TITLE: COVID-19 ARTIC v4.1 Illumina library construction and sequencing protocol - tailed method
AUTHORS: DNA Pipelines R&D, Benjamin Farr, Diana  Rajan, Emma  Betteridge, Lesley  Shirley, Michael  Quail, Naomi  Park, Nicholas Redshaw, Iraad F Bronner, Louise Aigrain, Scott  Goodwin, Scott  Thurston, Stefanie Lensing, James Bonfield, Keith James, Nicholas Salmon, Charlotte Beaver, Rachel Nelson, David K.  Jackson, Alex Alderton, Ian Johnston

[DESCRIPTION]
This SOP describes the procedure for generating cDNA from SARS-CoV-2 viral nucleic acid extracts and subsequently producing 400nt amplicons tiling the viral genome using V4.1 nCov-2019 primers (ARTIC) in multiplex PCR. Illumina-compatible sequencing libraries are then made directly from these amplicons in a second PCR step, obviating the need for conventional library preparation. The products of these PCRs are then equivolume pooled and quantitated, prior to sequencing on the Illumina NovaSeq.

It is an adaptation of the COVID-19 ARTIC v3 amplicon protocol which can be found here:
https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-bibtkann

Both the above protocols were adapted from the nCov-2019 sequencing protocol: 
https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w

[GUIDELINES]
It is vital cDNA setup is performed in a laboratory in which post-PCR COVID-19 amplicons are not present, to minimise any risk of sample contamination.

Note: Throughout the protocol we have indicated the liquid handling automation in use at Sanger for specific parts of the process. However, these steps could be performed on alternative liquid handlers or manually.

[STEPS]
SECTION: cDNA generation
1. Important! This step must be performed in a RNase free, pre-PCR environment in which post PCR COVID-19 amplicons are not present, to minimise risk of sample contamination.

Decontaminate bench surfaces, pipettes and gloves with RNase ZAP before starting work. Keep reagents and samples chilled throughout the process.
SECTION: cDNA generation
2. Defrost PCR plate containing 10 µL extracted RNA on ice.

SECTION: cDNA generation
3. 
 

Prepare RT mastermix in a dedicated UV treated pre-PCR area to minimise contamination risk.
 
     RT   Master Mix      VOL / RXN (µL)      96 RXN (µL) inc. excess        LunaScript Super Mix      4      461        Nuclease-free water      6      691        Total      10      1152   
 
Mix thoroughly by vortexing.
SECTION: cDNA generation
4. Use the SPT Labtech Dragonfly Discovery to dispense 10 µLof RT mastermix into the PCR plate containing 10 µL extracted RNA.

SECTION: cDNA generation
5. Seal plate and place on a BioShake plate shaker for 30 seconds at 1500rpm to mix. Briefly centrifuge plate.
SECTION: cDNA generation
6. Place plate on a thermocycler and run the following program:
 
     Temperature      Time        25°C      10 minutes        55°C      20 minutes        95°C      1 minute        4°C      ∞        Lid temp: Tracking          
 
SECTION: cDNA generation
7. PAUSE POINT cDNA can be stored at 4°C (same day) or -20°C (up to a week).


SECTION: cDNA amplification (PCR1)
8. 

 

 

 


Prepare the following mastermixes:
 
     PCR Primer   Pool 1 Master Mix      Vol/PCR RXN (µl)      Vol/96 plate (µl) inc.   excess        Q5 Hotstart 2X Master Mix      12.5      1440        Primer Pool 1 (11µM   total)      3.6      415        Nuclease-free water      6.9      795        Total      23      2650   

     PCR Primer Pool 2 Master Mix      Vol/PCR RXN (µl)      Vol/96 plate (µl)   inc. excess        Q5 Hotstart   2X Master Mix      12.5      1440        Primer Pool 2 (10.8µM total)      3.6      415        Nuclease-free   water      6.9      795        Total      23      2650   

 

Mix thoroughly by vortexing.
SECTION: cDNA amplification (PCR1)
9. Use the SPT Labtech Dragonfly Discovery to dispense 19 µLmastermix per well into 2x384 well plates.

