TITLE: Modified NEBNext® VarSkip Short SARS-CoV-2 Enrichment and library prep for Oxford Nanopore Technologies- adapted for wastewater samples
AUTHORS: Padmini  Ramachandran, Tamara Walsky, Amanda Windsor, Chris  Grim, Maria Hoffmann

[DESCRIPTION]
This protocol details methods for the preparation of  SARS-CoV-2 sequencing library using VSS primers from library preparation from NEBNext® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®), NEB #E7660S/L 24/96 reactions adapted for wastewater samples.

Standard Protocol with PCR Bead Cleanup: This protocol includes 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. These pools will likely achieve sufficient and equal coverage in less run time, but they take more hands-on time

This protocol also includes options for barcoding with dual indexing or single indexing of the samples.

Version updates V2: Recommending the addition of VSS v2b (spike-in primers) to the primer pool1 to increase the coverage across multiple regions of SARS-CoV2 genome. Optimized the input of cDNA. Optimized wash steps to get rid of shorter fragments of library. 

[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.

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.

[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. 

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: Before you start
1. 
 

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. 
 

SECTION: Targeted cDNA Amplification
8. Addition of spike-in to improve coverage across certain regions of SARS-COV2 genome.

 For 96 reaction kits: 
Thaw BA2 Spike-in Mix and VarSkip Short v2 Primer Mix 1.  
Spin down both the tubes.
Add 1 ul of the BA2 Spike-in Mix to the VarSkip Short v2 Primer Mix 1. 
Mix and quick spin updated VarSkip Short v2 Primer Mix 1. 

For 24 reaction kits: 
Thaw BA2 Spike-in Mix and VarSkip Short v2 Primer Mix 1.  
Spin down both the tubes. 
Add 1 ul of BA2 Spike-in Mix to 3ul 0.1x TE to make a ¼ dilution of the BA2 Spike-in Mix. 
Add 1 ul of the diluted BA2 Spike-in Mix to the VarSkip Short v2 Primer Mix 1. 
Mix and quick spin updated VarSkip Short v2 Primer Mix 1. 

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

 

SECTION: Cleanup of cDNA Amplicons
11. We highly recommend the clean up step using either NEBNext sample purification beads or Ampure beads.
 

SECTION: Cleanup of cDNA Amplicons
12. For each sample, combine pool A and pool B PCR Reactions. 
SECTION: Cleanup of cDNA Amplicons
13. Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.
SECTION: Cleanup of cDNA Amplicons
14. Add 20 µL to the combined PCR reaction.  Mix well by flicking the tube or pipetting up and down 10 times to mix and a very short 2-3 seconds quick centrifugation. Be sure to stop the centrifugation before the beads start to settle out.

SECTION: Cleanup of cDNA Amplicons
15. Incubate samples at Room temperature for 10 min.
SECTION: Cleanup of cDNA Amplicons
16. Place the tubes on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample 1 s to collect the liquid from the sides of the tube before placing on the magnetic stand.
SECTION: Cleanup of cDNA Amplicons
17. 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.
 


SECTION: Cleanup of cDNA Amplicons
18. Add 200 µL to the tube while in 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 cDNA Amplicons
19. Repeat previous step once for a total of two washes: 
Add 200 µL to the tube while in 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. 

Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube for 1 s, place back on the magnetic stand and remove traces of ethanol with a p10 pipette tip.

SECTION: Cleanup of cDNA Amplicons
20. Air dry the beads for up to 3 min while the tube is on the magnetic stand with the lid open. 
 

SECTION: Cleanup of cDNA Amplicons
21. Remove the tube from the magnetic stand. Elute the DNA target from the beads by adding 18 µL. 
SECTION: Cleanup of cDNA Amplicons
22. Mix well by flicking the tube or pipetting up and down 10 times to mix and followed by a very short centrifugation. Incubate for 2 min 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. 
SECTION: Cleanup of cDNA Amplicons
23. Place the tube on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 17 µL to clean PCR tubes.
SECTION: Cleanup of cDNA Amplicons
24. Assess the concentration of the DNA targets. We recommend using a Qubit fluorometer for concentration assessment. Use 1 µl of sample for the Qubit fluorometer. Amplicons may also be run on a Bioanalyzer® or Tape Station using High Sensitivity (HS) 5000 tape or HS 1000 tape to confirm ~560-650 bp size of amplicons. 
 



 

SECTION: Cleanup of cDNA Amplicons
25. 
 

SECTION: NEBNext End Prep
26. Use the Qubit readings from Step 24 to determine the amount of the VSS Amplicons. Dilute each amplicon sample into 50 ng/12.5 μl (4ng/ul) concentration using Nuclease-free water.  Add the following components to a PCR tube (End Prep Reaction and Buffer can be pre-mixed and stable on ice for 4 hours):
 



SECTION: NEBNext End Prep
27. Flick the tube or gently 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
28. 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: NEBNext End Prep
29. 
 

