Jul 10, 2025

Public workspacePacBio Library Prep (SMRTbell Prep Kit 3.0) - Standard Operating Procedure

This protocol is a draft, published without a DOI.
  • Adrianne Doran1,2,
  • Liam Anstiss1,2,
  • OceanOmics UWA3
  • 1University of Western Australia;
  • 2OceanOmics Centre;
  • 3OceanOmics centre
  • OceanOmics UWA: University of Western Australia; OceanOmics Centre;
OceanOmics UWA
Icon indicating open access to content
QR code linking to this content
Protocol Citation: Adrianne Doran, Liam Anstiss, OceanOmics UWA 2025. PacBio Library Prep (SMRTbell Prep Kit 3.0) - Standard Operating Procedure. protocols.io https://protocols.io/view/pacbio-library-prep-smrtbell-prep-kit-3-0-standard-gzfcbx3ix
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: May 15, 2025
Last Modified: July 10, 2025
Protocol Integer ID: 218308
Keywords: genome sequencing, pacbio library prep, pacbio system, libraries from high molecular weight, genome, oceanomics centre, pacbio, smrtbell prep kit, revio system
Abstract
To describe the workflow for constructing whole-genome sequencing (WGS) libraries from high molecular weight (HMW) DNA using the SMRTbell Prep Kit 3.0, for sequencing on PacBio systems.

This method is employed by the OceanOmics Centre to generate high-quality libraries for sequencing on the Revio system. It represents a compiled and modified protocol based on PacBio’s published methods, adapted into a streamlined, single-processing workflow.
Guidelines
All users must read and understand this protocol in full before commencing work. Ensure that all steps are performed with precision and consistency to maintain sample integrity and maximise sequencing success. Adhere to standard laboratory safety practices and wear appropriate personal protective equipment at all times. Equipment must be calibrated and maintained according to manufacturer specifications. Any deviations from the protocol, including reagent substitutions or workflow modifications, should be documented and approved by a supervisor or laboratory manager. Good record-keeping, labelling, and traceability are essential throughout the process.
Materials
  • SimpliAmp Thermal Cycler – ThermoFisher, Cat# A24811
  • DynaMag 96 Side – ThermoFisher, Cat# 12331D
  • SMRTbell Prep Kit 3.0 – PacBio, Cat# 102-182-700
  • SMRTbell Barcoded Adapter Plate 3.0 – PacBio, Cat# 102-009-200
  • SMRTbell Cleanup Beads – PacBio, Cat# 102-158-300
  • Molecular Grade Ethanol – Various suppliers
  • Nuclease-Free, Molecular Biology Grade Water – MilliQ
  • AMPure PB Bead Size Selection Kit – PacBio, Cat# 102-182-500
  • 1.5 mL DNA LoBind Tubes – Eppendorf, Cat# 0030108051
  • 0.5 mL DNA LoBind Tubes – Eppendorf, Cat# 0030108035
  • 0.2 mL PCR Tubes – Eppendorf, Cat# 0030124332
  • 10 µL XL Tips – Neptune, Cat# 034-BT10XL
  • 20 µL Tips – Neptune, Cat# 034-BT20
  • 100 µL Tips – Neptune, Cat# 034-BT100
  • 200 µL Tips – Neptune, Cat# 034-BT200
  • 1000 µL Tips – Neptune, Cat# 034-BT1000.96
  • Femto Pulse System – Integrated Sciences, Cat# M5330AA
  • Femto Pulse gDNA 165 kb Kit – Agilent, Cat# FP-1002-0275
  • Qubit Fluorometer – ThermoFisher, Cat# Q33238
  • Qubit 1X dsDNA HS Assay Kit – ThermoFisher, Cat# Q32854
  • MegaRuptor 3 System – Custom Science, Cat# B06010003
  • MegaRuptor 3 Shearing Kit – Custom Science, Cat# E07010003
Protocol materials
ReagentSRE KitPacBioCatalog #102-208-300
ReagentSMRTbell prep kit 3.0PacBioCatalog #102-182-700
Reagent Megaruptor 3 Shearing KitDiagenodeCatalog #E07010003
Troubleshooting
Safety warnings
This protocol involves the use of potentially hazardous biological materials, chemicals (including ethanol and enzymatic reagents), and electrical laboratory equipment. Always wear appropriate personal protective equipment (PPE), including a lab coat, gloves, and eye protection. Be familiar with the relevant Safety Data Sheets (SDS) and laboratory risk assessments before commencing work. Follow institutional safety guidelines and dispose of waste in accordance with local regulations. Exercise caution when handling sharp instruments, thermal cyclers, and high-speed centrifuges to prevent injury or equipment damage.
REQUIRED MATERIALS AND EQUIPMENT
ReagentSMRTbell prep kit 3.0PacBioCatalog #102-182-700

