TITLE: Modified NEBNext® VarSkip Long SARS-CoV-2 Enrichment and library prep (SMRTbell prep kit 3.0 Pacific Biosciences)- adapted for wastewater samples
AUTHORS: Kathryn Judy, Padmini  Ramachandran, Amanda Windsor, Tamara Walsky, Chris  Grim, Maria Hoffmann

[DESCRIPTION]
This protocol details methods for the preparation of SARS-CoV-2 sequencing library using VSL primers from NEB, adapted for wastewater samples. This protocol produces multiplexed amplicon libraries suitable for sequencing on PacBio systems (e.g., Sequel® IIe) using the SMRTbell® prep kit 3.0 and SMRTbell® barcoded adapter plate 3.0.

[BEFORE_START]
Note: 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 
Sequences and information on the NEBNext VarSkip Long primers can be found at https://github.com/nebiolabs/VarSkip. All other enzymes, buffers, beads and oligos required to convert cDNA into targeted, high quality libraries for next-generation sequencing on the PacBio platform are available. 

[STEPS]
SECTION: Before you start
1. 
 

 

SECTION: Targeted cDNA Amplification
2. 
 

SECTION: Targeted cDNA Amplification
3. Prepare master mixes fresh immediately before performing cDNA amplification. 

Q5 Hot Start High-Fidelity Polymerase should stay on ice at all times. Do not vortex.
Thaw Q5 Reaction Buffer, MgCl2, dNTPs, and water.
Mix thawed tubes, spin down, and place on ice.
Thaw VarSkip Long Primer Mix 1 and VarSkip Long Primer Mix 2.  
Mix by flicking and spin down both the tubes.
Keep on ice.

Prepare the split pool amplification reactions as described below:

For Pool set A:
Prepare the master mix below in sufficient volume for your samples.
 

For Pool Set B: 
Prepare the master mix below in sufficient volume for your samples.
 
Mix the two master mix tubes by flicking and spin down. Dispense 7.5 µl master mix from each tube into separate PCR tube strips (A and B), two PCR tubes (one for each master mix) per sample to amplify.

SECTION: Targeted cDNA Amplification
6. Gently flick the tube strips to mix and spin down briefly.
SECTION: Targeted cDNA Amplification
7. Incubate Pool A reactions in a thermocycler* with the following steps:
 

Incubate Pool B reactions in a thermocycler* with the following steps:

 

 

SECTION: Cleanup of cDNA Amplicons
8. We highly recommend this clean up step using AMPure® XP beads, though NEBNext sample purification beads can be used as well. 

This step replaces the input DNA quality control and cleanup step from the amplicon library preparation using SMRTbell® prep kit 3.0. It may be possible to omit this cleanup in favor of the PacBio initial cleanup, but this has not been tested.

 

SECTION: Cleanup of cDNA Amplicons
9. For each sample, combine pool A and pool B PCR products (amplicons), measuring the pooled volume.
SECTION: Cleanup of cDNA Amplicons
10. Vortex AMPure® XP beads for 30 s to resuspend.

SECTION: Cleanup of cDNA Amplicons
11. Add 0.6 X to the combined PCR product.  Mix well by flicking the tube 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
12. Incubate samples at Room temperature for 5 min.
SECTION: Cleanup of cDNA Amplicons
13. Quickly spin samples to collect the liquid from the sides of the tube before placing on the magnetic stand for 5 min to separate the beads from the supernatant.

SECTION: Cleanup of cDNA Amplicons
14. 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
15. 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
16. 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
17. 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
18. Remove the tube from the magnetic stand. Elute the DNA target from the beads by adding 18 µL. 
SECTION: Cleanup of cDNA Amplicons
19. Mix well by flicking the tube followed by a very short centrifugation. Incubate for 5 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
20. Place the tube on the magnetic stand. After 2 min (or when the solution is clear), transfer 17 µL to clean PCR tubes.

SECTION: Cleanup of cDNA Amplicons
21. 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 should also be run on Femto or Bioanalyzer® or Tape Station using High Sensitivity (HS) 5000 tape to confirm ~1500-1600 bp size of amplicons.
 



 

SECTION: Targeted cDNA Amplification
4. Add 4.5 µl cDNA into each pre-filled PCR tube, ensuring each sample to be amplified is added into exactly 1 tube in strip A and 1 tube in strip B.
SECTION: Targeted cDNA Amplification
5. While keeping the polymerase on ice, add 0.5 µL Q5 Hot Start High-Fidelity Polymerase to each tube.

SECTION: DNA Repair and A-tailing
22. 
 

Calculate the volume of each sample needed to bring forward at least 150 ng per sample. We recommend bringing forward approximately the same mass of DNA for each sample. DNA mass <150 ng may be usable but masses < 125 ng have not been tested.

Aliquot the volume of each sample calculated into fresh PCR tubes and make up each sample to 46 µL using SMRTbell Low TE buffer. Excess amplicons should be returned to 4 °C
SECTION: DNA Repair and A-tailing
23. Make the repair and A-tailing master mix by combining the reagents below in the order and amounts listed in the table. Adjust component volumes for your number of samples plus 20% overage.

