Jun 17, 2020
Version 1
Illumina Nextera DNA Flex library construction and sequencing for SARS-CoV-2: Adapting COVID-19 ARTIC protocol V.1
- Sureshnee Pillay1,
- Jennifer Giandhari1,
- Houriiyah Tegally1,
- Eduan Wilkinson1,
- Benjamin Chimukangara1,
- Richard Lessells1,2,
- Yunus Moosa2,
- Inbal Gazy1,
- Maryam Fish1,
- Lavanya Singh1,
- Khulekani Sedwell Khanyile1,
- Vagner Fonseca1,3,4,
- Marta Giovanetti4,
- Luiz Carols Alcantara3,4,
- Tulio de Oliveira1,5,6,
- Jennifer Giandhari1,5,6
- 1KwaZulu-Natal Research Innovation and Sequencing Platform (KRISP), School of Laboratory Medicine & Medical Sciences, University of KwaZulu-Natal, Durban, South Africa;
- 2Infectious Diseases Department, Nelson R Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa;
- 3Laboratorio de Genetica Celular e Molecular, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil;
- 4Laboratório de Flavivírus, Instituto Oswaldo Cruz Fiocruz, Rio de Janeiro, Brazil;
- 5Centre for Aids Programme of Research in South Africa (CAPRISA), Durban, South Africa;
- 6Department of Global Health, University of Washington, Seattle, Washington, USA
External link: https://www.krisp.org.za/publications.php?pubid=292
Protocol Citation: Sureshnee Pillay, Jennifer Giandhari, Houriiyah Tegally, Eduan Wilkinson, Benjamin Chimukangara, Richard Lessells, Yunus Moosa, Inbal Gazy, Maryam Fish, Lavanya Singh, Khulekani Sedwell Khanyile, Vagner Fonseca, Marta Giovanetti, Luiz Carols Alcantara, Tulio de Oliveira, Jennifer Giandhari 2020. Illumina Nextera DNA Flex library construction and sequencing for SARS-CoV-2: Adapting COVID-19 ARTIC protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bhjgj4jw
Manuscript citation:
Pillay S, Giandhari J, Tegally H, Wilkinson E, Chimukangara B, Lessells R, Mattison S, Moosa Y, Gazy I, Fish M, Singh L, Khanyile KS, Fonseca V, Giovanetti M, Alcantara LCJ, de Oliveira T. Whole Genome Sequencing of SARS-CoV-2: Adapting Illumina Protocols for Quick and Accurate Outbreak Investigation During a Pandemic. bioRxiv, doi: https://doi.org/10.1101/2020.06.10.144212 (2020).
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: June 16, 2020
Last Modified: June 17, 2020
Protocol Integer ID: 38216
Abstract
This protocol describes the procedure for generating cDNA from SARS-CoV-2 viral nucleic acid extracts and subsequently producing amplicons tiling the viral genome sequencing. It uses the V3 nCov-2019 primers from the ARTIC network. This is followed by library construction using Nextera Flex, which we found to save 9h of hands on time as compared with original protocol that use TrueSeq for library contstruction. It also describes the pooling of samples and quantitation, prior to sequencing on the Illumina Miseq and NextSeq.
It is adapted from the nCov-2019 sequencing protocol from Quick and colleagues, which can be found here:
CITATION
Guidelines
Introduction
This protocol describes a method for whole genome sequencing of the SARS-CoV-2 using a tiling PCR approach with overlapping primers and Nextera Flex libraries for Illumina sequencers. This method was produced in KRISP labs for the Network of Genomics Surveillance of South Africa (NGS-SA).
Briefly, primers are designed to be 20-30bp in length and to generate 400bp amplicons with a 70bp overlap. The primers are designed using an online tool called Primal Scheme (http://primal.zibraproject.org/).The amplicons generated can be sequenced on the on the Illumina MiSeq. This will produce next generation sequences covering the whole genome of the SARS-CoV-2 .
Purpose
The purpose of this document is to provide detailed instructions that should be followed when performing the sequencing of SARS-CoV-2 whole genomes from RNA samples using the Nextera DNA Flex Library Kit.