ReagentSRE KitPacBioCatalog #102-208-300

Reagent Megaruptor 3 Shearing KitDiagenodeCatalog #E07010003


Equipment
Femto Pulse System
NAME
Automated Gel electrophoresis
TYPE
Agilent
BRAND
M5330AA
SKU

Equipment
Qubit fluorometer
NAME
designed to measure DNA, RNA, and protein quantity or quality using the Invitrogen Qubit RNA IQ (integrity & quality) assay.
TYPE
ThermoFisher
BRAND
Q33238
SKU


Equipment
Megaruptor® 3
NAME
Diagenode
BRAND
B06010003
SKU
https://www.diagenode.com/en/p/megaruptor-3
LINK


Equipment
DynaMag-2
NAME
Magnet
TYPE
Invitrogen
BRAND
12321D
SKU
https://www.thermofisher.com/order/catalog/product/12321D#/12321D
LINK


Before You Begin
(Optional) If gDNA solution is highly viscous, this is a sign that the gDNA has not yet homogenized and two options should be explored, otherwise downstream processing will be negatively affected.
If post DNA extraction concentration came back with concentration > 200ng/µL, dilute sample to between 150-200 ng/µL with TE buffer (pH 8) or Buffer LTE. Pipette mix sample 10X with wide bore pipette tip, then leave on heating block at 37°C for an hour. If gDNA is still viscous, further mix with wide bore pipette tip and leave at room temperature for a day.
If sample concentration is not evidently high (i.e. not above >200ng/µL), pipette mix sample with wide bore pipette 10X times and incubate tube on heating block at 37°C for an hour. If gDNA is still viscous, further mix with wide bore pipette tip and leave at room temperature for a day.
Ensure gDNA meets quality requirements (Qubit concentration, Nanodrop purity ratios and Femto Pulse fragment size). Minimum sample quality metrics are listed in the table below. All sample QCs are to be evaluated on a sample-by-sample basis. Samples of high significance may require processing via a customized workflow (e.g., omitted fragment removal and/or shearing) despite not meeting QC requirements.
Qubit Yield >1,500 ng (25ng/ µL in 60 µL)
Nanodrop A260/280 Ratio >1.8
Nanodrop A260/230 Ratio >2.0
Qubit: Nanodrop Ratio >0.7
Fragment Size: 50% ≥30 kb (requirement for PacBio’s Short Read Eliminator Kit)
Set heat block to 50°C.
Remove Short Fragments
ReagentSRE KitPacBioCatalog #102-208-300