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

SECTION: Barcode Ligation
31. 
 

SECTION: Barcode Ligation
32. Flick the tube or gently 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
33. Place in a thermocycler, with the heated lid set to = 75°C, and run the following program: 
 25 °C for 20 min 
 65 °C for 10 min.
Place  on ice for 1 min.
SECTION: Barcode Ligation
34. Pool all barcoded samples into one 1.5 ml DNA LoBind Tube.
SECTION: Cleanup of Barcoded DNA
35. The following section is for cleanup of the ligation reaction. 
 

SECTION: Cleanup of Barcoded DNA
36. Vortex NEBNext Sample Purification Beads to resuspend.
SECTION: Cleanup of Barcoded DNA
37. Add 0.4X resuspended beads to pooled, barcoded samples (Step 30), for example, if you are pooling 12 samples with 2 barcode set up, which will be 24 libraries (which amounts to 480 µl total), add 192 µLto the 480 µl of pooled sample. Flick the tube or pipet up and down 10 times to mix to resuspend pellet. Perform a quick spin for 1 s to collect all liquid from the sides of the tube.

SECTION: Cleanup of Barcoded DNA
38. Incubate samples on bench top for 10 min at 65 Room temperature.
SECTION: Cleanup of Barcoded DNA
39. 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
40. 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.
 

SECTION: Cleanup of Barcoded DNA
41. 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 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
42. Place the tube on an appropriate magnetic stand for 4 minutes (or until the solution is clear) to separate the beads from the supernatant. Remove the supernatant.

SECTION: Cleanup of Barcoded DNA
43. Repeat previous 2 steps 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 4 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, place back on the magnetic stand and remove traces of SFB with a p10 pipette tip
SECTION: Cleanup of Barcoded DNA
44. 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. Repeat this wash step once for a total of two washes.
SECTION: Cleanup of Barcoded DNA
45. Perform a quick spin and place the sample tube on the magnetic stand, to remove any residual ethanol.
SECTION: Cleanup of Barcoded DNA
46. Air dry the beads for up to 3 min while the tube is on the magnetic stand with the lid open. 
 


SECTION: Cleanup of Barcoded DNA
47. Remove the tube from the magnetic stand. Elute the DNA target from the beads by adding 33 µL.
SECTION: Cleanup of Barcoded DNA
48. Resuspend the pellet by flicking the tube or pipetting up 10 times and down 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
49. 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 microcentrifuge DNA LoBind Tube or PCR tube.
SECTION: Cleanup of Barcoded DNA
50. We recommend assessing cDNA concentrations with a Qubit fluorometer. Use 1 µl for the Qubit fluorometer.
 

SECTION: Adapter Ligation
51. Add the following components into a 1.5 ml microcentrifuge DNA LoBind Tube or nuclease-free PCR tube:
 

SECTION: Adapter Ligation
52. 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
53. Incubate at 25 °C or at  20 Room temperature for 20 min.

SECTION: Adapter Ligation
54. Proceed to Cleanup of Adapter-ligated DNA in the next section.
SECTION: Cleanup of Adapter Ligated DNA
55. 
 

SECTION: Cleanup of Adapter Ligated DNA
56. Vortex NEBNext Sample Purification Beads to resuspend. 
SECTION: Cleanup of Adapter Ligated DNA
57. Add 40 µL to the ligation mix. Mix well by flicking the tube to mix followed by a quick spin for 1 s.
SECTION: Cleanup of Adapter Ligated DNA
58. Incubate samples for 15 min at 20 Room temperature.
SECTION: Cleanup of Adapter Ligated DNA
59. 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
60. 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 libraries.
 

SECTION: Cleanup of Adapter Ligated DNA
61. Wash the beads by adding 250 µL. Flick the tube 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.
SECTION: Cleanup of Adapter Ligated DNA
62. Wait for 5 minutes (or until the solution is clear) to separate the beads from the supernatant. Remove the supernatant.

SECTION: Cleanup of Adapter Ligated DNA
63. Repeat previous 2 steps once for a total of two washes: 
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.
Wait for 5 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
64. 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
65. Resuspend the pellet well in EB buffer by flicking the tube. Incubate for 15 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
66. Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 15 µL to a new DNA LoBind tube.
SECTION: Cleanup of Adapter Ligated DNA
67. Use Qubit to quantify 1 µL. Follow Oxford Nanopore Protocol SQK-LSK109 to prepare MinION® flow cell and DNA library sequencing mix and load the flow cell. We recommend not multiplexing more than 10 samples(9 samples + NTC) in one R9.4.1 flow cell. We highly recommend using a Negative template control and label them as 'water', 'negative', 'blank', 'ntc' if using our custom analysis pipeline for the data analysis.


The base calling options can be one among the three: Fast base calling, High accuracy base calling, or Super accurate base calling. We have observed improved average read quality with High accuracy and Super accurate base calling, but little difference in read numbers, or variant calling compared to Fast base calling, using our custom analysis pipeline.