TITLE: RSV Nested Amplicon Prep (from VarSkip Workflow)
AUTHORS: Parker Montfort

[DESCRIPTION]
Use this modified protocol to prepare RSV Tiled amplicons for sequencing using the VarSkip Reagents and Workflow, with cleanup steps.This original protocol details methods for the NEBNext® ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®), NEB #E7658S/L 24/96 reactions.

[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. It is advisable to set up your reactions in the hood.

[GUIDELINES]
Overview 
The NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit for Illumina contains the enzymes, buffers and oligos required to convert a broad range of total RNA into high quality, targeted, 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 indexed libraries on an 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. 

Figure 1. Workflow demonstrating the use of NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit.
  


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



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

 

SECTION: cDNA Amplification
3. 
 
Gently mix and spin down reagents. Prepare the split pool cDNA amplification reactions as described below: (Follow this protocol for Primer Pooling)

Tiled RSV Amplicon reaction setup
 

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

SECTION: cDNA Amplification
5. 
 

SECTION: Cleanup of First PCR Product
6. 
 
Vortex NEBNext Sample Purification Beads to resuspend.
SECTION: Cleanup of First PCR Product
7. Add 25 µL to each PCR Plate (evens or odds). 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. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out. 
SECTION: Cleanup of First PCR Product
8. Incubate samples at Room temperature for at least 5 min.
SECTION: Cleanup of First PCR Product
9. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant.
SECTION: Cleanup of First PCR Product
10. 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 First PCR Product
11. 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 First PCR Product
12. Repeat 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. 
SECTION: Cleanup of First PCR Product
13. 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 First PCR Product
14. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 25 µL. 
SECTION: Cleanup of First PCR Product
15. 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 First PCR Product
16. Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 23 µL to a new PCR tube.
 

SECTION: Fragmentation/End Prep
31. Historic RSV FS NEB Preps utilized a 5 min incubation period. 

TEST A SUBSET ON 5 MIN, AND RUN ON TAPESTATION TO CONFIRM ~300bp

 

SECTION: Fragmentation/End Prep
32. Ensure that the Ultra II FS Reaction Buffer is completely thawed. If a precipitate is seen in the buffer, pipette up and down several times to break it up, and quickly vortex to mix. Place on ice until use.

There are short videos on NEB.com about handling the FS reagents: 
Preparing FS Reagents: https://www.neb.com/tools-and-resources/video-library/quick-tips---preparing-the-nebnext-ultra-ii-fs-dna-reaction-buffer-and-enzyme-mix
Making FS master mix: https://www.neb.com/tools-and-resources/video-library/quick-tips---preparing-nebnext-ultra-ii-fs-dna-reaction-buffer-and-enzyme-mix-master-mix
SECTION: Fragmentation/End Prep
33. Vortex the Ultra II FS Enzyme Mix 5-8 seconds prior to use and place on ice. 
 

SECTION: Fragmentation/End Prep
34. Add the following components to a 0.2 ml thin wall PCR tube on ice:
 

SECTION: Fragmentation/End Prep
35. Vortex the reaction for 5 s and briefly spin in a microcentrifuge.

SECTION: Fragmentation/End Prep
36. In a thermocycler* run the following program:
 

 

SECTION: Adaptor Ligation
38. Add the following components directly to the FS Reaction Mixture:
 

 

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


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

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

SECTION: Cleanup of Adaptor-ligated cDNA
44. Vortex the NEBNext Sample Purification Beads to resuspend.
SECTION: Cleanup of Adaptor-ligated cDNA
45. Add 28 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 cDNA
46. Incubate samples at Room temperature for at least 5 min.

SECTION: Cleanup of Adaptor-ligated cDNA
47. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. 
SECTION: Cleanup of Adaptor-ligated cDNA
48. 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 cDNA
49. 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 Adaptor-ligated cDNA
50. 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 Adaptor-ligated cDNA
51. 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 cDNA
52. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 10 µL. 
SECTION: Cleanup of Adaptor-ligated cDNA
53. 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 Adaptor-ligated cDNA
54. Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 7.5 µL to a new PCR tube. 
 

