Effective Target Capture/Enrichment of Respiratory Viruses from Wastewater
Jannatul Ferdous, Cynthia Gibas, Jessica A Schlueter, wtaylo, April N Harris, Lauren Roppolo Brazell, Lolo Aboufoul
Abstract
Human respiratory viruses (HRVs) are highly communicable viral pathogens that present varying degrees of illness in infected individuals. The advent of the COVID-19 pandemic demonstrated the utility of wastewater-based epidemiology (WBE) methods that bypassed traditional clinical testing for the virus. This method yielded powerful results, informing communities of potential outbreaks and allowing them to make informed decisions for the health and well-being of their community. The success of WBE can be employed to screen wastewater for other HRVs. Herein we establish a protocol that prepares viral RNA from extracted wastewater for use in a HRV target capture/enrichment sequencing assay. This protocol addresses the unique challenge of low viral load input for library preparation by randomly priming and non-specifically amplifying viral RNA. By then enriching only the sequences of interest in subsequent target capture/hybridization reactions, this method conserves sequencing resources and results in high-quality sequence information. Following downstream bioinformatics analyses, this sequence information can be used to help communities take appropriate action in mitigating the negative effects of HRVs.
Steps
Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
SISPA-A: Reverse Transcription & 2nd Strand cDNA Synthesis
Total Nucleic Acids Library Preparation
Purify
Make a working stock of your SOL Primer A. Stock should be 100 pmol/1μL. Add 4μL of stock + 6μL molecular biology-grade H₂O; you now have 10μL of a 40pmol/μL working stock.
Vortex the pre-equilibrated, room-temperature DNA purification beads until well mixed.
Incubate at 37°C for 8 min.
Remove the plate from the magnetic rack and add 17μL buffer EB to each sample. Mix by pipetting until homogenized.
After 8 min incubation, add 1μL RNAse H to each sample.
Incubate at RT for 2 minutes.
Incubate at 37°C for 20 min. SISPA A is now complete and samples can be stored at -20°C.
Place the plate on a magnetic rack and let stand for 3 minutes, or until beads form a pellet.
Transfer 15μL of the clear supernatant containing the ligated libraries to a clean 96-well plate, making sure not to disturb the bead pellet.
Spin down the sample plate and immediately transfer to the thermocycler. Proceed to purification when the program has completed.
Add 1μL SOL Primer A working stock to 4μL extracted RNA. Heat to 65°C for 5 mins in a thermocycler and let cool at 4°C for 5 mins.
Add 60μL of homogenized (0.8x) DNA purification beads to each ligation sample, and mix well by vortexing.
While reaction is on the thermocycler, make a master mix for 1 reaction (scale up as needed) consisting of the following:
| A | B |
|---|---|
| 5x RT Buffer | 2μL |
| 10mM dNTP | 1μL |
| Molecular Biology-Grade H₂O | 1μL |
| 0.1M DTT | 0.5μL |
| SSIV RT | 0.5μL |
| TOTAL | 5μL |
Incubate the samples for 5 minutes at RT.
Add 5μL master mix to reaction. Incubate at 50°C for 10 mins.
Place the samples on a magnetic rack for 1 minute or until the supernatant is clear.
While reaction is on the thermocycler, make a master mix for 1 reaction (scale up as needed) consisting of the following:
| A | B |
|---|---|
| 5x Sequenase | 1μL |
| Molecular Biology-Grade H₂O | 3.8μL |
| Sequenase | 0.15μL |
| TOTAL | 4.95μL |
Without disturbing the bead pellet, remove and discard the supernatant.
After 10 minute incubation, add 5μL of Sequenase Mix #1 to the reaction.
Wash the bead pellet by gently adding 200μL of freshly prepared 80% EtOH, being careful not to disturb the pellet. Incubate for 1 minute, then remove and discard the EtOH.
Incubate at 37°C for 8 min.
Repeat the wash once, for a total of two washes, while keeping the samples on the magnetic rack.
While reaction is on the thermocycler, make a master mix for 1 reaction (scale up as needed) consisting of the following:
| A | B |
|---|---|
| Sequenase dilution buffer | 0.45μL |
| Sequenase | 0.15μL |
| TOTAL | 0.6μL |
Carefully remove all remaining EtOH with a 10μL pipette, making sure not to disturb the bead pellet.
After 8 minute incubation, add 0.6μL of Sequenase Mix #2 to the reaction.
Air dry the bead pellet on the magnetic rack for 5 minutes, or until the bead pellet is dry. Do not overdry the bead pellet.
SISPA B: PCR Amplification of Randomly Primed cDNA
Make a master mix for 1 reaction (scale up as needed) consisting of the following:
| A | B |
|---|---|
| 2X LongAmp Taq Master Mix | 44μL |
| SOL Primer B | 1μL |
| TOTAL | 45μL |
Add 5μL of product from SISPA A to 45μL master mix.
Run a thermocycler set with the following parameters:
| A | B | C | D |
|---|---|---|---|
| Initialization | 98°C | 30 seconds | 1 |
| Denaturation | 94°C | 15 seconds | 30 |
| Annealing | 50°C | 20 seconds | |
| Extension | 68°C | 2 minutes | |
| Final Extension | 68°C | 10 minutes | 1 |
| Hold | 4°C | ∞ | -- |
Purification of SISPA Product
Add 50μL of AMPure XP beads to 50μL SISPA product for a 1:1 purification ratio.
Resuspend samples in 50μL of buffer EB.
Incubate samples at RT for 5 minutes.
Transfer samples to magnetic rack. Transfer 48μL of eluted product to a new tube. Proceed to library prep protocol of your choice, or the samples may be stored at -20°C.
Incubate at RT for 10 minutes.
Spin down briefly and place on magnetic rack. Remove and discard supernatant once solution turns clear. Be sure not to disturb the bead pellets.
Wash beads with 200μL of freshly prepared 70% EtOH.
Remove EtOH without disturbing beads.
Repeat wash for a total of 2 washes. Remove EtOH without disturbing beads.
Remove samples from magnet and spin down briefly.
Remove residual EtOH.
Let air dry briefly. Do not overdry the bead pellet.
Total Nucleic Acids Library Preparation [2]
DNA Fragmentation, End Repair, and dA-Tailing
Program a thermocycler set with the following parameters:
| A | B | C | D |
|---|---|---|---|
| Hold | 4°C | -- | 1 |
| Incubation 1 | 37°C | 30 minutes | 1 |
| Incubation 2 | 65°C | 30 minutes | 1 |
| Hold | 4°C | ∞ | -- |
Set the heated lid to 105°C, and start the program to pre-chill the thermocycler.
When the thermocycler has completed the program and sample block has returned to 4°C, remove the samples and place them on ice.
When the thermocycler has completed and the sample block has returned to 4°C, remove the samples and place them on ice. Proceed to ligate adapters and purification.
Add 25μL of each cDNA pool into a well of a 96-well plate.
Spin down to ensure all solution is at the bottom of the tube and place on ice.
Spin down briefly to ensure all of the solution is at the bottom of the tube, and then place on ice.
Vortex the Frag/AT Buffer for 5 seconds, and spin down to collect all liquid in the bottom of the tube.
Vortex the Frag/AT Buffer for 5 seconds. Spin down to collect all liquid in the bottom of the tube.
Invert Frag/AT Enzymes a minimum of 10 times to homogenize, or briefly vortex to ensure complete mixing. Spin down to collect all liquid in the bottom of the tube.
Invert the Frag/AT Enzymes a minimum of 10 times to homogenize or briefly vortex to ensure complete mixing. Spin down to collect all liquid in the bottom of the tube.
Prepare an enzymatic fragmentation mix in a tube on ice consisting of the following:
| A | B |
|---|---|
| Molecular Biology Grade H₂O | 15μL |
| Frag/AT Buffer | 4μL |
| Frag/AT Enzymes | 6μL |
| TOTAL | 25μL |
Homogenize the master mix with moderate vortexing for 5s or pipetting a minimum of half the total volume up and down 10 times, avoiding the formation of bubbles.
Prepare an enzymatic fragmentation mix consisting of the following (scale up as needed):
| A | B |
|---|---|
| Molecular Biology-Grade H₂O | 15μL |
| Frag/AT Buffer | 4μL |
| Frag/AT Enzymes | 6μL |
| TOTAL | 25μL |
Homogenize the master mix with moderate vortexing for 5 seconds or pipetting a minimum of half the total volume up and down 10 times, avoiding the formation of bubbles.
Add 25μL enzymatic fragmentation mix to each 25μL sample well. Homogenize with moderate vortexing for 5s or by pipetting a minimum of half the total volume up and down 10 times, avoiding the formation of bubbles. Seal the plate and keep the reaction on ice.
Add 25μL enzymatic fragmentation mix to each 25μL sample. Homogenize with moderate vortexing for 5 seconds or by pipetting a minimum of half the total volume up and down 10 times, avoiding the formation of bubbles. Seal the samples and keep the reactions on ice.
Spin down and immediately transfer to pre-chilled thermocycler.
Spin down the samples and immediately transfer to the pre-chilled thermocycler.
Initiate the remaining steps of thermocycling program (refer to step 4.1). While the thermocycler is running, prepare the reagents for step 5 (below).
Initiate the remaining steps of the thermocycler program (from step 4.1). While thermocycler is running, prepare reagents for step 5.
Ligate Twist Universal Adapters and Purify
Ligate Twist Universal Adapters
Add 2.5μL of Twist Universal Adapters into each sample well containing the dA-tailed DNA fragments from the previous step. mix gently by pipetting, and keep on ice.
Add 2.5μL of Twist Universal Adapters into each sample containing the dA-tailed DNA fragments from the above step. Mix gently by pipetting and keep on ice.
Invert the Ligation Master Mix a minimum of 10 times until homogenized and place on ice. Do not vortex the Ligation Master Mix.
Invert the Ligation Master Mix a minimum of 10 times until homogenized and place on ice. Do not vortex the Ligation Master Mix.
Add 2.5μL of molecular biology-grade H₂O and 20μL of Ligation Master Mix to each sample from step 5.1. Pipette a minimum of half the total volume up and down 10 times to ensure complete mixing. Seal the sample plate and spin down to ensure all solution is at the bottom of the tubes.
Add 2.5μL molecular biology-grade H₂O and 20μL Ligation Master Mix to each sample. Pipette a minimum of half the total volume up and down 10 times to ensure complete mixing. Seal the samples and spin down to ensure all solution is at the bottom of the tube.
Incubate the ligation reaction at 20°C for 15 minutes in a thermocycler with the heated lid turned off, then move the samples to the bench top. While the thermocycler is running, prepare the reagents for step 7 (below).
Incubate the ligation reaction at 20°C for 15 minutes in a thermocycler with the heated lid off, then move the samples to the bench top and proceed to purify.
Purification of Product
Vortex the pre-equilibrated, RT DNA purification beads until well mixed.
Remove the samples from the magnetic rack and add 17μL of buffer EB to each sample. Mix by pipetting until homogenized.
Incubate at RT for 2 minutes.
Place the samples back on a magnetic rack and let stand for 3 minutes or until beads form a pellet.
Transfer 15μL of the clear supernatant containing the ligated libraries to a clean 96-well plate, making sure not to disturb the bead pellet. Proceed to index and amplify library.
Add 60μL of homogenized (0.8x) DNA purification beads to each ligation sample from the previous step. Mix well by vortexing.
Incubate the samples for 5 minutes at RT.
Place the samples on a magnetic rack for 1 minute. The DNA beads form a pellet, leaving a clear supernatant.
Without removing the samples from the magnetic rack, remove and discard the supernatant.
Wash the bead pellet by gently adding 200μL of freshly prepared 80% EtOH, being careful not to disturb the pellet. Incubate for 1 minute, then remove and discard the EtOH.
Repeat this wash once, for a total of two washes, while keeping the samples on the magnetic rack.
Carefully remove all remaining EtOH with a 10μL pipette, making sure not to disturb the bead pellet.
Air dry the bead pellet on the magnetic rack for 5 minutes or until the bead pellet is dry. Do not overdry the pellet.
PCR Amplify with Twist UDI Primers
PCR Amplify Using Twist UDI Primers
Program a thermocycler with the following parameters:
98°C initialization for 45s, followed by 12 cycles of 98°C for 15, 60°C for 30s, and 72°C for 30s, 72°C final extension for 1 minute, and a 4°C hold.
Set the heated lid to 105°C.
Program a thermocycler set with the following parameters:
| A | B | C | D |
|---|---|---|---|
| Initialization | 98°C | 45 seconds | 1 |
| Denaturation | 98°C | 15 seconds | 12 |
| Annealing | 60°C | 30 seconds | |
| Extension | 72°C | 30 seconds | |
| Final Extension | 72°C | 1 minute | 1 |
| Hold | 4°C | ∞ | -- |
Set the heated lid to 105°C.
Add 10μL of Twist UDI Primers from the provided 96-well plate to each of the cDNA libraries from the previous step, and mix well by gentle pipetting.
Add 10μL of Twist UDI Primers from the provided 96-well plate to each of the cDNA libraries from the previous step. Mix well by gentle pipetting.
Add 25μL of Equinox Library Amp Mix (2x) to the cDNA libraries from the previous step and mix well by gentle pipetting.
Invert, do not vortex, Equinox Library Amp Mix (2x) 5 times prior to use. Add 25μL Equinox Library Amp Mix (2x) to the cDNA libraries and mix well by gentle pipetting.
Spin sample down and immediately transfer to thermocycler. Start the program.
Sin samples down and immediately transfer to thermocycler, and start the program.
Remove the samples from the thermocycler block when complete, and proceed to purification.
Remove the samples from the thermocycler block when the program is complete, and proceed to purification.
Purify
Purification
Vortex the pre-equilibrated DNA purification beads until mixed.
Vortex the pre-equilibrated DNA purification beads until mixed.
Remove the samples from the magnetic plate and add 22μL of buffer EB to each sample. Mix by pipetting until homogenized.
Remove the samples from the magnetic rack and add 22μL of buffer EB to each sample. Mix by pipetting until homogenized.
Incubate at RT for 2 minutes.
Incubate at RT for 2 minutes.
Place the plate back on the magnetic rack and let stand for 3 minutes, or until the beads form a pellet.
Place the plate or tubes on a magnetic rack and let stand for 3 minutes or until the beads form a pellet.
Transfer 20μL of the clear supernatant containing the amplified indexed cDNA libraries to a clean 96-well plate, making sure not to disturb the pellet.
Transfer 20μL of the clear supernatant containing the amplified indexed libraries to a clean PCR tube, making sure not to disturb the pellet.
Add 50μL (1x) of homogenized DNA purification beads to each ligation sample post amplification. Mix well by vortexing.
Add 50μL (1x) homogenized DNA purification beads to each ligation sample from the above step. Mix well by vortexing.
Incubate the samples at RT for 5 minutes.
Incubate the samples for 5 minutes at RT.
Place the samples on a magnetic rack for 1 minute.
Place the samples on a magnetic rack for 1 minute.
The DNA purification beads form a pellet, leaving a clear supernatant. With plate still on the magnetic rack, remove and discard the supernatant.
The DNA purification beads form a pellet, leaving a clear supernatant. With plate still on the magnetic rack, remove and discard the supernatant.
Gently wash the bead pellet by adding 200μL of freshly prepared 80% EtOH, careful not to disturb the bead pellet. Incubate for 1 minute, then remove and discard the EtOH.
Gently wash the bead pellet by adding 200μL of freshly prepared 80% EtOH, careful not to disturb the bead pellet. Incubate for 1 minute, then remove and discard the EtOH.
Repeat this wash once, for a total of two washes, while keeping the samples on the magnetic rack.
Repeat this wash for a total of two washes, while keeping the samples on the magnetic rack.
Carefully remove all remaining EtOH with a 10μL pipette, making sure not to disturb the bead pellet.
Carefully remove all remaining EtOH with a 10μL pipette, making sure not to disturb the bead pellet.
Air dry the pellet on the magnetic rack for 5-10 minutes, or until it is dry. Do not overdry the pellet.
Air dry the pellet on the magnetic rack for 5-10 minutes, or until it is dry. Do not overdry the pellet.
Perform QC
Perform QC
At this point, it is best to quantify and validate the size of fragments in the library. A Qubit dsDNA Broad Range Quantitation Assay or Agilent DNA 7500 is a valid method for QC.
Quantify and validate the size range of each library using the Thermo Fisher Scientific Qubit dsDNA Broad Range Quantitation Assay.
Target Enrichment [3]
Aliquot and Dry Down Library
Use the concentration of each amplified, indexed library to calculate the volume (in μL) of each library needed for hybridization.
| A | B | C |
|---|---|---|
| 1 | 500 ng | 500 ng |
| 2 | 500 ng | 1,000 ng |
| 3 | 500 ng | 1,500 ng |
| 4 | 375 ng | 1,500 ng |
| 8 | 187.5 ng | 1,500 ng |
Transfer the calculated volumes from each amplified, indexed library to an indexed library pool reaction tube for each hybridization reaction being performed.
Spin down the library tubes to minimize the amount of bubbles present.
Dry the indexed library pools using a vacuum concentrator on a low or no heat setting. If not immediately proceeding to next step, store the dried indexed library pools at -20°C for 24 hours.
Hybridize Capture Probes with Pools
Heat the Hybridization Mx at 65°C in the heat block for 10 minutes, or until all precipitate is dissolved.
Prepare a probe solution for each pool in a PCR tube consisting of the following (scale up as needed):
| A | B |
|---|---|
| Hybridization Mix | 20μL |
| Twist Respiratory Panel | 4μL |
| Molecular Biology-Grade H₂O | 4μL |
| TOTAL | 28μL |
Mix by flicking tubes.
Resuspend the dried, indexed library pools by preparing the reagents as follows (scale up as needed):
| A | B |
|---|---|
| Dried, Indexed Library Pool | --- |
| Blocker Solution | 5μL |
| Universal Blockers | 7μL |
| TOTAL | 12μL |
Heat the probe solution to 95°C for 2 minutes in a thermocycler with the lid at 105°C, then immediately cool on ice for 5 minutes.
While probe solution is cooling on ice, heat the tube containing the resuspended, indexed library pool at 95°C for 5 minutes in a thermocycler with the lid at 105°C, then equilibrate both the probe solution and resuspended, indexed library pool to RT on the bench top for 5 minutes.
Vortex and spin down the probe solution, then transfer the entire volume to the resuspended indexed library pool. Mix well by vortexing.
Spin down again to ensure all solution is at the bottom of the tube(s).
Incubate the hybridization reaction at 70°C for 16 hours in a thermocycler with the lid at 85°C.
Bind Hybridized Targets to Streptavidin Beads
Vortex the pre-equilibrated streptavidin binding beads until mixed.
Place the tube(s) on a magnetic rack for 1 minute.
Remove and discard the clear supernatant including the hybridization enhancer. Do not disturb the bead pellet.
Remove the tube(s) from the magnetic stand and add 200μL 68°C standard wash buffer 1, and mix by pipetting.
Incubate the tubes for 5 minutes at 68°C.
Spin down to ensure all solution is at the bottom of the tube(s).
Transfer the entire volume from the above step (about 200μL) into a new 1.5 mL micro centrifuge tube, one per hybridization reaction. Place the tube(s) on a magnetic rack for 1 minute.
Remove and discard the clear supernatant. Make sure not to disturb the bead pellet. Remove the tube(s) from the magnetic rack and add 200μL of 48°C wash buffer 2. Mix by pipetting, then pulse-spin to ensure all solution is at the bottom of the tube(s).
Incubate the tube(s) for 5 minutes at 48°C.
Place the tube(s) on a magnetic rack for 1 minute.
Remove and discard the clear supernatant. Make sure not to disturb the bead pellet.
Add 100μL streptavidin binding beads to a 1.5mL micro centrifuge tube. Prepare one tube for each hybridization reaction.
Repeat the wash two more times, for a total of three washes.
After the final wash, use a 10μL pipette to remove all traces of supernatant. Proceed immediately to the next step, and do not allow the beads to dry.
Remove the tube(s) from the magnetic rack and add 45μL molecular biology-grade H₂O. Mix by pipetting until homogenized, then incubate this solution, hereafter referred to as the streptavidin binding bead slurry, on ice.
