Mar 17, 2026
KAPA HyperCap ViroCap3
This protocol is a draft, published without a DOI.
- Kristine Wylie1,
- Jane Schrimpf1,
- Madison Eschbach1
- 1Washington University
- HVP Pregnancy Postpartum
Protocol Citation: Kristine Wylie, Jane Schrimpf, Madison Eschbach 2026. KAPA HyperCap ViroCap3. protocols.io https://protocols.io/view/kapa-hypercap-virocap3-jwakcpacx
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 16, 2026
Last Modified: March 17, 2026
Protocol Integer ID: 313388
Keywords: roche kapa hypercapture reagent kit, hypercap virocap3 protocol
Funders Acknowledgements:
NIH NCCIH
Grant ID: U01 AT012970
Abstract
Protocol was adapted from the Roche KAPA HyperCapture Reagent Kit.
Troubleshooting
Before start
- Mixing beads and sample can be accomplished by vortexing and then spinning the tube briefly, or by pipetting up and down multiple times.
- All liquid waste should be placed in an appropriately labeled waste container to be disposed of by EHS. The lid of the thermocycler should be closed during use.
Hybridize the Sample Libraries to KAPA HyperCap Target Enrichment Probes
Make up 80% EtOH in molecular-grade water.
Make sure the KAPA HyperPure Beads are moved from 4°C to allow time for proper equilibration to room temperature.
Pool 10 μL of each library (maximum of 48 libraries/pool) in a 1.7 mL tube.
Reserve 10 μL of the un-captured pool for qPCR and store at -20°C.
Add 20 μL of COT Human DNA to each pool of libraries to be captured.
Vortex and spin down briefly.
Poke a hole in the lid with an 18 G needle and dry the sample in speed-vac at 65°C until <65 μL remains. Place a piece of tape over the hole in the lid.
Add PCR-grade water to a final volume of 65 μL.
Add 130 μL KAPA HyperPure Beads to each tube.
Vortex and spin down briefly or mix by pipetting.
Incubate at RT for 10 min. to allow DNA to bind to beads.
Place the tube on the magnet and incubate until the liquid is clear.
Carefully remove and discard the supernatant.
Keeping the tube on the magnet, add 300 μL 80% EtOH.
Incubate at RT for > 30 sec.
Carefully remove and discard the EtOH without disturbing the beads.
Quick spin the tube, place back on the magnet and remove any remaining EtOH.
Allow the beads to dry for 3-5 min, until all the EtOH has evaporated.
Note: Do not over-dry beads which may result in a dramatic yield loss.
Add 13.4 μL of the Universal Enhancing Oligos to the bead-bound DNA sample.
Remove from the magnet and mix thoroughly by pipetting or vortexing.
Prepare Hybridization Master Mix:
Add 43 μL of the Hybridization Master Mix to the tube from step 20.
Mix thoroughly and incubate at RT for 2 min.
Place the tube on the magnet and incubate until the liquid is clear.
Transfer 56.4 μL of the eluate (entire volume) to a new tube containing 4 μL of the ViroCap V3 probes.
Mix thoroughly and place in thermocycler using the following program:
Wash and Recover Captured DNA Sample
Thaw Hybridization Wash Buffers.
Dilute 10X Wash Buffers and 2.5X Bead Wash Buffer to create 1X working solutions in 1.7 mL tubes (volumes given are for one capture):
To pre-warm 1X Stringent Wash Buffer, make two aliquots of 200 μL in 1.7 mL tubes and place the tubes in a heat block set to 55°C.
To pre-warm 1X Wash Buffer I, place the tube containing 100 μL in a heat block set to 55°C.
Pre-warm buffers for a minimum of 15 min.
Allow the Capture Beads to equilibrate to RT prior to use.
Vortex Capture Beads for 15 sec. before immediate use to insure a homogeneous mixture.
Aliquot 100 μL of Capture Beads per capture reaction into a 0.2 mL or 1.7 mL tube. Beads for multiple capture reactions can be prepared in the same tube.
Place the tube on the magnet and incubate until the liquid is clear.
Remove and discard supernatant without disturbing the beads.
Keeping the tube on the magnet, add 1X the initial beads’ volume of 1X Bead Wash Buffer.
Remove tube from the magnet and mix thoroughly.
Place the tube on the magnet and incubate until the liquid is clear.
Remove and discard supernatant without disturbing the beads.
Wash a second time by repeating steps 37-40.
Add 1X the initial beads’ volume of 1X Bead Wash Buffer (ex. 100 μL/capture).
Remove tube from the magnet and mix thoroughly.
If preparing beads for multiple captures in a single tube, aliquot 100 μL of resuspended beads into a new 0.2 mL or 1.7 mL tube for each capture.