In a 1.5 mL Eppendorf DNA LoBind tube, dilute DNA to 25–150 ng/μL in 60 μL using TE buffer (pH 8), Buffer LTE, or water. Ensure the dilution is based on the Qubit DNA concentration.
Add 60 μL of Buffer SRE to the sample. Vortex to mix for 5 seconds at max speed.
Incubate the tube for 1 hour at 50°C in the heating block.
Load the tube into the centrifuge with the hinge facing towards the outside of the rotor.
Centrifuge at 10,000 x g for 30 mins at room temperature.
If using a centrifuge with temperature control (i.e., cooling function), turn this function off by setting the temperature to 29°C.
Carefully remove the supernatant from the tube without disturbing the pellet. Place the pipette tip on the thumb lip side of the tube. The DNA pellet will have formed on the bottom of the tube under the hinge region but may not be visible.
Leaving up to 10 μL is acceptable to be sure the pellet is not disturbed.
Add 121 μL of Low TE Buffer to the tube and incubate at 50°C for 20 minutes.
After incubation, pipette-mix 20 times with a wide-bore pippete tip and vortex the tube for 5s to ensure that the DNA is properly resuspended and mixed.
Assess DNA recovery using a Qubit Fluorometer with the High Sensitivity (HS) dsDNA Assay Kit. A loss of up to 50% in yield is typical following short fragment removal. If recovery is lower than expected, thoroughly pipette-mix the sample again, allow it to rest overnight at room temperature, and repeat the Qubit measurement. Poor recovery may be due to incomplete re-homogenisation of the DNA.
This is a safe stopping point.
DNA can be stored at 4°C for several months. Long-term storage at -20°C or -80°C can be used if necessary. Avoid freeze/thaw cycles since this can degrade high molecular weight DNA.
Pause
DNA Shearing and Clean-up
In a MegaRuptor Hydro tube, make a 120 µL aliquot of 39 ng/µL concentration HMW DNA in Low TE Buffer. To calculate the volumes needed, use the following equations:
HMW DNA input (µL) = (39×120) ÷ Qubit conc.
Low TE Buffer (µL) = 120 − HMW DNA input µL.
For a visual demonstration of proper instrument setup and operation, refer to the following instructional video on using the MegaRuptor system:
https://www.youtube.com/watch?v=zVhqnkQL8zo
Using the MegaRuptor 3 , shear samples using the below parameters (these parameters can be altered as needed):
a) Speed: 31
b) Volume: 120 µL
c) Concentration: 39 ng/µL
Whilst MegaRuptor is running, remove SMRTbell clean-up beads from the fridge so that the beads reach room temperature before use.
Once the MegaRuptor has finished, recover the sheared DNA into a 1.5 mL Eppendorf Lo-bind tube and measure the volume. Typical volume loss is 5 – 10 µL.
Clean up sheared DNA using 1X SMRTbell clean-up beads.
Add a 1:1 ratio of resuspended, vortexed, room-temperature SMRTbell clean-up beads to each tube of sheared DNA (e.g., if 115 µL DNA recovered, add 115 µL beads).
Tap / flick the beads until evenly distributed and quick spin to collect liquid.
Leave at room temperature for 10 minutes to allow the beads to bind.
During these 10 minutes, freshly prepare 5mL of 80% EtOH.
Place tube onto magnetic rack for 2 minutes to separate beads from solution.
Without disturbing the beads, slowly pipette off and discard the clear supernatant.
Slowly dispense 200 µL of the freshly prepared 80% EtOH into each tube. After 30 seconds, pipette off and discard the EtOH.
Repeat previous step for a second ethanol wash.
To remove residual EtOH:
a) Remove tube from magnet rack and quick spin.
b) Place tube on magnetic rack and allow beads to separate.
c) Pipette off residual EtOH.
Remove samples from magnetic rack and after 30 seconds, add 47 µL of Low TE Buffer to each tube.
Tap to mix until beads are evenly distributed.
Quick spin to collect liquid. Leave at room temperature for 5 minutes (this elutes the DNA). To increase yield, after 2 minutes tap the tube every 30 seconds.
Place the sample tube on the magnetic rack for 2 minutes. During these 2 minutes, prepare a new 1.5mL Eppendorf LoBind tube to collect the SRE + sheared DNA.
Without disturbing the beads, transfer the supernatant (47 µL) into a 1.5mL Eppendorf LoBind tube. Discard the tube containing beads.
Prepare a 1:10 dilution by combining 9 µL of Low TE buffer with 1 µL of the DNA sample. This aliquot will be used for quality control assessments. The remaining undiluted sample should be stored at 4 °C if it will be used for library preparation within the next week. For longer-term storage, keep the sample at −20 °C to maintain DNA integrity.
Evaluate sample quality after short-read elimination and shearing using the following instruments:
  1. Qubit Fluorometer with the High Sensitivity (HS) dsDNA Assay Kit to assess DNA concentration.
  2. Femto Pulse System using the Genomic DNA 165 kb Kit to assess fragment size distribution.

This is a safe stopping point.
Store the cleaned, sheared DNA at 4 °C until proceeding with library preparation.
Pause
Library Preparation - Repair and A-tailing
Ensure the sample is in a 0.2 µL PCR strip-tube and the volume is 46 µL.
Remove the following items from SMRTbell Prep Kit 3.0 (stored at -20°C) and thaw as indicated below:





With the samples on ice, add 8 µL of Repair Buffer (Purple) to each sample.
Add 4 µL of End Repair Mix (Blue) to each sample.
Add 2 µL of DNA Repair Mix (Dark Green) to each sample.
Pipette to mix and spin down in a microcentrifuge to collect liquid.
Place samples onto the thermal cycler and run the ‘Repair and A-tailing’ program. This program has the following settings:




Whilst the thermocycler is running, take the following items out of the freezer and let them slowly thaw:





Continue with Adapter Ligation and Clean-up.
Adapter Ligation and Clean-up
Add 4 µL of unique SMRTbell barcoded adapter 3.0 (96 well plate) to each of the A-tailed samples. Record which adapter well has been added to each sample.
Add 30 µL of Ligation Mix (yellow) to each sample. Do not make a master mix.
Add 1 µL Ligation enhancer (red) to each sample. Do not make a master mix.
Pipette to mix and spin down in a microcentrifuge to collect liquid. Do not try flicking the tubes as the 0.2ml PCR tubes are likely to open.
Place samples onto the thermal cycler and run the ‘Adapter Ligation’ program. This has the following settings:



