Feb 13, 2026
PacBio Ampli-Fi - Amplifying genomic DNA for SMRTbell library preparation and HiFi sequencing
- CHIARA NATALI1,
- Claudio Ciofi1
- 1Department of Biology, University of Florence
- Biodiversity Genomics Europe
Protocol Citation: CHIARA NATALI, Claudio Ciofi 2026. PacBio Ampli-Fi - Amplifying genomic DNA for SMRTbell library preparation and HiFi sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3nxzev25/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
We use this protocol and it's working
Created: January 20, 2026
Last Modified: February 13, 2026
Protocol Integer ID: 238944
Keywords: BGE, pacbio, smrtbell library prep, smrtbell, long read library, amplifi, ampli-fi, uli, low input, ultra low input, hifi sequencing, Biodiversity Genomics Europe, amplifying genomic dna for smrtbell library preparation, preparing pacbio smrtbell library, pacbio smrtbell libraries with prep kit, smrtbell library preparation, ultra low input dna sample, genomic dna, incubation times at adapter ligation, dna sample, incubation times at nuclease treatment, amplifying, nuclease treatment
Funders Acknowledgements:
Biodiversity Genomics Europe receives funding from the European Union's Horizon Europe Research and Innovation Action
Grant ID: 101059492
Disclaimer
The duration times presented are estimations only.
Abstract
This is a modified protocol for preparing PacBio SMRTbell libraries with Prep kit 3.0 of ultra low input DNA samples.
The protocol is based on Ampli-Fi - Amplifying genomic DNA for SMRTbell library preparation and HiFi sequencing (103-648-000 REV04 JUL2025).
The main differences are:
- adding MgCl2 and changing incubation times at adapter ligation
- changing reagent amounts and incubation times at nuclease treatment
Attachments
Materials
- PacBio SMRTbell Prep Kit 3.0
- SMRTbell Clean-up beads
- Freshly prepared 80% ethanol
- Pipettes, standard and wide-bore pipette tips
- Microcentrifuge tubes, strips
- MgCl2
- Qubit fluorometer + Qubit 1X dsDNA HS Assay
- Megaruptor 2 system + Long Hydropores - or an equivalent shearing method
- Pippin Pulse Gel Electrophoresis + Quick-Load 1 kb Extend DNA Ladder - or an equivalent method of determining size distribution
- Minicentrifuge
- Magnetic separation rack
- 5200 Fragment Analyzer System - or an equivalent method of determining size distribution
- Thermocycler
- Vortexer
Troubleshooting
Safety warnings
The operator must wear a lab coat, powder-free nitrile gloves and safety specs to perform the laboratory procedures in this protocol.
Waste needs to be collected in a suitable container and disposed of in accordance with local regulations.
Liquid waste needs to be collected in a suitable container and disposed of in accordance with local regulations.
See best practices, thawing and handling instructions, and safety instructions for all the kit reagents, as well as instructions and safety data sheets for other reagents used in this protocol before starting.
Input DNA quality control
8h 40m
Bring the Qubit 1X dsDNA HS working solution and standards to room temperature.
Pulse vortex or pipette mix each sample to homogenize the DNA in solution.
Quick spin each sample to collect liquid.
Take a 1 µl aliquot from each sample and measure DNA concentration with a Qubit fluorometer using the 1X dsDNA HS kit.
Required
Concentration [ng/µL]Yield [ng]
Resolve DNA size distribution with Pippin Pulse Gel Electrophoresis using 50 - 100 ng of DNA (gDNA) and Quick-Load 1 kb Extend DNA Ladder (size range: 0.5 kb to 48.5 kb).
Note
Also other suitable methods can be used to determine the size distribution.
8h
Proceed to the next step of the protocol if sample quality is acceptable.
SAFE STOPPING POINT - Store at 4°C
Shearing genomic DNA using Megaruptor 2 system + QC
3h
If using the Megaruptor 2, ensure that the system is thoroughly washed before shearing. Since this workflow requires amplification, contamination introduced during shearing will be amplified which complicates assembly.
Note
Also other suitable methods can be used to shear the genomic DNA.
Use 100 ng of gDNA in a 51 µl sample volume for shearing.
Shear gDNA using Long Hydropores and have the Long Hydropore option selected in the Megaruptor software.