SECTION: cDNA amplification (PCR1)
10. Use the Agilent Bravo to add 6 µL of cDNA template to each primer pool reaction and mix.

 

SECTION: cDNA amplification (PCR1)
11. 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.     


 


 
     Temperature      Time        98°C      30 seconds        95°C      15 seconds        65°C      5 minutes        Repeat steps 2 &   3 for a total of 35 cycles        4°C      ∞   
 
SECTION: cDNA amplification (PCR1)
12. PAUSE POINT Amplified cDNA can be stored at 4°C (overnight) or -20°C (up to a week).
SECTION: Library construction from amplified cDNA (PCR2)
13. 

 


 

 

SECTION: Library construction from amplified cDNA (PCR2)
14. Defrost two UDI tag plates (one containing each tail primer pool), both of which should contain the same i5 and i7 barcodes per well.

SECTION: Library construction from amplified cDNA (PCR2)
15. Use the SPT Labtech Mosquito LV to transfer 100 nl of amplified pool 1 cDNA into the UDI tag plate containing the pool 1 tailed primers  and 100 nlof amplified pool 2 cDNA into the UDI tag plate containing the pool 2 tailed primers, maintaining the same well locations throughout. Immediately proceed to the next step.

SECTION: Library construction from amplified cDNA (PCR2)
16. Use the SPT Labtech Dragonfly Discovery to dispense 6.25 µL of Kapa HiFi 2X Mastermix into each well of both UDI tag plates, and place on iceimmediately. The dispense is sufficient to mix all the reagents.

 

SECTION: Library construction from amplified cDNA (PCR2)
17. Heat seal and place the two plates onto a thermocycler and run the following program. 
Important! Heat seal to minimise evaporation.
 
     Temperature      Time        95°C      5 minutes        98°C      30 seconds        65°C      30 seconds        72°C      2 minutes        Repeat 4 times        72°C      5 minutes        4°C      ∞   
 
 

SECTION: Library construction from amplified cDNA (PCR2)
18. PAUSE POINT Amplified cDNA can be stored at 4°C (overnight) or -20°C (up to a week).
SECTION: Construction of equivolume pool
19. 
In a post-PCR lab, use the Agilent Bravo to combine and mix  5 µL of pool 1 and pool 2 PCR2 reactions per sample into one plate.
SECTION: Construction of equivolume pool
20. Use the Hamilton STAR to combine 3 µL of each sample to form an equivolume pool of 384 samples.
SECTION: Equivolume pool SPRI bead cleanup
21. Allow AMPure XP beads to equilibrate to room temperature (~30 minutes). Ensure solution is homogenous prior to use, mixing gently by inversion.

The Hamilton STAR will perform a 0.8X SPRI clean-up and elute the final pool in 1ml elution buffer as follows:
SECTION: Equivolume pool SPRI bead cleanup
21.1. Add 0.8X volume of SPRI beads per pool tube, mix well by pipetting.
SECTION: Equivolume pool SPRI bead cleanup
21.2. Incubate for 6 min at 20 Room temperature.

SECTION: Equivolume pool SPRI bead cleanup
21.3. Transfer the tube to a magnet, allow 4 minfor the beads to form a pellet.

SECTION: Equivolume pool SPRI bead cleanup
21.4. Carefully remove and discard the supernatant, taking care not to disturb the bead pellet.
SECTION: Equivolume pool SPRI bead cleanup
21.5. Wash the beads with 500 µL 75% ethanol for 15 s then carefully remove ethanol and discard.
(First wash)

SECTION: Equivolume pool SPRI bead cleanup
21.6. Wash the beads with 500 µL 75% ethanol for 15 s then carefully remove ethanol and discard.
(Second wash)

SECTION: Equivolume pool SPRI bead cleanup
21.7. Wash the beads with 500 µL 75% ethanol for 15 s then carefully remove ethanol and discard.
(Third wash)
SECTION: Equivolume pool SPRI bead cleanup
21.8. Allow beads to dry for 5 min.

SECTION: Equivolume pool SPRI bead cleanup
21.9. Remove tube from magnet and resuspend beads in 200 µL elution buffer, mix well by pipetting.

