Last updated: 10 April 2026
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.
To see how these steps come together in practice, watch this short video explaining how HiFi sequencing works:
A visual overview of the steps in the PacBio sequencing workflow and how HiFi reads are generated.
Although each sequencing project is unique, there are five main steps to go from DNA to discovery with SMRT sequencing:
What are the steps of HiFi sequencing?
The PacBio sequencing workflow includes five main steps:
- Sample preparation – extracting high-quality HMW DNA
- Library preparation – creating SMRTbell templates
- Sequencing – generating HiFi reads in real time
- Data analysis – assembling and interpreting sequence data
- Biological interpretation – turning data into discovery
Step 1: Sample prep
Similar to cooking, for the best results, start with the best ingredients. The ideal sequencing starter is high molecular weight (HMW) DNA. PacBio Nanobind technology supports high-quality sample preparation by enabling efficient extraction of HMW DNA from a wide range of sample types, including blood and animal tissue, while preserving long fragment lengths.
Nanobind uses a silica disk-based approach to gently bind and release ultra-long DNA molecules, minimizing shearing while maximizing yield. Solutions like the Nanobind PanDNA kit are designed to support diverse sample inputs in a single workflow, helping streamline project setup. These kits are also compatible with automation, enabling scalable and reproducible sample preparation for high-throughput sequencing projects.
Expert sample wrangler Olga Pettersson (@OlgaVPettersson) of SciLifeLab at Uppsala University, also advises: “Aim for getting molecules as long as you can, as pure as you can, as fresh as you can.”
When in doubt, you can always outsource the task to experts at other labs or sequencing centers, such as our certified service providers.
Step 2: Library prep
Library preparation for all of the major next generation sequencing (NGS) platforms requires the ligation of specific adapter oligos to fragments of the DNA to be sequenced. The DNA has to be fragmented to the optimal length determined by the sequencing technology you are using.
PacBio uses a SMRTbell library format, in which DNA fragments are capped on both sides with ligated hairpin adapters, where the sequencing primers attach. This creates a circular template for the polymerase to navigate. These can be created for libraries of varying insert lengths — from 250 bp to greater than 25,000 bp. Samples can also be barcoded and multiplexed to increase throughput. Learn more about kits for fast and easy library preparation.
PacBio now offers a range of library preparation options tailored to different throughput and application needs. Scalable workflows like HiFi Prep + Plex kits enable efficient processing of up to 96 libraries at a time. For specialized applications, solutions such as Kinnex kits for RNA sequencing support full-length transcript analysis, while ultra-low input workflows like Ampli-Fi enable sequencing from limited starting material. Together, these options provide flexibility across a wide range of experimental designs within the PacBio sequencing workflow.
Step 3: Sequencing
Once your library is prepared, sequencing is performed on PacBio systems like the high-throughput Revio system or the benchtop Vega system, depending on the scale of your project. At the heart of HiFi sequencing is the SMRT Cell, which contains millions of tiny wells called zero-mode waveguides (ZMWs). Single molecules of DNA are immobilized in these wells, and as the polymerase incorporates each nucleotide, light is emitted, and nucleotide incorporation is measured in real time. The reactions are recorded in a format that can then be analyzed using on-instrument tools, as well as additional software.
During sequencing, the system not only determines the DNA sequence but it also captures kinetic information, subtle variations in the timing of nucleotide incorporation. These kinetic signals enable direct detection of base modifications, including 5mC, 5hmC, and 6mA methylation, without additional sample preparation.
HiFi sequencing also extends into multiomic applications, including transcriptomics and chromatin accessibility with Fiber-seq, providing a more comprehensive view of genome function.
SMRT Cells remain the core consumable of HiFi sequencing. The latest HiFi chemistry, SPRQ-Nx, enables multiple uses of the same SMRT Cell, significantly increasing operational efficiency and reducing cost. At scale, this enables HiFi sequencing at approximately $345 per human genome at 20x coverage, making high-accuracy long-read sequencing more accessible and potentially amounting to millions of dollars in cost savings for large projects compared to other sequencing technologies.
Step 4: Data analysis
As mentioned, initial analysis occurs within the instrument itself to provide the sequence output. Secondary analysis can then be performed with SMRT Link software, which features a suite of analytical applications and visualization tools.
Unique to HiFi sequencing is the ability to sequence the same DNA molecule multiple times generating highly accurate long reads, or HiFi reads. For users, like Jeremy Schmutz of the HudsonAlpha Institute of Biotechnology, this is quite beneficial.
“With HiFi reads, we can take the reads and do something with them right away. We don’t have to go through an enormous amount of downstream computation and processing to get to the point of having some sequence that we can evaluate,” he stated.
Depending on what you want to do with your data, there are also a range of tertiary analysis tools you can use, many of which have been developed by users. A growing ecosystem of computational tools and workflows supports downstream analysis, enabling researchers to assemble genomes, detect variants, phase haplotypes, and analyze full-length transcripts with confidence.
Explore example datasets to see the data for yourself.
Step 5: Understanding biology
The final and most exciting part of all good sequencing projects is taking your study from data to discovery by using it to improve understanding of biology. Members of the PacBio community have already greatly expanded knowledge across a huge range of fields. Check out our blog and customer success stories, to see how researchers are using HiFi sequencing to drive new discoveries with meaningful impact.
HiFi sequencing is already delivering important insights across disease research and beyond. In cancer research for example, HiFi data has revealed that double mutations in cis are associated with increased oncogenicity and treatment response in breast cancer, and has enabled the identification of isoforms that improve tumor control and survival in melanoma models.
Together, these examples highlight how the steps of the PacBio sequencing workflow work in concert to translate high-quality data into meaningful biological discovery.
Want to learn more?
Sign up for the latest updates on SPRQ-Nx chemistry and connect with a PacBio scientist directly
Explore other posts in the Sequencing 101 series
Watch the updated HiFi how it works video
Understand the HiFi difference and debunk common sequencing myths
See customer success stories for yourself