- Choose at target shear size of 10 kb in the Megaruptor software setting.
- A single sample requires a volume of 51 μl.
Take a 1 µl aliquot from each sample and measure DNA concentration with a Qubit fluorometer using the 1X dsDNA HS kit.
Required
Concentration [ng/µl]Yield [ng]
Measure DNA size distribution with a 5200 Fragment Analyzer System. If the DNA size is >11 kb,
repeat step 8.
Note
Also other suitable methods can be used to resolve the sample's size distribution.
Note
It is recommended to confirm gDNA is sheared to the appropriate size range (<11 kb) prior to proceeding. If the DNA is under-sheared, a second shear with the same parameters can be repeated. The same Megaruptor consumables can be used if a secondary shear is required.
Proceed to the “Repair & A-tailing” section.
SAFE STOPPING POINT - Store at 4°C
Repair and A-tailing
45m
Use PCR strip tubes throughout this procedure.
Use the following table to set up a reaction using around 100 ng of input sheared gDNA.
For each sample to be processed, add the following components to a single PCR tube of an 8-tube strip on ice:
| Reagent | Volume | |
| Sheared DNA | 49.0 μl | |
| Repair Buffer | 8.0 μl | |
| End repair mix | 2.0 μl | |
| DNA repair mix | 1.0 μl | |
| Total volume | 60.0 μl |
Reagents for Repair and A-tailing
Pipette-mix 10 times each sample with wide-bore pipette tips and quick-spin the tube strip to collect liquid.
Run the repair and A-tailing thermocycler program 00:36:00 .
| Time | Temperature | |
| 30 min | 37°C | |
| 5 min | 65°C | |
| Hold | 4°C |
Repair and A-tailing thermocycler program
36m
Proceed to the next step of the protocol.
Ligation of amplification adapter and clean-up
3h 15m
For individual preps, with the reaction on ice, add the components below in the order listed, directly to the sample in Reaction Mix 1:
| Reagent | Volume / sample | |
| Twist universal adapter | 2 μl | |
| Ligation Mix | 15.02 µl | |
| Ligation enhancer | 1 μl | |
| MgCl2 | 4.98 µl (final concentration 3mM) |
Reagents for amplification adapter ligation
The total reaction volume should now be 83 μl.
Pipette mix 10 times with wide-bore pipette tips and quick-spin the tube strip to collect liquid.
Run the adapter ligation thermocycler program02:32:00 .
| Time | Temperature | |
| 140 min | 20°C | |
| 10 min | 65°C | |
| Hold | 4°C |
Adapter ligation thermocycler program
Add 83 μl of SMRTbell cleanup beads to each sample and gently pipette mix 10 times. Perform a quick spin to collect all liquid from the sides of the tube.
Note
SMRTbell Clean-up beads must be brought to room temperature for 30 to 60 mins prior to use.
Incubate at 25°C for 00:15:00 .
15m
Place on a magnet stand and wait until supernatant is clear. Use a P200 pipette to remove the supernatant.
While on magnet, wash two times with 200 μl of freshly prepared 80% ethanol. After removal of second wash of 200 μl of ethanol, spin the tube strip briefly, return to magnetic stand and remove residual ethanol with a P20 pipette. Do not let the beads to dry out.
Remove the tube strip from the magnetic stand. Immediately add 25 μl of elution buffer and pipette mix 10 times to resuspend. Do not let the beads to dry out. Quick spin to collect all liquid from the sides of the tube. Place at 25°C for 00:07:00 to elute the DNA from the beads.
7m
Place the tube on the magnetic stand to separate the beads from the supernatant. When the supernatant is clear, transfer 25 μl of the purified eluted sample into a to a new tube and set aside on ice until ready to use. Discard old tube strip with beads.
Take a 1 µl aliquot from each sample and measure DNA concentration with a Qubit fluorometer using the 1X dsDNA HS kit to define number of PCR cycles.
Required
Concentration [ng/µl]Yield [ng]
| gDNA input | # PCR cycles | |
| 1 ng | 14 cycles | |
| 5 ng | 12 cycles | |
| 10 ng | 11 cycles | |
| 20 ng | 10 cycles | |
| 50 ng | 8 cycles |
Table to decide the number of PCR cycles based on the input amount.