Thaw Repair Buffer at room temperature, then vortex and spin down briefly.
Thaw End Repair and DNA Repair mixes on ice, spin down briefly, and return to ice. Do not vortex.

 
Mix the master mix components by pipetting or gentle flicking and quickly centrifuge.


SECTION: DNA Repair and A-tailing
24. Add 14 µL to each sample for a total reaction volume of 60 µl per sample.

SECTION: DNA Repair and A-tailing
25. Gently mix samples by flicking and quickly spin to collect liquid.
SECTION: DNA Repair and A-tailing
26. Incubate samples in a thermocycler* with the following settings:

 

SECTION: Adapter Ligation and Cleanup
27. 
 

Add 4 µL to each sample tube from the previous step, using a different barcode for each sample. Add only one barcode to each sample.

SECTION: Adapter Ligation and Cleanup
28. Make the adapter ligation master mix by combining the reagents below in the amounts and volumes listed in the table. Adjust component volumes to your number of samples plus 20% overage.

Thaw Ligation mix and Ligation enhancer on ice. Do not vortex. 

 
Mix components by pipetting or gently flicking the tube, then centrifuge briefly.
SECTION: Adapter Ligation and Cleanup
29. Add 31 µL to each sample for a total volume of 95 µl per sample.

SECTION: Adapter Ligation and Cleanup
30. Gently mix samples by flicking and quickly spin to collect liquid.
SECTION: Adapter Ligation and Cleanup
31. Incubate samples in a thermocycler* with the below settings:

 

SECTION: Adapter Ligation and Cleanup
32. Thaw elution buffer at room temperature. 
Room temperature SMRTbell cleanup beads should also be stored at room temperature until the protocol is completed or paused overnight at a safe stopping point.

Add 124 µL (1.3X) to each adapter-ligated sample and mix by gently flicking followed by a short spin to collect the liquid. Stop the centrifugation before the beads begin to settle.

SECTION: Adapter Ligation and Cleanup
33. Leave samples at room temperature for 10 min to bind DNA to the beads.

SECTION: Adapter Ligation and Cleanup
34. Place tube strip in an appropriate magnetic separation rack until the beads have separated, usually within 3 min 

SECTION: Adapter Ligation and Cleanup
35. Carefully pipette off the supernatant without disturbing the beads, discarding the supernatant. Do not discard the beads, which contain your DNA target.
SECTION: Adapter Ligation and Cleanup
36. Slowly dispense 200 µL to each sample tube. After 30 s, pipette off the ethanol and discard. Do not discard the beads.

SECTION: Adapter Ligation and Cleanup
37. Repeat the previous step once for a total of two washes:
Slowly dispense 200 µL to each sample tube. After 30 s, pipette off the ethanol and discard. Do not discard the beads.
SECTION: Adapter Ligation and Cleanup
38. To remove residual ethanol, quickly spin samples and return the tubes to the magnetic rack, allowing beads to separate fully. Pipette off residual ethanol with a P20 pipette and discard. Do not discard the beads.
SECTION: Adapter Ligation and Cleanup
39. Remove samples from the magnetic rack and immediately add 40 µL. Resuspend beads by flicking, then quickly spin to collect liquid.

SECTION: Adapter Ligation and Cleanup
40. Leave samples at room temperature for 5 min to elute DNA.

SECTION: Adapter Ligation and Cleanup
41. Place samples on the magnetic rack until beads separate fully from the solution, usually less than 5 min 

SECTION: Adapter Ligation and Cleanup
42. Slowly pipette 40 µL of clear supernatant without disturbing the beads and transfer to a new PCR tube strip. Discard the old sample tubes with beads. Do not discard the supernatant.


 



SECTION: Nuclease Treatment and Cleanup
43. 
 
Make the nuclease treatment master mix by adding the reagents listed below in the following order and amounts. Adjust the volumes as needed for your number of samples plus 20% overage.

Thaw Nuclease buffer at room temperature, then vortex and spin down briefly.
Thaw Nuclease mix on ice, spin down briefly, and return to ice. Do not vortex.

 
Mix components by pipetting or gently flicking the tube, then quickly centrifuge to mix.
SECTION: Nuclease Treatment and Cleanup
44. Add 10 µL to each sample for a total volume of 50 µl per sample.

SECTION: Nuclease Treatment and Cleanup
45. Gently flick the tubes to mix and briefly spin down.
SECTION: Nuclease Treatment and Cleanup
46. Incubate samples in a thermocycler* with the below settings:

 

SECTION: Nuclease Treatment and Cleanup
47. Thaw elution buffer at room temperature. 
Room temperature SMRTbell cleanup beads should also be stored at room temperature until the protocol is completed or paused overnight at a safe stopping point.

Add 65 µL (1.3X) to each nuclease-treated sample and mix by gently flicking followed by a short spin to collect the liquid. Stop the centrifugation before the beads begin to settle.
SECTION: Nuclease Treatment and Cleanup
48. Leave samples at room temperature for 10 min to bind DNA to the beads.

SECTION: Nuclease Treatment and Cleanup
49. Place tube strip in an appropriate magnetic separation rack until the beads have separated, usually within 3 min 

SECTION: Nuclease Treatment and Cleanup
50. Carefully pipette off the supernatant without disturbing the beads, discarding the supernatant. Do not discard the beads, which contain your DNA target.
SECTION: Nuclease Treatment and Cleanup
51. Slowly dispense 200 µL to each sample tube. After 30 s, pipette off the ethanol and discard. Do not discard the beads.

SECTION: Nuclease Treatment and Cleanup
52. Repeat the previous step once for a total of two washes:
Slowly dispense 200 µL to each sample tube. After 30 s, pipette off the ethanol and discard. Do not discard the beads.
SECTION: Nuclease Treatment and Cleanup
53. To remove residual ethanol, quickly spin samples and return the tubes to the magnetic rack, allowing beads to separate fully. Pipette off residual ethanol with a P20 pipette and discard. Do not discard the beads.
SECTION: Nuclease Treatment and Cleanup
54. Remove samples from the magnetic rack and immediately add 15 µL. Resuspend beads by flicking, then quickly spin to collect liquid.

SECTION: Nuclease Treatment and Cleanup
55. Leave samples at room temperature for 5 min to elute DNA.

SECTION: Nuclease Treatment and Cleanup
56. Place samples on the magnetic rack until beads separate fully from the solution, usually less than 5 min 

SECTION: Nuclease Treatment and Cleanup
57. Slowly pipette 15 µL of clear supernatant without disturbing the beads and transfer to a new PCR tube strip. Discard the old sample tubes with beads. Do not discard the supernatant.
SECTION: Pooling and Concentrating Barcoded Samples
59. 
 

Based on the Qubit values determined in step 58, combine an equal mass of each sample together in a single pool within a 1.5mL DNA LoBind tube. The total mass of the pooled samples should be at least 100 ng.

SECTION: Nuclease Treatment and Cleanup
58. Dilute 1 µL from each sample in 9 µL elution buffer or water, then measure DNA concentration with a Qubit Fluorometer using the 1x dsDNA HS kit.

 

SECTION: Pooling and Concentrating Barcoded Samples
60. Add 1.3 X to the sample pool and mix by gently flicking followed by a short spin to collect the liquid. Stop the centrifugation before the beads begin to settle.

SECTION: Pooling and Concentrating Barcoded Samples
61. Leave pool at room temperature for 10 min to bind DNA to the beads.

SECTION: Pooling and Concentrating Barcoded Samples
62. Place the tube in an appropriate magnetic separation rack until the beads have separated, usually within 3 min 

SECTION: Pooling and Concentrating Barcoded Samples
63. Carefully pipette off the supernatant without disturbing the beads, discarding the supernatant. Do not discard the beads, which contain your DNA target.
SECTION: Pooling and Concentrating Barcoded Samples
64. Slowly dispense 200 µL into the tube. After 30 s, pipette off the ethanol and discard. Do not discard the beads.

SECTION: Pooling and Concentrating Barcoded Samples
65. Repeat the previous step once for a total of two washes:
Slowly dispense 200 µL into the tube. After 30 s, pipette off the ethanol and discard. Do not discard the beads.
SECTION: Pooling and Concentrating Barcoded Samples
66. To remove residual ethanol, quickly spin the tube before returning it to the magnetic rack, allowing beads to separate fully. Pipette off residual ethanol with a P20 pipette and discard. Do not discard the beads.
SECTION: Pooling and Concentrating Barcoded Samples
67. Remove pool from the magnetic rack and immediately add 15 µL. Resuspend beads by flicking, then quickly spin to collect liquid.

SECTION: Pooling and Concentrating Barcoded Samples
68. Leave pool at room temperature for 5 min to elute DNA.

SECTION: Pooling and Concentrating Barcoded Samples
69. Place tube on the magnetic rack until beads separate fully from the solution, usually less than 5 min 

SECTION: Pooling and Concentrating Barcoded Samples
70. Slowly pipette 15 µL of clear supernatant without disturbing the beads and transfer to a new tube. Discard the old LoBind tube with beads. Do not discard the supernatant.
SECTION: Pooling and Concentrating Barcoded Samples
71. Dilute 1 µL of the concentrated library in 9 µL elution buffer or water, then measure DNA concentration with a Qubit Fluorometer using the 1x dsDNA HS kit.

We also recommend running the library on Tape Station using High Sensitivity (HS) 5000 tape to confirm ~1500-1600 bp size of the library.

  
 

SECTION: End Protocol
72. Use SMRTLink Sample Setup with 150 pM on plate concentration and Binding kit 3.1 to prepare library(ies) for sequencing. Use SMRT Cell 8M tray and Sequel II sequencing kit 2.0 to sequence on the Sequel IIe instrument.