PacBio is a biotechnology company that developed a DNA sequencing platform. This technology allows scientists to perform sequencing, the process of determining the exact order of the four nucleotides that make up a DNA molecule. By reading the sequence of these nucleotides—adenine (A), cytosine (C), guanine (G), and thymine (T)—researchers gain insight into an organism’s genetic makeup.
Single-Molecule, Real-Time (SMRT) Sequencing
PacBio’s technology is centered on a method called Single-Molecule, Real-Time (SMRT) Sequencing. The process begins by preparing DNA into a circular template known as a SMRTbell. These SMRTbells are loaded onto a SMRT Cell, a chip housing millions of microscopic wells called Zero-Mode Waveguides (ZMWs). Within each ZMW, a single DNA polymerase, the enzyme responsible for copying DNA, is anchored to the bottom.
A single DNA molecule is threaded through the immobilized polymerase, which synthesizes a new, complementary DNA strand. To do this, it incorporates nucleotides tagged with different colored fluorescent dyes. Each time a nucleotide is added, it emits a flash of light that is recorded by a high-speed camera in real time.
This continuous recording of light pulses allows the system to read the sequence of a single DNA molecule directly. Because the DNA template is circular, the polymerase can circle it multiple times. This repeated sequencing of the same molecule generates highly accurate long reads, known as HiFi reads, which have an accuracy rate of over 99.9%.
The Advantage of Long-Read Sequencing
The extensive length of data from PacBio systems provides an advantage in genomic research. While older technologies generate short fragments, PacBio’s long reads can span tens of thousands of nucleotides. This capability is like assembling a puzzle where short reads are tiny, individual pieces, and long reads are large sections already pieced together. This makes final assembly simpler and more accurate.
The ability to read long stretches of DNA is effective for assembling complete genomes. Many genomes contain repetitive sections that confound short-read technologies, leaving gaps in the final assembly. Long reads span these complex regions, enabling scientists to construct a more continuous and gap-free view of an organism’s genetic blueprint.
Long-read sequencing also excels at identifying large-scale genetic changes called structural variants. These changes, such as insertions, deletions, or inversions of large DNA segments, are often missed by short-read methods. Because structural variants are frequently associated with diseases, their accurate detection is useful for genetic research. Long reads can also distinguish between genes inherited from each parent, a process known as phasing haplotypes.
Applications in Scientific Research
Data from PacBio sequencing has many applications in science and medicine. In human genomics, the technology is used to create accurate reference genomes, such as in the Human Pangenome Project, which aims to capture the full spectrum of human genetic diversity. It is also used to diagnose rare genetic disorders by identifying complex mutations that other technologies might not detect. Researchers use HiFi long-read whole-genome sequencing to study pediatric patients with genetic conditions to improve diagnostic rates.
In the field of transcriptomics, which studies the complete set of RNA molecules in a cell, an application called Iso-Seq allows for the sequencing of full-length RNA transcripts. This provides scientists with a clear picture of all the different versions of proteins that a single gene can produce, offering deeper insights into gene function and regulation.
The technology also offers a direct way to study epigenetics, the chemical modifications on DNA that influence gene activity. PacBio sequencing can detect these modifications, such as methylation, during the standard sequencing process without extra lab work. This provides an integrated view of both the genetic sequence and its regulatory landscape. In metagenomics, the technology is used to identify the various species within a complex biological sample, like microbial communities in the human gut or an environmental sample.