During this cycle, bring SMRTbell clean-up beads and Elution Buffer (EB) aliquots to room temperature.
Removed samples from thermal cycler and let them sit at room temperature for 2 minutes.
Add 95 µL of re-suspended, room temperature SMRTbell beads to each sample.
Tap / flick the beads until evenly distributed and quick spin to collect liquid.
Leave at room temperature for 10 minutes to allow the beads to bind.
During these 10 minutes, freshly prepare 5mL of 80% EtOH.
Place the sample tubes on a magnetic rack for 2 minutes to separate beads from solution.
Without disturbing the beads, slowly pipette off and discard the clear supernatant.
Slowly dispense 200 µL of the freshly prepared 80% EtOH into each tube. After 30 seconds, pipette off and discard the EtOH.
Repeat Previous step.
To remove residual EtOH:
Remove tube from magnetic rack and quick spin.
Place tube back on magnetic rack, allow beads to separate out and pipette off residual EtOH.
Remove sample tubes from the magnetic rack and after 30 seconds add 41 µL of Elution Buffer (EB) to each tube. Pipette 10 times to resuspend tubes or until evenly distributed.
Quick spin tube to collect liquid and leave at room temperature for 5 minutes (this elutes the DNA).
Place sample tubes on a magnetic rack for 2 minutes to separate beads from solution.
Without disturbing the beads, slowly transfer 41 µL clear supernatant into a new 0.2 µL tube. This is the Adapter Ligated sample. Discard the tube containing beads.
Optional: Measure DNA concentration of your adapter-ligated samples using a Qubit Fluorometer with the High Sensitivity (HS) dsDNA Assay Kit. This allows calculation of DNA recovery (total nanograms) before and after nuclease treatment. It is not needed to take a DNA concentration measurement at this point, but it helps inform users on the quality of the DNA when comparing it to the post nuclease treatment yield. Additionally it helps create a database for knowing how well samples are expected to perform.

This is a safe stopping point
Store the cleaned, adapter-ligated samples at 4 °C until further processing
Nuclease Treatment
Take the following items out of the freezer and let them slowly thaw:




With the samples on ice, add 5 µL of Nuclease buffer to each of the Adapter ligated samples.
Add 5 µL of Nuclease mix to each sample. Do not make a master mix.
Pipette to mix and spin down in a microcentrifuge to collect liquid.
Place samples onto thermal cycler and run the ‘Nuclease Treatment’ program. This program has the following settings:



AMPure PB Beads Size Selection
Make a 35% dilution of AMPure PB beads by adding 1.75mL of resuspended, room temperature AMPure beads to 3.25mL of elution buffer. This dilution needs to be very accurate, so it is recommended to add 3 x 1000 µL, 1 x 200 µL and 1 x 50 µL. The 35% dilution can be stored at 4 degrees for 30 days.
Add 155 µL of the resuspended, room temperature 35% bead dilution to each of the nuclease treated samples.
Pipette to mix and spin down in a microcentrifuge to collect liquid.
Leave at room temperature for 20 minutes to allow DNA to bind to the beads.
During these 20 minutes, freshly prepare 5mL of 80% EtOH (if not freshly prepared prior).
Place sample tubes on a magnetic rack for 2 minutes to separate beads from solution.
Without disturbing the beads, slowly pipette off the clear supernatant. It is recommended to save this in another tube in case of poor recovery.
Slowly dispense 200 µL of the freshly prepared 80% EtOH into each tube. After 30 seconds, pipette off and discard the EtOH.
Repeat Previous step.
To remove residual EtOH:
Remove tube from magnet and quick spin.
Place tube back on magnet, allow beads to separate out and pipette off residual EtOH.
Remove sample tube from the magnetic rack and after 30 seconds add 15 µL of elution buffer to each tube. Pipette 10 times to resuspend tubes or until evenly distributed. Note: If expecting low final library concentration can reduce elution buffer input down to 10 µL.
Quick spin tube to collect liquid and leave at room temperature for 5 minutes (this elutes the DNA).
Place sample tubes on a magnetic rack for 2 minutes to separate beads from solution. During these 2 minutes, prepare a new 0.5 µL low-bind tube to collect the final SMRTbell library. Pre-label these tubes.
Without disturbing the beads, slowly pipette the clear supernatant into the new 0.5 µL library tube. Discard the tube containing beads.
Take a 1  µL aliquot from each final library and dilute it with 9  µL of Elution Buffer to prepare a 1:10 dilution for quantification. Measure DNA concentration using a Qubit Fluorometer with the High Sensitivity (HS) dsDNA Assay Kit. Calculate the total DNA yield using the formula: Qubit result × 10 (dilution factor) × 14 (remaining sample volume in µL).
Assess fragment size using the Femto Pulse System with the Genomic DNA 165 kb Kit and record the average fragment length
Proceed with sequencing on a PacBio platform.

THIS IS A SAFE STOPPING POINT.
Store SMRTbell libraries at 4°C for a maximum of 7 days or 20°C for long-term storage.
Pause