TITLE: SARS-CoV-2 Whole Genome Sequencing on Illumina 
AUTHORS: Guerrino Macori, Lauren Russell, Seamus Fanning

[DESCRIPTION]
This SOP describes the procedure for generating cDNA from SARS-CoV-2 viral nucleic acid extracts and subsequently obtaining, through the amplicons tiling, the whole viral genome using V3 nCov-2019 primers (ARTIC). This is followed by library construction and pooling of samples and quantitation, prior to sequencing on the Illumina MiSeq. 
The SOP is adapted from the nCoV-2019 sequencing protocol: https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w, and it was used in this study:
Lucey M, Macori G, Mullane N, Sutton-Fitzpatrick U, Gonzalez G, Coughlan S, Purcell A, Fenelon L, Fanning S, Schaffer K. Whole-genome Sequencing to Track Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Transmission in Nosocomial Outbreaks. Clinical Infectious Diseases. 2020.
https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa1433/5909421

[STEPS]
SECTION: SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription
1. cDNA/Reverse Transcription Section        Date/Initials:_________________



In this section, the nucleic acid is extracted and used for the qPCR diagnostic test as starting material for sequencing.


SECTION: SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription
1.1. [   ]  In a PCR hood, mix the following reagents in a 0.2 mL PCR tube or PCR plate:
 

 

 
Lot# _______________ Exp. Date _______________

 
Lot# _______________ Exp. Date _______________

 

SECTION: SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription
1.2. [   ]  Mix gently and briefly centrifuge to spin down the components, and return  on ice. 


SECTION: SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription
1.3. [   ]  Preheat Thermocycler to 65 °C, with heated lid at 105 °C


SECTION: SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription
1.4. [   ]  Incubate the reaction at 65 °C for 5 min, followed by an immediate snap-cool on ice for at least 1 min.

SECTION: SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription
1.5. [   ]  In a clean 1.5 mL LoBind tube (96 well plates can also be used), mix together the following reagents:

 

 

 
Lot# _______________ Exp. Date _______________

 
Lot# _______________ Exp. Date _______________

 

SECTION: SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription
1.6. [   ]  Add the above mastermix (7 µL) to the annealed DNA (13 µL) giving a total volume 20 µL 

SECTION: SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription
1.7. [   ]  Cap the tube (or seal the plate), mix and then briefly centrifuge the contents.
SECTION: SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription
1.8. [   ]  Preheat thermocycler to 42 °C , with heated lid at 105 °C 
SECTION: SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription
1.9. [   ]  Incubate sample using the following reverse transcription program:

 

*PAUSE POINT cDNA can be stored at 4 °C (same day) or -20 °C (up to a week).*

SECTION: SARS-CoV-2 WvGS protocol - ARTIC protocol - Tiled PCR
2. Tiled PCR Section        Date/Initials:_________________

This section outlines the process for the tiled PCR approach from the ARTIC protocol. 


 

If required, resuspend lyophilised primers at a concentration of 100 µM each.
Prepare the primer working solution diluting to 10 micromolar (µM) using 0.1 % volume TE buffer.
SECTION: SARS-CoV-2 WvGS protocol - ARTIC protocol - Tiled PCR
2.1. [   ]  Set up two individual reactions using primer pool 1 (set 1) and primer pool 2 (set 2) in 0.2 mL PCR tubes according to the following table:


 



 
Lot# _______________ Exp. Date _______________

SECTION: SARS-CoV-2 WvGS protocol - ARTIC protocol - Tiled PCR
2.2. [   ]  Aliquot 22.5 µL from the mastermix into 2 96-well PCR plates or 2 sets of PCR tubes.
SECTION: SARS-CoV-2 WvGS protocol - ARTIC protocol - Tiled PCR
2.3. [   ]  Add 2.5 µL of sample cDNA (from step 1.9) to each pool giving a total volume 25 µL and mix by pipetting. Spin briefly.
SECTION: SARS-CoV-2 WvGS protocol - ARTIC protocol - Tiled PCR
2.4. [   ]  Heat seal and place the plates onto a thermocycler and run the following program. 
Important! Heat seal to minimise evaporation.

