TITLE: COVID-19 ARTIC v3 Illumina library construction and sequencing protocol - high throughput 384 format
AUTHORS: DNA Pipelines R&D, Benjamin Farr, Diana Rajan, Emma Betteridge, Lesley Shirley, Michael Quail, Naomi Park, Nicholas Redshaw, Iraad Bronner, Louise Aigrain, Scott Goodwin, Scott Thurston, Stefanie Lensing, Carol Scott, Nicholas Salmon, Charlotte Beaver, Rachel Nelson, Alex Alderton, Ian Johnston

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">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 V3 nCov-2019 primers (ARTIC). This is followed by library construction, equivolume pooling of samples and quantitation, prior to sequencing on the Illumina NovaSeq. </div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">It offers the benefit of higher density sample processing in 384 format, whilst matching the data quality achieved in 96 format described in the original protocol:</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><a href="https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-bgxjjxkn" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-bgxjjxkn</span></a></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block">Both the above protocols were adapted from the nCov-2019 sequencing protocol: </div><div class = "text-block"><div class = "justify" style = "text-align:left"><a href="https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w</span></a></div></div></div>

[STEPS]
?. [cDNA generation]
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.
?. [cDNA generation]
Defrost PCR plate containing  extracted RNA .
10 µl
on ice
?. [cDNA generation]
Prepare RT mastermix in a dedicated UV treated pre-PCR area to minimise contamination risk.  ABC1RT Master MixVol / RXN (µL)Vol/384 RXN (µL) inc. excess2  LunaScript Super Mix    4  18433  Nuclease-free water    6  27654  Total    10  4608Mix thoroughly by vortexing.
ABC1RT Master MixVol / RXN (µL)Vol/384 RXN (µL) inc. excess2  LunaScript Super Mix    4  18433  Nuclease-free water    6  27654  Total    10  4608
?. [cDNA generation]
Use the SPT Labtech Dragonfly Discovery to dispense of RT mastermix into the PCR plate containing  extracted RNA.
10 µl
10 µl
?. [cDNA generation]
Seal plate and place on a BioShake plate shaker for 30 seconds at 1500rpm to mix. Briefly centrifuge plate.
?. [cDNA generation]
Place plate on a thermocycler and run the following program:  AB1  Temperature    Time  2  25°C  2 minutes3  55°C    20 minutes  4  95°C    1 minute  5  4°C    ∞  6  Lid temp: Tracking
AB1  Temperature    Time  2  25°C  2 minutes3  55°C    20 minutes  4  95°C    1 minute  5  4°C    ∞  6  Lid temp: Tracking
?. [cDNA generation]
PAUSE POINT cDNA can be stored at 4°C (same day) or -20°C (up to a week).
?. [cDNA amplification]
Prepare the following mastermixes:  ABC1Weighted PCR Primer Pool 1 Master Mix  Vol/PCR RXN (µl)  Vol/384 plate (µl) inc. excess2  Q5 Hotstart 2X Master Mix    12.5  57603Primer Pool 1 (mean 102nM)  3.6  16594  Nuclease-free water  2.913365  Total  198755  ABC1Weighted PCR Primer Pool 2 Master Mix  Vol/PCR RXN (µl)  Vol/384 plate (µl) inc. excess2Q5 Hotstart 2X Master Mix  12.5  57603Primer Pool 2 (mean 102nM)  3.6  16594Nuclease-free water2.913365  Total  198755Mix thoroughly by vortexing.
Primer pool sequences (v3) can be found here: https://github.com/joshquick/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V3/nCoV-2019.tsvWhere an alt primer is available, the non alt version is omitted.
