TITLE: Sequencing SARS-CoV-2 from Animal Samples with NEBNext® ARTIC SARS-CoV-2 Companion Kit 
AUTHORS: Isabel Gautreau, Brianna Stenger

[DESCRIPTION]

This protocol is adapted for animal samples and is based on the methods for the NEBNext® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®), NEB #E7660S/L 24/96 reactions.

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.

Changes in this version:
Removal of the human primers
Combining multiple barcoding reactions (step 15 and 21)
Addition of Loading Flongle Flow Cell section 
Addition of Analysis section

[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 and all reactions are set-up in a 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.

[GUIDELINES]
Overview 
The NEBNext ARTIC SARS-CoV-2 Companion Library Prep Kit (Oxford Nanopore Technologies) contains the enzymes, buffers, beads and oligos required to convert a broad range of total RNA input amounts into targeted, high quality libraries for next-generation sequencing on the Oxford Nanopore platform. Primers targeting the human EDF1 and NEDD8 genes are supplied as optional internal controls. The fast, user-friendly workflow also has minimal hands-on time. 

Each kit component must pass rigorous quality control standards, and for each new lot the entire set of reagents is functionally validated together by construction and sequencing of an indexed library on the Oxford Nanopore sequencing platform. 

For larger volume requirements, customized and bulk packaging is available by purchasing through the OEM/Bulks department at NEB. Please contact OEM@neb.com for further information.

Figure 1. Workflow demonstrating the use of NEBNext ARTIC SARS-CoV-2 Companion Library Prep Kit for Oxford Nanopore.
  

[STEPS]
SECTION: cDNA Synthesis
1. The presence of genomic DNA or 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.
SECTION: cDNA Synthesis
2. Gently mix 10 times by pipetting and spin down the LunaScript RT SuperMix reagents (contains primers). Prepare the cDNA synthesis reaction as described below:
 
 

SECTION: cDNA Synthesis
3. Flick the tube or pipet up and down 10 times to mix followed by a quick spin. 
SECTION: cDNA Synthesis
4. For no template controls, mix the following components:  
 

SECTION: cDNA Synthesis
5. Flick the tube or pipet up and down 10 times to mix followed by a quick spin.
SECTION: cDNA Synthesis
6. Incubate reactions in a thermocycler  with lid temperature at 105°C with the following steps:
 

 

SECTION: Targeted cDNA Amplification
7. 

 

Gently mix Q5 Hot Start High Fidelity 2X Master Mix 10 times by pipetting and spin down reagents. Prepare the split pool amplification reactions as described below:

For Pool Set A: .
 

For Pool Set B: 
 

SECTION: Targeted cDNA Amplification
8. Flick the tubes or pipet up and down 10 times to mix followed by a quick spin. 
SECTION: Targeted cDNA Amplification
9. Incubate reactions in a thermocycler** with the following steps:
 

 

SECTION: PCR Reaction Pooling
10. 
 

SECTION: PCR Reaction Pooling
11. For each sample, combine pool A and pool B PCR Reactions. 
 

SECTION: NEBNext End Prep
12. 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): 
 

SECTION: NEBNext End Prep
13. 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. 
 

SECTION: NEBNext End Prep
14. Place in a thermocycler, with the heated lid set to ≥ 75°C, and run the following program: 
10 min @ 20 °C
10 min @ 65 °C 
Hold at 4 °C
 

SECTION: Barcode Ligation
15. Add the following components directly to a sterile nuclease-free PCR tube:
 

SECTION: Barcode Ligation
16. 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. 
 

SECTION: Barcode Ligation
17. Incubate at 25 °C for 20 min. 
Incubate at 65 °C for 10 min.
Place on ice for 1 min.
SECTION: Barcode Ligation
18. Pool all barcoded samples into one 1.5 ml DNA LoBind Tube.
SECTION: Cleanup of Barcoded DNA
19. 
 

SECTION: Cleanup of Barcoded DNA
20. Vortex NEBNext Sample Purification Beads to resuspend.
SECTION: Cleanup of Barcoded DNA
21. Add 0.4X resuspended beads to pooled, barcoded samples (Step 20) 

There is a maximum of 480uL of pooled samples. So multiple barcoded samples can be pooled.
As an example, for seven samples, including four duplicated samples: 
40+40+40+40+20+20+20uL = 220 µL with 88 µL of resuspended Sample Purification beads                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              
 

Mix well by flicking the tube or pipetting up and down 10 times. Perform a quick spin for 1 s to collect all liquid from the sides of the tube.

SECTION: Cleanup of Barcoded DNA
22. Incubate samples on bench top for 10 min at 65 Room temperature.
SECTION: Cleanup of Barcoded DNA
23. 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. 
SECTION: Cleanup of Barcoded DNA
24. After 3 min (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
 


SECTION: Cleanup of Barcoded DNA
25. Wash the beads by adding 250 µL. Flick the tube or pipet up and down 10 times to mix to resuspend pellet. If necessary, quickly spin the sample for 1 s to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.
SECTION: Cleanup of Barcoded DNA
26. Place the tube on an appropriate magnetic stand for 3 min 3 minutes (or until the solution is clear) to separate the beads from the supernatant. Remove the supernatant.

