Nov 10, 2021
Sub-ARTIC Illumina SARS-CoV-2 Spike sequencing protocol (LoCost) V3.2
- Gavin Horsburgh1,
- Kathryn Maher1,
- Steve Paterson2,
- Terry Burke1,
- Paul J Parsons3
- 1NERC Environmental Omics Facility, Ecology & Evolutionary Biology, School of Biosciences, University of Sheffield, Sheffield, UK;
- 2NERC Environmental Omics Facility, Department of Evolution, Ecology and Behaviour, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, UK;
- 3University of Sheffield
Protocol Citation: Gavin Horsburgh, Kathryn Maher, Steve Paterson, Terry Burke, Paul J Parsons 2021. Sub-ARTIC Illumina SARS-CoV-2 Spike sequencing protocol (LoCost) V3.2. protocols.io https://dx.doi.org/10.17504/protocols.io.btpjnmkn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: March 26, 2021
Last Modified: November 10, 2021
Protocol Integer ID: 48587
Keywords: SARS-CoV-2, COVID, variant, sequencing, Illumina, ARTIC , SubARTIC, amplicon
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Abstract
This protocol describes a procedure for sequencing the Spike region of SARS-CoV-2 using short amplicons (146-208bp). The method has proved to successful with both clinical RNA samples and degraded wastewater samples. The primers are unique to this method. The library prep procedure has been heavily adapted from the ncov-2019 sequencing v3 (ARTIC) protocol by Josh Quick (https://www.protocols.io/view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye) and the "low cost" method from the NEOF Liverpool Illumina ARTIC protocol.
Materials
Primers are listed in the attached 
SubArtic primers v3-2 091121.csv
SubArtic primers v3-2 091121.csv LunaScript® RT SuperMix Kit, New England BioLabs, Cat# E3010L
Q5® Hot Start High-Fidelity 2X Master Mix, New England BioLabs, Cat# M0494L
NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, New England BioLabs, Cat# E7645L
(contains NEBNext Ultra II End Prep Mix and buffer, Ultra II Q5 Master Mix, Ligation Enhancer, Ultra II Ligation Master Mix)
NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1), NewEngland BioLabs, Cat# E7600S (contains i5/i7 indexes, Adaptor for Illumina and User enzyme)
NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 2), NewEngland BioLabs, Cat# E7780S (contains i5/i7 indexes, Adaptor for Illumina and User enzyme)
AMPure XP® paramagnetic beads for PCR purification, Beckman Coulter, Cat# A63881
Kapa Library Quantification Kit for Illumina®, Complete Kit (ROX Low), Roche, Cat# KK4873 (for Quantstudio 12k)
Before start
Before starting, generate the "odd" and "even" primer pools as follows:
1. Fully resuspend lyophilised oligonucleotides in 1x TE to a concentration of 100 micromolar (µM) , vortex thoroughly and spin down
2. Sort the odd and even primer sets into separate batches and label two 1.5-ml tubes
3. Starting with the even primer set add the volume (μl) given overleaf to the pooled 1.5-ml tube (between 7.5 - 15). Repeat with the odds. Vortex and spin down. These are your pooled stocks.
4. Dilute the pools one in ten across several aliquots with molecular grade water. Vortex and spin down. These are your working primer pools.SubArtic primers v3-2 091121.csv
SubArtic primers v3-2 091121.csv cDNA prep
cDNA prep
1h 25m
1h 25m
In a freshly bleached Pre-PCR hood (ideally in an isolated clean room), that has been subjected to UV irradiation for 00:40:00 , add LunaScript and RNA sample to a tube/well as follows (making sure to include negative controls):
| A | B | |
| Component | Vol/Rxn | |
| LunaScript Super Mix | 2 µl | |
| RNA sample | 8 µl |
1h
Mix thoroughly by pipetting up and down several times, seal plate, and centrifuge briefly.
10m
Incubate the reaction as follows (with heated lid):
| A | B | |
| Temperature | Time | |
| 25°C | 2 min | |
| 55°C | 10 min | |
| 95°C | 1 min | |
| 4°C | Hold |
15m
Multiplex PCR
Multiplex PCR
4h 10m
4h 10m
Primers are separated into two pools, odd and even, depending on where they sit across the Spike region. See the guidelines section for further details. In a clean pre-PCR hood set up two PCR reactions, one per pool as follows:
| A | B | C | |
| Component | Rxn 1 | Rxn 2 | |
| Pool Even | 0 µl | 1.75 µl | |
| Pool Odd | 1.75 µl | 0 µl | |
| Q5 Hot Start Hi-Fi 2x Master Mix | 6.25 µl | 6.25 µl | |
| Total | 8 µl | 8 µl |
30m
Add 4.5 µL of cDNA to respective wells to give a total volume of 12.5 µL . Include negative controls. Mix by pipetting, seal plate and centrifuge briefly.