Add 200μL binding buffer to the tube(s) and mix by pipetting.
Place the tube(s) on a magnetic rack for 1 minute, then remove and discard the clear supernatant. Make sure not to disturb the bead pellet. Remove the tube from the magnetic rack. Repeat the wash two more times for a total of three washes.
After removing the clear supernatant from the third wash, add a final 200μL binding buffer and resuspend the beads by vortexing until homogenized.
Heat the resuspended beads at 68°C for at least 10 minutes before continuing to the next step.
Once the hybridization is complete, open the thermocycler lid and directly transfer the volume of each hybridization reaction into a corresponding tube of preheated streptavidin binding beads from the above step. Mix by pipetting and flicking.
Incubate the tube(s) of the hybridization reaction with the streptavidin binding beads for 5 minutes on a heated mixer set to 68°C.
Remove the tube(s) containing the hybridization reaction with the streptavidin binding beads from the mixer and pulse-spin to ensure all solution is at the bottom of the tube(s).
Post-Capture PCR Amplify
Program a thermocycler set with the following parameters:
| A | B | C | D |
|---|---|---|---|
| Initialization | 98°C | 45 seconds | 1 |
| Denaturation | 98°C | 15 seconds | 15 |
| Annealing | 60°C | 30 seconds | |
| Extension | 72°C | 30 seconds | |
| Final Extension | 72°C | 1 minute | 1 |
| Hold | 4°C | ∞ | -- |
Set the heated lid to 105°C.
If the streptavidin binding bead slurry has settled, mix by pipetting.
Transfer 22.5μL of the streptavidin binding bead slurry to a PCR tube. Keep on ice until ready to use in the next step.
Prepare a PCR mixture for each streptavidin binding bead aliquot consisting of the following (scale up as needed):
| A | B |
|---|---|
| Streptavidin Binding Bead Slurry | 22.5μL |
| Amplification Primers, ILMN | 2.5μL |
| Equinox Library Amp Mix (2x) | 25μL |
| TOTAL | 50μL |
Mix by pipetting.
Spin down the tubes, and transfer them to the thermocycler. Start the thermocycling program.
When the thermocycler program is complete, remove the tube(s) from the block and immediately proceed to the next step.
Purification
Vortex the pre-equilibrated DNA purification beads until well mixed.
Remove the tubes from the magnetic rack and add 32μL buffer EB to each capture reaction. Mix by pipetting until homogenized.
Incubate at RT for 2 minutes.
Place tubes on a magnetic rack and let stand for 3 minutes or until beads fully pellet.
Transfer 30μL of the clear supernatant containing the enriched library to a clean PCR tube, making sure not to disturb the bead pellet. If not proceeding to sequence immediately, the enriched library may be stored at -20°C for up to 24 hours.
Add 90μL (1.8x) homogenized DNA purification beads to the tube(s) from the previous step. Mix well by vortexing.
Incubate at RT for 5 minutes.
Place the tubes on a magnetic rack for 1 minute or until the supernatant is clear.
The DNA purification beads form a pellet, leaving a clear supernatant. Without removing the plate or tubes from the magnetic rack, remove and discard the clear supernatant.
Wash the bead gently by adding 200μL of freshly prepared 80% EtOH, careful not to disturb the pellet. Incubate for 1 minute, then remove and discard the EtOH.
Repeat this wash, for a total of two washes while keeping the tube on the magnetic rack.
Carefully remove all remaining EtOH using a 10μL pipette, making sure not to disturb the bead pellet.
Air Dry the bead pellet on the magnetic rack for 5 minutes, or until the bead pellet is dry. Do not overdry the bead pellet.
Perform QC
Quantify and validate the concentration and/or fragment size range of each library using the Thermo Fisher Scientific Qubit dsDNA Broad Range Quantitation Assay or an Agilent DNA 7500 assay.
Sequence Prepared Libraries [4]
Sequencing on an Illumina NextSeq 2000 Platform
Subsequent sequencing preparation steps, including thawing of flow cell/reagent cartridge, diluting libraries to starting concentration, and setting up/loading a sequencing run into the NextSeq 2000 instrument can be found in Illumina's NextSeq 1000/2000 Product Documentation in the Sequencing Protocol section.