SECTION: Cleanup of Barcoded DNA
27. Repeat Step 26 and 27 once for a total of two washes: 
Wash the beads by adding 250 µL. Flick the tube or pipet up and down to mix to resuspend pellet. If necessary, quickly spin the sample for 3 s 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 3 min  (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. 
SECTION: Cleanup of Barcoded DNA
28. Add 500 µL to the tube while on the magnetic stand. Incubate at Room temperature for 30 s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

SECTION: Cleanup of Barcoded DNA
29. 
Repeat the previous step once to make it a total of 2 washes.

SECTION: Cleanup of Barcoded DNA
30. Perform a quick spin and place the sample tube on the magnetic stand, remove any residual ethanol.
SECTION: Cleanup of Barcoded DNA
31. Air dry the beads for 30 s while the tube is on the magnetic stand with the lid open. 
 

SECTION: Cleanup of Barcoded DNA
32. Remove the tube from the magnetic stand. Elute the DNA target from the beads by adding 33 µL.
SECTION: Cleanup of Barcoded DNA
33. 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 1 s to collect the liquid from the sides of the tube before placing back on the magnetic stand.
SECTION: Cleanup of Barcoded DNA
34. Place the tube on the magnetic stand. After 2 minutes (or when the solution is clear), transfer 32 µL to a new 1.5 ml Eppendorf DNA LoBind Tube or PCR tube.
SECTION: Cleanup of Barcoded DNA
35. Assess the concentration of DNA targets using 1 µL of sample with a DeNovix with FX fluorometer module. Use 

It is also recommend using a Qubit fluorometer for concentration assessment. (Nanodrop is NOT recommended since it may overestimate the DNA concentration). 
 


SECTION: Adapter Ligation
36. Use the Qubit readings from the previous Step to dilute purified Native barcoded DNA pool in nuclease-free water. Dilute the sample to a concentration of 60 ng total in a final volume of 30 μl (or 2 ng/µl in 30 µl). Add the following components into a 1.5 ml Eppendorf DNA LoBind Tube or nuclease-free PCR tube:
 

SECTION: Adapter Ligation
37. Flick the tube to mix solution. Perform a quick spin for 1 s to collect all liquid from the sides of the tube. 
 

SECTION: Adapter Ligation
38. Incubate at 25 °C for 20 min.
SECTION: Adapter Ligation
39. Proceed to Cleanup of Adapter-ligated DNA in the next section.
SECTION: Cleanup of Adapter Ligated DNA
40. Vortex NEBNext Sample Purification Beads to resuspend. 
SECTION: Cleanup of Adapter Ligated DNA
41. Add 50 µL to the ligation mix. Mix well by flicking the tube followed by a quick spin for 3 s.
SECTION: Cleanup of Adapter Ligated DNA
42. Incubate samples for 10 min at 20 Room temperature.
SECTION: Cleanup of Adapter Ligated DNA
43. 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.
SECTION: Cleanup of Adapter Ligated DNA
44. 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.
 

SECTION: Cleanup of Adapter Ligated DNA
45. Wash the beads by adding 250 µL. 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.
SECTION: Cleanup of Adapter Ligated DNA
46. Wait for 3 min (or until the solution is clear) to separate the beads from the supernatant. Remove the supernatant.

SECTION: Cleanup of Adapter Ligated DNA
47. Repeat Step 46 and 47 once for a total of two washes: 
Wash the beads by adding 250 µL. 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. 
SECTION: Cleanup of Adapter Ligated DNA
48. Remove the tube from the magnetic stand. Elute the DNA target from the beads by adding 15 µL provided in SQK-LSK109 kit from Oxford Nanopore. 
SECTION: Cleanup of Adapter Ligated DNA
49. Resuspend the pellet well in EB buffer by flicking. Incubate for 10 min at 20 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. 
SECTION: Cleanup of Adapter Ligated DNA
50. Place the tube/plate on the magnetic stand. After 3 min (or when the solution is clear), transfer 15 µL to a new DNA LoBind tube.

SECTION: Cleanup of Adapter Ligated DNA
51. Determine DNA concentration by quantifying 1 µL.


SECTION: Loading Flongle Flow Cell
52. Follow Oxford Nanopore Protocol SQK-LSK109 to prepare MinION® flow cell and DNA library sequencing mix using up to 20 ng adapter-ligated DNA sample (previous step).

SECTION: Loading Flongle Flow Cell
52.1. Combine 117 µLof Flush Buffer + 3 µL Flush Tether in a LoBind tube and mix by pipetting

SECTION: Loading Flongle Flow Cell
52.2. Prime the flongle flow cell by slowly pipetting the majority of the previous mix, avoid pushing too fast and bubbles
SECTION: Loading Flongle Flow Cell
52.3. In another LoBind tube, combine 15 µL of Sequencing Buffer II + 10 µLof freshly vortexed Loading Beads II +5 µLof ~20fmol DNA library (if volume was less than 5uL, bring up to 5uL with Elution Buffer.

 

SECTION: Loading Flongle Flow Cell
52.4. Load ~30uL on the flongle flow cell, avoid bubbles.  
SECTION: Analysis
53. 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            Used the EPI2ME Fastq + ARTIC + NextClade workflow for analyses.