10m
Perform PCR using the following program (with heated lid):
| A | B | C | D | |
| Step | Temperature | Time | Cycles | |
| Initial Denaturation | 98°C | 30 s | 1 | |
| Denaturation | 98°C | 15 s | 35 | |
| Annealing & Extension | 60°C | 5 min | ||
| Hold | 4°C | Hold |
Note
Fewer PCR cycles can be used for samples of higher concentration.
3h 30m
PCR pooling
PCR pooling
30m
30m
In a freshly bleached post-PCR hood that has been subjected to UV for 00:40:00 , combine Pool Even and Pool Odd to give 20 µL in each tube/well. Add 20 µL nuclease-free water to dilute products 1 in 2.
Note
At least a 1 in 5 dilution is advised when using highly concentrated clincial samples subjected to a 35x cycle PCR.
20m
Check the quality and concentration of negatives and a selection of samples using a fluorometer and/or Agilent TapeStation.
Note
Samples can be normalised at this point if even barcode representation is required, but at the cost of time.
10m
NEBNext Ultra II End Prep
NEBNext Ultra II End Prep
50m
50m
Prepare a master mix of the reagents as below by multiplying volumes by the number of samples; add 10% to allow for pipetting error.
| A | B | |
| Component | Vol/PCR Rxn | |
| NEBNext Ultra II End Prep Enzyme Mix | 0.6 µl | |
| NEBNext Ultra II End Prep Reaction Buffer | 1.4 µl | |
| Total | 2 µl |
5m
Mix well by pipetting and centrifuge briefly.
5m
Combine 2 µL of End Prep master mix with 10 µL of amplified cDNA and mix by pipetting. Spin down, seal, place in a thermocycler and run the following program:
| A | B | |
| Temperature | Time | |
| 20°C | 15 min | |
| 65°C | 15 min | |
| 4°C | Hold |
40m
Adapter Ligation
Adapter Ligation
45m
45m
Prepare adapter ligation master mix by adding volumes as detailed below for each sample; adding 10% to allow for pipetting error.
| A | B | |
| Component | Vol/PCR rxn | |
| NEBNext Ultra II Ligation Master Mix | 6 µl | |
| NEBNext Ligation Enhancer | 0.2 µl | |
| NEBNext Adapter | 0.5 µl | |
| Total | 6.7 µl |
10m
Add 6.7 µL ligation mix to each 12 µL amplified cDNA/mastermix and mix by pipetting. Incubate at 20 °C for 00:15:00 .
25m
Add 1 µL of USER enzyme to the ligation mixture. Mix by pipetting, centrifuge briefly and incubate at 37 °C for 00:15:00 .
10m
Magnetic Bead clean up
Magnetic Bead clean up
1h 15m
1h 15m
Increase volume of sample from 19.7 µL to 25 µL by the addition of nuclease-free water.
5m
Perform a 0.9x Ampure XP bead clean by adding 22.5 µL of Ampure XP beads and mix by pepetting. Incubate for 00:05:00 at Room temperature .
10m
Transfer tube/plate to magnet and allow beads to clump for 00:05:00 . Remove supernatant taking care not to disturb pellet, and discard.
15m
Wash the beads with 100 µL 80% ethanol for 00:00:30 then remove ethanol by pipetting. Repeat one more time making sure to remove any residual ethanol.
10m
Allow beads to air dry for around 00:05:00 ; ensure that all the ethanol has evaporated. Do not let beads dry to the point of cracking, as this could affect the amount of DNA recovered.
5m
Add 17 µL of nuclease-free water to each well and mix by pipetting. Allow to stand for 00:05:00 then transfer to magnet and allow beads to clump for 00:05:00 . Remove 15 µL into a fresh low-bind tube.
20m
Perform Qubit assay to determine concentration.
10m
Addition of NEBNext indexes
Addition of NEBNext indexes
1h 5m
1h 5m
Add the following components to separate wells of a PCR plate. Make sure to use the indexes in appropriate wells so that each sample has unique i5 and i7 indexes. If using less than 12 samples, the i5 primer can be replaced by the Universal PCR primer.
| A | B | |
| Component | Vol/Rxn | |
| Adapter ligated cDNA fragments | 7.5 µl | |
| NEBNext Ultra II Q5 Master Mix | 12.5 µl | |
| Index Primer i7 | 2.5 µl | |
| Index Primer i5* | 2.5 µl | |
| Total | 25 µl |
* if using 12 samples or fewer replace index primer i5 with the Universal primer.