Library amplification by PCR and clean-up
4h 15m
To prepare Amplification master mix, add the following components (see table) in the order and volume listed to a new tube. Adjust component volumes for the number of samples being prepared, plus 10% overage.
For individual preps, on ice, add components directly to the sample from the previous step at the specified volumes:
| Reagent | Volume | V / sample into a master mix | |
| 2x Xtreme Buffer | 50 µl | 55 μl | |
| dNTP (2 mM each) | 20 µl | 22 μl | |
| KOD Xtreme Hot Start DNA polymerase | 2 µl | 2.2 μl | |
| Twist UDI Primers | 4 µl | 4.4 μl | |
| Total volume / sample | 76 μl |
Reagents for amplification by PCR, and recipe for Amplification master mix
If you've prepared Amplification master mix:
- Pipette mix Amplification master mix
- Quick spin Amplification master mix to collect liquid.
- On ice, add 76 μl of the Amplification master mix to 24 µl of sample for a total volume of 100 µl.
Pipette mix 10 times with wide-bore pipette tips and then perform a quick spin to collect all liquid from the sides of the tube
Place in a thermocycler and run the following program (lid 105°C).
The PCR reactions may be left at 4°C overnight.
| Time | Temperature | # PCR cycles | |
| 2 minutes | 94°C | 1 | |
| 10 seconds | 98°C | ||
| 30 seconds | 58.8°C | 8-14 | |
| 10 minutes | 68°C | ||
| 7 minutes | 68°C | 1 | |
| Hold | 4°C |
PCR program for Library amplification by PCR. The times and temperatures for the part of the program that repeats for 8-14 cycles, based on the input amount, are in bold.
Add 100 μl of SMRTbell cleanup beads to the 100 μl of PCR and gently pipette mix 10 times. Perform a quick spin to collect all liquid from the sides of the tube.
Note
SMRTbell cleanup beads must be brought to room temperature for 30 to 60 mins prior to use.
Incubate at 25°C for 00:15:00 .
15m
Place on a magnet stand and wait until supernatant is clear. Use a P200 pipette to remove supernatant While on magnet, wash two times with 200 μl of freshly prepared 80% ethanol. After removal of second wash of 200 μl of ethanol, spin the tube strip briefly, return to magnetic stand and remove residual ethanol with a P20 pipette. Do not let the beads to dry out.
Remove the tube strip from the magnetic stand. Immediately add 51 μl of elution buffer (preheated at 37°C) and pipette mix 10 times to resuspend. Do not let the beads to dry out. Quick spin to collect all liquid from the sides of the tube. Incubate at 25°C for 00:07:00 to elute the DNA from the beads.
7m
Place the tube on the magnetic stand to separate the beads from the supernatant. When the supernatant is clear, transfer 51 μl of eluted amplified DNA reaction to a new tube and set in ice until ready to use.
Take a 1 µl aliquot from each sample and measure DNA concentration with a Qubit fluorometer using the 1X dsDNA HS kit.
Required
Concentration [ng/μl]Yield [ng]
Measure DNA size distribution with a 5200 Fragment Analyzer System.
Note
Also other suitable instruments can be used to measure DNA size distribution.
Proceed to the next step of the protocol if sample quality and mass is acceptable.
SAFE STOPPING POINT - Store at 4°C
Repair and A-tailing of amplified DNA
40m
Add the following components from the SMRTbell prep kit 3.0 to a microcentrifuge tube. Adjust
component volumes for the number of libraries being prepared, plus 15% overage.
For individual preps, add components directly to the 49 µl amplified DNA sample from the previous section:
| Reagent | Volume per sample | V / sample into a master mix | |
| Repair buffer | 8 µl | 9.2 µl | |
| End repair mix | 2 µl | 2.3 µl | |
| DNA repair mix | 1 µl | 1.15 µl | |
| Total volume / sample | 11 µl |
Reagents for Repair and A-tailing, and recipe for a master mix
The total reaction volume should now be 60 µl.
Pipette mix, and run the Repair and A-tailing thermocycler program00:37:00 .
| Time | Temperature | |
| 30 min | 37°C | |
| 5 min | 65°C | |
| Hold | 4°C |
Repair and A-tailing thermocycler program
37m
Proceed to the next step of the protocol.
