Modified NEBNext® VarSkip Long SARS-CoV-2 Enrichment and library prep (SMRTbell prep kit 3.0 Pacific Biosciences)- adapted for wastewater samples
Padmini Ramachandran, Tamara Walsky, Amanda Windsor, Maria Hoffmann, Christopher Grim, Kathryn Judy
Disclaimer
Abstract
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.
Steps
Before you start
Targeted cDNA Amplification
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.
| A | B |
|---|---|
| COMPONENT | VOLUME |
| Q5 Reaction Buffer | 2.5 µl |
| 50mM Magnesium Chloride | 0.5 µl |
| Deoxynucleotide (dNTP) Solution | 0.75 µl |
| Nuclease-free water | 1.75 µl |
| NEBNext VarSkip Long Primer Mix 1 | 2.25 µl |
| Total Volume | 7.5 µl |
For Pool Set B:
Prepare the master mix below in sufficient volume for your samples.
| A | B |
|---|---|
| COMPONENT | VOLUME |
| Q5 Reaction Buffer | 2.5 µl |
| 50mM Magnesium Chloride | 0.5 µl |
| Deoxynucleotide (dNTP) Solution | 0.75 µl |
| Nuclease-free water | 1.75 µl |
| NEBNext VarSkip Long Primer Mix 2 | 2.25 µl |
| Total Volume | 7.5 µl |
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.
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.
While keeping the polymerase on ice, add 0.5µL Q5 Hot Start High-Fidelity Polymerase to each tube.
Gently flick the tube strips to mix and spin down briefly.
Incubate Pool A reactions in a thermocycler* with the following steps:
| A | B | C | D |
|---|---|---|---|
| CYCLE STEP | TEMP | TIME | CYCLES |
| Initial Denaturation | 98°C | 30 seconds | 1 |
| Denature | 95°C | 15 seconds | 38 |
| Annealing | 59°C | 1 minute | |
| Extension | 72°C | 2 minutes | |
| Hold | 4°C | ∞ | 1 |
- Set heated lid to 105°C.
Incubate Pool B reactions in a thermocycler* with the following steps:
| A | B | C | D |
|---|---|---|---|
| CYCLE STEP | TEMP | TIME | CYCLES |
| Initial Denaturation | 98°C | 30 seconds | 1 |
| Denature | 95°C | 15 seconds | 38 |
| Annealing | 61°C | 45 seconds | |
| Extension | 72°C | 2 minutes | |
| Hold | 4°C | ∞ | 1 |
- Set heated lid to 105°C.
Cleanup of cDNA Amplicons
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.
For each sample, combine pool A and pool B PCR products (amplicons), measuring the pooled volume.
Vortex AMPure® XP beads for 0h 0m 30s to resuspend.
Add 0.6X 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.
Incubate samples at Room temperature for 0h 5m 0s.
Quickly spin samples to collect the liquid from the sides of the tube before placing on the magnetic stand for 0h 5m 0s to separate the beads from the supernatant.
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.
Add 200µL to the tube while in the magnetic stand. Incubate at Room temperature for 0h 0m 30s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Repeat previous step once for a total of two washes:
Add 200µL to the tube while in the magnetic stand. Incubate at Room temperature for 0h 0m 30s, 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 , place back on the magnetic stand and remove traces of ethanol with a p10 pipette tip. 0h 0m 1s, place back on the magnetic stand and remove traces of ethanol with a p10 pipette tip.
Air dry the beads for up to 0h 3m 0s while the tube is on the magnetic stand with the lid open.
Remove the tube from the magnetic stand. Elute the DNA target from the beads by adding 18µL.
Mix well by flicking the tube followed by a very short centrifugation. Incubate for 0h 5m 0s 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.
Place the tube on the magnetic stand. After 0h 2m 0s (or when the solution is clear), transfer 17µL to clean PCR tubes.
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.
DNA Repair and A-tailing
Calculate the volume of each sample needed to bring forward at least 150ng 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 46µL using SMRTbell Low TE buffer. Excess amplicons should be returned to 4°C
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.
| A | B |
|---|---|
| Component | Volume per Sample |
| Repair buffer | 8 µl |
| End Repair mix | 4 µl |
| DNA Repair mix | 2 µl |
| Total volume | 14 µl |
Mix the master mix components by pipetting or gentle flicking and quickly centrifuge.
Add 14µL to each sample for a total reaction volume of 60 µl per sample.
Gently mix samples by flicking and quickly spin to collect liquid.
Incubate samples in a thermocycler* with the following settings:
| A | B | C |
|---|---|---|
| TEMP | TIME | CYCLES |
| 37°C | 30 minutes | 1 |
| 65°C | 5 minutes | 1 |
| 4°C | ∞ | 1 |
- Set heated lid to 75°C
Adapter Ligation and Cleanup
Add 4µL to each sample tube from the previous step, using a different barcode for each sample. Add only one barcode to each sample.