Achieving more even genome coverageA hypothetical 'ideal' multiplex primer pool would generate the same number of reads from each amplicon, so the fraction of reads due to each amplicon would be 1/n, where n is the number of primer pairs in the multiplex pool. In reality this is not achievable, and the fraction of reads observed for each amplicon varies widely.The ratio [actual observed read fraction/‘ideal’ read fraction] can be calculated for each individual amplicon, as indicated by the differently-coloured dots on the box-and-whisker plots below. This tells us whether a particular amplicon is under-represented (ratio 1x).By changing the weights of each primer pair within the primer pool ('rebalancing') the number of reads obtained for each amplicon can be modified, and the effect of the process is illustrated below. The plots show the distribution per amplicon prior to rebalancing primer pair concentrations (above) and after (below). More amplicons cluster around 1x after rebalancing and the distance between the maximum and minimum ratios is also markedly reduced.Weight to apply per primer pairA more detailed description of the process is provided in this document:
ABC1Weighted PCR Primer Pool 1 Master Mix  Vol/PCR RXN (µl)  Vol/384 plate (µl) inc. excess2  Q5 Hotstart 2X Master Mix    12.5  57603Primer Pool 1 (mean 102nM)  3.6  16594  Nuclease-free water  2.913365  Total  198755
ABC1Weighted PCR Primer Pool 2 Master Mix  Vol/PCR RXN (µl)  Vol/384 plate (µl) inc. excess2Q5 Hotstart 2X Master Mix  12.5  57603Primer Pool 2 (mean 102nM)  3.6  16594Nuclease-free water2.913365  Total  198755
The equivolume primer pools used in the standard protocol are of    cumulative concentration, therefore each of the 98 primers in each pool is at   in the pool and at   in the final reaction. With the rebalanced primer pools, for equivalency we dilute them such that the average primer concentration is   , and therefore the average concentration of each primer in the final reaction is also   .
?. [cDNA amplification]
Use the SPT Labtech Dragonfly Discovery to dispense mastermix per well into 2x384 well plates.
19 µl
?. [cDNA amplification]
Use the Agilent Bravo to add  of cDNA template to each primer pool reaction and mix.
6 µl
It is recommended to use filtered tips for this transfer to reduce risk of cross sample contamination via aerosolisation.
?. [cDNA amplification]
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.       ABC1Step  Temperature    Time  21  98°C    30 seconds  32  95°C    15 seconds  4363°C  5 minutes  54Repeat steps 2 &amp; 3 for a total of 35 cycles65  4°C    ∞
Critical step: We strongly recommend performing a gradient PCR to determine the optimal annealing temperature for your thermocycler. Subtle differences in thermocycler calibration can result in specific amplicons dropping out. Reducing our annealing temperature from 65°C to 63°C for identical cDNA input recovered amplicon #64 as shown in the image below.
ABC1Step  Temperature    Time  21  98°C    30 seconds  32  95°C    15 seconds  4363°C  5 minutes  54Repeat steps 2 &amp; 3 for a total of 35 cycles65  4°C    ∞
?. [cDNA amplification]
PAUSE POINT Amplified cDNA can be stored at 4°C (overnight) or -20°C (up to a week).
?. [Amplified cDNA SPRI]
Allow AMPure XP beads to equilibrate to room temperature (~30 minutes). Ensure solution is homogenous prior to use, mixing gently by inversion.
?. [Amplified cDNA SPRI]
Centrifuge amplified cDNA plates.
Centrifuge: 1000 34, 1 min
?. [Amplified cDNA SPRI]
Use the SPT LabTech HV Mosquito to combine   of primer 1 PCR and  of primer 2 PCR reactions per sample into a new plate. Store the unused portion of primer 1 and primer 2 PCR plates at  . Proceed as follows with the recombined plate.
5 µl
5 µl
-20 °C
?. [Amplified cDNA SPRI]
Use the Hamilton STAR with a 384 well multichannel head to perform the following steps:
?. [Amplified cDNA SPRI]
Add   nuclease-free water to each sample and mix well by pipetting.
10 µl
?. [Amplified cDNA SPRI]
Add 0.8X volume of SPRI beads per sample (  SPRI :   amplified cDNA), mix well by pipetting.
16 µl
20 µl
?. [Amplified cDNA SPRI]
Incubate for  at
0 Room temperature
?. [Amplified cDNA SPRI]
Transfer the plate to the magnet, allow   for the beads to settle.
?. [Amplified cDNA SPRI]
Carefully remove and discard the supernatant without disturbing the bead pellet.
?. [Amplified cDNA SPRI]
Wash the beads with  75% freshly prepared ethanol for  , then remove ethanol and discard.(First wash)
45 µl
?. [Amplified cDNA SPRI]
Wash the beads with  75% freshly prepared ethanol for  , then remove ethanol and discard.(Second wash)
45 µl
?. [Amplified cDNA SPRI]
Allow beads to dry
?. [Amplified cDNA SPRI]
Remove plate from magnet, add  nuclease-free water and resuspend by mixing well.