Note: Amplification should ideally be performed in a different lab to minimise the risk of contaminating other samples.
 

 

 

SECTION: SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection
3. Section for Clean-Up and Size Selection        Date/Initials:_________________

Reagent preparation: 
Allow AMPure XP beads to equilibrate to room temperature (~30 minutes). Ensure solution is homogenous prior to use, mixing gently by inversion.

 


 
Prepare the 80 % volume ethanol (EtOH) using the following calculation:

0.360 mL  x (# Sample + 1: ________________) = ________ mL total volume (EtOH 100%)


mL total volume x 0.8 = ________ mL EtOH


Total volume _______mL  - _______mL EtOH = ________mL H2O

SECTION: SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection
3.1. [   ]  Combine the entire volumes of pool 1 and pool 2 PCR reactions (50 µL in total) into one clean PCR plate (or  PCR tubes set). 
SECTION: SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection
3.2. [   ]  Add 0.8X volume of SPRI beads per sample (40 µL SPRI : 50 µL amplified cDNA), mix well by pipetting.
Incubate 10 min at Room temperature.
SECTION: SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection
3.3. [   ] Transfer the plate on the magnet and incubate for  5 min at Room temperature.
SECTION: SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection
3.4. [   ] Keep the plate on the magnet and remove the superanatant by pipetting from the bottom.

 

SECTION: SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection
3.5. [   ] Wash the beads in the magnet with 180 µL of freshly prepared 80 % volume EtOH without disturbing the pellet and incubate for 30 s and remove the EtOH.
SECTION: SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection
3.6. [   ]  Repeat previous step (total 2 washes).
SECTION: SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection
3.7. [   ] Spin down and place the tubes back on the magnet. Pipette off any residual ethanol with a P10 pipette and allow to dry for approximately 10 min.
 

SECTION: SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection
3.8. [   ] Remove the plate from the magnet and add 30 µL of nuclease-free water, resuspend the beads pipetting up and down at least 10 times or vortex at 1800 rpm for 1 min
SECTION: SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection
3.9. [   ] Incubate at room temperature for 2 min

SECTION: SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection
3.10. [   ] Transfer the plate on the magnet and incubate for 5 min at Room temperature
SECTION: SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection
3.11. [   ]  Carefully transfer the supernatant (28μl) into a new plate, taking care not to disturb the bead pellet.


 

SECTION: SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection
3.12. [   ]  Quantify the  sample on Qubit fluorometer or similar instrument and store completed PCR amplified cDNA prep at -20 °C
 

 


SECTION: NEBNext library preparation protocol - Fragmentation/End prep
4. 
 






This section is an adaptation protocol for FS DNA Library Prep Kit (E7805, E6177) with Inputs ≥ 100 ng

 



SECTION: NEBNext library preparation protocol - Fragmentation/End prep
4.1. [   ]  Prepare enzyme Master Mix using the following table:

 

 

SECTION: NEBNext library preparation protocol - Fragmentation/End prep
4.2. [   ] Add 4.5 µL of prepared mastermix (above) to each well.  Add 13 µL  of purified DNA to the PCR tube or to the wells of the PCR plate.Vortex the reaction for 5 seconds and briefly spin in a microcentrifuge.

SECTION: NEBNext library preparation protocol - Adapter ligation
5. [   ]  Add the following components directly to the FS Reaction Mixture:

 

 

SECTION: NEBNext library preparation protocol - Adapter ligation
5.1. [   ]  Incubate at 20 °C for 15 min in a thermocycler with the heated lid off.
SECTION: NEBNext library preparation protocol - Adapter ligation
5.2. [   ]  Add 1.5 µL  µl of USER Enzyme to the ligation mixture from Step 5.1. Vortex for 10 s  & spin briefly. 

SECTION: NEBNext library preparation protocol - Adapter ligation
5.3. [   ]  Mix well and incubate in thermocycler at 37 °C for 15 min  with the heated lid set to ≥ 47 °C 

 

SECTION: Cleanup of Adaptor-ligated DNA
5.4. 

