TITLE: NEBNext® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®) E7660 -- Express Protocol without PCR Bead Cleanup and Varskip Primers
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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. </div><br/><div class = "text-block">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.</div><br/><div class = "text-block"><span>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 </span><a href="mailto:info@neb.com" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">info@neb.com</span></a><span>. </span></div><br/><div class = "text-block"><span>For other NEBNext® ARTIC SARS-CoV-2 protocols, please see the </span><a href="https://www.protocols.io/edit/nebnext-artic-protocols-collection-bw2apgae" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">NEBNext ARTIC Protocols Collection</span></a><span>. </span></div></div>

[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:COMPONENTVOLUMERNA Sample8 µl
(lilac) LunaScript RT SuperMix2 µlTotal Volume10 µl
COMPONENTVOLUMERNA Sample8 µl
(lilac) LunaScript RT SuperMix2 µlTotal Volume10 µl
?. [cDNA Synthesis]
Flick the tube or pipet up and down 10 times to mix followed by a quick spin.
?. [cDNA Synthesis]
For no template controls, mix the following components:  COMPONENTVOLUME(white) Nuclease-free Water8 µl
(lilac) LunaScript RT SuperMix2 µlTotal Volume10 µl
COMPONENTVOLUME(white) Nuclease-free Water8 µl
(lilac) LunaScript RT SuperMix2 µlTotal Volume10 µl
?. [cDNA Synthesis]
Flick the tube or pipet up and down 10 times to mix followed by a quick spin.
?. [cDNA Synthesis]
Incubate reactions in a thermocycler  with lid temperature at 105°C with the following steps:CYCLE STEPTEMPTIMEPrimer Annealing25°C2 minutescDNA Synthesis55°C20 minutesHeat Inactivation95°C1 minuteHold4°C∞
CYCLE STEPTEMPTIMEPrimer Annealing25°C2 minutescDNA Synthesis55°C20 minutesHeat Inactivation95°C1 minuteHold4°C∞
Samples can be stored at   for up to a week.
?. [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  with , vortex and spin down reagents. If using a 96 reaction kit, combine  with , vortex and spin down reagents. Use  for each Pool Set A reaction.COMPONENTVOLUMEcDNA (Step 5) 4.5 µl(lilac) Q5 Hot Start High-Fidelity 2X Master Mix6.25 µlNEBNext VarSkip Short SARS-CoV-2 Primer Mix 1*1.75 µlTotal Volume12.5 µlFor Pool Set B: If using the NEBNext ARTIC Human Control Primer Pairs and a 24 reaction kit, combine  with , vortex and spin down reagents. If using 96 reaction kit, combine  with . Use  for each Pool Set B reaction.COMPONENTVOLUMEcDNA (Step 5) 4.5 µl(lilac) Q5 Hot Start High-Fidelity 2X MM6.25 µlNEBNext VarSkip Short SARS-CoV-2 Primer Mix 2*1.75 µlTotal Volume12.5 µl
4.5 µl cDNA input 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.Use of the NEBNext ARTIC Human Control Primer Pairs 1 and 2 are optional. If used, the appropriate NEBNext ARTIC Human Control Primer Pairs and NEBNext VarSkip Short SARS-CoV-2 Primer Mix should be combined prior to use. More specifically, NEBNext ARTIC Human Control Primer Pairs 1 should be combined with NEBNext VarSkip Short SARS-CoV-2 Primer Mix 1 and NEBNext ARTIC Human Control Primer Pairs 2 with NEBNext VarSkip Short SARS-CoV-2 Primer Mix 2. Mixing directions are listed below.
[NEBNext ARTIC Human Control Primer Pairs 1]
[NEBNext VarSkip Short SARS-CoV-2 Primer Mix 1]
[ NEBNext ARTIC Human Control Primer Pairs 1]
[NEBNext VarSkip Short  SARS-CoV-2 Primer Mix 1]
[combined mix]
COMPONENTVOLUMEcDNA (Step 5) 4.5 µl(lilac) Q5 Hot Start High-Fidelity 2X Master Mix6.25 µlNEBNext VarSkip Short SARS-CoV-2 Primer Mix 1*1.75 µlTotal Volume12.5 µl
[NEBNext ARTIC Human Control Primer Pairs 2]
[NEBNext VarSkip Short  SARS-CoV-2 Primer Mix 2]
[NEBNext ARTIC Human Control Primer Pairs 2]
[NEBNext VarSkip Short  SARS-CoV-2 Primer Mix 2]
[combined mix]
COMPONENTVOLUMEcDNA (Step 5) 4.5 µl(lilac) Q5 Hot Start High-Fidelity 2X MM6.25 µlNEBNext VarSkip Short SARS-CoV-2 Primer Mix 2*1.75 µlTotal Volume12.5 µl
?. [Targeted cDNA Amplification]
Flick the tubes or pipet up and down 10 times to mix followed by a quick spin.
