Sep 25, 2021
Version 1
NEBNext® Varskip Short ARTIC SARS-CoV-2 RT-PCR Module E7626 V.1
- 1New England Biolabs
- New England Biolabs (NEB)
- Coronavirus Method Development Community
External link: https://www.neb.com/products/e7626-nebnext-artic-sars-cov-2-rt-pcr-module#Product%20Information
Protocol Citation: Isabel Gautreau 2021. NEBNext® Varskip Short ARTIC SARS-CoV-2 RT-PCR Module E7626. protocols.io https://dx.doi.org/10.17504/protocols.io.byfdpti6
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: September 21, 2021
Last Modified: September 25, 2021
Protocol Integer ID: 53445
Keywords: NEBNext, NEB, ARTIC, SARS-CoV-2, Library Prep, Illumina
Abstract
This protocol details methods for the NEBNext® ARTIC SARS-CoV-2 RT-PCR Module, NEB #E7626S/L 24/96 reactions.
cDNA Synthesis and Targeted cDNA Amplification with NEBNext VarSkip Short Primer Mixes:
This protocol follows an alternate variant-tolerant approach for targeting SARS-CoV-2 by utilizing NEBNext VarSkip Short SARS-CoV-2 Primer Mixes. The NEBNext VarSkip Short SARS-CoV-2 Primer mixes cannot be added to the same cDNA amplification reaction as the NEBNext ARTIC SARS-CoV-2 Primer Mixes. If downstream applications include sequencing, performing RNA input normalization prior to cDNA synthesis and targeted amplification promotes more even distribution of reads across sequencing libraries.
To obtain instructions for using NEBNext ARTIC SARS-CoV-2 Primer Mix and the NEBNext® ARTIC SARS-CoV-2 RT-PCR Module please see the NEBNext ARTIC SARS CoV2 RT PCR Module Manual.
Guidelines
Overview
The NEBNext ARTIC SARS-CoV-2 RT-PCR Module contains the enzymes and buffers required to convert a broad range of total RNA into high quality, targeted cDNA amplicons with minimal hands-on time.
The NEBNext VarSkip Short SARS-CoV-2 Primer Mixes included provide an alternate variant-tolerant approach for targeting SARS-CoV-2. The NEBNext VarSkip Short SARS-CoV-2 Primer Mixes cannot be added to the same cDNA amplification reaction as the NEBNext ARTIC SARS-CoV-2 Primer Mixes.
Workflow demonstrating the use of NEBNext ARTIC SARS-CoV-2 RT-PCR Module.
Materials
The Library Kit Includes
The volumes provided are sufficient for preparation of up to 24 reactions (NEB #E7658S) and 96 reactions (NEB #E7658L).
Store at –20°C.
(lilac) LunaScript® RT SuperMix
(lilac) Q5® Hot Start High-Fidelity 2X Master Mix
(Orange) NEBNext VarSkip Short SARS-CoV-2 Primer Mix 1
(Orange)NEBNext VarSkip Short SARS-CoV-2 Primer Mix 2
(lilac) NEBNext ARTIC SARS-CoV-2 Primer Mix 1
(lilac) NEBNext ARTIC SARS-CoV-2 Primer Mix 2
(white) 0.1X TE Buffer
(white) Nuclease-free Water
Required Materials Not Included
- 80% Ethanol (freshly prepared)
- DNA LoBind Tubes (Eppendorf® #022431021)
- Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific, Inc.® Q32851)
- Magnetic rack/stand (NEB #S1515S; Alpaqua®, cat. #A001322 or equivalent)
- Thermal cycler
- Vortex Mixer
- Microcentrifuge
- Agilent® Bioanalyzer® or similar fragment analyzer and associated consumables
- DNase RNase free PCR strip tubes (USA Scientific 1402-1708)
Kit Components
NEB #E7658S Table of Components
| A | B | C | |
| NEB # | PRODUCT | VOLUME | |
| E7651A | LunaScript RT SuperMix | 0.048 ml | |
| E7652A | Q5 Hot Start High-Fidelity 2X Master Mix | 0.3 ml | |
| E7725A | NEBNext ARTIC SARS-CoV-2 Primer Mix 1 | 0.042 ml | |
