NEBNext® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®) E7660 -- Express Protocol without PCR Bead Cleanup and Varskip Primers
Isabel Gautreau
Abstract
This protocol details methods for the NEBNext® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®), NEB #E7660S/L 24/96 reactions and the NEBNext VarSkip Short SARS-CoV-2 Primer Mix 1 and 2 released in Sep 2021.
This protocol does not include a cleanup and normalization step for each sample after cDNA synthesis. Performing the cleanup and normalization step creates library pools where the reads for each library are more evenly distributed. Skipping these steps reduces hands on time, but may require a longer sequencing run to obtain sufficient coverage for each sample. To obtain more even sample to sample coverage, we recommend normalizing the RNA samples prior to starting the protocol.
To obtain instructions for using NEBNext VarSkip Short SARS-CoV-2 Primer Mix and the NEBNext® ARTIC SARS-CoV-2 Companion Kit STANDARD workflow (with cleanup after PCR) please contact NEB using info@neb.com.
For other NEBNext® ARTIC SARS-CoV-2 protocols, please see the NEBNext ARTIC Protocols Collection.
Before start
Note: 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. It is advisable to set up your reactions in the hood.
Note: : If sample Ct is between 12-15, then it is recommended per the nCoV 2019 sequencing protocol v3 LoCost to dilute the sample 100-fold in water, if between 15-18 then dilute 10-fold in water. This will reduce the likelihood of PCR inhibition.
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.
Steps
cDNA Synthesis
Gently mix 10 times by pipetting and spin down the LunaScript RT SuperMix reagent (contains primers). 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 |
Flick the tube or pipet up and down 10 times to mix followed by a quick spin.
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 |
Flick the tube or pipet up and down 10 times to mix followed by a quick spin.
Incubate reactions in a thermocycler with lid temperature at 105°C with the following steps:
| A | B | C |
|---|---|---|
| CYCLE STEP | TEMP | TIME |
| Primer Annealing | 25°C | 2 minutes |
| cDNA Synthesis | 55°C | 20 minutes |
| Heat Inactivation | 95°C | 1 minute |
| Hold | 4°C | ∞ |
Targeted cDNA Amplification
Gently mix and spin down reagents. Prepare the split pool cDNA amplification reactions as described below:
For Pool Set A:
If using the NEBNext ARTIC Human Primer Pairs and a 24 reaction kit , combine 0.7µL with 42µL, vortex and spin down reagents. If using a 96 reaction kit , combine 2.8µL with 168µL, vortex and spin down reagents. Use 1.75µL for each Pool Set A reaction.
| A | B |
|---|---|
| COMPONENT | VOLUME |
| cDNA (Step 5) | 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 |
- If using NEBNext ARTIC Human Control Primer Pairs 1, add 1.75 μl of the combined NEBNext ARTIC Human Control Primer Pairs 1 and NEBNext VarSkip Short SARS-CoV2 Primer Mix 1
For Pool Set B:
If using the NEBNext ARTIC Human Control Primer Pairs and a 24 reaction kit , combine 0.7µL with 42µL, vortex and spin down reagents. If using 96 reaction kit , combine 2.8µL with 168µL. Use 1.75µL for each Pool Set B reaction.
| A | B |
|---|---|
| COMPONENT | VOLUME |
| cDNA (Step 5) | 4.5 µl |
| (lilac) Q5 Hot Start High-Fidelity 2X MM | 6.25 µl |
| NEBNext VarSkip Short SARS-CoV-2 Primer Mix 2* | 1.75 µl |
| Total Volume | 12.5 µl |
- If using NEBNext ARTIC Human Control Primer Pairs 2, add 1.75 µl of the combined NEBNext ARTIC Human Control Primer Pairs 2 and NEBNext VarSkip Short SARS-CoV2 Primer Mix 2.
Flick the tubes or pipet up and down 10 times to mix followed by a quick spin.
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.
PCR Reaction Pooling
For each sample, combine pool A and pool B PCR Reactions.
Figure 11: VarSkip Short SARS-CoV-2 cDNA amplicons generated from 1000 total viral copies. 1/10 diluted cDNA amplicons without bead cleanup run on a TapeStation.
NEBNext End Prep
Add the following components to a PCR tube (End Prep Reaction and Buffer can be pre-mixed and master mix is stable On ice for 4 hours):
| A | B |
|---|---|
| COMPONENT | VOLUME |
| Targeted cDNA Amplicons (Step 10) | 1 µl |
| (white) Nuclease-free water | 11.5 µl |
| (green) NEBNext Ultra II End Prep Reaction Buffer | 1.75 µl |
| (green) NEBNext Ultra II End Prep Enzyme Mix | 0.75 µl |
| Total Volume | 15 µl |
Flick the tube or pipet up and down 10 times to mix the solution. Perform a quick spin to collect all liquid from the sides of the tube.
Place in a thermocycler, with the heated lid set to ≥ 75°C, and run the following program:
0h 10m 0s @ 20°C
0h 10m 0s @ 65°C
Hold at 4°C
Barcode Ligation
Add the following components directly to a sterile nuclease-free PCR tube:
| A | B |
|---|---|
| COMPONENT | VOLUME |
| (white) Nuclease-free water | 6 µl |
| End-prepped DNA (Previous Step) | 1.5 µl |
| Native Barcode* | 2.5 µl |
| (red) Blunt/TA Ligase Master Mix** | 10 µl |
| Total Volume | 20 µl |
- Native Barcodes are provided in Oxford Nanopore Technologies Native Barcoding Expansion 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114) or 1-96 (EXP-NBD196)** Mix the Blunt/TA Ligase Master Mix by pipetting up and down several times prior to adding to the reaction.
