What Is HiFi Sequencing and Why Is It Important?

High-Fidelity (HiFi) sequencing, a technology from Pacific Biosciences (PacBio), generates DNA data that is both long and highly accurate. This method provides a more complete and precise view of an organism’s genetic makeup than was previously possible, setting a new standard for genomic research.

The Mechanics of HiFi Sequencing

At the core of HiFi sequencing is a technology called Single Molecule, Real-Time (SMRT) sequencing. The process takes place on a specialized microchip known as a SMRT Cell, which houses millions of microscopic wells called Zero-Mode Waveguides (ZMWs). Inside each ZMW, a single DNA molecule is anchored along with a DNA polymerase, the enzyme responsible for DNA replication. This structure allows for the observation of DNA synthesis as it happens in real time.

To prepare for sequencing, a sample of double-stranded DNA is converted into a circular template called a SMRTbell by attaching hairpin adaptors to both ends. The SMRTbell template is then placed into the ZMWs where the polymerase begins to replicate the circular DNA. As the polymerase adds nucleotides to the new strand, each base emits a distinct fluorescent light, which is recorded to determine the DNA sequence.

The key to “Hi-Fi” quality is the Circular Consensus Sequencing (CCS) method. Because the DNA template is circular, the polymerase can circle it multiple times. A single sequencing run on a molecule can be up to 25,000 bases long, allowing for numerous passes on a shorter DNA insert. By combining the data from these multiple passes, random errors are corrected, generating a final consensus sequence with an accuracy greater than 99.9%.

Achieving High Accuracy with Long Reads

The combination of long read lengths and high accuracy is a significant technological leap. For years, genomic researchers faced a trade-off: short-read technologies offered high accuracy but struggled to assemble complex regions, while early long-read technologies could span these regions but had higher error rates. HiFi sequencing effectively eliminates this compromise, providing reads that are tens of thousands of bases long with an accuracy comparable to the most precise short-read methods.

This data type overcomes some of the most persistent challenges in genomics. Long, accurate reads can span large, repetitive sections of a genome that often confuse assemblers that use shorter reads. This capability is instrumental in creating complete and correct genome assemblies. It allows scientists to resolve large-scale structural variations, which are rearrangements of the genome that can be thousands of bases in size and are often missed by other methods.

HiFi reads are also able to phase haplotypes, meaning they can distinguish between the genetic variants inherited from the maternal and paternal chromosomes. This level of detail is important for understanding how combinations of variants contribute to traits and diseases. The uniform coverage of HiFi sequencing ensures that even difficult-to-sequence regions are accurately represented.

Impactful Uses of HiFi Sequencing

HiFi sequencing is used across a wide range of applications. In de novo genome assembly, it is used to construct high-quality reference genomes from scratch. This has been applied in projects like the human pangenome project, which aims to create a more comprehensive reference that represents human genetic diversity, and in assembling the complete genomes of other species.

The technology is also applied in several other fields:

• Medical genetics: It excels at comprehensive variant detection, identifying everything from single nucleotide variants (SNVs) to large structural variants (SVs), which is valuable for rare disease and cancer research.
• Transcriptomics: A method called Iso-Seq sequences full-length messenger RNA (mRNA) molecules, allowing scientists to identify and quantify different transcript isoforms without computational assembly.
• Epigenetics: HiFi sequencing can directly detect epigenetic modifications like DNA methylation during the sequencing process, eliminating the need for chemical treatments like bisulfite conversion.
• Metagenomics: It helps characterize complex microbial communities by enabling the assembly of complete bacterial genomes directly from mixed environmental samples.

Positioning HiFi in Modern Genomics

HiFi sequencing bridges the gap between the high throughput of short-read sequencing, like that from Illumina, and the extreme read length of other long-read technologies, such as Oxford Nanopore. While short-read sequencing is a cost-effective choice for applications that require counting molecules, its inability to resolve complex genomic regions is a limitation.

HiFi sequencing is often the preferred method when the goal is to achieve a complete genome assembly, characterize complex structural variants, or phase haplotypes. Its high accuracy per individual read distinguishes it from other long-read methods, reducing the computational burden required to achieve a reliable consensus.

The technology is not always a replacement for other methods but can be a complement. In many research projects, a combination of short and long-read technologies is used to leverage the strengths of each. HiFi sequencing’s role continues to expand as its costs decrease and throughput increases with newer platforms like the Revio system, advancing biological research and moving personalized medicine closer to reality.