Jul 23, 2020
NEBnext library construction and sequencing for SARS-CoV-2: Adapting COVID-19 ARTIC protocol
- Jennifer Giandhari1,
- Sureshnee Pillay1,
- 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
- 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
- Coronavirus Method Development Community
- KRISP
External link: https://www.krisp.org.za/publications.php?pubid=292
Protocol Citation: Jennifer Giandhari, Sureshnee Pillay, 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 2020. NEBnext library construction and sequencing for SARS-CoV-2: Adapting COVID-19 ARTIC protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bhu2j6ye
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 24, 2020
Last Modified: July 23, 2020
Protocol Integer ID: 38522
Keywords: COVID-19, Illumina, ARTIC,
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 uses TruSeq for library contstruction. It also describes the pooling of samples and quantitation, prior to sequencing on the Illumina Miseq.
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 NEBNext Ultra II Library Preparation Kit 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 NEBNext Ultra II Library Preparation 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
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
NEBNext Ultra II Library Preparation KitNew England BiolabsCatalog #NEB #E7103
NEBNext Multiplex OligosNew England BiolabsCatalog #E6442S
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 | 13 |
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 pool.
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 5X Q5 Reaction Buffer 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 End Repair
Library Preparation End Repair
Note
For a 400bp insert, use 200ng input DNA. Input amounts lower than those specified results in low yield and increased duplicates
Note
Starting Material: Cleaned-up DNA diluted to 1 – 5 ng/μl, in at least 50 μl volume.
Prepare the end repair master mix.
Multiply each volume by the number of samples being processed
| Component | Volume (μl) per sample | |
| NEBNext Ultra II End Prep Enzyme Mix (green top) | 3 | |
| NEBNext Ultra II End Prep Reaction Buffer (green top) | 7 | |
| Total | 10 |
Table 8. End Repair Master mix
Please scale this step as needed.
For 50 sample :
3 µL NEBNext Ultra II End Prep Enzyme Mix (green top)
7 µL NEBNext Ultra II End Prep Reaction Buffer (green top)
----
60 µL Total
Add 50μl of DNA to the respective wells, for a total reaction volume of 60 μl.
Mix well on a vortex mixer or by pipetting up and down 10 times.
Perform a quick spin to collect all liquid from the sides of the tube.
Note
It is important to mix well. The presence of a small amount of bubbles will not interfere with performance.
Place in a thermocycler, with the heated lid set to ≥75°C, and run the following program:
Seal the plate with a plate sealer, place on the preprogrammed thermal cycler, and run the end repair program.
| Temperature (°C) | Time | |
| 20 | 30 minutes | |
| 65 | 30 minutes | |
| 10 | Hold |
PCR – End repair conditions
Note
If necessary, samples can be stored at –20°C; however, a slight loss in yield (~20%) may be observed. We recommend continuing with adaptor ligation before stopping.
Adapter Ligation
Adapter Ligation
Make up the following Mastermix and add 31μl to the respective wells.
Add 35μl of the End Prep DNA to the respective wells.
Add 2.5μl of NEBNext Adpaters for Illumina to the respective wells.
Mix well on a vortex mixer or by pipetting up and down 10 times.
Perform a quick spin to collect all liquid from the sides of the tube. Incubate at 20°C for 15 minutes in a thermocycler with the heated lid open.
Note
The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time and is stable for at least 8 hours @ 4°C. We do not recommend adding adaptor to a premix in the Adaptor Ligation Step.
Note
The NEBNext Ultra II Ligation Master Mix is very viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interfere with performance).
Note: Samples can be stored overnight at –20°C.
Clean-up of Adapter Ligated DNA
Clean-up of Adapter Ligated DNA
Note
Allow AMPure XP Beads to warm to room temperature for at least 30 minutes before use.
Vortex AMPure Beads thoroughly to resuspend.
Add 57μl (~0.8X) of resuspended beads to the adaptor ligation reaction.
Mix well by vortexing for 3 – 5 seconds or by pipetting up and down 10 times.
Centrifuge briefly. Be sure to stop the centrifugation before the beads start to settle out.
Incubate samples on bench top for at least 5 minutes at room temperature.
Place the tube/plate on an appropriate magnetic stand for 5 minutes to separate the beads from the supernatant.
Carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the DNA targets.
Incubate at room temperature for 30 seconds.
Carefully remove and discard the supernatant without disturbing the pellet.
Add 200μl of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. 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.
Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
Safety information
Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.
Remove the tube/plate from the magnetic stand.
Add 17μl of nuclease free water to elute the DNA target from the beads.
Mix well on a vortex mixer or by pipetting up and down 10 times.
Incubate at room temperature for at 2 minutes.
Quickly spin to collect the liquid from the sides of the tube or plate wells.
Place the tubes/ plate on the magnetic stand and incubate for at least 5 minutes at room temperature.
Transfer 15μl (i.e. 7.5μl twice) of the supernatant to a new tubes/ plate for amplification.
Note
Samples can be stored at –20°C.
PCR Enrichment
PCR Enrichment
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 | |
| NEBNext Ultra II Q5 Master Mix (blue top) | 25 | |
| Universal PCR primer (blue top) | 5 | |
| Total | 30 |
Table 13. PCR Master Mix
Note
The PCR will take approximately 30 minutes
Add 30μl of the master mix into newly labelled tubes/ plate.
Add 15.0μl of adaptor-ligated DNA to the respective wells.
Mix well on a vortex mixer or by pipetting up and down 10 times.
Perform a quick spin to collect all liquid from the sides of the tube.
Place the tubes/plate on a thermocycler and perform PCR using the following conditions:
| Temperature (°C) | Time | Cycles | |
| 98 | 3 minutes | 1 | |
| 98 | 10 seconds | 8 | |
| 60 | 30 seconds | ||
| 65 | 45 seconds | ||
| 65 | 5 minutes | 1 | |
| 4 | Hold |
Table 13. PCR Conditions
Clean-up of Enrichment PCR
Clean-up of Enrichment PCR
Note
If using AMPure XP Beads, allow the beads to warm to room temperature for at least 30 minutes before use
Vortex AMPure Beads thoroughly to resuspend.
Add 45μl (0.9X) resuspended AMPure beads to the PCR reaction.
Mix well by vortexing for 3 – 5 seconds or by pipetting up and down 10 times.
Centrifuge very briefly. Be sure to stop the centrifugation before the beads start to settle out.
Incubate samples on bench top for at least 5 minutes at room temperature.
Place the tube/plate on an appropriate magnetic stand for 5 minutes to separate the beads from the supernatant.
Carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the DNA targets.
Add 200μl of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand.
Incubate at room temperature for 30 seconds.
Carefully remove and discard the supernatant without disturbing the pellet.
Add 200μl of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. 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.
Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
Note
Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.
Remove the tube/plate from the magnetic stand.
Add 33μl of nuclease free water to elute the DNA target from the beads.
Mix well on a vortex mixer or by pipetting up and down 10 times.
Quickly spin to collect the liquid from the sides of the tube or plate wells.
Incubate the tubes/ plate for 2 minutes at room temperature.
Place the tubes/ plate on the magnetic stand and incubate for at least 5 minutes.
Transfer 30μl (i.e. 15.5μl twice) of the supernatant to a new tubes/ plate.
Assess the concentration of the libraries using a Qubit fluorometer.
Assess the library fragments using the LabChip GX Touch.
Note
Samples can be stored at –20°C after clean-up.
Normalization of DNA
Normalization of DNA
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