Last updated: 03 April 2026
Welcome to the Sequencing 101 blog series — where we will provide introductions to sequencing technology, genomics, and much more. If you’re not immersed in the field of DNA sequencing, it can be challenging to keep up with the rapid evolution among all the platforms and technologies on the market. Let’s start with a quick overview of how these different technologies came about — and how each is used today.
The evolution of PacBio HiFi sequencing technology, from early SMRT systems to recent advances in throughput, accessibility, and sequencing chemistry.
First generation sequencing — starting the era of genomics
The process of Sanger sequencing.
DNA sequencing as we know it originated in the late 1970s, when Frederick Sanger at the MRC Centre in Cambridge developed a gel-based method that combined a DNA polymerase with a mixture of standard and chain-terminating nucleotides, known as ddNTPs. Mixing dNTPS with ddNTPs causes random early termination of sequencing reactions during PCR. Four reactions are run, each with the chain-terminating version of only one base (A, T, G or C). When visualized with gel electrophoresis, one reaction per lane, the fragments are sorted by length, allowing the DNA sequence to be read off base by base. This technique was revolutionary at the time, enabling sequencing of 500–1,000 bp fragments. However, since the original method used radioactive ddNTPs and X-rays, it was less than ideal for widespread use.
By the 1980s, Sanger’s original method had been automated by scientists at Caltech and commercialized by Applied Biosystems. Radioactive ddNTPs were replaced with dye-labelled nucleotides and large slab gels were replaced with acrylic-finer capillaries. Scientists could now simply feed prepared DNA into a machine and view the results of fluorescence-based reactions on an electropherogram. This technology, which was continuously improved over the years, served as the bedrock of the Human Genome Project. Today, automated Sanger sequencing is still in use, primarily in clinical labs where it is common to have low throughput, higher per-sample costs, and sequencing reads 500–1,000 bp in length.
But even after the Human Genome Project, the cost of automated Sanger sequencing — also known as capillary electrophoresis — remained too high to enable the kind of large-scale sequencing projects envisioned by scientists. By the mid-2000s, remarkable efforts had been made to bring down the costs of sequencing. Driven largely by grants from the National Human Genome Research Institute (NHGRI), labs around the world tested out new methods for higher-throughput sequencing, using concepts as diverse as electronics, physics, and magnetics.
Second generation sequencing — short reads become fast and efficient
One key player in the advent of next-generation sequencing (NGS) was a UK-based company called Solexa, which was later acquired by Illumina. The key innovation of the Illumina platform was “bridge amplification,” which allows the formation of dense clusters of amplified fragments across a silicon chip. Amplification of the original single molecule into a large cluster of many copies is what makes it possible to detect a fluorescent signal as a single dNTP is added one at a time, as sequencing proceeds by synthesis. Over time, the number of clusters that could be read simultaneously grew tremendously, and Illumina instruments became the first commercially available massively parallel sequencing technology. Other tools developed around the same time, such as the Ion Torrent platform, became part of the NGS landscape as well. NGS platforms are the dominant type of sequencing technology used today. Their high capacity allows for sequencing at very low cost. They are limited, however, in read length; NGS platforms typically produce reads of ~50–500 bp in length. This makes them an excellent fit for resequencing projects, SNP calling, and targeted sequencing of very short amplicons.
Third generation sequencing — the rise of long reads
However, short reads are not suitable for all sequencing projects. Another approach that was supported by the so-called $1,000 genome grants from NHGRI was Single Molecule, Real-Time (SMRT) sequencing from PacBio. This technique uses miniaturized wells, known as zero-mode waveguides, in which a single polymerase incorporates labeled nucleotides and light emission is measured in real time. A different single-molecule approach to long-read sequencing, using pore-forming proteins and electrical detection, was adopted by Oxford Nanopore Technologies (ONT).
Watch this short video to learn how SMRT sequencing works.
SMRT sequencing has a number of advantages. Most notable, perhaps, is its ability to produce long reads — tens of thousands of bases long — in a single read. These long reads make it possible to span large structural variants and challenging repetitive regions that confound short-read sequencers because their short snippets cannot be differentiated from each other during assembly. Another advantage is low GC bias, which allows PacBio systems to sequence through extreme-GC at AT regions that cannot be amplified during cluster generation on short read platforms. A third advantage is the ability to detect DNA methylations while sequencing, since no amplification is done on the instrument.
