Jun 04, 2026

PacBio Circular Consensus Sequencing (CCS) on the Revio platform

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvmdomov3p/v1
  • Stephanie Durgin1,
  • Erin LaRoche1,
  • Evan Mullaney1,
  • Desiree Byron1,
  • Garnette Goorahlal1,
  • Mujahed Kazi1,
  • Michelle Cipicchio1,
  • Evan McDaid1,
  • Corey Nolet1,
  • Ally Day1,
  • Catie Ramnarine1,
  • Michael DaSilva1,
  • Niall Lennon1
  • 1Broad Clinical Labs
  • Broad Clinical Labs
Michael DaSilva
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvmdomov3p/v1
Protocol Citation: Stephanie Durgin, Erin LaRoche, Evan Mullaney, Desiree Byron, Garnette Goorahlal, Mujahed Kazi, Michelle Cipicchio, Evan McDaid, Corey Nolet, Ally Day, Catie Ramnarine, Michael DaSilva, Niall Lennon 2026. PacBio Circular Consensus Sequencing (CCS) on the Revio platform. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmdomov3p/v1
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
Created: June 01, 2026
Last Modified: June 04, 2026
Protocol  Integer ID: 318312
Keywords: pacbio circular consensus sequencing, workflow for pacbio circular consensus sequencing, revio platform this protocol, revio sequencer plate preparation, revio platform, revio loading, cc, initial genomic dna, processing steps from initial genomic dna
Abstract
This protocol describes the workflow for PacBio Circular Consensus Sequencing (CCS) on the Revio platform. This version of the protocol includes the processing steps from initial genomic DNA (gDNA) quality control, automated library construction, Revio sequencer plate preparation, and Revio loading.
Materials
**Instruments**

- Galaxy MiniStar - Microcentrifuge, VWR
- Plate Centrifuge 5804, Eppendorf, 022622501
- Pipette (10μL, 20μL, 200μL), Gilson/Rainin, Pipetman/Pipet-lite
- Vortex Genie 2, Scientific Industries, SI-0236
- Femto Pulse, Agilent Technologies, M5330AA
- Bravo, Agilent Technologies, G5509A
- Hamilton STARlet, Hamilton Company, 173000
- Lunatic, Unchained Labs
- Megaruptor 3, Diagenode, B06010003
- Revio, Pacific BioSciences, 102-962-600 REV03
- PippinHT, Sage Sciences, HTP0001

**Reagents and consumables**

- Megaruptor 3 Shearing Kit, Diagenode, E07010003
- Barcoded 0.5mL tubes, Computype Inc, 18266901
- Clear 96-well Plate, Eppendorf, 477-44-116
- Semi Skirt Plate, VWR, 47744-110
- 1mL Deep well plate, Fisherbrand / Agilent, 12566120 / P60-20
- 250mL conical tubes, VWR, 21008-771
- Low TE Buffer (10mM Tris pH8, 0.1mM EDTA), Agilent Technologies, FP-1002-0275
- 2mL reagent tubes, VWR, 101093-780
- Abgene 96 Well 0.8mL Polypropylene DeepWell plate, ThermoFisher, AB0859
- Hamilton filtered Tips 50μL, Hamilton Company, 235948
- Hamilton filtered Tips 300μL, Hamilton Company, 235903
- Hamilton filtered Tips 1000μL, Hamilton Company, 235905
- 70μL 384w Bravo Tips, Agilent Technologies, 19133-102
- 50μL 384w Bravo Tips, Agilent Technologies, 19133-142
- Lunatic Plate, Unchained Labs, 701-2019
- 1000μL Pipette tips, Thomas Scientific LLC, 7740G73
- 200μL Pipette tips, VWR, TX1059-0089BR
- 50μL Pipette tips, Thomas Scientific LLC, 1142W03
- 10μL Pipette tips, VWR, TX1051-0389BRI
- Screw cap tubes, Thermo Scientific, 3744
- SMRTbell prep kit v3.0, Pacific Biosciences, 102-141-700
- SMRTbell Barcoded Adapter Plate 3.0, Pacific Biosciences, 102-009-200
- SMRTbell Cleanup Beads, Pacific Biosciences, 102-158-300
- PippinHT kit, 0.75% agarose, Sage Sciences, HPE7550
- Revio Polymerase Kit, Pacific Biosciences, 102-739-100
- Revio SMRTcell tray, 25M, Pacific Biosciences, 102-202-200
- Revio Sequencing Plate, Pacific Biosciences, 102-587-400