20 µl
?. [Amplified cDNA SPRI]
Incubate for  at
0 Room temperature
?. [Amplified cDNA SPRI]
Transfer the plate to the magnet, allow   for the beads to settle.
?. [Amplified cDNA SPRI]
Carefully transfer supernatant into a new plate, taking care not to disturb the bead pellet.
?. [Amplified cDNA SPRI]
PAUSE POINT Purified amplified cDNA can be stored at -20°C for several weeks prior to library preparation.
?. [Amplified cDNA quantification]
Purified amplified cDNA is quantified with a fluorescence based assay. We use the AccuClear Ultra High Sensitivity dsDNA Quantitation kit with 7 DNA standards (Biotium) according to manufacturer’s instructions. To streamline the workflow, we do not normalise sample input for library preparation. Instead we confirm samples are in the range of 50ng-1ug per 20µl sample and take the entire volume into library preparation.
?. [Amplified cDNA quantification]
Pipette of each DNA standard into wells A1 – G1 of a PCR plate. Add nuclease-free water to H1.
20 µl
?. [Amplified cDNA quantification]
Dilute the AccuClear dye (100X) to working concentration by mixing  dye with  AccuClear buffer in a 50ml Falcon. Mix thoroughly by vortexing and transfer to a 384 well reservoir.
300 µl
30 mL
?. [Amplified cDNA quantification]
Use the SPT Labtech Mosquito LV to stamp 1µl of amplified cDNA and 1µl of known standards into a 384 assay plate. Immediately proceed to the next step.
?. [Amplified cDNA quantification]
Use the Agilent Bravo 384ST to add 1X AccuClear dye from the reservoir to the assay plate, mix thoroughly by pipetting.
50 µl
?. [Amplified cDNA quantification]
Measure fluorescence values on a BMG FLUOstar Omega plate reader calibrated for use with AccuClear dye.
?. [Amplified cDNA quantification]
Confirm known standards are performing as expected.
?. [Amplified cDNA quantification]
Dilute any samples >125ng/µl with nuclease free water so they are in the range of 10 - 125ng/µl and repeat quantitation.
?. [Amplified cDNA quantification]
Ensure all samples (20µl total volume) are in the range of 2.5-50ng/µl prior to proceeding with library preparation.
?. [Library preparation for Illumina sequencing]
We use the NEB NEBNext® Ultra™ II DNA Library Prep Kit for Illumina, which we have automated on the Agilent Bravo platform with some modifications. 200ng is our standard input for library preparation, an acceptable range is 50ng – 1ug per sample. We use a custom adapter set, however any TruSeq adapters are suitable.
?. [Library preparation for Illumina sequencing]
Prepare end prep mastermix :  ABC1NEBNext End Prep  Vol/PCR RXN (µl)  Vol/384 plate (µl) inc. excess2  NEBNext Ultra II End Prep Enzyme Mix    1.2  5523  NEBNext Ultra II End Prep Reaction Buffer    2.8  12884  Total    4  1840 Mix well by pipetting.
on ice
ABC1NEBNext End Prep  Vol/PCR RXN (µl)  Vol/384 plate (µl) inc. excess2  NEBNext Ultra II End Prep Enzyme Mix    1.2  5523  NEBNext Ultra II End Prep Reaction Buffer    2.8  12884  Total    4  1840
?. [Library preparation for Illumina sequencing]
The Bravo will combine  of end prep mastermix with amplified cDNA and mix by pipetting.
4 µl
20 µl
?. [Library preparation for Illumina sequencing]
Seal and transfer the plate to a thermocycler and run the following program:  AB1  Temperature    Time  2  20°C    30 minutes  3  65°C    30 minutes  4  4°C    ∞
AB1  Temperature    Time  2  20°C    30 minutes  3  65°C    30 minutes  4  4°C    ∞
?. [Library preparation for Illumina sequencing]
Prepare adapter ligation mastermix :  ABC1Adapter Ligation  Vol/PCR RXN (µl)  Vol/384 plate (µl) inc. excess2  NEBNext Ultra II Ligation Master Mix    12  55203  NEBNext Ligation Enhancer    0.4  1844  TruSeq adapter (10µM)    1  4605  Total    13.4  6164 Mix well by pipetting.
on ice
ABC1Adapter Ligation  Vol/PCR RXN (µl)  Vol/384 plate (µl) inc. excess2  NEBNext Ultra II Ligation Master Mix    12  55203  NEBNext Ligation Enhancer    0.4  1844  TruSeq adapter (10µM)    1  4605  Total    13.4  6164
?. [Library preparation for Illumina sequencing]
The Bravo will add  adapter ligation mastermix to each sample and mix by pipetting.