Adapter ligation and clean-up
3h 5m
Add the following components in the order and volume listed below to a new microcentrifuge tube. Adjust component volumes for the number of samples being prepared, plus 10% overage. For individual preps, add components directly to each sample from the previous section in the order and volume listed below and skip step 43.
| Reagent | Volume per sample | V/sample into a master mix | |
| SMRTbell adapter | 4 µl | 4.4 µl | |
| Ligation mix | 14.9 µl | 16.39 µl | |
| Ligation enhancer | 1 µl | 1.1 µl | |
| MgCl2 | 5.1 µl (final concentration 3mM) | 4.64 µl | |
| Total volume/sample | 25 µl |
Reagents for Adapter ligation, and recipe for RM2 master mix
If you've prepared a master mix:
- Pipette mix the master mix.
- Quick spin the master mix to collect liquid.
- Add 25 µl of master mix to each sample from previous step.
Total volume should be 85 µl.
Pipette mix each sample and quick spin the tube strip to collect liquid.
Run the adapter ligation thermocycler program02:31:00 .
| Time | Temperature | |
| 140 min | 20°C | |
| 10 min | 65°C | |
| Hold | 4°C |
Adapter ligation thermocycler program
Add 85 µl of resuspended, room-temperature SMRTbell cleanup beads to each sample.
Pipette mix the beads until evenly distributed.
Quick spin the tube strip in a microcentrifuge to collect all liquid from the sides of the tubes.
Incubate at 25°C for 00:15:00 .
15m
Place tube strip in a magnetic separation rack until beads separate fully from the solution.
Slowly pipette off the cleared supernatant without disturbing the beads. Discard the supernatant.
Slowly dispense 200 µl, or enough to cover the beads, of freshly prepared 80% ethanol into each tube. After 30 seconds, pipette off the 80% ethanol and discard.
Repeat the previous step.
Remove residual 80% ethanol:
• Remove tube strip from the magnetic separation rack.
• Quick spin tube strip in a microcentrifuge.
• Place tube strip back in a magnetic separation rack until beads separate fully from the solution.
• Pipette off residual 80% ethanol and discard.
Remove tube strip from the magnetic rack. Immediately add 26 µl of elution buffer to each tube and resuspend the beads.
Quick spin the tube strip.
Incubate at 25°C for 00:07:00 to elute the DNA from the beads..
7m
Place tube strip in a magnetic separation rack until beads separate fully from the solution.
Slowly pipette off the cleared supernatant without disturbing the beads. Transfer supernatant to a new tube strip. Discard old tube strip with beads.
Take a 1 µl aliquot from each sample and measure DNA concentration with a Qubit fluorometer using the 1X dsDNA HS kit.
Required
Concentration [ng/µl]Yield [ng]
Proceed to the next step of the protocol.
SAFE STOPPING POINT - Store at 4°C
Nuclease treatment of SMRTbell library
15m
Adjust component volumes for the number of samples being prepared, plus 10% overage. For individual preps, add components directly to each sample from the previous section, then skip RM3 step 63.
| Reagent | Volume per sample | V/sample into a master mix | |
| Nuclease buffer | 2.5 µl | 2.75 µl | |
| Nuclease mix | 2.5 µl | 2.75 µl | |
| Total volume/sample | 5 µl |
Reagents for Nuclease treatment, and recipe for Nuclease master mix
If you've prepared Nuclease master mix:
- Pipette mix Nuclease master mix.
- Quick spin Nuclease master mix to collect liquid.
- Add 5 µl of Nuclease master mix to each sample.
The total reaction volume should now be 31 μl.
Pipette mix each sample and quick spin the strip tube to collect liquid.
Run the nuclease treatment thermocycler program 00:08:00 .
| Time | Temperature | |
| 7 min | 37°C | |
| Hold | 4°C |
Nuclease treatment thermocycler program
Take a 1 µl aliquot from each sample and measure DNA concentration with a Qubit fluorometer using the 1X dsDNA HS kit.
Required
Concentration [ng/µl]Yield [ng]
Proceed to the next step of the protocol.