 



SECTION: Cleanup of Adaptor-ligated DNA
5.5. [   ]  Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.
SECTION: Cleanup of Adaptor-ligated DNA
5.6. [   ]  Add 28 µL (FS fragmentation) or 43 µL(end repair) of the Ampure XP Beads to the ligation reaction mixture and mix well by pipetting up and down, or vortex. Spin briefly. 

SECTION: Cleanup of Adaptor-ligated DNA
5.7. [   ]  Incubate at room temperature for 5 min 
SECTION: Cleanup of Adaptor-ligated DNA
5.8. [   ]  Place the plate on magnetic block for 5 min
SECTION: Cleanup of Adaptor-ligated DNA
5.9. [   ]  Carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets




SECTION: Cleanup of Adaptor-ligated DNA
5.10. [   ]  Wash the beads adding 200 µL of freshly prepared 80% ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for  30 s, and then carefully remove and discard the supernatant.
Be careful not to disturb the beads that contain DNA targets.
SECTION: Cleanup of Adaptor-ligated DNA
5.11. [   ]  Repeat Step 5.10 once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
SECTION: Cleanup of Adaptor-ligated DNA
5.12. [   ]  Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.

 

SECTION: Cleanup of Adaptor-ligated DNA
5.13. [   ]   Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 10 µL 0.1 % volume  TE (dilute 1X TE Buffer 1:10 in water).

SECTION: Cleanup of Adaptor-ligated DNA
5.14. [   ]  Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

SECTION: Cleanup of Adaptor-ligated DNA
5.15. [   ]  Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 7.5 µL to a new PCR tube.

 

SECTION: NEBNext library preparation protocol - PCR Enrichment of Adapter-ligated DNA
6. [   ]  Add the following reagents to each well from step 5.15

 

 

Index set no.  _______________ 

Index Range (A) _______________        Index Range (B) _______________                   
SECTION: NEBNext library preparation protocol - PCR Enrichment of Adapter-ligated DNA
6.1. [   ]  Set a 100 µl or 200 µl pipette to 40 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
SECTION: NEBNext library preparation protocol - PCR Enrichment of Adapter-ligated DNA
6.2. [   ]  Place the tube/plate on a thermocycler with the heated lid set to 105 °C  and perform PCR amplification using the following PCR cycling conditions:



 

 

SECTION: NEBNext library preparation protocol - Clean up of PCR reaction
7. Allow the Ampure XP beads to warm to room temperature for at least 30 minutes before use. 

[   ]  Vortex SPRIselect to resuspend.  
SECTION: NEBNext library preparation protocol - Clean up of PCR reaction
7.1. [   ]  Add 22.5 µL (0.9X) resuspended beads to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.
SECTION: NEBNext library preparation protocol - Clean up of PCR reaction
7.2. [   ]  Incubate samples on bench top for at least 5 min  at Room temperature

SECTION: NEBNext library preparation protocol - Clean up of PCR reaction
7.3. [   ]  Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
SECTION: NEBNext library preparation protocol - Clean up of PCR reaction
7.4. [   ]  After 5 min (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets (Caution: do not discard the beads).

SECTION: NEBNext library preparation protocol - Clean up of PCR reaction
7.5. [   ]  Add 200 µL of 80 % volume freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for 30 s , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
SECTION: NEBNext library preparation protocol - Clean up of PCR reaction
7.6. [   ]  Repeat Step 7.5. once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
SECTION: NEBNext library preparation protocol - Clean up of PCR reaction
7.7. [   ]  Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
 

SECTION: NEBNext library preparation protocol - Clean up of PCR reaction
7.8. [   ]  Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 17 µL  of 0.1 % (v/v) TE (dilute 1X TE Buffer 1:10 in water).

SECTION: NEBNext library preparation protocol - Clean up of PCR reaction
7.9. [   ]  Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min  at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

SECTION: NEBNext library preparation protocol - Clean up of PCR reaction
7.10. [   ]  Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 15 µL  to a new PCR tube and store at -20 °C.

SECTION: Assess Library quality
8. Set up dilutions and standards as laid out in the kit protocol for dsDNA high sensitivity kit.
Record Qubit readings before normalization.

 

SECTION: Assess Library quality
8.1. [   ]  Run Samples on Agilent Bioanalyser or Agilent Tapestation to check that the library shows a narrow distribution with an expected peak size based on fragmentation time and size selection. Record the the average peak bp size.
 