SECTION: Equivolume pool SPRI bead cleanup
21.10. Incubate for 3 min at 20 Room temperature

SECTION: Equivolume pool SPRI bead cleanup
21.11. Transfer tube to magnet, allow 5 minfor the beads to form a pellet.

SECTION: Equivolume pool SPRI bead cleanup
21.12. Carefully transfer supernatant into a new tube, taking care not to disturb the bead pellet. 
SECTION: Equivolume pool quantification
22. 
 

qPCR
Quantify samples in triplicate using the KAPA Complete kit (Universal) for Illumina (KK4824) plus the KAPA Library Quantification Dilution Control (KK4906). 

We use the SPT Labtech Mosquito LV to stamp library pools in triplicate into a 384 assay plate, and the Agilent Bravo to setup the qPCR reactions (1:1600 dilution). 

qPCR is performed on the Roche LightCycler 480. 

Agilent Bioanalyzer 
Prepare 3 dilutions of the equivolume pool (1:10, 1:100, 1:1000). Run 1µl of each dilution in triplicate using the High Sensitivity DNA assay kit.

Confirm size distribution is as expected, check there is no primer-dimer present.

Agilent TapeStation
Run 1µl of each pool in triplicate using the Agilent D5000 ScreenTape System.
Confirm size distribution is as expected, check there is no primer-dimer present.
Set the region size range to 250-1500bp to quantify the pool.


SECTION: Sequencing
23. 
 

SECTION: Sequencing
24. The following protocol is for loading a NovaSeq. We currently plex up to 384 samples per NovaSeq SP lane. 
SECTION: Sequencing
25. Steps must be performed within a given timeframe or data quality may be affected. Therefore, ensure the instrument is washed, waste containers emptied and ready for use prior to beginning step 46.
SECTION: Sequencing
26. Defrost Illumina NovaSeq SP SBS and cluster reagent cartridges for 2-4 hours in a 20 Room temperature water bath. Use a lint free tissue to blot any water present on the foil seal. Gently mix cartridges 10X by inversion. Gently tap the bottom of the cartridges on the bench to reduce air bubbles.

SECTION: Sequencing
27. Defrost components DPX1, DPX2 and DPX3 from a NovaSeq XP-2 lane kit, then keep on ice

SECTION: Sequencing
28. Bring flow cell to 20 Room temperature (~10 minutes) prior to use.

SECTION: Sequencing
29. 18 µL of each 1 nanomolar (nM) pool is required per SP lane. 
Denature pools by adding4 µL 0.2N NaOH per 18µl. Vortex briefly to mix.

SECTION: Sequencing
30. Incubate at 20 Room temperature for 8 min
SECTION: Sequencing
31. Add 5 µL 400mM Tris-HCl, pH8.0 to each tube to neutralise the reaction. Vortex briefly to mix, then keep on ice.

 

SECTION: Sequencing
32. Important! Use mastermix within 60 minof preparation for optimal sequencing performance.

Prepare ExAmp mastermix on ice:
 
     ExAmp Master Mix      Volume per SP flow cell (µl)        DPX1      126        DPX2      18        DPX3      66        Total      210   

 Vortex30 sto mix, then centrifuge briefly up to 280 x g
 

SECTION: Sequencing
33. Add 63 µL ExAmp mastermix to each denatured pool, mix well by pipetting.

SECTION: Sequencing
34. Prepare the flowcell for sample loading by placing into the flow cell dock with the 2-lane manifold clamped in place.
SECTION: Sequencing
35. Pipette 80 µL of library + ExAmp pool mix per manifold well. Wait for approximately 2 minutes to allow the solution to fill the lane. 

SECTION: Sequencing
36. Important! The sequencing run must be started within 30 min of libraries being loaded onto the flow cell.

SECTION: Sequencing
36.1. Unclamp the flow cell dock and discard the manifold. Load the flow cell onto the NovaSeq flow cell stage.
SECTION: Sequencing
36.2. Load the SBS and cluster reagent cartridges.
SECTION: Sequencing
36.3. Start sequencing run (250PE).