Size selection of SMRTbell library
20h
For constructing SMRTbell libraries from ultra-low DNA input, PacBio recommends size-selection
using the BluePippin or the PippinHT systems (from Sage Science). In this protocol we describe how we use BluePippin.
The average size distribution of the final size-selected library is approximately 10 kb - 11 kb. Typical recovery yields after size-selection are 20% to 30% (from input of purified SMRTbell library) and are highly dependent on the size distribution of the starting SMRTbell library.
Prepare SMRTbell libraries in a final volume of 30 μl of elution buffer for each BluePippin lane.
Bring the Loading solution to room temperature and then, add 10 μl of Loading solution to each 30 μl sample remaining from the previous step above. Mix well by gentle pipetting and spin briefly to collect the contents at the bottom of the tube.
Follow the manufacturer’s recommendations to set up a run protocol.
• Select the “0.75% DF 3-10 kb Marker S1- Improved Recovery” Cassette Definition File for your sample.
• Using the “Range” selection mode, enter a desired “BP Start” value of 7000 and a “BP End” value of 17000.
• Be sure to assign a marker lane.
Note
We do not recommend running lanes with <400 ng of SMRTbell library material.
Load S1 marker and samples into the BluePippin gel cassette and start the run. Run time is approximately 4.0 hours.
Collect Eluate. To maximize recovery of eluted DNA unselect “End Run when Elution is Completed” and wait overnight after the run is finished before removing the sample from the elution chamber. After collecting the eluate, wash the eluate chamber with 40 μl of Sage Science’s 0.1% Tween-20 Wash Solution and then combine the recovered wash solution with the eluted sample. Washing the elution well may further increase recovery yields by approximately 10-20%.
Measure the volume of your size-selected sample (eluate plus wash). Take a 1 µl aliquot from each sample and measure DNA concentration with a Qubit fluorometer using the 1X dsDNA HS kit.
Required
Concentration [ng/µl]Yield [ng]
Measure DNA size distribution with a 5200 Fragment Analyzer System.
Note
Also other suitable instruments can be used to measure DNA size distribution.
Proceed to the next section if sample quality is acceptable.
Purification of size-selected SMRTbell library
35m
Add 1X SMRTbell cleanup beads to each sample from the previous section.
Pipette mix the beads until evenly distributed.
Quick spin the tube strip in a microcentrifuge to collect all liquid.
Leave at room temperature 00:10:00 to allow DNA to bind beads.
10m
Place tube strip in a magnetic separation rack until beads separate fully from the solution.
Slowly pipette off the cleared supernatant without disturbing the beads. It is recommended to save the supernatant in another tube strip in case of poor DNA recovery.
Slowly dispense 200 µl, or enough to cover the beads, of freshly prepared 80% ethanol into each tube. After 30 seconds, pipette off the 80% ethanol and discard.
Repeat the previous step.
Remove residual 80% ethanol:
• Remove tube strip from the magnetic separation rack.
• Quick spin tube strip in a microcentrifuge.
• Place tube strip back in a magnetic separation rack until beads separate fully from the solution.
• Pipette off residual 80% ethanol and discard.
Remove tube strip from the magnetic rack. Immediately add 15 µl of elution buffer to each tube and resuspend the beads by pipetting 10 times or until evenly distributed.
Quick spin the tube strip in a microcentrifuge to collect liquid.
Leave at room temperature for 00:05:00 5 minutes to elute DNA.
5m
Place tube strip in a magnetic separation rack until beads separate fully from the solution.
Slowly pipette off the cleared supernatant without disturbing the beads. Transfer supernatant to a new tube strip. Discard old tube strip with beads.
Take a 1 µl aliquot from each sample and measure DNA concentration with a Qubit fluorometer using the 1X dsDNA HS kit.
Required
Concentration [ng/µl]Required
Yield [ng]Proceed to SMRT Link Sample Setup to prepare the SMRTbell library for sequencing.
Note
Store SMRTbell libraries at 4°C if sequencing within the week. Long-term storage should be at -20°C. Minimize freeze-thaw cycles when handling SMRTbell libraries.
Protocol references
The protocol is based on Ampli-Fi - Amplifying genomic DNA for SMRTbell library preparation and HiFi sequencing (PN 103-648-000 REV04 JUL2025).