Short-read sequencing produces reads 50–500 base pairs in length, which can lead to sequence gaps and incomplete assemblies, known as draft genomes. Highly accurate long-read sequencing from PacBio produces reads tens of kilobases in length, creating overlaps which allow for the generation of complete genome assemblies.
As scientists began to work with SMRT sequencing — sometimes known as third-generation sequencing — they realized that it had particular value for applications including de novo genome sequencing, phasing, detection of structural variants, epigenetic characterization, and sequencing of the transcriptome without the need for assembly. Technology improvements over time increased the throughput and accuracy of SMRT sequencing platforms, bringing their costs in line with NGS platforms for many types of projects. Now, SMRT sequencing delivers industry-leading accuracy through HiFi sequencing, and it is being used around the world to produce reference-grade genomes for microbes, plants, animals, and people.
Comparison of short-read sequencing and PacBio HiFi long-read sequencing, showing how long, high-accuracy reads reduce gaps and enable more comprehensive variant detection.
What’s new in long-read and HiFi sequencing
Since the inception of SMRT sequencing, the pace of innovation has only continued to accelerate. Recent advances from PacBio are building on this foundation to further improve accuracy, scale, and accessibility, while enabling new applications that were not previously possible.
The evolution of PacBio HiFi sequencing technology, from early SMRT systems to recent advances in throughput, accessibility, and sequencing chemistry.
New systems expand scale and accessibility
Recent platform developments have made HiFi sequencing more scalable and accessible across a wider range of research environments.
The Revio system supports high-throughput sequencing at scale, enabling large studies such as population genomics and cohort-based research by significantly increasing data output to up to 480 Gb per run with only 500 ng of input.
Alternatively, the benchtop Vega system expands access to HiFi sequencing by offering a more flexible and approachable platform for labs looking to bring long-read sequencing in-house. Powered by the same HiFi technology, the Vega system enables multiomic insight with every run, including on-instrument methylation detection without additional library prep. Together, these systems help extend the reach of high-accuracy long-read sequencing across both large centers and smaller labs.
Scientific recognition reflects growing adoption
The impact of long-read sequencing continues to gain recognition across the scientific community. Nature Methods named long-read sequencing its 2022 Method of the Year, underscoring its importance in advancing genomics research.
As adoption increases, HiFi sequencing is being used to study complex genomic regions, structural variation, and epigenetic modifications with greater resolution and confidence.
Advances improve cost efficiency and expand applications
Ongoing innovation in sequencing chemistry is further increasing efficiency and expanding what researchers can achieve with HiFi sequencing.
The latest SPRQ-Nx sequencing chemistry is designed to deliver HiFi whole-genome sequencing at approximately $345 per 20x human genome. By enabling multiple uses per SMRT Cell while maintaining output per run, SPRQ-Nx improves efficiency and lowers cost.
At the same time, continued development is expanding multiomic capabilities, including support for 5hmC detection for epigenetic profiling. These advances also complement emerging approaches such as Fiber-seq, which enables single-molecule analysis of chromatin structure and genome organization.
Recent studies highlight real-world impact
A growing body of recent research demonstrates how HiFi sequencing is delivering measurable impact across applications including clinical research and translational genomics.
Children’s Mercy Kansas City recently implemented HiFi sequencing as a single assay to help accelerate answers for pediatric patients with suspected genetic disease. In their landmark study in JAMA Pediatrics, these researchers demonstrate how HiFi sequencing can transform pediatric disease discovery by delivering 10% higher success over all prior testing methods, helping to provide families with findings in less than one month compared to three months, and reducing the need for multiple stressful and costly rounds of testing.
As another example, a multi-center study from the HiFi Solves EMEA Consortium showed that HiFi sequencing can accurately resolve some of the most challenging regions of the genome. Across 86 individuals and 125 known pathogenic variants, HiFi sequencing identified all clinically relevant variants, including those in complex and previously inaccessible regions. This work underscores the potential for a single HiFi genome to replace multiple targeted tests, helping to streamline workflows while improving diagnostic confidence.
Together, these studies reflect a broader trend as HiFi sequencing continues to move from research into real-world applications, enabling faster answers, greater accuracy, and more comprehensive insights for researchers and healthcare teams alike.
Want to learn more?
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