Materials
MATERIALS
Q5 Hot Start High-Fidelity DNA Polymerase - 100 unitsNew England BiolabsCatalog #M0493S
Qubit™ Assay TubesInvitrogen - Thermo FisherCatalog #Q32856
Qubit dsDNA HS Assay kit Thermo Fisher ScientificCatalog #Q32854
SuperScript™ IV Reverse TranscriptaseThermo Fisher ScientificCatalog #18090050
Random Hexamers (50 µM)Thermo FisherCatalog #N8080127
dNTP Mix (10 mM each)Thermo FisherCatalog #R0192
AMPure XP Beckman CoulterCatalog #A63881
RNaseOUT Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
Artic Primers-specific for 2019-nCoV according to Primal Scheme
Nextera DNA Flex Library Prep KitIllumina, Inc.
Nextera™ DNA UD Indexes (96 Indexes 96 Samples)
MiSeq Reagent Nano Kit v2 (500 cycles)Illumina, Inc.Catalog #MS-103-1003
DNA High Sensitivity Reagent KitPerkin ElmerCatalog #CLS760672
DNA 1K / 12K / Hi Sensitivity Assay LabChipPerkin ElmerCatalog #760517
General PCR laboratory equipment and consumables
cDNA
cDNA
Prepare the cDNA mastermix in the pre-PCR clean room. The mastermix hood must be decontaminated before and after use with 10% extran, and 70% ethanol, and sterilised with ultraviolet light (UV).
Mix the following components in a labeled 1.5ml Component:
| Component | Volume (ul) | |
| 50μM Random Hexamers 1 | 1 | |
| 10mM dNTPs mix (10mM each) 1 | 1 | |
| Template RNA | 11 | |
| Total | 12 |
Table 1. cDNA synthesis mastermix 1
Add 1 µL 50μM Random Hexamers 1 to a labeled 1.5ml eppendorf tube.
Add 1 µL 10mM dNTPs mix (10mM each) 1 .
Add 11 µL Template RNA .
Note
The total volume in the tube should now be 13 µL .
Gently mix by pipetting and pulse-spin the tube to collect the liquid at the bottom of the tube.
Aliquot the mastermix in labelled PCR strip tubes.
Note
PCR master mixes (shown in Tables 1 and 3) can be prepared at the same time, in the pre-PCR area before starting amplifications.
Incubate the reaction as follows in a thermal cycler.
| Temperature (°C) | Time | |
| 65 | 5 minutes | |
| 4 | 1 minute |
Table 2. PCR conditions
Spin down the tubes with the RNA and primers to get all liquid to the bottom.
Prepare the following mastermix in the clean mastermix room.
Mix the following components in a labeled 1.5ml eppendorf tube:
| Component | Volume (μl) | |
| SSIV Buffer | 4 | |
| 100mM DTT | 1 | |
| RNaseOUT RNase Inhibitor | 1 | |
| SSIV Reverse Transcriptase | 1 | |
| Total | 7 |
Table 3. cDNA synthesis mastermix 2
Add 4 µL SSIV Buffer to a labeled 1.5ml eppendorf tube.
Add 1 µL 100mM DTT .
Add 1 µL RNaseOUT RNase Inhibitor .
Add 1 µL SSIV Reverse Transcriptase .
Note
The total volume should now be 7 µL .
The mastermix must be added to the 13 µL denatured RNA for a 20 µL total volume .
Gently mix by pipetting and pulse-spin the tube to collect the liquid at the bottom of the tube.
Incubate the reaction as follows in a thermal cycler.
| Temperature (°C) | Time | |
| 42 | 50 minutes | |
| 70 | 10 minutes | |
| 5 | Hold |
Table 4. PCR conditions
Primer Pool Preparation
Primer Pool Preparation
Primers must be diluted and pooled using nuclease free water in a clean mastermix hood. The mastermix hood must be decontaminated before and after use with 10% extran, and 70% ethanol, and sterilised with ultraviolet light (UV).
If required, resuspend lyophilised primers at a concentration of 100 µM each.
Note
2019- nCoV primers for this protocol were designed using Primal Scheme to generate overlapping 400 nucleotide amplicons.
To generate 100 µM primer pool stocks , add 5 µL of each primer pair (named pool 1 or pool 2) to a 1.5ml eppendorf tube labeled either “Pool 1 (100μM)” or “Pool 2 (100μM)”.