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.
| A | B |
|---|---|
| Component | Volume per Sample |
| Ligation mix | 30 µl |
| Ligation enhancer | 1 µl |
| Total volume | 31 µl |
Mix components by pipetting or gently flicking the tube, then centrifuge briefly.
Add 31µL to each sample for a total volume of 95 µl per sample.
Gently mix samples by flicking and quickly spin to collect liquid.
Incubate samples in a thermocycler* with the below settings:
| A | B | C |
|---|---|---|
| TEMP | TIME | CYCLES |
| 20°C | 30 minutes | 1 |
| 4°C | ∞ | 1 |
- Set heated lid to 75°C.
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 124µL (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.
Leave samples at room temperature for 0h 10m 0s to bind DNA to the beads.
Place tube strip in an appropriate magnetic separation rack until the beads have separated, usually within 0h 3m 0s
Carefully pipette off the supernatant without disturbing the beads, discarding the supernatant. Do not discard the beads, which contain your DNA target .
Slowly dispense 200µL to each sample tube. After 0h 0m 30s, pipette off the ethanol and discard. Do not discard the beads.
Repeat the previous step once for a total of two washes:
Slowly dispense 200µL to each sample tube. After 0h 0m 30s, pipette off the ethanol and discard. Do not discard the beads.
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.
Remove samples from the magnetic rack and immediately add 40µL. Resuspend beads by flicking, then quickly spin to collect liquid.
Leave samples at room temperature for 0h 5m 0s to elute DNA.
Place samples on the magnetic rack until beads separate fully from the solution, usually less than 0h 5m 0s
Slowly pipette 40µL 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.
Nuclease Treatment and Cleanup
- 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.
| A | B |
|---|---|
| Component | Volume per Sample |
| Nuclease buffer | 5 µl |
| Nuclease mix | 5 µl |
| Total volume | 10 µl |
Mix components by pipetting or gently flicking the tube, then quickly centrifuge to mix.
Add 10µL to each sample for a total volume of 50 µl per sample.
Gently flick the tubes to mix and briefly spin down.
Incubate samples in a thermocycler* with the below settings:
| A | B | C |
|---|---|---|
| TEMP | TIME | CYCLES |
| 37°C | 15 minutes | 1 |
| 4°C | ∞ | 1 |
- Set heated lid to 75°C.
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 65µL (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.
Leave samples at room temperature for 0h 10m 0s to bind DNA to the beads.
Place tube strip in an appropriate magnetic separation rack until the beads have separated, usually within 0h 3m 0s
Carefully pipette off the supernatant without disturbing the beads, discarding the supernatant. Do not discard the beads, which contain your DNA target .
Slowly dispense 200µL to each sample tube. After 0h 0m 30s, pipette off the ethanol and discard. Do not discard the beads.
Repeat the previous step once for a total of two washes:
Slowly dispense 200µL to each sample tube. After 0h 0m 30s, pipette off the ethanol and discard. Do not discard the beads.
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.
Remove samples from the magnetic rack and immediately add 15µL. Resuspend beads by flicking, then quickly spin to collect liquid.
Leave samples at room temperature for 0h 5m 0s to elute DNA.
Place samples on the magnetic rack until beads separate fully from the solution, usually less than 0h 5m 0s
Slowly pipette 15µL 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.
Dilute 1µL from each sample in 9µL elution buffer or water, then measure DNA concentration with a Qubit Fluorometer using the 1x dsDNA HS kit.
Pooling and Concentrating Barcoded Samples
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 100ng.
Add 1.3X 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.
Leave pool at room temperature for 0h 10m 0s to bind DNA to the beads.
Place the tube in an appropriate magnetic separation rack until the beads have separated, usually within 0h 3m 0s
Carefully pipette off the supernatant without disturbing the beads, discarding the supernatant. Do not discard the beads, which contain your DNA target .
Slowly dispense 200µL into the tube. After 0h 0m 30s, pipette off the ethanol and discard. Do not discard the beads.
Repeat the previous step once for a total of two washes:
Slowly dispense 200µL into the tube. After 0h 0m 30s, pipette off the ethanol and discard. Do not discard the beads.
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.
Remove pool from the magnetic rack and immediately add 15µL. Resuspend beads by flicking, then quickly spin to collect liquid.
Leave pool at room temperature for 0h 5m 0s to elute DNA.
Place tube on the magnetic rack until beads separate fully from the solution, usually less than 0h 5m 0s
Slowly pipette 15µL 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.
Dilute 1µL of the concentrated library in 9µL 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.
End Protocol
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.