| E7726A | NEBNext ARTIC SARS-CoV-2 Primer Mix 2 | 0.042 ml | |
| E8005A | NEBNext VarSkip Short SARS-CoV-2 Primer Mix 1 | 0.042 ml | |
| E8006A | NEBNext VarSkip Short SARS-CoV-2 Primer Mix 2 | 0.042 ml | |
| E7657A | 0.1X TE | 1.3 ml | |
| E7667A | Nuclease free-Water | 1.5 ml |
NEB #E7658L Table of Components
| A | B | C | |
| NEB # | PRODUCT | VOLUME | |
| E7651AA | LunaScript RT SuperMix | 0.192 ml | |
| E7652AA | Q5 Hot Start High-Fidelity 2X Master Mix | 1.2 ml | |
| E7725AA | NEBNext ARTIC SARS-CoV-2 Primer Mix 1 | 0.168 ml | |
| E7726AA | NEBNext ARTIC SARS-CoV-2 Primer Mix 2 | 0.168 ml | |
| E8005AA | NEBNext VarSkip Short Primer Mix 1 | 0.168 ml | |
| E8006AA | NEBNext VarSkip Short Primer Mix 2 | 0.168 ml | |
| E7657AA | 0.1X TE | 5.2 ml | |
| E7667AA | Nuclease free-Water | 1.5 ml |
Companion Products
| A | B | C | |
| NEB # | PRODUCT | VOLUME | |
| T2010S | Monarch® Total RNA Miniprep Kit | 50 preps |
NEBNext ARTIC SARS-CoV-2 Primers
NEBNext ARTIC SARS-CoV-2 Primers for SARS-CoV-2 genome amplification are based on hCoV-2019/nCoV-2019 Version 3 (v3) sequences with balanced primer concentrations. Sequence information can be found at: https://github.com/joshquick/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V3/nCoV-2019.tsv
NEBNext VarSkip Short SARS-CoV-2 Primer Mix 1 and 2
NEBNext VarSkip Short SARS-CoV-2 Mix 1 and 2 for SARS-CoV-2 genome amplification were designed to reduce the impact of variants on amplification efficiency. Sequence information can be found at: https://github.com/nebiolabs/VarSkip
Safety warnings
Please refer to Safety Data Sheets (SDS) for health and environmental hazards.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
Before start
The amount of RNA required for detection depends on the abundance of the RNA of interest. In general, we recommend, using > 10 copies of the SARS-CoV-2 viral genome as input. In addition, we recommend setting up a no template control reaction and that reactions are set-up in a hood.
The presence of carry-over products can interfere with sequencing accuracy, particularly for low copy targets. Therefore, it is important to carry out the appropriate no template control (NTC) reactions to demonstrate that positive reactions are meaningful.
cDNA Synthesis
cDNA Synthesis
Gently mix and spin down the LunaScript RT SuperMix reagent. Prepare the cDNA synthesis reaction as described below:
| A | B | |
| COMPONENT | VOLUME | |
| RNA Sample | 8 µl | |
| (lilac) LunaScript RT SuperMix | 2 µl | |
| Total Volume | 10 µl |
For no template controls, mix the following components:
| A | B | |
| COMPONENT | VOLUME | |
| (white) Nuclease-free Water | 8 µl | |
| (lilac) LunaScript RT SuperMix | 2 µl | |
| Total Volume | 10 µl |
Incubate reactions in a thermocycler* with the following steps:
| A | B | C | D | |
| CYCLE STEP | TEMP | TIME | CYCLES | |
| Primer Annealing | 25°C | 2 minutes | 1 | |
| cDNA Synthesis | 55°C | 20 minutes | ||
| Heat Inactivation | 95°C | 1 minute | ||
| Hold | 4°C | ∞ |
*Set heated lid to 105°C
Note
Samples can be stored at -20 °C for up to a week.
cDNA Amplification
cDNA Amplification
Note
4.5 µl of cDNA is recommended. If using less than 4.5 µl of cDNA, add nuclease-free water to a final volume of 4.5 µl. We recommend setting up the cDNA synthesis and cDNA amplification reactions in different rooms to minimize cross-contamination of future reactions.