Note
Total volume will be 490 µL for Pool 1 (100uM) and 490 µL for Pool 2 (100uM). These are now 100 µM stocks of each primer.
Dilute the 100μM primer pool 1:10 in molecular grade water, to generate 10 µM primer stocks .
Note
It is recommended that multiple aliquots of each primer pool are made in case of degradation or contamination.
Note
Primers need to be used at a final concentration of 0.015 µM per primer . In this case both pools have 98 primers in, so the requirement is 3.6 µL primer pools (10μM) per 25 µL reaction .
Tiling PCR
Tiling PCR
Prepare the PCR mastermix in the clean mastermix room.
The mastermix hood must be decontaminated before and after use with 10% extran, and 70% ethanol, and sterilised with ultraviolet light (UV).
A mastermix for each pool must be made up in the mastermix hood.
Mix the following components in a labeled 1.5ml eppendorf tube:
| Component | Pool 1 volumes (μl) | Pool 2 volumes (μl) | |
| 5X Q5 Reaction Buffer | 5 | 5 | |
| 10mM dNTPs | 0.5 | 0.5 | |
| Q5 Hot Start DNA Polymerase | 0.25 | 0.25 | |
| Primer Pool 1 or 2 (10μM) | 3.6 | 3.6 | |
| Nuclease-free water | 10.65 | 10.65 | |
| Total | 20 | 20 |
Table 5. PCR mastermix
Add 5 µL Table 5. PCR mastermix to a labeled 1.5ml eppendorf tube.
Add 0.5 µL 10mM dNTPs .
Add 0.25 µL Q5 Hot Start DNA Polymerase .
Add 3.6 µL Primer Pool 1 or 2 (10μM) .
Add 10.65 µL Nuclease-free water .
Note
The total volume should now be 20 µL .
Aliquot the mastermix in labelled PCR strip tubes.
Add 5 µL of cDNA under the extraction hood or general lab hood, which has been decontaminated using with 10% extran, and 70% ethanol, and sterilised with ultraviolet light (UV).
Gently mix by pipetting and pulse-spin the tube to collect the liquid at the bottom of the tube.
Incubate the reaction as follows in a thermal cycler.
| Step | Temperature (°C) | Time | Cycles | |
| Heat Activation | 98 | 30 seconds | 1 | |
| Denaturation | 98 | 15 seconds | 35 | |
| Annealing | 65 | 5 minutes | ||
| Hold | 4 | ∞ |
Table 6. PCR conditions
*Cycle number should be 25 for Ct 18-21, and up to a maximum of 35 cycles for Ct 35.
PCR Clean-up
PCR Clean-up
Combine the entire contents of “Pool 1” and “Pool 2” PCR reactions for each biological sample into a single 1.5 ml eppendorf tube.
Vortex Ampure beads thoroughly to ensure they are well resuspended; the solution should be a homogenous brown colour.
Add an equal volume (1:1) of Ampure beads to the pooled sample tube and mix gently by either flicking or pipetting.
Note
For example, add 50 µL Ampure beads to a 50 µL reaction .
Pulse centrifuge to collect all liquid at the bottom of the tube.
Incubate for 00:05:00 at Room temperature .
Place on magnetic rack and incubate for 00:02:00 or until the beads have pelleted and the supernatant is completely clear.
Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
Add 200 µL of freshly prepared 70% ethanol (at Room temperature ) to the pellet.
Carefully remove and discard ethanol, being careful not to touch the bead pellet.
Add 200 µL of freshly prepared 70% ethanol (at Room temperature ) to the pellet.
Carefully remove and discard ethanol, being careful not to touch the bead pellet.
Pulse centrifuge to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible using a P10 pipette.
With the tube lid open incubate for 00:01:00 or until the pellet loses its shine.
Note
If the pellet dries completely it will crack and become difficult to resuspend
Resuspend pellet in 30 µL Elution Buffer (EB) , mix gently by either flicking or pipetting and incubate for 00:02:00 .
Place on magnetic stand and transfer sample to a clean 1.5mL eppendorf tube ensuring no beads are transferred into this tube.
***Sample concentration can be determined using the Qubit and the size of amplicons can be visualized using the LabChip Fragment Analyzer.