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

SECTION: PCR Enrichment of Adaptor-ligated DNA
56. 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
57. Place the tube on a thermocycler and perform PCR amplification using the following PCR cycling conditions:
 

SECTION: Cleanup of PCR Reaction
58. The following section is for cleanup of the PCR reaction.
 

SECTION: Cleanup of PCR Reaction
59. Vortex NEBNext Sample Purification Beads to resuspend. 
SECTION: Cleanup of PCR Reaction
60. 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
61. Incubate samples on bench top for at least 5 min at Room temperature.
SECTION: Cleanup of PCR Reaction
62. 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
63. 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
64. 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
65. 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
66. 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
67. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 17 µL. 
SECTION: Cleanup of PCR Reaction
68. 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
69. 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
70. Assess the library size distribution with TapeStation high sensitivity DNA 1000 reagents. The sample may need to be diluted before loading. A peak size of 200–250 bp is expected.
 

SECTION: cDNA Synthesis
1.1. Add 3 µL to each reaction well of a midskirt plate in the clean room. Be sure to add cDNA NTC.

SECTION: cDNA Synthesis
1.2. Bring cDNA plate into Template Addition (TA/ Low Titer). Add 12 µL to each reaction well and seal with PCR seal.

 

SECTION: cDNA Synthesis
1.3. Centrifuge plate and load on thermocycler. It is ok to use the BioRad cycler in TA for cDNA synthesis.
SECTION: Cleanup of First PCR Product
12.1. Be sure to remove all visible liquid after the second wash. You may briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
SECTION: Adaptor Ligation
37. Determine whether adaptor dilution is necessary, by following the table below;

 

SECTION: cDNA Amplification
3.1. RSV A Mastermix: scale as necessary
Combine 12.5 µL , 1.67 µL , and 4.58 µL . Plate into alignment with each RSV A sample on the plate map.

SECTION: cDNA Amplification
3.2. RSV B Mastermix: scale as necessary
Combine 12.5 µL , 1.67 µL , and 4.58 µL . Plate into alignment with each RSV B sample on the plate map.

SECTION: cDNA Amplification
3.3. Add 6.25 µL  to both Pool 1 and Pool 2 reactions. Mix, seal, and centrifuge. 

SECTION: Nested PCR
17. 
 
Gently mix and spin down reagents. Prepare the split pool cDNA amplification reactions as described below: (Follow this protocol for Primer Pooling)

Tiled RSV Amplicon reaction setup
 
 
 

SECTION: Nested PCR
17.1. RSV A Mastermix: scale as necessary
Combine 12.5 µL , 1.67 µL , and 9.83 µL . Plate into alignment with each RSV A sample on the plate map.
SECTION: Nested PCR
17.2. RSV B Mastermix: scale as necessary
Combine 12.5 µL , 1.67 µL , and 9.83 µL . Plate into alignment with each RSV B sample on the plate map.
SECTION: Nested PCR
17.3. Add 1 µL  to both even and odd reactions. Mix, seal, and centrifuge. 
SECTION: Nested PCR
18. Incubate reactions in a thermocycler with the following steps:
 
 
 

 

 

SECTION: Cleanup of Nested PCR Product
19. 
 
Vortex NEBNext Sample Purification Beads to resuspend.
SECTION: Cleanup of Nested PCR Product
20. Add 25 µL to each PCR Plate (evens or odds). 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. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out. 
SECTION: Cleanup of Nested PCR Product
21. Incubate samples at Room temperature for at least 5 min.
SECTION: Cleanup of Nested PCR Product
22. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant.
SECTION: Cleanup of Nested PCR Product
23. 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 Nested PCR Product
24. 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 Nested PCR Product
25. Repeat 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. 
SECTION: Cleanup of Nested PCR Product
25.1. Be sure to remove all visible liquid after the second wash. You may briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
SECTION: Cleanup of Nested PCR Product
26. 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 Nested PCR Product
27. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 17 µL. 
SECTION: Cleanup of Nested PCR Product
28. 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 Nested PCR Product
29. 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.

SECTION: Cleanup of Nested PCR Product
30. Combine the paired Pool 1 and Pool 2 PCR reactions for each sample.