Flick the tube or pipet up and down 10 times to mix solution. Perform a quick spin to collect all liquid from the sides of the tube.
Incubate at 25°C for 0h 20m 0s.
Incubate at 65°C for 0h 10m 0s.
Place 65On ice for 0h 1m 0s.
Pool all barcoded samples into one 1.5 ml DNA LoBind Tube.
Cleanup of Barcoded DNA
Vortex NEBNext Sample Purification Beads to resuspend.
Add 0.4X resuspended beads to pooled, barcoded samples (Step 20) (for example, if you are pooling 24 libraries [which amounts to 480 µl total], add 192µL to the 480 µl of pooled sample). Mix well by flicking the tube or pipetting up and down 10 times. Perform a quick spin for 0h 0m 1s to collect all liquid from the sides of the tube.
Incubate samples on bench top for 0h 10m 0s at 65Room temperature.
Place the tube on a 1.5 ml magnetic stand (such as NEB S1506) to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
After 0h 3m 0s (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Wash the beads by adding 250µL. Flick the tube or pipet up and down 10 times to mix to resuspend pellet. If necessary, quickly spin the sample for 0h 0m 1s to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.
Place the tube on an appropriate magnetic stand for 0h 3m 0s 3 minutes (or until the solution is clear) to separate the beads from the supernatant. Remove the supernatant.
Repeat Step 25 and 26 once for a total of two washes:
Wash the beads by adding 250µL. Flick the tube or pipet up and down to mix to resuspend pellet. If necessary, quickly spin the sample for 0h 0m 3s to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.
Place the tube on an appropriate magnetic stand for 0h 3m 0s (or until the solution is clear) to separate the beads from the supernatant. Remove the supernatant.
Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube, place back on the magnetic stand and remove traces of SFB with a p10 pipette tip.
Add 500µL to the tube while on the magnetic stand. Incubate at Room temperature for 0h 0m 30s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Repeat the previous step once to make it a total of 2 washes:
Add 500µL to the tube while on the magnetic stand. Incubate at Room temperature for 0h 0m 30s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Perform a quick spin and place the sample tube on the magnetic stand, remove any residual ethanol.
Air dry the beads for 0h 0m 30s while the tube is on the magnetic stand with the lid open.
Remove the tube from the magnetic stand. Elute the DNA target from the beads by adding 33µL.
Resuspend the pellet by flicking the tube or pipetting up and down 10 times to mix. Incubate for at least 2 minutes at Room temperature. If necessary, quickly spin the sample for 0h 0m 1s to collect the liquid from the sides of the tube before placing back on the magnetic stand.
Place the tube on the magnetic stand. After 2 minutes (or when the solution is clear), transfer 32µL to a new 1.5 ml Eppendorf DNA LoBind Tube or PCR tube.
Assess the concentration of purified barcoded DNA sample. We recommend using a Qubit fluorometer for concentration assessment. (Nanodrop is NOT recommended since it may overestimate the DNA concentration). Use 1µL for the Qubit fluorometer.
Adapter Ligation
Use the Qubit readings from the previous step to dilute 75 ng purified Native barcoded DNA pool with nuclease-free water to a final volume of 30 μl (or ~ 2.5 ng/µl). Add the following components into a 1.5 ml Eppendorf DNA LoBind Tube or nuclease-free PCR tube:
| A | B |
|---|---|
| COMPONENT | VOLUME |
| Native barcoded and purified DNA (Step 34, up to 75 ng) | 30 µl |
| (red) NEBNext Quick Ligation Reaction Buffer * | 10 µl |
| Adapter Mix II (AMII)** | 5 µl |
| (red) NEBNext Quick T4 Ligase | 5 µl |
| Total Volume | 50 µl |
- Mix the NEBNext Quick Ligation Reaction Buffer by pipetting up and down several times prior to adding to the reaction. ** Adapter Mix II is provided by Oxford Nanopore Technologies Native Barcoding Expansion 1-12 (EXP-NBD104), 13-24 (EXP-NBD114) and 1-96 (EXP-NBD-196) kits.
Flick the tube to mix solution. Perform a quick spin for 0h 0m 1s to collect all liquid from the sides of the tube.
Incubate at 25°C or room temperature for 0h 20m 0s.
Cleanup of Adapter Ligated DNA
Vortex NEBNext Sample Purification Beads to resuspend.
Add 50µL to the ligation mix. Mix well by flicking the tube followed by a quick spin for 0h 0m 1s.
Incubate samples for 0h 10m 0s at 20Room temperature.
Place the tube on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
After 3 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Wash the beads by adding 250µL. Flick the tube to resuspend pellet. 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. Place the tube on an appropriate magnetic stand.
Wait for 0h 3m 0s (or until the solution is clear) to separate the beads from the supernatant. Remove the supernatant.
Repeat Step 44 and 45 once for a total of two washes:
Wash the beads by adding 250µL. Flick the tube or pipet up and down to mix to resuspend pellet. 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. Place the tube on an appropriate magnetic stand.
Wait for 3 minutes (or when the solution is clear) to separate the beads from the supernatant. Remove the supernatant.
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 SFB with a p10 pipette tip.
Remove the tube from the magnetic stand. Elute the DNA target from the beads by adding 15µL provided in SQK-LSK109 kit from Oxford Nanopore.
Resuspend the pellet well in EB buffer by flicking . Incubate for 0h 10m 0s at 20Room 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.
Place the tube/plate on the magnetic stand. After 0h 3m 0s (or when the solution is clear), transfer 15µL to a new DNA LoBind tube.
Use Qubit to quantify 1µL. Follow Oxford Nanopore Protocol SQK-LSK109 to prepare MinION® flow cell and DNA library sequencing mix using up to 30 ng adapter-ligated DNA sample (previous step).