?. [Targeted cDNA Amplification]
Incubate reactions in a thermocycler* with the following steps:CYCLE STEPTEMPTIMECYCLESInitial Denaturation98°C30 seconds1Denature95°C15 seconds35Annealing/Extension63°C5 minutesHold4°C∞1
CYCLE STEPTEMPTIMECYCLESInitial Denaturation98°C30 seconds1Denature95°C15 seconds35Annealing/Extension63°C5 minutesHold4°C∞1
Samples can be stored at   for up to a week.
?. [PCR Reaction Pooling]
Please note, there is also a protocol that includes a cleanup and normalization step at this point . Performing the cleanup and normalization step creates library pools where the reads for each library are more evenly distributed. These pools will likely achieve sufficient coverage in less run time.
?. [PCR Reaction Pooling]
For each sample, combine pool A and pool B PCR Reactions.
?. [PCR Reaction Pooling]
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  for 4 hours): COMPONENTVOLUMETargeted cDNA Amplicons (Step 10)1 µl(white) Nuclease-free water11.5 µl(green) NEBNext Ultra II End Prep Reaction Buffer1.75 µl(green) NEBNext Ultra II End Prep Enzyme Mix 0.75 µlTotal Volume 15 µl
on ice
COMPONENTVOLUMETargeted cDNA Amplicons (Step 10)1 µl(white) Nuclease-free water11.5 µl(green) NEBNext Ultra II End Prep Reaction Buffer1.75 µl(green) NEBNext Ultra II End Prep Enzyme Mix 0.75 µlTotal Volume 15 µl
?. [NEBNext End Prep]
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.
It is important to mix well. The presence of a small amount of bubbles will not interfere with performance.
?. [NEBNext End Prep]
Place in a thermocycler, with the heated lid set to ≥ 75°C, and run the following program:  @  @ Hold at
20 °C
65 °C
4 °C
If necessary, samples can be stored at   for a few days; however, a slight loss in yield (~20%) may be observed. We recommend continuing with barcode ligation before stopping.
?. [Barcode Ligation]
Add the following components directly to a sterile nuclease-free PCR tube:COMPONENTVOLUME(white) Nuclease-free water 6 µlEnd-prepped DNA (Previous Step)1.5 µlNative Barcode*2.5 µl(red) Blunt/TA Ligase Master Mix**10 µlTotal Volume20 µl
COMPONENTVOLUME(white) Nuclease-free water 6 µlEnd-prepped DNA (Previous Step)1.5 µlNative Barcode*2.5 µl(red) Blunt/TA Ligase Master Mix**10 µlTotal Volume20 µl
?. [Barcode Ligation]
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.
Caution: The Blunt/TA Ligase 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.
?. [Barcode Ligation]
Incubate at  for . Incubate at  for .Place  for .
25 °C
65 °C
on ice
?. [Barcode Ligation]
Pool all barcoded samples into one 1.5 ml DNA LoBind Tube.
?. [Cleanup of Barcoded DNA]
Note: Use the pooled barcoded DNA samples (from previous step), up to 480 µl for bead cleanup. Remaining pooled DNA can be stored at  .
?. [Cleanup of Barcoded DNA]
Vortex NEBNext Sample Purification Beads to resuspend.
?. [Cleanup of Barcoded DNA]
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   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  to collect all liquid from the sides of the tube.
[of resuspended Sample Purification beads]
?. [Cleanup of Barcoded DNA]
Incubate samples on bench top for  at .
65 Room temperature
?. [Cleanup of Barcoded DNA]
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.
?. [Cleanup of Barcoded DNA]
After  (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Caution: do not discard the beads.
?. [Cleanup of Barcoded DNA]
Wash the beads by adding . Flick the tube or pipet up and down 10 times to mix to resuspend pellet. If necessary, quickly spin the sample for  to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.
[Short Fragment buffer (SFB)]
?. [Cleanup of Barcoded DNA]
Place the tube on an appropriate magnetic stand for  3 minutes (or until the solution is clear) to separate the beads from the supernatant. Remove the supernatant.
