TITLE: Modified NEBNext® VarSkip Short SARS-CoV-2 Library Prep Kit for Illumina Platforms - adapted for wastewater samples
AUTHORS: Padmini   Ramachandran, Tamara  Walsky, Amanda  Windsor, Maria  Hoffmann, Chris   Grim

[DESCRIPTION]
PURPOSE: 
This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater​​.  Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR, library prep, genome sequencing, quality control checks, and data submission to NCBI.  

This modified protocol details methods for cDNA synthesis and library preparation for sequencing of wastewater samples containing SARS-CoV-2.  The protocol is based primarily on the NEBNext® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®), NEB #E7650S/L 24/96 reactions, with a few modifications.  Primarily, VarSkip Short primers are used in place of the ARTIC V3 primers.  These primers are available in the NEBNext®ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®); however, for optimal variant detection from wastewater, sequenced fragments should be as large as possible, so we discourage fragmentation prior to end prep.

There are a couple of decision points in this protocol.  Examining cDNA amplicon samples on an Agilent TapeStation system or similar fragment analyzer is extremely helpful in making these decisions. 



[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, however, results may vary depending on the quality of the 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.

[GUIDELINES]
Overview 
The NEBNext SARS-CoV-2 Library Prep Kit (Illumina) contains the enzymes, buffers and oligos required to convert a broad range of total RNA input amounts into targeted, high quality libraries for next-generation sequencing on the Illumina platform. Primers targeting the human EDF1 (NEBNext ARTIC Human Primer Mix 1) and NEDD8 (NEBNext ARTIC Human Primer Mix 2) 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 Illumina 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.

  


[STEPS]
SECTION: cDNA Synthesis
2. Gently mix and spin down the LunaScript RT SuperMix reagent. Prepare the cDNA synthesis reaction as described below:
 

For no template controls, mix the following components:
 

SECTION: cDNA Synthesis
4. Incubate reactions in a thermocycler* with the following steps:
 


 

SECTION: Targeted cDNA Amplification
5. 
 


SECTION: Targeted cDNA Amplification
6. Gently mix and spin down reagents. Prepare the split pool cDNA amplification reactions as described below:

For Pool Set A: 

 

For Pool Set B:


 

SECTION: Targeted cDNA Amplification
8. Incubate reactions in a thermocycler* with the following steps:
 

SECTION: Targeted cDNA Amplification
9. Combine the Pool A and Pool B PCR reactions for each sample.
 

SECTION: Cleanup of cDNA Amplicons.
12. 
 

SECTION: Cleanup of cDNA Amplicons.
13. Vortex the NEBNext Sample Purification Beads to resuspend.
SECTION: Cleanup of cDNA Amplicons.
14. Add 20 µL to the combined 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.

SECTION: Cleanup of cDNA Amplicons.
15. Incubate samples at -20 Room temperature for at least 5 min.

SECTION: Cleanup of cDNA Amplicons.
16. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. 
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/plate while in the magnetic stand. Incubate at -20 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. Perform second Ethanol wash:

Add 200 µL to the tube/plate while in the magnetic stand. Incubate at -20 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/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
SECTION: Cleanup of cDNA Amplicons.
20. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open. 
 
  

SECTION: Cleanup of cDNA Amplicons.
21. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 28 µL. If not assessing cDNA (Step 24) elute DNA in 27 µL.
SECTION: Cleanup of cDNA Amplicons.
22. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 minutes 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 cDNA Amplicons.
23. Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 26 µL to a new PCR tube. If not assessing cDNA (Step 24) transfer 25 µL to a new PCR tube.
SECTION: Cleanup of cDNA Amplicons.
24. We recommend assessing cDNA amplicon (from Step 23) concentrations with a Qubit fluorometer using Qubit HS DNA assay kit. 

 


 

SECTION: NEBNext End Prep
28. Add the following components to a sterile nuclease-free tube:
 

SECTION: NEBNext End Prep
29. Set a 100 µl or 200 µl pipette to 25 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
 

SECTION: NEBNext End Prep
30. Place in a thermocycler* and run the following program:
 

 

SECTION: Adaptor Ligation
31. Add the following components directly to the End Prep Reaction Mixture:
 

 

SECTION: Adaptor Ligation
32. Set a 100 µl or 200 µl pipette to 40 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
 

SECTION: Adaptor Ligation
33. Incubate at 20 °C for 15 min in a thermocycler with the heated lid off.
SECTION: Adaptor Ligation
34. Add 1.5 µL to the ligation mixture from the previous step.

 

SECTION: Adaptor Ligation
35. Mix well and incubate at 37 °C for 15 min with the heated lid set to ≥ 47 °C.
 

SECTION: Cleanup of Adaptor-ligated DNA
36. The following section is for cleanup of the ligation reaction. 
 

SECTION: Cleanup of Adaptor-ligated DNA
37. Vortex the NEBNext Sample Purification Beads to resuspend.
SECTION: Cleanup of Adaptor-ligated DNA
38. Add 43 µL to the Adaptor Ligation 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.

SECTION: Cleanup of Adaptor-ligated DNA
39. Incubate samples on bench top for at least 5 min at 47 Room temperature.

SECTION: Cleanup of Adaptor-ligated DNA
40. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. 
SECTION: Cleanup of Adaptor-ligated DNA
41. 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 Adaptor-ligated DNA
42. Add 200 µL to the tube/ plate while in the magnetic stand. Incubate at 47 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 Adaptor-ligated DNA
43. Repeat the previous step once for a total of two washes:

Add 200 µL to the tube/ plate while in the magnetic stand. Incubate at 47 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/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
SECTION: Cleanup of Adaptor-ligated DNA
44. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
 
  

SECTION: Cleanup of Adaptor-ligated DNA
45. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 10 µL.
SECTION: Cleanup of Adaptor-ligated DNA
46. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 min at 47 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 Adaptor-ligated DNA
47. Place the tube/plate on the magnetic stand. After 5 min (or when the solution is clear), transfer 7.5 µL to a new PCR tube.
 