SECTION: Assess Library quality
8.2. [   ]  Calculate the dilutions required to normalise each sample to a 4nM concentration using the following formula:
  

 

SECTION: Assess Library quality
9. [   ]  Run Samples on a bioanalyser or tapestation and check that the library shows a narrow distribution with an expected peak size based on fragmentation time and size selection. Record the the average peak bp size
 

SECTION: MiSeq Sequencing
10. Pooling and Library Denaturation        Date/Initials:_________________


This section demonstrates how to generate a pooled library for V2 reagents on the MiSeq.
 

 

SECTION: MiSeq Sequencing
10.1. [   ]  Pool 5 µL of each normalised sample into an eppendorf tube. This will be (1) pooled library.
SECTION: MiSeq Sequencing
10.2. [   ]  Combine the following volumes in a microcentrifuge tube (2): 
5 µL 4nM pooled library and 5 µL of 0.2 N NaOH.

[   ]  Vortex briefly and then centrifuge at 280 x g for 1 minute. 
[   ]  Incubate at room temperature for 5 min 
SECTION: MiSeq Sequencing
10.3. [   ]  Add 990 µL of pre-chilled HT1 to the tube containing the denatured library (2). The result is 1 mL of a 20 pM denatured library.
SECTION: MiSeq Sequencing
10.4. [   ]  Dilute the 20 pM library to the desired concentration, see table below:

 

 


[   ]  Invert to mix and then pulse centrifuge




 

SECTION: MiSeq Sequencing
10.5. [   ]  Dilute stock PhiX to 4nM by combining: 
- 2 µLof 10 nanomolar (nM) PhiX library 
- 3 µLof  10 millimolar (mM) Tris-Cl, pH 8.5 with 0.1% Tween 20

SECTION: MiSeq Sequencing
10.6. Denature the PhiX control by adding the following volumes in a microcentrifuge tube: 
- 5 µL of 4 nanomolar (nM) PhiX library 
- 5 µL of  0.2 nanomolar (nM) NaOH 

 

SECTION: MiSeq Sequencing
10.7. [   ]  Vortex briefly to mix and centrifuge at   for1 min. 
[   ]  Incubate at8 Room temperature for 5 min



SECTION: MiSeq Sequencing
10.8. [   ]  Dilute denatured PhiX library to 20 pM by adding 990 uL pre-chilled HT1 to the PhiX tube. Invert to mix.

 

SECTION: MiSeq Sequencing
10.9. [   ]  Combine library and PhiX control according to the table below:

 



SECTION: MiSeq Sequencing
10.10. [   ]  Set aside on ice until you are ready to load it onto the reagent cartridge.
SECTION: MiSeq Sequencing
10.11. [   ]  Mix reagents of the MiSeq cartridge thoroughly by inverting several times. 

[   ]  Using a fresh 1000 µL pipette tip, transfer the denatured and library (with PhiX spiked) into position 17.

SECTION: MiSeq Sequencing
10.12. [   ]  Load the sample sheet and reagents according to onscreen instructions in the MiSeq Control software.
SECTION: NEBNext library preparation protocol - Fragmentation/End prep
4.3. [   ]  In a Thermocycler, with the heated lid set to 75°C, run the following program:
     ABC1StepTempTime2Tagmentation55°C15 min3Hold10°CHold


 

 

SECTION: NEBNext library preparation protocol - Fragmentation/End prep
4.4. 
Steps 4.1 to 4.3 detailed enzymatic fragmentation. The following steps (4.4 to 4.6) detail the end repair option. 

*If you have carried out steps 4.1 to 4.3, this protocol continues from step 5*


[   ] Prepare the following mastermix in a sterile nuclease-free tube: 


 

SECTION: NEBNext library preparation protocol - Fragmentation/End prep
4.5. 
[   ] Add 5 µL of mastermix (above) to each well. Add 25 µL  of purified DNA to the PCR tube or to the wells of the PCR plate.Vortex the reaction for 5 seconds and briefly spin in a microcentrifuge.


SECTION: NEBNext library preparation protocol - Fragmentation/End prep
4.6. 
[   ] In a thermocycler, with the heated lid set to 75 °C, run the following program: