TITLE: Effective Target Capture/Enrichment of Respiratory Viruses from Wastewater
AUTHORS: Lauren Roppolo Brazell, wtaylo, Lolo Aboufoul, Jannatul Ferdous, Aharr206, Jessica A Schlueter, Cynthia Gibas

[DESCRIPTION]
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]
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
1. SISPA-A: Reverse Transcription & 2nd Strand cDNA Synthesis
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
1.1. 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.
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
1.2. 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. 
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
1.3. While reaction is on the thermocycler, make a master mix for 1 reaction (scale up as needed) consisting of the following:   

 

SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
1.4. Add 5μL master mix to reaction. Incubate at 50°C for 10 mins.
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
1.5. While reaction is on the thermocycler, make a master mix for 1 reaction (scale up as needed) consisting of the following:

 

SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
1.6. After 10 minute incubation, add 5μL of Sequenase Mix #1 to the reaction.
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
1.7. Incubate at 37°C for 8 min.
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
1.8. While reaction is on the thermocycler, make a master mix for 1 reaction (scale up as needed) consisting of the following:

 

SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
1.9. After 8 minute incubation, add 0.6μL of Sequenase Mix #2 to the reaction.
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
1.10. Incubate at 37°C for 8 min.
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
1.11. After 8 min incubation, add 1μL RNAse H to each sample.
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
1.12. Incubate at 37°C for 20 min. SISPA A is now complete and samples can be stored at -20°C. 
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
2. SISPA B: PCR Amplification of Randomly Primed cDNA
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
2.1. Make a master mix for 1 reaction (scale up as needed) consisting of the following:

 

SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
2.2. Add 5μL of product from SISPA A to 45μL master mix.
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
2.3. Run a thermocycler set with the following parameters:

 


SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
3. Purification of SISPA Product
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
3.1. Add 50μL of AMPure XP beads to 50μL SISPA product for a 1:1 purification ratio. 
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
3.2. Incubate at RT for 10 minutes. 
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
3.3. Spin down briefly and place on magnetic rack. Remove and discard supernatant once solution turns clear. Be sure not to disturb the bead pellets. 
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
3.4. Wash beads with 200μL of freshly prepared 70% EtOH.
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
3.5. Remove EtOH without disturbing beads. 
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
3.6. Repeat wash for a total of 2 washes. Remove EtOH without disturbing beads. 
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
3.7. Remove samples from magnet and spin down briefly. 
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
3.8. Remove residual EtOH. 
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
3.9. Let air dry briefly. Do not overdry the bead pellet. 
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
3.10. Resuspend samples in 50μL of buffer EB.
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
3.11. Incubate samples at RT for 5 minutes. 
SECTION: Sequence Independent, Single-Primer Amplification [Moreno et al.; 1]
3.12. 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.
SECTION: Total Nucleic Acids Library Preparation [2]
4.3. Spin down to ensure all solution is at the bottom of the tube and place on ice. 
SECTION: Total Nucleic Acids Library Preparation
1. Purify
SECTION: Total Nucleic Acids Library Preparation
1.1. Vortex the pre-equilibrated, room-temperature DNA purification beads until well mixed.
SECTION: Total Nucleic Acids Library Preparation
1.2. Add 60μL of homogenized (0.8x) DNA purification beads to each ligation sample, and mix well by vortexing. 
SECTION: Total Nucleic Acids Library Preparation
1.3. Incubate the samples for 5 minutes at RT.
SECTION: Total Nucleic Acids Library Preparation
1.4. Place the samples on a magnetic rack for 1 minute or until the supernatant is clear. 
SECTION: Total Nucleic Acids Library Preparation
1.5. Without disturbing the bead pellet, remove and discard the supernatant.
SECTION: Total Nucleic Acids Library Preparation
1.6. 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. 
SECTION: Total Nucleic Acids Library Preparation
1.7. Repeat the wash once, for a total of two washes, while keeping the samples on the magnetic rack. 
SECTION: Total Nucleic Acids Library Preparation
1.8. Carefully remove all remaining EtOH with a 10μL pipette, making sure not to disturb the bead pellet.
SECTION: Total Nucleic Acids Library Preparation
1.9. 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. 
SECTION: Total Nucleic Acids Library Preparation
1.10. Remove the plate from the magnetic rack and add 17μL buffer EB to each sample. Mix by pipetting until homogenized. 
SECTION: Total Nucleic Acids Library Preparation
1.11. Incubate at RT for 2 minutes. 
SECTION: Total Nucleic Acids Library Preparation
1.12. Place the plate on a magnetic rack and let stand for 3 minutes, or until beads form a pellet. 
SECTION: Total Nucleic Acids Library Preparation
1.13. Transfer 15μL of the clear supernatant containing the ligated libraries to a clean 96-well plate, making sure not to disturb the bead pellet. 
SECTION: Total Nucleic Acids Library Preparation
1.14. Spin down the sample plate and immediately transfer to the thermocycler. Proceed to purification when the program has completed. 
2. 
SECTION: Total Nucleic Acids Library Preparation
7.4. Spin sample down and immediately transfer to thermocycler. Start the program. 
SECTION: Total Nucleic Acids Library Preparation
7.5. Remove the samples from the thermocycler block when complete, and proceed to purification. 
SECTION: Total Nucleic Acids Library Preparation
8. Purify
SECTION: Total Nucleic Acids Library Preparation
8.1. Vortex the pre-equilibrated DNA purification beads until mixed.
SECTION: Total Nucleic Acids Library Preparation
8.2. Add 50μL (1x) of homogenized DNA purification beads to each ligation sample post amplification. Mix well by vortexing. 
SECTION: Total Nucleic Acids Library Preparation
8.3. Incubate the samples at RT for 5 minutes. 
SECTION: Total Nucleic Acids Library Preparation
8.4. Place the samples on a magnetic rack for 1 minute.
SECTION: Total Nucleic Acids Library Preparation
8.5. The DNA purification beads form a pellet, leaving a clear supernatant. With plate still on the magnetic rack, remove and discard the supernatant. 
SECTION: Total Nucleic Acids Library Preparation
8.6. 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.
SECTION: Total Nucleic Acids Library Preparation
8.7. Repeat this wash once, for a total of two washes, while keeping the samples on the magnetic rack. 
SECTION: Total Nucleic Acids Library Preparation
8.8. Carefully remove all remaining EtOH with a 10μL pipette, making sure not to disturb the bead pellet.
SECTION: Total Nucleic Acids Library Preparation
8.9. Air dry the pellet on the magnetic rack for 5-10 minutes, or until it is dry. Do not overdry the pellet. 
SECTION: Total Nucleic Acids Library Preparation
8.10. Remove the samples from the magnetic plate and add 22μL of buffer EB to each sample. Mix by pipetting until homogenized. 
SECTION: Total Nucleic Acids Library Preparation
8.11. Incubate at RT for 2 minutes. 
SECTION: Total Nucleic Acids Library Preparation
8.12. Place the plate back on the magnetic rack and let stand for 3 minutes, or until the beads form a pellet. 
SECTION: Total Nucleic Acids Library Preparation
8.13. 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. 
SECTION: Total Nucleic Acids Library Preparation
9. Perform QC
SECTION: Total Nucleic Acids Library Preparation
9.1. 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. 
SECTION: Total Nucleic Acids Library Preparation
7.3. Add 25μL of Equinox Library Amp Mix (2x) to the cDNA libraries from the previous step and mix well by gentle pipetting. 
SECTION: Total Nucleic Acids Library Preparation
7.2. 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. 
SECTION: Total Nucleic Acids Library Preparation
7.1. 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.
SECTION: Total Nucleic Acids Library Preparation
7. PCR Amplify with Twist UDI Primers
6. 
SECTION: Total Nucleic Acids Library Preparation
5.4. 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). 
SECTION: Total Nucleic Acids Library Preparation
5.3. 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. 
SECTION: Total Nucleic Acids Library Preparation
5.2. Invert the Ligation Master Mix a minimum of 10 times until homogenized and place on ice. Do not vortex the Ligation Master Mix. 
SECTION: Total Nucleic Acids Library Preparation
5.1. 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. 
SECTION: Total Nucleic Acids Library Preparation
5. Ligate Twist Universal Adapters and Purify
SECTION: Total Nucleic Acids Library Preparation
4.10. When the thermocycler has completed the program and sample block has returned to 4°C, remove the samples and place them on ice.
SECTION: Total Nucleic Acids Library Preparation
4.9. Initiate the remaining steps of thermocycling program (refer to step 4.1). While the thermocycler is running, prepare the reagents for step 5 (below). 
SECTION: Total Nucleic Acids Library Preparation
4.8. Spin down and immediately transfer to pre-chilled thermocycler.
SECTION: Total Nucleic Acids Library Preparation
4.7. 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. 
SECTION: Total Nucleic Acids Library Preparation
4.6. Prepare an enzymatic fragmentation mix in a tube on ice consisting of the following:
 
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. 
SECTION: Total Nucleic Acids Library Preparation
4.5. 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. 
SECTION: Total Nucleic Acids Library Preparation
4.4. Vortex the Frag/AT Buffer for 5 seconds, and spin down to collect all liquid in the bottom of the tube.
SECTION: Total Nucleic Acids Library Preparation
4.3. Spin down briefly to ensure all of the solution is at the bottom of the tube, and then place on ice. 
SECTION: Total Nucleic Acids Library Preparation [2]
4.2. Add 25μL of each cDNA pool into a well of a 96-well plate.
SECTION: Total Nucleic Acids Library Preparation [2]
4.1. Program a thermocycler set with the following parameters:

 
 
Set the heated lid to 105°C, and start the program to pre-chill the thermocycler. 
SECTION: Total Nucleic Acids Library Preparation [2]
4. DNA Fragmentation, End Repair, and dA-Tailing


SECTION: Total Nucleic Acids Library Preparation [2]
4.4. Vortex the Frag/AT Buffer for 5 seconds. Spin down to collect all liquid in the bottom of the tube. 
SECTION: Total Nucleic Acids Library Preparation [2]
4.5. 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. 
SECTION: Total Nucleic Acids Library Preparation [2]
4.6. Prepare an enzymatic fragmentation mix consisting of the following (scale up as needed):

 

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. 
SECTION: Total Nucleic Acids Library Preparation [2]
4.7. 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. 
SECTION: Total Nucleic Acids Library Preparation [2]
4.8. Spin down the samples and immediately transfer to the pre-chilled thermocycler. 
SECTION: Total Nucleic Acids Library Preparation [2]
4.9. Initiate the remaining steps of the thermocycler program (from step 4.1). While thermocycler is running, prepare reagents for step 5. 
SECTION: Total Nucleic Acids Library Preparation [2]
4.10. 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. 
SECTION: Total Nucleic Acids Library Preparation [2]
5. Ligate Twist Universal Adapters
SECTION: Total Nucleic Acids Library Preparation [2]
5.1. 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. 
SECTION: Total Nucleic Acids Library Preparation [2]
5.2. Invert the Ligation Master Mix a minimum of 10 times until homogenized and place on ice. Do not vortex the Ligation Master Mix. 
SECTION: Total Nucleic Acids Library Preparation [2]
5.3. 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. 
SECTION: Total Nucleic Acids Library Preparation [2]
5.4. 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. 
SECTION: Total Nucleic Acids Library Preparation [2]
6. Purification of Product
SECTION: Total Nucleic Acids Library Preparation [2]
6.1. Vortex the pre-equilibrated, RT DNA purification beads until well mixed. 
SECTION: Total Nucleic Acids Library Preparation [2]
6.2. Add 60μL of homogenized (0.8x) DNA purification beads to each ligation sample from the previous step. Mix well by vortexing. 
SECTION: Total Nucleic Acids Library Preparation [2]
6.3. Incubate the samples for 5 minutes at RT. 
SECTION: Total Nucleic Acids Library Preparation [2]
6.4. Place the samples on a magnetic rack for 1 minute. The DNA beads form a pellet, leaving a clear supernatant. 
SECTION: Total Nucleic Acids Library Preparation [2]
6.5. Without removing the samples from the magnetic rack, remove and discard the supernatant. 
SECTION: Total Nucleic Acids Library Preparation [2]
6.6. 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. 
SECTION: Total Nucleic Acids Library Preparation [2]
6.7. Repeat this wash once, for a total of two washes, while keeping the samples on the magnetic rack. 
SECTION: Total Nucleic Acids Library Preparation [2]
6.8. Carefully remove all remaining EtOH with a 10μL pipette, making sure not to disturb the bead pellet. 
SECTION: Total Nucleic Acids Library Preparation [2]
6.9. Air dry the bead pellet on the magnetic rack for 5 minutes or until the bead pellet is dry. Do not overdry the pellet. 
SECTION: Total Nucleic Acids Library Preparation [2]
6.10. Remove the samples from the magnetic rack and add 17μL of buffer EB to each sample. Mix by pipetting until homogenized. 
SECTION: Total Nucleic Acids Library Preparation [2]
6.11. Incubate at RT for 2 minutes. 
SECTION: Total Nucleic Acids Library Preparation [2]
6.12. Place the samples back on a magnetic rack and let stand for 3 minutes or until beads form a pellet. 
SECTION: Total Nucleic Acids Library Preparation [2]
6.13. 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. 
SECTION: Total Nucleic Acids Library Preparation [2]
7. PCR Amplify Using Twist UDI Primers
SECTION: Total Nucleic Acids Library Preparation [2]
7.1. Program a thermocycler set with the following parameters:

 

Set the heated lid to 105°C.
SECTION: Total Nucleic Acids Library Preparation [2]
7.2. 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. 
SECTION: Total Nucleic Acids Library Preparation [2]
7.3. 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.
SECTION: Total Nucleic Acids Library Preparation [2]
7.4. Sin samples down and immediately transfer to thermocycler, and start the program. 
SECTION: Total Nucleic Acids Library Preparation [2]
7.5. Remove the samples from the thermocycler block when the program is complete, and proceed to purification. 
SECTION: Target Enrichment [3]
10. Aliquot and Dry Down Library
SECTION: Target Enrichment [3]
10.1. Use the concentration of each amplified, indexed library to calculate the volume (in μL) of each library needed for hybridization. 

 

SECTION: Target Enrichment [3]
10.2. Transfer the calculated volumes from each amplified, indexed library to an indexed library pool reaction tube for each hybridization reaction being performed. 
SECTION: Target Enrichment [3]
10.3. Spin down the library tubes to minimize the amount of bubbles present. 
SECTION: Target Enrichment [3]
10.4. 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. 
SECTION: Target Enrichment [3]
11. Hybridize Capture Probes with Pools
SECTION: Target Enrichment [3]
11.1. Heat the Hybridization Mx at 65°C in the heat block for 10 minutes, or until all precipitate is dissolved. 
SECTION: Target Enrichment [3]
11.2. Prepare a probe solution for each pool in a PCR tube consisting of the following (scale up as needed):

 
Mix by flicking tubes.

SECTION: Target Enrichment [3]
11.3. Resuspend the dried, indexed library pools by preparing the reagents as follows (scale up as needed):
 

SECTION: Target Enrichment [3]
11.4. 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. 
SECTION: Target Enrichment [3]
11.5. 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. 
SECTION: Target Enrichment [3]
11.6. Vortex and spin down the probe solution, then transfer the entire volume to the resuspended indexed library pool. Mix well by vortexing. 
SECTION: Target Enrichment [3]
11.7. Spin down again to ensure all solution is at the bottom of the tube(s).
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11.8. Incubate the hybridization reaction at 70°C for 16 hours in a thermocycler with the lid at 85°C.
SECTION: Target Enrichment [3]
12. Bind Hybridized Targets to Streptavidin Beads
SECTION: Target Enrichment [3]
12.1. Vortex the pre-equilibrated streptavidin binding beads until mixed. 
SECTION: Target Enrichment [3]
12.2. Add 100μL streptavidin binding beads to a 1.5mL micro centrifuge tube. Prepare one tube for each hybridization reaction. 
SECTION: Target Enrichment [3]
12.3. Add 200μL binding buffer to the tube(s) and mix by pipetting. 
SECTION: Target Enrichment [3]
12.4. 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.
SECTION: Target Enrichment [3]
12.5. After removing the clear supernatant from the third wash, add a final 200μL binding buffer and resuspend the beads by vortexing until homogenized. 
SECTION: Target Enrichment [3]
12.6. Heat the resuspended beads at 68°C for at least 10 minutes before continuing to the next step.
SECTION: Target Enrichment [3]
12.7. 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. 
SECTION: Target Enrichment [3]
12.8. Incubate the tube(s) of the hybridization reaction with the streptavidin binding beads for 5 minutes on a heated mixer set to 68°C. 
SECTION: Target Enrichment [3]
12.9. 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). 
SECTION: Target Enrichment [3]
12.10. Place the tube(s) on a magnetic rack for 1 minute.
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12.11. Remove and discard the clear supernatant including the hybridization enhancer. Do not disturb the bead pellet.
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12.12. Remove the tube(s) from the magnetic stand and add 200μL 68°C standard wash buffer 1, and mix by pipetting. 
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12.13. Incubate the tubes for 5 minutes at 68°C. 
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12.14. Spin down to ensure all solution is at the bottom of the tube(s). 
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12.15. 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. 
SECTION: Target Enrichment [3]
12.16. 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). 
SECTION: Target Enrichment [3]
12.17. Incubate the tube(s) for 5 minutes at 48°C. 
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12.18. Place the tube(s) on a magnetic rack for 1 minute. 
SECTION: Target Enrichment [3]
12.19. Remove and discard the clear supernatant. Make sure not to disturb the bead pellet. 
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12.20. Repeat the wash two more times, for a total of three washes. 
SECTION: Target Enrichment [3]
12.21. 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. 
SECTION: Target Enrichment [3]
12.22. 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. 
SECTION: Target Enrichment [3]
13. Post-Capture PCR Amplify
SECTION: Target Enrichment [3]
13.1. Program a thermocycler set with the following parameters:

 

Set the heated lid to 105°C.
SECTION: Target Enrichment [3]
13.2. If the streptavidin binding bead slurry has settled, mix by pipetting. 
SECTION: Target Enrichment [3]
13.3. Transfer 22.5μL of the streptavidin binding bead slurry to a PCR tube. Keep on ice until ready to use in the next step. 
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13.4. Prepare a PCR mixture for each streptavidin binding bead aliquot consisting of the following (scale up as needed):

 

