrop and run an RABV RT-PCR to determine the Ct value. Sequencing does not work well if extraction quality is low or the Ct is > 30.

2.2.

Incubate at Room temperature for 0h 20m 0s.

2.3.

Briefly centrifuge the tube to remove drops from the inside of the lid.

2.4.

Add 560µL ethanol (96–100%) to the sample, and mix by pulse-vortexing for 0h 0m 15s. After mixing, briefly centrifuge the tube to remove drops from inside the lid.

2.5.

Carefully apply 630µL 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 8000rpm,0h 0m 0s for 0h 1m 0s. Place the QIAamp Mini column into a clean 2 mL collection tube, and discard the tube containing the filtrate.

2.6.

Carefully open the QIAamp Mini column, and repeat the step.

Note

If the original sample volume exceeds 140µL, repeat this step until the lysate has been loaded onto the spin column.

2.7.

Carefully open the QIAamp Mini column, and add 500µL Buffer AW1. Close the cap, and centrifuge at 8000rpm,0h 0m 0s for 0h 1m 0s. Place the QIAamp Mini column in a clean 2 mL collection tube (provided), and discard the tube containing the filtrate.

2.8.

Carefully open the QIAamp Mini column, and add 500µL Buffer AW2. Close the cap and centrifuge at full speed 14.000rpm,0h 0m 0s for 0h 3m 0s.

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 60µL of Buffer AVE. Close the cap and incubate at Room temperature for 0h 1m 0s.

Note

If a very low viral load is expected, such as in a salivary sample, it is advised to elute in approximately 20µL.

cDNA Conversion

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.

Note

Use nuclease-free water as a Negative Control in one or more wells depending on the user's requirement.

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 0h 1m 0s.

Centrifuge at 1000 rpm for 0h 1m 0s.

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

65°C for 0h 3m 0s

Hold at 4°C

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- 720µL

RVT- 80µL

Add 8 µl master mix to each well of the CDNA plate.

Seal and shake at 1600 rpm for 0h 1m 0s.

Centrifuge at 1000 rpm for 0h 1m 0s.

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

Choose the preheat lid option

Set the reaction volume to 25µL

25°C 0h 5m 0s

50°C 0h 10m 0s

80°C 0h 5m 0s

Hold at 4°C

Targeted Amplification of cDNA

Preparation of RABV Primer Pools

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

RABV_PrimerPool.xlsx

Note

1. The RABV_Primer Pools were designed using Primal Scheme ( 1. The RABV_Primer Pools were designed using Primal Scheme ( https://primalscheme.com/ ) )2. The RABV_PrimerPool.xlsx contains two sheets, for odd and even primal pool mix. The quantity of 100micromolar (µM) stock solution to be taken for generating a primer pool and the resulting final concentration in (uM) is provided in the the excel sheet.

5.2.

Prepare two master mixes as follows.

1. Odd Rabies PrimerPool Master Mix

IPM (Illumina PCR Mix):1260µL

Odd PrimerPool Mix:361.20µL

Nuclease Free Water:394.8µL

2. Even Rabies PrimerPool Master Mix

IPM (Illumina PCR Mix):1260µL

Odd PrimerPool Mix:361.20µL

Nuclease Free Water:394.8µL

Note

The Master mix is calculated to accommodate 96 reactions. If using lesser samples calculate accordingly.

5.3.

Amplification PCR

Label two PCR plates as

(i) RABV_Odd plate

(ii) RABV_Even Plate

Add 20µL of Odd Rabies PrimerPool Master Mix to each well of the RABV_Odd plate

Add 5µL of cDNA synthesised in the previous step to the corresponding well of the RABV_Odd plate.

Add 20µL of Even Rabies PrimerPool Master Mix to each well of the RABV_Even plate

Add 5µL of cDNA synthesised in the previous step to the corresponding well of the RABV_even plate.

Seal and shake at 1600 rpm for 0h 1m 0s.

Centrifuge at 1000 rpm for 0h 1m 0s.

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

Choose the preheat lid option

Set the reaction volume to 25µL

98°C for 0h 3m 0s

35 cycles of:

     `98°C` for `0h 0m 15s`



     `63°C` for `0h 5m 0s`

Hold at 4°C

Note

If you are stopping, seal the plate and store at -20°C for up to 3 days

Tagment of PCR Amplicons

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.

6.1.

Thaw EBLTS and TB1 Buffer at Room temperature

Note

If the RABV_Odd plate and RABV_Even Plate were stored thaw at Room temperature, shake the plates at 1600 rpm for 1 minute and centrifuge at 1000 x g for 1 minute before starting.

Label a new PCR plate as TAG.