**Reagents Generated In House**

- EB (10mM Tris-HCl pH 8)
- 1X Low TE
- 80% Ethanol
- 0.1% Tween in EB
gDNA inputs
≥ 4 µg HMW gDNA
DQN40kb ≥ 5
Required Materials
Instruments
InstrumentSupplierProduct #
Galaxy MiniStar - MicrocentrifugeVWR
Plate Centrifuge 5804Eppendorf 022622501
Pipette (10μL, 20μL, 200μL) Gilson/ Rainin Pipetman/Pipet-lite
Vortex Genie 2Scientific Industries SI-0236
Femto Pulse Agilent Technologies M5330AA
Bravo Agilent Technologies G5509A
Hamilton STARletHamilton Company 173000
LunaticUnchained Labs
Megaruptor 3DiagenodeB06010003
RevioPacific BioSciences102-962-600 REV03
PippinHTSage SciencesHTP0001

Reagents and Consumables
ItemSupplierProduct #
Megaruptor 3 Shearing KitDiagenodeE07010003
Barcoded 0.5mL tubesComputype Inc18266901
Clear 96-well PlateEppendorf477-44-116
Semi Skirt PlateVWR47744-110
1mL Deep well plateFisherbrand / Agilent12566120 / P60-20
250mL conical tubesVWR21008-771
Low TE Buffer (10mM Tris pH8, 0.1mM EDTA)Agilent TechnologiesFP-1002-0275
2mL reagent tubesVWR101093-780
Abgene 96 Well 0.8mL Polypropylene DeepWell plateThermoFisherAB0859
Hamilton filtered Tips 50μLHamilton Company 235948
Hamilton filtered Tips 300μLHamilton Company 235903
Hamilton filtered Tips 1000μL Hamilton Company 235905
70µL 384w Bravo Tips Agilent Technologies19133-102
50µL 384w Bravo TipsAgilent Technologies19133-142
Lunatic PlateUnchained Labs701-2019
1000µL Pipette tipsThomas Scientific LLC7740G73
200µL Pipette tipsVWRTX1059-0089BR
50µL Pipette tipsThomas Scientific LLC1142W03
10µL Pipette tipsVWRTX1051-0389BRI
Screw cap tubesThermo Scientific 3744
SMRTbell prep kit v3.0Pacific Biosciences102-141-700
SMRTbell Barcoded Adapter Plate 3.0Pacific Biosciences102-009-200
SMRTbell Cleanup Beads Pacific Biosciences102-158-300
PippinHT kit, 0.75% agaroseSage SciencesHPE7550
Revio Polymerase KitPacific Biosciences102-739-100
Revio SMRTcell tray, 25MPacific Biosciences102-202-200
Revio Sequencing Plate Pacific Biosciences102-587-400

Reagents Generated In House
Item
EB (10mM Tris-HCl pH 8)
1X Low TE
80% Ethanol
0.1% Tween in EB

Incoming DNA Quality Control
Volume Check and Quantification
A minimum of 4 μg of DNA is required per attempt of SMRTbell Library Construction.
  1. Perform a volume check using a P200 pipette with wide-bore tips (Fisher Scientific, 21-236-1A)
  2. Using a Bravo liquid handling platform (Agilent Technologies, G5509A) & 70μL Agilent tips (Agilent Technologies Inc, 19133-102), transfer 3 μL aliquots of the samples to Lunatic chips (Unchained Labs, 7012019).
  3. Quantify DNA using the Lunatic UV/Vis Spectrometer (A260 dsDNA turbidity assay) to determine concentration.
  4. Calculate total mass; ensure ≥ 4 μg is available.