Expected result
The expected amplicon size is 400bp.
Library Preparation Tagment Amplicon DNA
Library Preparation Tagment Amplicon DNA
| Item | Storage | Instructions | |
| BLT(bead-linked transposomes) | 2°C to 8°C | Bring to room temperature. Vortex to mix. Do not centrifuge before pipetting. | |
| TB1(Tagmentation buffer) | -25°C to -15°C | Bring to room temperature. Vortex to mix. |
Table 7. Preparation of reagents
Add 2 µL –30 µL DNA to each well of a 96-well PCR plate / 0.2ml strip tubes so that the total input amount is 100–500 ng.
If DNA volume < 30 μl, add nuclease-free water to the DNA samples to bring the total volume to 30 μl.
Vortex BLT vigorously for 00:00:10 to resuspend.
Vortex in between adding BLT as necessary.
Prepare the tagmentation master mix.
Multiply each volume by the number of samples being processed
| Component | Volume (μl) per sample | |
| BLT | 11 | |
| TB1 | 11 | |
| Total | 22 |
Table 8. Tagmentation Master Mix
Please scale this step as needed.
For 1 sample :
11 µL BLT
11 µL TB1
----
22 µL Total
Vortex the tagmentation master mix thoroughly.
Transfer 20 µL tagmentation master mix to each well of the plate containing a sample.
Note
Use fresh tips for each sample column.
Resuspend by pipetting each sample 10 times.
Seal the plate with a plate sealer, place on the preprogrammed thermal cycler, and run the tagmentation program.
| Temperature (°C) | Time | |
| 55 | 15 minutes | |
| 10 | Hold |
PCR – Tagmentation conditions
Post Tagmentation Clean-up
Post Tagmentation Clean-up
| Item | Storage | Instructions | |
| TSB (Tagment stop buffer) | 15°C to 30°C | If precipitates are observed, heat at 37°C for 10 minutes, and then vortex until precipitates are dissolved. Use at room temperature. | |
| TWB (Tagment wash buffer) | 15°C to 30°C | Use at room temperature. |
Table 10. Preparation of Reagents
| Temperature (°C) | Time | |
| 37 | 15 minutes | |
| 10 | Hold |
Table 11. Post Tagmentation Cleanup
Add 10 µL TSB to the tagmentation reaction.
Resuspend the beads by slowly pipetting each well/ tube 10 times.
Seal the plate with / tubes, place on the preprogrammed thermal cycler, and run the post tagmentation cleanup program.
Place the plate on the magnetic stand for approximately 00:03:00 until liquid is clear.
Using a multichannel pipette, remove and discard supernatant.
Remove the sample plate from the magnetic stand and use a deliberately slow pipetting technique to add 100 µL TWB directly onto the beads.
Note
This slow pipetting technique minimizes the potential of TWB foaming to avoid incorrect volume aspiration and incomplete mixing.
Slowly pipette until beads are fully resuspended.
Place the plate on the magnetic stand for approximately 00:03:00 until liquid is clear.
Using a multichannel pipette, remove and discard supernatant.
Remove the plate from the magnetic stand and use a deliberately slow pipetting technique to add 100 µL TWB directly onto the beads.
Slowly pipette each well/tube to resuspend the beads.
Place the plate on the magnetic stand for approximately 00:03:00 until liquid is clear.
Using a multichannel pipette, remove and discard supernatant.
Remove the plate from the magnetic stand and use a deliberately slow pipetting technique to add 100 µL TWB directly onto the beads.
Slowly pipette each well/tube to resuspend the beads.
Seal the plate and keep on the magnetic stand until step 69 of the Procedure section in Amplify Tagmented DNA.
The TWB remains in the wells to prevent overdrying of the beads.
Amplify Tagment DNA
Amplify Tagment DNA
| Item | Storage | Instructions | |
| EPM (enhanced PCR MIx) | -25°C to -15°C | Thaw on ice. Invert to mix, then briefly centrifuge. | |
| Index Adapters (Plates) | -25°C to -15°C | Thaw at room temperature. Spin briefly before use. |
Table 12. Preparation of Reagents
Combine the following volumes to prepare the PCR master mix. Multiply each volume by the number of samples being processed.
| Component | Volume (μl) per sample | |
| EPM | 22 | |
| NFW | 22 | |
| Total | 44 |
Table 13. PCR Master Mix
Note
Reagent overage is included in the volume to ensure accurate pipetting.