20m
Mix thoroughly by pipetting and centrifuge briefly.
5m
Place on thermocycler and perform PCR using the following program:
| A | B | C | D | |
| Cycle Step | Temperature | Time | Cycles | |
| Initial Denaturation | 98°C | 30 s | 1 | |
| Denaturation | 98°C | 10 s | 3 - 15* | |
| Annealing/Extension | 65°C | 75 s | ||
| Final Extension | 65°C | 5 mins | 1 | |
| Hold | 4°C | Hold |
*Refer to NEBNext protocol for number of cycles required. (Examples: 100 ng = ~3 cycles, 50 ng = ~3–4 cycles, 10 ng = ~6–7 cycles)
40m
Pool
Pool
1h 15m
1h 15m
Pool 5 µL of each sample together in a fresh low-binding microfuge tube and mix. This will give a final volume of 60 µL for twelve samples. If larger numbers of samples are used then pool 5 µL of each and mix. Split into aliquots of about 120 µL .
10m
Perform a 0.7x bead clean by adding 42 µL or 84 µL magnetic beads, depending on sample quantity. Leave for 00:05:00 to allow beads to bind DNA.
10m
Place on a magnet and allow beads to clump for 00:05:00 . Remove supernatant and discard.
10m
Wash pellet with 100 µL 80% ethanol for 00:00:30 and remove. Repeat one more time. Allow the ethanol to dry off for about 00:05:00 , making sure the pelleted beads do not dry out, which can reduce DNA recovery.
10m
Add 30 µL of nuclease-free water and mix by pipetting. Leave for 00:05:00 at Room temperature to allow DNA to elute from the beads.
10m
Place the tube/plate on the magnet and let the beads clump for 00:05:00 . Elute DNA into a fresh low-binding tube.
10m
Perform TapeStation assay to determine if primer dimers are present. If present, perform another 0.7x bead clean and check again.
15m
qPCR
qPCR
2h
2h
Perform qPCR by using a Kapa Library Quant kit for Illumina from Roche. Reactions are scaled down to 10 µL . Use the appropriate Kapa kit for the type of qPCR machine used, see https://pim-eservices.roche.com/eLD/api/downloads/ca670ceb-fb38-eb11-0291-005056a71a5d?countryIsoCode=pi
1h 30m
Enter the values obtained from qPCR into the KAPA Library Quantification Data Analysis Template.
15m
Dilute sample to 4 nanomolar (nM) .
15m
Library dilution
Library dilution
14m
14m
Take 5 µL of 4 nanomolar (nM) dilution of library and add 5 µL of 0.1 Molarity (M) NaOH to denature the DNA and incubate at Room temperature for 00:05:00 .
5m
Neutralise with 5 µL of 200 millimolar (mM) Tris-HCl 7.0 for 00:01:00 .
5m
Add 985 µL cold HT1 (hybridisation buffer) to give 20 picomolar (pM) denatured library
2m
Take35 µL of 20 picomolar (pM) sample and add 465 µL of HT1. This will give 500 µL of 1.4 picomolar (pM) sequencing mix.
2m
PhiX control
PhiX control
18m
18m
Thaw a tube of 10 nanomolar (nM) PhiX stock .
5m
Combine the following volumes in a microcentrifuge tube:
10 nanomolar (nM) PhiX (10 μl) and RSB (Resuspension Buffer) (15 μl). The total volume is 25 μl at 4 nanomolar (nM) .
Vortex briefly and then pulse centrifuge. The 4 nanomolar (nM) PhiX can be stored frozen for 3 months.
5m
Combine 5 µL of 4 nanomolar (nM) PhiX with 5 μl of 0.1 Molarity (M) NaOH. Vortex briefly and pulse centrifuge. Incubate at Room temperature for 00:05:00
5m
Add 5 µL of 200 nanomolar (nM) Tris-HCl (7.0 ), vortex briefly, pulse centrifuge and incubate at Room temperature for 00:01:00
1m
Add 985 µL of HT1 (hybridisation buffer) to give a concentration of 20 picomolar (pM) . Vortex briefly and pulse centrifuge.
1m
PhiX control at 20 picomolar (pM) can be frozen at -20°C for up to two weeks. After this time cluster numbers tend to decrease.
1m
MiniSeq Sequencing
MiniSeq Sequencing
5m
5m
Combine your library with PhiX control to give a final dilution of PhiX of 5%
5m