NEBNext VarSkip Short Primer Mixes cannot be combined with NEBNext ARTIC Primer Mixes in the same targeted amplification reaction.
Gently mix and spin down reagents. Prepare the split pool cDNA amplification reactions as described below:
For Pool Set A:
| A | B | |
| COMPONENT | VOLUME | |
| cDNA (Previous Step) | 4.5 µl | |
| (lilac) Q5 Hot Start High-Fidelity 2X Master Mix | 6.25 µl | |
| NEBNext VarSkip Short SARS-CoV-2 Primer Mix 1 | 1.75 µl | |
| Total Volume | 12.5 µl |
For Pool Set B:
| A | B | |
| COMPONENT | VOLUME | |
| cDNA (Step 2) | 4.5 µl | |
| (lilac) Q5 Hot Start High-Fidelity 2X Master Mix | 6.25 µl | |
| NEBNext VarSkip Short SARS-CoV-2 Primer Mix 2 | 1.75 µl | |
| Total Volume | 12.5 µl |
Incubate reactions in a thermocycler* with the following steps:
| A | B | C | D | |
| CYCLE STEP | TEMP | TIME | CYCLES | |
| Initial Denaturation | 98°C | 30 seconds | 1 | |
| Denature | 95°C | 15 seconds | 35 | |
| Annealing/Extension | 63°C | 5 minutes | ||
| Hold | 4°C | ∞ | 1 |
*Set heated lid to 105°C
Combine the Pool A and Pool B PCR reactions for each sample.
Note
Samples can be stored at –20°C for up to a week.
Please consider the downstream application before proceeding. The next section lists instructions for a standard bead cleanup, however, it may not be necessary for your application.
Cleanup of cDNA Amplicons
Cleanup of cDNA Amplicons
7m
7m
Note
The volume of NEBNext Sample Purification Beads provided here are for use with the sample composition at this step (25 μl; Step 5). These bead volumes may not work properly for a cleanup at a different step in the workflow, or if this is a second cleanup at this step. For cleanups of samples contained in different buffer conditions, the volumes may need to be experimentally determined.
Vortex NEBNext Sample Purification Beads to resuspend.
Add 20 µL (0.8 X) resuspended beads to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.
Incubate samples on bench top for at least 00:05:00 at Room temperature .
5m
Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant.
After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Note
Caution: do not discard the beads.
Add 200 µL 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at Room temperature for 00:00:30 , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Repeat the previous step once for a total of two washes:
Add 200 µL 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at Room temperature for 00:00:30 , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
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
Caution: Do not over-dry the beads. This may result in lower recovery of DNA. 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. Elute the DNA target from the beads by adding 16 µL 0.1X TE . Elution volume can be adjusted for specific applications.
Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 00:02:00 at Room temperature . If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.
2m
Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 14 µL to a new PCR tube. If elution volume was adjusted, the transfer volume should also be adjusted.
We recommend assessing the cDNA amplicon concentrations with a size distribution with Qubit® fluorometer.
Samples can be stored at –20°C for up to a week.
Note
Amplicons may also be run on a Bioanalyzer or TapeStation® to confirm ~550 bp size of amplicons. To run on a TapeStation, dilute amplicon 10-fold with 0.1X TE Buffer and run 2 μl on a DNA High Sensitivity ScreenTape®. (See Figure 17 below for example of amplicon size profile on a Bioanalyzer).
Figure 17 VarSkip Short SARS-CoV-2 cDNA amplicons generated from 1,000 total viral copies.