Please scale this step as needed.
For 1 sample :
22 µL EPM
22 µL NFW
------
44 µL Total
Vortex and centrifuge the PCR master mix at 280 x g, 00:00:10 .
With the plate on the magnetic stand, use a 200 μl multichannel pipette to remove and discard supernatant. (from step 65 of post tagmentation clean-up)
Note
Foam that remains on the well walls does not adversely affect the library.
Remove from the magnet.
Immediately add 40 µL PCR master mix directly onto the beads in each sample well/ tube.
Pipette mix until the beads are fully resuspended.
Note
Alternatively, seal the plate and use a plate shaker at 1600 rpm for 00:01:00 .
Seal the sample plate and centrifuge at 280 x g, 00:00:03 .
Add 10 µL of the appropriate index adapters to each sample.
Using a pipette set to 40 μl, pipette 10 times to mix.
Note
Alternatively, seal the plate/ tubes and use a plate shaker at 1600 rpm for 00:01:00 .
Centrifuge at 280 x g, 00:00:30 .
Place on the thermal cycler and run the Enrichment PCR program.
| Temperature (°C) | Time | ||
| 68 | 3 minutes | ||
| 98 | 3 minutes | ||
| 98 | 45 seconds | 8 cycles | |
| 62 | 30 seconds | ||
| 68 | 2 minutes | ||
| 68 | 1 minute | ||
| 10 | Hold |
Table 13. PCR Conditions
SAFE STOPPING POINT
If you are stopping, store at 2 °C to 8 °C for up to 72:00:00 (3 days).
Clean-up Libraries
Clean-up Libraries
| Item | Storage | Instructions | |
| SPB (Sample Purification Beads) | 2°C to 8°C | Let stand at room temperature for 30 minutes. Vortex and invert to mix. | |
| RSB (Resuspension Buffer) | -25°C to -15°C | Thaw and bring to room temperature. Vortex to mix. |
Table 14. Preparation of Reagents
Prepare fresh 80 % EtOH from absolute ethanol.
Centrifuge at 280 x g, 00:01:00 to bring all contents to the bottom.
Place the plate/ tubes on a magnetic stand for approximately 00:05:00 until liquid is clear.
Transfer 45 µL supernatant from each well of the PCR plate/ tubes to the corresponding well of a new plate/ tubes.
Vortex and invert SPB multiple times to resuspend.
Add 40 µL nuclease-free water to each well/ tube.
Add 45 µL SPB to each well/ tube.
Mix well by pipetting 10 times.
Note
Alternatively, seal the plate and use a plate shaker at 1600 rpm for 00:01:00 .
Incubate at Room temperature for 00:05:00 .
Place on the magnetic stand for approximately 00:05:00 until the liquid is clear.
During incubation, thoroughly vortex the SPB (undiluted stock tube), and then add 15 µL to each well of a new plate/tubes.
Transfer 125 µL supernatant from each well of the first plate/ tubes into the corresponding well of the second plate/ tubes (containing 15 μl undiluted SPB).
Mix well by pipetting 10 times.
Note
Alternatively, seal the plate and use a plate shaker at 1600 rpm for 00:01:00 .
Discard the first plate/ tubes.
Pipette each well 10 times to mix.
Incubate at Room temperature for 00:05:00 .
Place on the magnetic stand for approximately 00:05:00 until the liquid is clear.
Without disturbing the beads, remove and discard supernatant.
Wash two times as follows:
Add 200 µL freshly prepared 80% ethanol with the plate on the magnetic stand.
Incubate for 00:00:30 .
Without disturbing the beads, remove and discard the supernatant.
Add 200 µL freshly prepared 80% ethanol with the plate on the magnetic stand.
Without disturbing the beads, remove and discard the supernatant.
Use a 20 μl pipette to remove any residual ethanol.
Air-dry on the magnetic stand for 00:05:00 .
Remove from the magnetic stand.
Add 32 µL RSB to each well/ tube.
Resuspend by pipette mixing.