Fragment Analysis
  1. Perform fragment analysis using the Agilent Genomic DNA 165 kb Kit (FP-1002-0275) on the Femto Pulse instrument (Agilent Technologies, M5330AA) according to the manufacturer’s instructions.
  2. Conduct a smear analysis using ProSize software (v3.0; Agilent Technologies) to calculate the percentage of DNA fragments ≥ 40 kb.
  3. Evaluate samples against the standard long-read sequencing specification: ≥ 50% of DNA > 40 kb.
  • If target of ≥ 50% of DNA > 40kb is met, proceed to standard workflow.
  • If target of ≥ 50% of DNA > 40kb is not met, reach out to PacBio Tech Support for guidance.
DNA Shearing
  1. Using a STARlet liquid handler (Hamilton Company, 173000), transfer a mass of 4 µg in a volume of 110 µL into barcoded 0.5 mL shearing tubes: Normalize with Low TE buffer if necessary to meet the 4µg input.
  2. Attach a hydropore 6 syringe assembly (Diagenode, E07010003) to each 0.5mL shearing tube
  3. Load shear tubes onto the Megaruptor 3 (Diagenode, B06010003) and start run with the following parameters: Concentration: 49 ng/μL; Volume: 100 μL; Speed: 31.
  4. When the shearing run is complete, transfer samples from the shearing tubes to a barcoded Abgene 96 Well 0.8 mL Polypropylene DeepWell plate.
  5. Perform a post-shear size QC using the FemtoPulse Agilent Genomic DNA 165 kb Kit (FP-1002-0275) on the Femto Pulse instrument (Agilent Technologies, M5330AA) according to the manufacturer’s instructions - Target shear size: 15-20 kb.
SMRTbell Library Construction
SMRTbell Library Construction utilizes the PacBio SMRTbell Prep Kit 3.0 (Pacific Biosciences, 102-141-700) and is fully automated on the STARlet liquid handler equipped with Hamilton filtered Tips 50 μL (Hamilton Company, 235948), Hamilton filtered Tips 300 μL (Hamilton Company, 235903), Hamilton filtered Tips 1000 μL (Hamilton Company, 235905), an Alpaqua magnet (Alpaqua SKU: A000400), 37°C heat block, 67°C heat block, shaker, and tube chiller. Master mixes were made and added to the tube chiller according to PacBio’s SMRTbell prep protocol.

Perform a 1.0X volume over volume (v/v) of SMRTbell Cleanup Beads (Pacific Biosciences, 102-158-300)
  1. Add 110 μL of SMRTbell beads to the MIDI plate of sheared DNA.
  2. Shake for 30 seconds on the shaker block.
  3. Incubate for 10 minutes at room temperature.
  4. Transfer the MIDI plate to an Alpaqua Magnet.
  5. Allow beads to separate until liquid is clear.
  6. Remove the supernatant.
  7. Add 200 μL of 80% Ethanol to the plate.
  8. Incubate for 30 seconds and remove.
  9. Repeat ethanol wash in steps 13g and 13h.
  10. Move the MIDI plate off the Alpaqua magnet.
  11. Add 47 μL of 1X Low TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8).
  12. Shake for 30 seconds on the shaker block.
  13. Incubate for 5 minutes at room temperature.
  14. Transfer the MIDI plate to an Alpaqua Magnet.
  15. Allow beads to separate until sample is clear.
  16. Transfer eluate to a clear 96-well Plate (Eppendorf, 477-44-116).

DNA Repair and A-Tailing
  1. Add 14 μL of DNA repair master mix (consisting of Repair buffer, End repair mix, DNA repair mix in a 4:2:1 ratio) to each DNA sample.
  2. Mix thoroughly using the 96-head.
  3. Move plate to the 37°C heat block.
  4. Incubate for 30 minutes.
  5. Move plate to the 65°C heat block.
  6. Incubate for 5 minutes.

Adapter Ligation
  1. Add 4 μL of SMRTbell adapters from SMRTbell barcoded adapter plate 3.0 (PacBio 102-009-200) to each sample well.
  2. Add 31 μL of ligation master mix (consisting of ligation mix and ligation enhancer in a 30:1 ratio).
  3. Shake for 30 seconds on the shaker block.
  4. Incubate for 30 minutes at 20°C.

1X SMRTbell Bead Cleanup
  1. Perform a 1.0x v/v bead cleanup using a 40 μL final elution volume.

Nuclease Treatment
  1. Add 10 μL of nuclease master mix (consisting of nuclease buffer and nuclease mix in a 1:1 ratio) was added to each sample well.
  2. Shake for 30 seconds on the shaker block.
  3. Incubate for 15 minutes at 37°C.
  4. Perform a 1.0x v/v bead cleanup using a 25 μL final elution volume of 10 mM Tris-HCl pH 8.0 buffer.
  5. Transfer the SMRTbell libraries to a rack of matrix tubes (Thermo Fisher Scientific, 3744).