?. [Cleanup of Barcoded DNA]
Repeat Step 25 and 26 once for a total of two washes: Wash the beads by adding . Flick the tube or pipet up and down to mix to resuspend pellet. If necessary, quickly spin the sample for  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   (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.
[Short Fragment buffer (SFB)]
?. [Cleanup of Barcoded DNA]
[80% freshly prepared ethanol]
0 Room temperature
?. [Cleanup of Barcoded DNA]
[80% freshly prepared ethanol]
0 Room temperature
?. [Cleanup of Barcoded DNA]
Perform a quick spin and place the sample tube on the magnetic stand, remove any residual ethanol.
?. [Cleanup of Barcoded DNA]
Air dry the beads for  while the tube is on the magnetic stand with the lid open.
Caution: 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.
?. [Cleanup of Barcoded DNA]
Remove the tube from the magnetic stand. Elute the DNA target from the beads by adding .
[Nuclease-free water]
?. [Cleanup of Barcoded DNA]
Resuspend the pellet by flicking the tube or pipetting up and down 10 times to mix. Incubate for at least 2 minutes at . If necessary, quickly spin the sample for  to collect the liquid from the sides of the tube before placing back on the magnetic stand.
0 Room temperature
?. [Cleanup of Barcoded DNA]
Place the tube on the magnetic stand. After 2 minutes (or when the solution is clear), transfer  to a new 1.5 ml Eppendorf DNA LoBind Tube or PCR tube.
32 µl
?. [Cleanup of Barcoded DNA]
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  for the Qubit fluorometer.
1 µl
Samples can be stored at   if they are not used immediately.
?. [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:COMPONENTVOLUMENative barcoded and purified DNA (Step 34, up to 75 ng)30 µl(red) NEBNext Quick Ligation Reaction Buffer *10 µlAdapter Mix II (AMII)**5 µl(red) NEBNext Quick T4 Ligase5 µlTotal Volume50 µl
COMPONENTVOLUMENative barcoded and purified DNA (Step 34, up to 75 ng)30 µl(red) NEBNext Quick Ligation Reaction Buffer *10 µlAdapter Mix II (AMII)**5 µl(red) NEBNext Quick T4 Ligase5 µlTotal Volume50 µl
?. [Adapter Ligation]
Flick the tube to mix solution. Perform a quick spin for  to collect all liquid from the sides of the tube.
Caution: The NEBNext Quick Ligation Buffer is 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.
?. [Adapter Ligation]
Incubate at  or room temperature for .
25 °C
?. [Cleanup of Adapter Ligated DNA]
Vortex NEBNext Sample Purification Beads to resuspend.
?. [Cleanup of Adapter Ligated DNA]
Add  to the ligation mix. Mix well by flicking the tube followed by a quick spin for .
[(1X) resuspended beads]
?. [Cleanup of Adapter Ligated DNA]
Incubate samples for  at .
20 Room temperature
?. [Cleanup of Adapter Ligated DNA]
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.
?. [Cleanup of Adapter Ligated DNA]
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.
Caution: do not discard the beads.
?. [Cleanup of Adapter Ligated DNA]
Wash the beads by adding . 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.
[Short Fragment Buffer (SFB)]
?. [Cleanup of Adapter Ligated DNA]
Wait for  (or until the solution is clear) to separate the beads from the supernatant. Remove the supernatant.
?. [Cleanup of Adapter Ligated DNA]
Repeat Step 44 and 45 once for a total of two washes: Wash the beads by adding . 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.
[Short Fragment Buffer (SFB)]
?. [Cleanup of Adapter Ligated DNA]
Remove the tube from the magnetic stand. Elute the DNA target from the beads by adding  provided in SQK-LSK109 kit from Oxford Nanopore.
[Elution Buffer (EB)]
?. [Cleanup of Adapter Ligated DNA]
Resuspend the pellet well in EB buffer by flicking. Incubate for  at . 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.
20 Room temperature
?. [Cleanup of Adapter Ligated DNA]
Place the tube/plate on the magnetic stand. After  (or when the solution is clear), transfer  to a new DNA LoBind tube.
15 µl
?. [Cleanup of Adapter Ligated DNA]
Use Qubit to quantify . 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).
[DNA sample]
After normalizing the DNA to 30 ng, if the volume is less than 12 µl, then top off the sample volume to 12 µl with EB.