Incubate at Room temperature for 00:02:00 .
Place the plate/ tubes on the magnetic stand for approximately 00:02:00 .
Transfer 30 µL supernatant to a new 96-well PCR plate/ tubes.
SAFE STOPPING POINT
If you are stopping, seal the plate, and store at -25 °C to -15 °C for up to 30 days.
Normalization of DNA
Normalization of DNA
Quantify the DNA as described using the Qubit and determine the fragment length using the LabChip.
Using the Qubit concentrations and fragment length normalize the libraries to equimolar 4nM by diluting with RSB buffer.
Calculate appropriate amount of diluent in an excel sheet to add to respective sample libraries in order to achieve a 4nm library concentration, using the following formula: Nanomolar concentration = (ng/μl /660 x 500) x 10^6
Pipette mix 5 times.
Use a multi-channel pipette to transfer 5 µL of the diluted sample library to an 8 strip-tube and spin briefly.
Pool the library samples from the 8-strip tubes to a labelled Pooled Amplicon Library (PAL) 2ml eppendorf tube.
Proceed to library denaturation.
Library Denaturation
Library Denaturation
Remove the tube of HT1 (Hybridization Buffer) from the freezer (-15°C to -25°C) and set aside at Room temperature to thaw.
When thawed, store at 2 °C to 8 °C until you are ready to dilute denatured libraries.
Prepare 500μl of 0.2 N NaOH by combining the following volumes in a 1.5ml microcentrifuge tube: 490 µL laboratory-grade water and 10 µL Stock 1.0 N NaOH .
Refer to the formula below:
x = 10 µL NaOH + 490 µL laboratory-grade water
Note
A fresh dilution of 0.2N NaOH is required for the denaturation process in preparing sample DNA and a PhiX control.
Invert the tube several times to mix.
Combine the following volumes of pooled sample DNA and freshly diluted 0.2 N NaOH in a micro-centrifuge tube, by adding 5 µL of 4nM sample DNA to 5 µL of 4nM sample DNA .
Discard the remaining dilution of 0.2 N NaOH or set aside to prepare a PhiX control within the next 12:00:00 .
Vortex briefly to mix the sample solution, and then centrifuge the sample solution to 280 x g, 00:01:00 .
Incubate for 00:05:00 at Room temperature to denature the DNA into single strands.
Add 10 µL of 4nM sample DNA to 990 µL of pre-chilled HT1 .
Note
The result is a 20pM denatured library in 1 mM NaOH.
Place the denatured DNA On ice or at 4 °C until you are ready to proceed to the final dilution.
Dilution of Denatured Library
Dilution of Denatured Library
Use the following instructions to dilute the 20pM DNA further to give 600μl of the desired input concentration.
Dilute the denatured DNA to the desired concentration using the following example (if using 5% PhiX):
| Final Concentration | 20pM denatured DNA | 5% PhiX | Pre-chilled HT1 | |
| 12pM | 356.4ul | 3.6ul | 240ul |
*This was found to be the optimal loading concentration when using a Miseq V2 Nano 500 cycle kit
Invert several times to mix and then pulse centrifuge.
To dilute PhiX to 4nM concentration, combine the following volumes in a microcentrifuge tube:
• 2 µL of 10nM PhiX library
• 3 µL of 10mM Tris-Cl , 8.5 with 0.1 % Tween 20
Note
If not prepared within the last 12 hours, prepare a fresh dilution of 0.2 N NaOH.
Combine the following volumes in a micro-centrifuge tube:
• 5 µL of 4 nM PhiX library
• 5 µL of 0.2 N NaOH
Vortex briefly to mix.
Centrifuge at 280 x g, 00:01:00 .
Incubate at Room temperature for 00:05:00 .
Dilute denatured PhiX to 20pM by adding pre-chilled HT1 to the denatured PhiX library as follows:
• 10 µL denatured PhiX library
• 990 µL pre-chilled HT1
Invert to mix.
Combine Library and PhiX Control.
Mix this solution well and briefly centrifuge.
Keep On ice or at 4 °C until it is ready to be loaded onto the MiSeq reagent cartridge.
Citations
Josh Quick. nCoV-2019 sequencing protocol
dx.doi.org/10.17504/protocols.io.bdp7i5rn