13.4 µl
?. [Library preparation for Illumina sequencing]
The plate is incubated on deck at  for , however this step may also be performed on a thermocycler.
20 °C
?. [Library preparation for Illumina sequencing]
Note: We use alternative TruSeq compatible adapters, which do not require the USER enzyme incubation step. If using NEBNext adapters, follow the steps in the NEB protocol to add USER enzyme to the ligation reaction.
?. [Library preparation for Illumina sequencing]
A 0.8X SPRI is performed post-ligation.Ensure AMPure XP beads have been equilibrated to room temperature (~30 minutes) and the solution is homogenous prior to use.The Bravo will perform a 0.8X SPRI clean-up and elute sample in 25 µl nuclease-free water as follows:
?. [Library preparation for Illumina sequencing]
Add 0.8X volume of SPRI beads per sample, mix well by pipetting.
?. [Library preparation for Illumina sequencing]
Incubate for at .
20 Room temperature
?. [Library preparation for Illumina sequencing]
Transfer the plate to the magnet, allow  for the beads to settle.
?. [Library preparation for Illumina sequencing]
Carefully remove and discard the supernatant without disturbing the bead pellet.
?. [Library preparation for Illumina sequencing]
Wash the beads with 75% freshly prepared ethanol for , then remove ethanol and discard.(First wash)
45 µl
?. [Library preparation for Illumina sequencing]
Wash the beads with 75% freshly prepared ethanol for , then remove ethanol and discard.(Second wash)
45 µl
?. [Library preparation for Illumina sequencing]
Allow beads to dry for
?. [Library preparation for Illumina sequencing]
Remove plate from magnet, add  nuclease free water and resuspend by mixing well.
25 µl
?. [Library preparation for Illumina sequencing]
Incubate for  at.
20 Room temperature
?. [Library preparation for Illumina sequencing]
Transfer the plate to the magnet, allow  for the beads to settle.
?. [Library preparation for Illumina sequencing]
Carefully transfer supernatant into a new plate, taking care not to disturb the bead pellet.   of this eluate is used as input for library PCR.
10 µl
?. [Library PCR]
KAPA HiFi HotStart ReadyMix is required per 384 plate (including excess).
We use KAPA HiFi HotStart ReadyMix and unique dual indexed (UDI) tag plates for library PCR. Note: this deviates from the standard NEB protocol which uses NEBNext Ultra II Q5 Master Mix and different cycling conditions.
6900 µl
?. [Library PCR]
The Bravo will add  KAPA HiFi HotStart ReadyMix and sample into a   plate of UDIs and mix thoroughly by pipetting. The final concentration of each UDI in the PCR reaction is 2μM.
15 µl
10 µl
5 µl
?. [Library PCR]
Seal and transfer the plate to a thermocycler and run the following program:  AB1  Temperature    Time  2  95°C    5 minutes  3  98°C    30 seconds  4  65°C    30 seconds  5  72°C    2 minutes  6  Repeat 4 times  7  72°C    5 minutes  8  4°C    ∞
AB1  Temperature    Time  2  95°C    5 minutes  3  98°C    30 seconds  4  65°C    30 seconds  5  72°C    2 minutes  6  Repeat 4 times  7  72°C    5 minutes  8  4°C    ∞
?. [Construct equivolume pool]
In a post-PCR lab, use the Hamilton STAR to combine   of each sample per plate to form an equivolume pool of 384 samples.
3 µl
?. [Equivolume pool SPRI]
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 200μl elution buffer as follows:
?. [Equivolume pool SPRI]
Add 0.8X volume of SPRI beads per pool tube, mix well by pipetting.
?. [Equivolume pool SPRI]
Incubate for  at .