Mix by pipetting. 
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13.5. Spin down the tubes, and transfer them to the thermocycler. Start the thermocycling program.
SECTION: Target Enrichment [3]
13.6. When the thermocycler program is complete, remove the tube(s) from the block and immediately proceed to the next step.
SECTION: Target Enrichment [3]
14. Purification
SECTION: Target Enrichment [3]
14.1. Vortex the pre-equilibrated DNA purification beads until well mixed. 
SECTION: Target Enrichment [3]
14.2. Add 90μL (1.8x) homogenized DNA purification beads to the tube(s) from the previous step. Mix well by vortexing. 
SECTION: Target Enrichment [3]
14.3. Incubate at RT for 5 minutes. 
SECTION: Target Enrichment [3]
14.4. Place the tubes on a magnetic rack for 1 minute or until the supernatant is clear. 
SECTION: Target Enrichment [3]
14.5. 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. 
SECTION: Target Enrichment [3]
14.6. 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.
SECTION: Target Enrichment [3]
14.7. Repeat this wash, for a total of two washes while keeping the tube on the magnetic rack. 
SECTION: Target Enrichment [3]
14.8. Carefully remove all remaining EtOH using a 10μL pipette, making sure not to disturb the bead pellet. 
SECTION: Target Enrichment [3]
14.9. 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. 
SECTION: Target Enrichment [3]
14.10. Remove the tubes from the magnetic rack and add 32μL buffer EB to each capture reaction. Mix by pipetting until homogenized. 
SECTION: Target Enrichment [3]
14.11. Incubate at RT for 2 minutes. 
SECTION: Target Enrichment [3]
14.12. Place tubes on a magnetic rack and let stand for 3 minutes or until beads fully pellet. 
SECTION: Target Enrichment [3]
14.13. 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. 
SECTION: Target Enrichment [3]
15. Perform QC
SECTION: Target Enrichment [3]
15.1. 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.
SECTION: Sequence Prepared Libraries [4]
16. Sequencing on an Illumina NextSeq 2000 Platform
SECTION: Total Nucleic Acids Library Preparation [2]
8. Purification
SECTION: Total Nucleic Acids Library Preparation [2]
9. Perform QC
SECTION: Total Nucleic Acids Library Preparation [2]
8.1. Vortex the pre-equilibrated DNA purification beads until mixed.
SECTION: Total Nucleic Acids Library Preparation [2]
8.2. Add 50μL (1x) homogenized DNA purification beads to each ligation sample from the above step. Mix well by vortexing. 
SECTION: Total Nucleic Acids Library Preparation [2]
8.3. Incubate the samples for 5 minutes at RT.
SECTION: Total Nucleic Acids Library Preparation [2]
8.4. Place the samples on a magnetic rack for 1 minute. 
SECTION: Total Nucleic Acids Library Preparation [2]
8.5. The DNA purification beads form a pellet, leaving a clear supernatant. With plate still on the magnetic rack, remove and discard the supernatant. 
SECTION: Total Nucleic Acids Library Preparation [2]
8.6. 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.
SECTION: Total Nucleic Acids Library Preparation [2]
8.7. Repeat this wash for a total of two washes, while keeping the samples on the magnetic rack. 
SECTION: Total Nucleic Acids Library Preparation [2]
8.8. Carefully remove all remaining EtOH with a 10μL pipette, making sure not to disturb the bead pellet. 
SECTION: Total Nucleic Acids Library Preparation [2]
8.9. Air dry the pellet on the magnetic rack for 5-10 minutes, or until it is dry. Do not overdry the pellet. 
SECTION: Total Nucleic Acids Library Preparation [2]
8.10. Remove the samples from the magnetic rack and add 22μL of buffer EB to each sample. Mix by pipetting until homogenized. 
SECTION: Total Nucleic Acids Library Preparation [2]
8.11. Incubate at RT for 2 minutes. 
SECTION: Total Nucleic Acids Library Preparation [2]
8.12. Place the plate or tubes on a magnetic rack and let stand for 3 minutes or until the beads form a pellet. 
SECTION: Total Nucleic Acids Library Preparation [2]
8.13. Transfer 20μL of the clear supernatant containing the amplified indexed libraries to a clean PCR tube, making sure not to disturb the pellet. 
SECTION: Total Nucleic Acids Library Preparation [2]
9.1. Quantify and validate the size range of each library using the Thermo Fisher Scientific Qubit dsDNA Broad Range Quantitation Assay. 
SECTION: Sequence Prepared Libraries [4]
16.1. 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.