SECTION: PCR Enrichment of Adaptor-ligated DNA
48. 


 

SECTION: PCR Enrichment of Adaptor-ligated DNA
49. Add the following components to a sterile strip tube:
 

SECTION: PCR Enrichment of Adaptor-ligated DNA
50. Set a 100 μl pipette to 20 μl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
SECTION: PCR Enrichment of Adaptor-ligated DNA
51. Place the tube on a thermocycler** and perform PCR amplification using the following PCR cycling conditions:
 

SECTION: Cleanup of PCR Reaction
52. 
 

SECTION: Cleanup of PCR Reaction
53. Vortex NEBNext Sample Purification Beads to resuspend.
SECTION: Cleanup of PCR Reaction
54. Add 22.5 µL 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.

SECTION: Cleanup of PCR Reaction
55. Incubate samples on bench top for at least 5 min at Room temperature.

SECTION: Cleanup of PCR Reaction
56. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. 
SECTION: Cleanup of PCR Reaction
57. 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 PCR Reaction
58. Add 200 µL to the tube/plate 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 PCR Reaction
59. Repeat the previous step once for a total of two washes:

Add 200 µL to the tube/plate 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/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
SECTION: Cleanup of PCR Reaction
60. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
 
  

SECTION: Cleanup of PCR Reaction
61. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 17 µL.
SECTION: Cleanup of PCR Reaction
62. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 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 PCR Reaction
63. Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 15 µL to a new PCR tube and store at -20 °C.
SECTION: Cleanup of PCR Reaction
64. Check the size distribution on an Agilent Bioanalyzer or TapeStation. The sample may need to be diluted before loading. A peak size of ~520 bp is expected (Figure 56).

 
  

SECTION: PCR Enrichment of Adaptor-ligated DNA
49. Add the following components to a sterile strip tube:
 

SECTION: PCR Enrichment of Adaptor-ligated DNA
50. Set a 100 μl pipette to 20 μl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
SECTION: PCR Enrichment of Adaptor-ligated DNA
51. Place the tube on a thermocycler and perform PCR amplification using the following PCR cycling conditions:
 

SECTION: PCR Enrichment of Adaptor-ligated DNA
52. Proceed to Cleanup of PCR Amplification in the next section.
SECTION: Cleanup of PCR Reaction
53. 
 

SECTION: Cleanup of PCR Reaction
54. Vortex NEBNext Sample Purification Beads to resuspend.
SECTION: Cleanup of PCR Reaction
55. Add 22.5 µL 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.

SECTION: Cleanup of PCR Reaction
56. Incubate samples on bench top for at least 5 min at Room temperature.

SECTION: Cleanup of PCR Reaction
57. Place the tube/plate 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 PCR Reaction
58. 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 PCR Reaction
59. Add 200 µL to the tube/plate 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 PCR Reaction
60. Repeat the previous step once for a total of two washes:

Add 200 µL to the tube/plate 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/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
SECTION: Cleanup of PCR Reaction
61. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
 

SECTION: Cleanup of PCR Reaction
62. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 17 µL.
SECTION: Cleanup of PCR Reaction
63. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 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 PCR Reaction
64. Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 15 µL to a new PCR tube and store at -20 °C.
SECTION: Cleanup of PCR Reaction
65. Check the size distribution on an Agilent Bioanalyzer or TapeStation. The sample may need to be diluted before loading. A peak size of ~520 bp is expected (Figure 61).

 


  

SECTION: cDNA Synthesis
3. Flick the tubes or pipet up and down 10 times to mix followed by a quick spin.
SECTION: Targeted cDNA Amplification
7. Flick the tubes or pipet up and down 10 times to mix followed by a quick spin.
SECTION: Targeted cDNA Amplification
10. Assess the quantity of the cDNA amplicon using the Qubit HS kit. (1 µL 

SECTION: Targeted cDNA Amplification
12. Assess the size of the cDNA amplicon using Agilent TapeStation System, Bioanalyzer, or similar size fragment analyzer.


     
  
     

SECTION: Targeted cDNA Amplification
11. 
 




SECTION: NEBNext End Prep
27. This is library preparation Option 1, and the most common that will be used.  You will use the Ultra II DNA workflow as described in the NEBNext® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®) (cat# E7650S/L), with NEBNext®Multiplex Oligos for Illumina®(96 Unique Dual Index Primer Pairs) (cat# E6440S/L).
SECTION: Cleanup of PCR Reaction
65. Sample Sheet generation for NEBNext® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®) with E6440 Index plate.  

SECTION: cDNA Synthesis
1. 
 

SECTION: Quality checks on cleaned amplicons before proceeding to library prep
25. We have observed the following output for VSS amplicons based on viral titer in the wastewater samples:

1)Moderate to high viral titer samples: The cleaned amplicon quantity is expected to be 0.5 ng/µl to 8 ng/µl
2)Low viral titer samples: The cleaned amplicon quantity is expected to be 0 - 0.2 ng/µl

We recommend to proceed to library preparation for samples at 0.2 ng/µl or higher.  Cleaned VSS amplicons below this value have not yielded sequencing results.

SECTION: Quality checks on cleaned amplicons before proceeding to library prep
26. We recommend assessing cDNA amplicon (from Step 23) on Tape Station or Bioanalyzer using High Sensitivity kit.