Place the tube on the magnet and incubate until the liquid is clear.
Remove and discard supernatant without disturbing the beads.
Note: Small amounts of residual 1X Bead Wash Buffer will not interfere with binding of the DNA to the Capture Beads.
Transfer each hybridization sample to a single tube with prepared Capture Beads.
Mix beads and sample thoroughly.
Incubate the capture reaction by placing the tube in a thermocycler set to 55°C for 15 min.
Add 100 μL pre-warmed 1X Wash Buffer I to the beads + DNA and mix thoroughly.
Place the tube on the magnet and incubate until the liquid is clear.
Remove and discard supernatant without disturbing the beads.
Add 200 μL pre-warmed 1X Stringent Wash Buffer to the beads + DNA, remove from magnet, and mix thoroughly.
Place the tubes in the thermocycler pre-heated to 55°C and incubate for 5 min.
Place the tube on the magnet and incubate until the liquid is clear.
Remove and discard supernatant without disturbing the beads.
Wash a second time by repeating steps 53-56.
Add 200 μL 1X Wash Buffer I at RT, remove from magnet, and mix thoroughly.
Incubate at RT for 1 min.
Place the tube on the magnet and incubate until the liquid is clear.
Remove and discard supernatant without disturbing the beads.
Add 200 μL 1X Wash Buffer II at RT, remove from magnet, and mix thoroughly.
Transfer contents to a new tube.
Note: This step is essential.
Incubate at RT for 1 min.
Place the tube on the magnet and incubate until the liquid is clear.
Remove and discard supernatant without disturbing the beads.
Add 200 μL 1X Wash Buffer III at RT, remove from magnet, and mix thoroughly.
Incubate at RT for 1 min.
Place the tube on the magnet and incubate until the liquid is clear.
Remove and discard supernatant without disturbing the beads.
Incubate at RT for 10 min.
Meanwhile, add 20 μL 1M Tris-HCl, pH 7.5 to a new 1.7 mL tube.
Place the tube on the magnet and incubate until the liquid is clear.
Transfer the bead eluate to the tube containing Tris-HCl and mix by pipetting.
Clean up DNA with Ampure beads using 60 μL (1.5X) volume.
Wash 2X with 1000 μL freshly-made 70% ethanol.
After second wash, quick spin, put back on magnet, and remove as much ethanol as possible.
Dry at RT for 10-15 min. Beads should look cracked and not shiny.
Resuspend beads in 40 μL EB by pipetting. Incubate 2 min. at RT and place tube back on magnet.
After 5 min. on magnet, transfer supernatant to new 1.7 mL tube. The captured DNA can be stored at -20°C at this point or amplified immediately.
Amplify Enriched DNA Sample
Prepare the Post-Capture PCR Master Mix:
Place in thermocycler using the following program:
Pause the program, collect 5 μL of the reaction, and restart the program at the following points: after 7, 10, 13, 16, 19, and 22 cycles.
Run all products on 1.5% agarose gel to determine the optimal number of cycles, i.e. just before the products are first visible on the gel.
Gel Example: Captured libraries should appear as a smear on the gel typically 300-600 basepairs in length, depending on insert size. Each well is labeled with the PCR cycle number. For this pool, 12 cycles was chosen for the PCR because the product is visible
Once optimal number of cycles is determined, make PCR master mix for 5 reactions for each captured sample:
Add 45 μL master mix and 5 μL captured library to each 0.2 mL PCR tube and mix by pipetting.
Cycle using determined optimal number of cycles.
Pool the reactions in a new 1.7 mL tube.
Clean up with 375 μL (1.5X) Ampure beads, washing 2X with 1000 μL freshly-made 70% EtOH.
After second wash, quick spin, put back on magnet, and remove as much ethanol as possible.
Dry at RT for 10-15 min. Beads should look cracked and not shiny.
Resuspend beads in 33 μL EB by pipetting. Incubate 2 min. at RT and place tube back on magnet.
After 5 min. on magnet, transfer 30 μL supernatant to new 1.7 mL tube.
Use dsDNA HS Qubit to determine DNA concentration.
Dilute post-capture pools to 3 ng/μL in EB in a total volume of 40 μL.
Choose two NSC primer pairs to use in PCR assay:
Note: Recommended NSC qPCR Assays. Forward and reverse primer sequences for four different NSC control locus assays are shown here.
Add the following to a 0.2 mL PCR tube for NSC assay: (15 μL total reaction volume):
Place in Real-Time PCR System thermocycler using the following program.
Analyze data:
Send 30 μL of remaining 3 ng/μL diluted captured pools for sequencing.