Post SMRTbell Prep QC
  1. Using an Agilent Bravo (G5509A) and 70 μL 384w Bravo Tips (19133-102), transfer 3 μL of the final SMRTbell products to the wells of the Lunatic plate (Unchained Labs, 701-2019).
  2. Open the Lunatic software and choose “Nucleic Acids”.
  3. Choose application “A260 dsDNA (turbidity)”.
  4. Select “Lunatic plate”.
  5. Name your experiment.
  6. Scan Lunatic plate ID.
  7. Select “Samples” tab and highlight the wells that contain sample.
  8. Choose the appropriate “blank” (see manufacturer guidelines).
  9. Load plate  press “Start” button.
  10. Analyze quants:
If ≥15 ng/µL, proceed to Size Selection.
If samples are <15 ng/µL, reach out to PacBio Tech Support for guidance.
Size Selection (Depletes samples of fragments 10kb)
  1. Calibrate PippinHT: Cover LED detectors with manufacturer provided fixtures and run calibration.
  2. Using an Agilent Bravo (G5509A), load samples into wells of the PippinHT 0.75% agarose cassette (Sage Sciences, HPE7550): Remove 150 μL of buffer from the upper buffer chambers & elution wells and replace it with fresh buffer; Place a strip seal from the kit over the elution wells; Perform continuity test; Add 25 μL of sample into the sample wells; Add DNA ladder into the last cassette well.
  3. Load cassette onto the PippinHT (Sage Sciences, HTP0001), Run settings: Cassette definition: 6-10 kb High Pass Marker 75E; BPstart value: 10,000 bp; BPend value: 50,000 bp; Select “Range”; Run time: 1 hour 20 minutes @ 75V.
  4. Allow cassettes to rest at room temperature for 45 minutes to increase yield.
  5. Unload samples from the cassettes using the Bravo: Transfer 30 uL out of the elution wells into sample tubes; Rinse elution wells with 30 uL of 0.1% Tween20 in low TE pH 8.0 (0.1% Tween 20, 10 mM Tris, 0.1 mM EDTA) as a wash and transfer this buffer into the sample tubes; Repeat the rinse step one time for a total sample volume of 90 uL.
  6. Perform a final 1.0X v/v SMRTbell cleanup (95 μL of SMRTBell Beads) on the STARlet liquid handler as described in “SMRTbell Library Construction” to purify size selected libraries.
Final Library Construction QC
Repeat “Post SMRTbell Prep QC” steps to obtain a final library quant.
Normalization & Pooling
Targeting a loading concentration of 215-250pM, use EB (10 mM Tris HCl pH 8.0) to normalize the samples according to PacBio’s recommendations in “Sample Setup” in SMRTlink.
PacBio Automated Annealing, Binding, and Cleanup (ABC)
  1. Using the Hamilton STARlet the Revio polymerase kit (Pacific Biosciences,102-739-100), add 25 μL of annealing mastermix (sequencing primer and buffer in a 1:1 ratio) to each sample well.
  2. Incubate at room temperature for 15 minutes.
  3. Add 1 μL of polymerase and 49 μL polymerase buffer to each sample.
  4. Incubate at room temperature for 15 minutes.
  5. Add 120 μL of SMRTbell beads to each sample well for 1.2X cleanup.
  6. Incubate sample with beads for 10 minutes at room temperature.
  7. Move sample plate to an Alpaqua magnet.
  8. Allow beads to separate out of the solution and remove the supernatant.
  9. Elute in 100 μL of Revio loading buffer.
  10. Incubate beads in elution buffer for 5 minutes.
  11. Move samples back to the Alpaqua magnet.
  12. Allow beads to separate out and transfer sample to a 96-well PCR plate (Eppendorf, 477-44-116).
  13. Manually dilute the Internal Control Complex (ICC) (PacBio, 103-508-800) and add to sample following PacBio’s guidelines.
  14. Store final ABC product in the dark at +4°C for up to 24 hours prior to sequencer loading.
Sequencing Plate Preparation & Revio Loading
  1. Thaw Revio Sequencing plate (102-587-400).
  2. Transfer 95 μL ABC product into the empty wells (A01-D01) of the Revio Sequencing Plate using the Bravo.
  3. Scan samples into the Revio sequencing plate using the QR code on the card associated with the PacBio plate.
  4. Manually seal the sample wells with the foil seals provided within the kit (Pacific Biosciences, 100-667-400).
  5. Generate a run design using SMRTlink software or by uploading a csv file with desired run parameters.
  6. Load the Revio sequencer as described within the manufacturer’s protocol.