Transfer 10µLfrom each well of the RABV_Odd plate to the corresponding well of the TAG plate.

Transfer 10µLfrom each well of the RABV_Even Plate to the corresponding well of the TAG plate

6.2.

Prepare Tagmentation Master Mix in a 15 ml tube as follows.

TB1 1008µL

EBLTS 336µL

Nuclease Free Water 1680µL

Note

Ensure that the beads are uniformly mixed before use. Pipette mix if needed and pulse centrifuge before use.

Add 30 μl master mix to each well in the TAG plate.

Seal and shake at 1600 rpm for 0h 1m 0s

Centrifuge at 1000 rpm for 0h 1m 0s

6.3.

Set up PCR (TAG program) as follows:

Choose the preheat lid option

Set the reaction volume to 50 μl

55°C for 0h 5m 0s

Hold at 10°C

Post Tagmentation Clean Up

This step washes the adapter-tagged amplicons before PCR amplification.

7.1.

Vortex ST2 (Stop Tagment Buffer 2) and TWB (Tagmentation Wash Buffer) before use.

Centrifuge the TAG plate at 500x g,0h 0m 0s for 0h 1m 0s.

Add 10µL ST2 to each well of the TAG plate.

Seal and shake at 1600 rpm for 1 minute.

Incubate at Room temperature for 0h 5m 0s.

Centrifuge at ,0h 0m 0s for 0h 1m 0s.

Place on the magnetic stand and wait until the liquid is clear (0h 3m 0s).

Note

If the liquid is not clear at this point continue to place it on the magnetic stand for another ~2 minutes. Inspect for bubbles on the seal. If present, centrifuge at 500 x g for 1 minute, and then place on the magnetic stand (~3 minutes).

7.2.

Wash the beads as follows:

Remove from the magnetic stand.

Add 100µL TWB to each well.

Seal and shake at 1600 rpm for 0h 1m 0s.

Centrifuge ,0h 0m 0s for 0h 1m 0s.

Place on the magnetic stand and wait until the liquid is clear (~3 minutes).

Note

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.

Amplify Tagmented Amplicons and Indexing

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

8.1.

Prepare the Master mix as follows, in a 15 ml tube.

EPM (Enhanced PCR Mix): 2016µL

Nuclease-free water: 2016µL

Note

Centrifuge the TAG plate, keep the on magnetic stand and remove remaining TWB. Do not leave any residual TWB in the wells.

8.2.

Add 40µL PCR Master Mix to each well.

Add 10µL index adapters to each well of the PCR plate.

Seal and shake at 1600 rpm for 0h 1m 0s

If the liquid is visible on the seal, centrifuge at 500x g,0h 0m 0sfor 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 50µL

72°C for 0h 3m 0s

98°C for 0h 3m 0s

7 cycles of:

 `98°C` for `0h 0m 20s`



 `60°C` for `0h 0m 30s`



`72°C` for `0h 1m 0s`

72°C for 0h 3m 0s

Hold at 10°C

Pool and Clean Up Libraries

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.

9.1.

Centrifuge the TAG plate at 500x g,0h 0m 0s for 0h 1m 0s.

Place on the magnetic stand and wait until the liquid is clear (~3 minutes).

Transfer 5µL 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.

Note

Assuming that 5µL from 96 wells were pooled, give a total volume of 480µL add 432µL of IPB (480x 0.9).

Vortex to mix.

Incubate at Room temperaturefor 0h 5m 0s.

Centrifuge briefly.

Place on the magnetic stand and wait until the liquid is clear (~5 minutes).

Remove and discard all supernatant.

9.2.

Wash beads as follows.

Keep on the magnetic stand and add 1000µLfresh 80% Ethanol.

Incubate at Room temperature 0h 0m 30s.

Remove and discard all supernatant.

Wash beads a second time.

Centrifuge briefly.

Use a 20 µl pipette to remove all residual EtOH.

Note

The final

9.3.

Add 55µL Resuspension Buffer (RSB)

Vortex to mix, and then centrifuge briefly.

Incubate at room temperature for 0h 2m 0s.

Place on the magnetic stand and wait until the liquid is clear (~2 minutes).

Transfer 50µL supernatant to a fresh new microcentrifuge tube.

Note

The final library can be at -20°C for up to 30 days.We check the quality of the final library prepared using a Tapestation and the library is quantified using a Qubit Fluorometer.

Rabies Virus Sequencing using Illumina- MiSeq

Abstract

Before start

Steps

Samples and Extraction

cDNA Conversion

Targeted Amplification of cDNA

Tagment of PCR Amplicons

Post Tagmentation Clean Up

Amplify Tagmented Amplicons and Indexing

Pool and Clean Up Libraries

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