20 Room temperature
?. [Equivolume pool SPRI]
Transfer the tube to a magnet, allow for the beads to form a pellet.
?. [Equivolume pool SPRI]
Carefully remove and discard the supernatant, taking care not to disturb the bead pellet.
?. [Equivolume pool SPRI]
Wash the beads with  75% ethanol for  then carefully remove ethanol and discard.(First wash)
500 µl
?. [Equivolume pool SPRI]
Wash the beads with  75% ethanol for  then carefully remove ethanol and discard.(Second wash)
500 µl
?. [Equivolume pool SPRI]
Wash the beads with  75% ethanol for  then carefully remove ethanol and discard.(Third wash)
500 µl
?. [Equivolume pool SPRI]
Allow beads to dry for .
?. [Equivolume pool SPRI]
Remove tube from magnet and resuspend beads in  elution buffer, mix well by pipetting.
200 µl
?. [Equivolume pool SPRI]
Incubate for  at
20 Room temperature
?. [Equivolume pool SPRI]
Transfer tube to magnet, allow for the beads to form a pellet.
?. [Equivolume pool SPRI]
Carefully transfer supernatant into a new tube, taking care not to disturb the bead pellet.
?. [Equivolume pool quantification]
qPCRQuantify pools 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 or adapter-dimer present.
Equivolume pools may be quantified either by qPCR or on an Agilent Bioanalyzer. Pools are then diluted to 1nM for sequencing.
?. [Sequencing]
We currently sequence samples on an Illumina NovaSeq SP flow cell, using the XP workflow.Alternatively, samples may be sequenced on an Illumina MiSeq using either v2 (500 cycle) or v3 (600 cycle) reagent kits. We have plexed up to 96 samples per run, this could be increased further depending on coverage requirements. Loading concentration will need to be optimised for MiSeq. MiSeq run parameters: Read length 212 paired end + 16bp.
?. [Sequencing]
The following protocol is for loading a NovaSeq. We currently plex up to 384 samples per NovaSeq SP lane.
?. [Sequencing]
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.
?. [Sequencing]
Defrost Illumina NovaSeq SP SBS and cluster reagent cartridges for 2-4 hours in a  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.
20 Room temperature
?. [Sequencing]
Defrost components DPX1, DPX2 and DPX3 from a NovaSeq XP-2 lane kit, then keep
on ice
?. [Sequencing]
Bring flow cell to  (~10 minutes) prior to use.
20 Room temperature
?. [Sequencing]
of each  pool is required per SP lane. Denature pools by adding 0.2N NaOH per 18µl. Vortex briefly to mix.
18 µl
4 µl
?. [Sequencing]
Incubate at  for
20 Room temperature
?. [Sequencing]
Add  400mM Tris-HCl, pH8.0 to each tube to neutralise the reaction. Vortex briefly to mix, then keep .
5 µl
on ice
For the following steps, keep samples and mastermix   until ready for loading onto the flow cell.
?. [Sequencing]
Important! Use mastermix within of preparation for optimal sequencing performance.Prepare ExAmp mastermix on ice:  AB1  ExAmp Master Mix    Volume per SP flow cell (µl)  2  DPX1    126  3  DPX2    18  4  DPX3    66  5  Total    210   Vortexto mix, then centrifuge briefly up to
AB1  ExAmp Master Mix    Volume per SP flow cell (µl)  2  DPX1    126  3  DPX2    18  4  DPX3    66  5  Total    210
Centrifuge: 280 34
?. [Sequencing]
Add  ExAmp mastermix to each denatured pool, mix well by pipetting.
63 µl
?. [Sequencing]
Prepare the flowcell for sample loading by placing into the flow cell dock with the 2-lane manifold clamped in place.
?. [Sequencing]
Pipette  of library + ExAmp pool mix per manifold well. Wait for approximately 2 minutes to allow the solution to fill the lane.
80 µl
?. [Sequencing]
Important! The sequencing run must be started within  of libraries being loaded onto the flow cell.
?. [Sequencing]
Unclamp the flow cell dock and discard the manifold. Load the flow cell onto the NovaSeq flow cell stage.
?. [Sequencing]
Load the SBS and cluster reagent cartridges.
?. [Sequencing]
Start sequencing run (250PE).