THE QUALITY OF YOUR RESULTS STARTS HERE
Variants, phasing, methylation — every downstream answer depends on the library you build. SMRTbell prep kits produce PCR-free, automation-ready libraries that preserve native DNA and epigenetic signal. From 500 ng of input to sequencing-ready library in 3.5 hours.
Learn more about WGS Explore datasets
Overview + prep kits
PacBio SMRTbell WGS library prep is a streamlined workflow that can be completed in as little as 3.5 hours using as little as 500 ng of genomic DNA.
Automation
PacBio WGS library prep is designed for automation across leading NGS liquid handling platforms.
Specifications
Check PacBio protocols for the latest guidance on each application and view specifications.
FAQs + resources
Frequencty asked questions about HiFi WGS library prep plus additional resources.
OVERVIEW OF PACBIO WGS LIBRARY PREP
PacBio SMRTbell WGS library prep is a streamlined workflow that can be completed in as little as 3.5 hours using as little as 500 ng of genomic DNA.
The result:
- Highly accurate, long HiFi reads
- Improved genome completeness
- Superior structural variant detection
- Haplotype phased reads
- Native methylation detection with no additional assay required
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| DNA input | Total assay time* | Hands-on time* | Methylation |
| 500 ng | ~3.5 hours | 1–1.5 hours | 5mC, 5hmC, and 6mA for Fiber-seq |
| *Based on manual preparation of ≤8 samples using SMRTbell prep kit 3.0. QC not included. Optional SRE size selection requires additional time. | |||
How does HiFi WGS library prep work?
PacBio libraries use hairpin adapters (SMRTbell adapters) to create circular DNA templates. This circular structure allows the sequencing polymerase to make multiple passes across both strands of a DNA fragment, producing a highly accurate HiFi read.
Blog
Sequencing 101: from DNA to discovery — the steps of SMRT sequencing
Starting a sequencing project can be daunting. First of all, there are several types of sequencing technologies, each based on unique processes. At PacBio, we use a technology called Single Molecule, Real-Time (SMRT) sequencing, now widely recognized as HiFi sequencing for its ability to deliver highly accurate long reads.
In this post, we walk through the PacBio workflow, highlighting the key steps involved in generating HiFi reads from DNA to discovery.
How to choose the right HiFi WGS prep kit
All PacBio prep kits produce the same SMRTbell template and are compatible with Revio, Vega, and Sequel II/IIe systems. The key differences are throughput, multiplexing flexibility, and workflow scale.
Automation-ready from start to finish
PacBio WGS library prep is designed for automation across leading NGS liquid handling platforms.
Automated in-tip DNA shearing
Genomic DNA for PacBio long-read workflows can be mechanically fragmented (aka sheared) directly using many common NGS liquid handler systems. This simplifies the workflow and saves on both expensive consumables and time on the bench.
Automated, in-tip DNA shearing has been qualified on systems from Hamilton, Tecan, Eppendorf, Opentrons, INTEGRA and Beckman Coulter.
“In-tip” DNA shearing provides:
- Consistent fragment size distributions for WGS
- Reduced hands-on time
- Improved reproducibility
- Seamless integration into automated workflows
Please reach out to your local PacBio team and automation provider or visit our Partner page for more information on DNA shearing protocols.
Qualified automation partners for library prep
PacBio-qualified systems support workflows ranging from 1–96 samples per run.
Qualified vendors and platforms include:
- Beckman Coulter – Biomek i7 Hybrid Workstation
- Eppendorf – epMotion 5075t
- Hamilton -NGS STAR MOA, STARlet, and STAR V
- Tecan – DreamPrep NGS series
- Opentrons – Flex
- Revvity – SciClone G3 NGSx
- SPT Labtech – firefly
- Volta Labs – Callisto
- INTEGRA – Miro Canvas
All systems and protocols are validated through the PacBio Compatible Program to ensure sequencing performance.
Automation buyer’s guide Automated workflow selector
Not ready for full automation?
The Hamilton Microlab Prep, available directly through PacBio, automates upfront sample prep steps:
• SRE
• In-tip shearing
• Post-shear bead cleanup
• Up to 24 samples per run
All protocols are developed and supported by PacBio.
WGS library prep specifications
Check PacBio protocols for the latest guidance on each application
| Feature | SMRTbell prep kit 3.0 | HiFi prep kit 96 | HiFi plex prep kit 96 |
|---|---|---|---|
| Genomic DNA input | 500–3000 ng | 500–2000 ng | 50–300 ng |
| DNA quality1 | 70% ≥10 kb | 70% ≥10 kb | 70% ≥10 kb |
| Workflow time | 3.5 hr (≤8 samples) | 6.5 hr (96 samples) | 6 hr (96 automated) |
| PCR-free | Yes | Yes | Yes |
| Native methylation | Yes | Yes | Yes |
| Recommended shearing | Mechanical or in-tip | Automated in-tip | Automated in-tip |
| Mean WGS read length | 15-20 kb | 15-20 kb | 15-20 kb |
| Multiplexing | ≥24 | ≥96 | 24–384 |
| Automation qualified | Yes | Yes | Yes |
| Instrument compatibility | All HiFi systems | All HiFi systems | All HiFi systems |
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1 It is recommend to use a field inversion electrophoresis system like the Agilent Femto Pulse System or the Yourgene Health Lightbench Discover to measure the fraction of high molecular weight DNA. 2 DNA shearing and library prep workflow time on the Hamilton NGS STAR MOA. Doesn't include time for SRE or ABC protocol. |
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Frequently asked questions about HiFi WGS library prep
PacBio SMRTbell library preparation converts DNA into circular molecules that are optimized for Single Molecule, Real-Time sequencing on PacBio systems. This unique approach enables highly accurate long-read sequencing (HiFi reads), helping researchers unlock deeper insights across genomes, metagenomes, and targeted regions.
HiFi reads combine long read lengths with exceptional accuracy (~99.9%). This allows you to:
- Resolve complex genomic regions
- Detect structural variants with confidence
- Generate highly complete, contiguous genome assemblies
The result is a clearer, more comprehensive view of your biology.
PacBio library prep workflows support a wide range of applications, including:
- Whole genome sequencing (WGS)
- Shotgun metagenomics
- Amplicon sequencing
- Variant detection and genome assembly
These workflows are compatible with multiple sample types, from microbes to human genomes.
DNA input depends on the workflow, but typical requirements include:
- 500 ng – 3 µg for standard whole genome sequencing.
- Lower inputs (ng-level) supported with specialized workflows
High-quality, high-molecular-weight DNA is recommended for optimal results since read length is directly proportional to total HiFi base yield.
Long-read sequencing requires accurate estimations of DNA molarity, which requires:
- A double-stranded DNA quantification assay to measure concentration.
- DNA sizing using automated electrophoresis systems.
Pulse field or field inversion systems provide the most accurate estimation because of the ability to separate high molecular weight DNA. Direct current approaches can provide more of a qualitative assessment but do not accurately measure the size of DNA >15 kb.
Automated high-throughput workflows using in-tip shearing may skip DNA sizing QC because of the reliability and predictability of the DNA shearing once DNA concentration is normalized.
PacBio library preparation is designed to be efficient:
- ~3.5–6 hours total workflow time depending on sample number
- Ability to go from DNA to sequencing-ready library in a single day
Automation options can further reduce hands-on time.
Yes. PacBio supports multiplexing using indexed adapters or indexed templates via primer barcodes, allowing multiple samples to be pooled and sequenced together. This increases throughput and reduces cost per sample
- SMRTbell prep kit 3.0: Flexible workflow for WGS, metagenomics, and amplicons
- HiFi prep kit 96: Streamlined, high-throughput workflow for preparing up to 96 libraries. Designed with extra volumes for automation.
- HiFi plex prep kit 96: Enables multiplexed library prep and barcoding in a single workflow. Designed with extra volumes for automation.
All kits are optimized for generating high-quality HiFi sequencing data
No specialized equipment is strictly required. PacBio workflows support upfront short fragment depletion using SRE and post-library bead-based size selection, which simplifies the process and eliminates the need for gel-based systems while maintaining high performance.
Yes. PacBio workflows are automation-ready, enabling:
- High-throughput processing (up to 96 samples)
- Reduced hands-on time
- Improved reproducibility
Automation makes it easier to scale sequencing projects