A Review of Current Sequencing Technologies

The sequencing landscape has dramatically changed over the last few years. With the significant drop in sequencing costs and the introduction of innovative instruments and companies, selecting an appropriate sequencer has become an increasingly complex task. To better navigate the current offerings of next-generation sequencing (NGS) technologies, we provide a review of leading instrument providers alongside the emerging groups.

Oxford Nanopore Technologies

Oxford Nanopore Technologies (ONT) offers a unique approach to sequencing by directly reading DNA or RNA sequences through nanopores. This enables the detection of simple variants such as single nucleotide variants, but also more complex ones including structural variation and methylation, all from a single experiment. The ease of library preparation and their non-reliance on complex optical systems makes them accessible and affordable technologies, explained Rosemary Sinclair Dokos, SVP of Product and Programme Management at Oxford Nanopore Technologies.

“Our key strengths are the ultra-rich data we provide and the scale at which we can do this, as well as our speed and accessibility,” Dokos emphasized. Their technology, adaptable from the MinION to the PromethION P48, enables diverse project scales. Furthermore, the real-time data analysis allows for rapid results, while the various upgrades offered in consumables and software ensure affordability and accessibility.

Applications and advances

Due to its speed and flexibility, Dokos noted that Nanopore sequencing applications fully cover the spectrum of life science research. In particular, their long-read capabilities make Nanopore sequencing a strong fit for producing telomere-to-telomere genome assemblies, characterizing structural variants, and profiling isoforms. The portability of ONT devices also makes them well-suited for field work, like metagenomic studies, or clinical research wherever samples are collected, such as for cancer or rare disease investigations. Their unique ability to directly detect DNA and RNA epigenetic modifications and sequence native RNA molecules differentiates them from other sequencing platforms.

After a decade of constant innovation, Dokos explained that ONT has notably improved their technology by achieving over 99% simplex and 99.9% duplex read accuracies, and routinely exceeding 100 Gb data outputs per experiment with the PromethION. In addition, Dokos highlighted the continuous enhancement of their EPI2ME analysis platform and the introduction of new products like the updated MinION sequencer and their complete automation solution named Project TurBOT.

Complete Genomics

“Accuracy, flexibility for the user, and lower sequencing cost are the differentiators of our technologies,” stated Rob Tarbox, VP of Product and Marketing at Complete Genomics. The DNA Nanoball technology (DNBSEQ) employed on their instruments utilizes rolling circle replication, which prevents errors common in PCR-based methods. This approach, Tarbox shared, offers high-fidelity amplification without optical duplicates or index hopping, achieving a sequencing accuracy in the Q40 range.

Complete Genomics offers a range of sequencers like the G99, G400, and T7 that provide batch size flexibility with multiple flow cells, enable efficient runs with varied sample numbers, and reduce waiting times and reagent use. “The core strength of our technology is the high-quality sequencing data that is helping to drive down overall cost per sample and make sequencing more readily accessible to groups who could otherwise not take advantage of it,” Tarbox shared.

Applications and advances

The group’s DNBSEQ instruments enable a wide range of applications for scientists including whole-genome sequencing (WGS), whole-exome sequencing (WES), targeted disease sequencing, population genomics studies (human and agriculture), infectious disease research, metagenomic analysis, spatial analysis with STOmics technology, single cell analysis (with third party sample prep from 10x Genomics or Parse Bioscience), and epigenetic applications.

“We continuously strive to innovate sequencing and are introducing a combined instrument and reagent upgrade that will enable Q40 quality sequencing across our fleet of platforms,” Tarbox noted. This year, Complete Genomics is expanding their line of instruments across the full range of sequencing output, from the compact E25 to the mid-output G800 for longer reads, up to the large-scale T20 X2. Additionally, they are introducing the FLT-L50, an instrument that combines MGI liquid handling automation with DNBSEQ sequencers and performs extractions, sample prep, sequencing, and analysis all on the same platform.

Pacific Biosciences

An important feature of Pacific Biosciences’ (PacBio) technology is its HiFi sequencing. Mark Van Oene, Chief Operating Officer at PacBio, noted that HiFi sequencing offers highly accurate, long reads of 15,000–20,000 bases, making it a leading technology for complex genomic studies due to its consistency and 99.9% read accuracy. “The combination of read length and accuracy in HiFi reads provides a more complete view of biology across genomes, transcriptomes, and epigenomes,” stated Van Oene. HiFi sequencing is the core method of PacBio’s Revio long-read sequencing system. For libraries from 1–25 kb, this instrument is capable of providing 90% of bases at Q30+ with simultaneous data on DNA methylation status.

In addition, PacBio offers Onso, a unique short-read technology. Its sequencing-by-binding (SBB™) chemistry allows it to deliver extremely low levels of sequencing errors (Q40+ specification). The system, with its low error rates and innovative SBB chemistry, excels at detecting rare variants and sequencing traditionally challenging areas like homopolymer repeats.

Applications and advances

Van Oene explained that Revio's cutting-edge applications include creating detailed genome assemblies, identifying genetic variants, exploring gene isoforms, and detecting epigenetic modifications. It's also been instrumental in projects like the complete human genome and studying tandem repeats. Revio’s launch in 2023 led to the enhanced scale and lowered cost of HiFi sequencing, enabling the sequencing of 1,300 human genomes annually at less than $1,000 each. The recent SMRT Link v13.0 update on the system introduced adaptive loading, run preview, and support for broader applications.

In contrast, Van Oene noted that Onso excels in detecting low-level variants in liquid biopsies, refining reference genomes, and identifying rare sequences in diverse samples. This is particularly beneficial in fields such as metagenomics and CRISPR editing with Onso’s low false positive rates and efficient sequencing. PacBio's recent acquisition of Apton Biosystems is expediting the launch of their future high-throughput, short-read sequencer, which utilizes SBB chemistry and achieves billions of Q40+ reads.

Illumina

As one of the first entrants into the field of next-generation sequencing, Illumina continues to be a leading force in the life sciences sector. Today, the company offers a wide array of instruments from the iSeq to the NovaSeq. Each platform is built on their well-known sequencing by synthesis (SBS) technology that produces high-quality, reliable results. Their devices have been adopted in labs around the world and have enabled countless scientific breakthroughs.

Applications and advances

Illumina's sequencing platforms support a wide array of applications making them suitable tools across diverse research fields. However, some more prominent applications run from single-cell sequencing to population genomics. The scalability of Illumina sequencers also allows for the deep sequencing required by many in-depth genomic studies. This includes studying cell-free DNA, circulating tumor DNA, and some clinical operations. Through extensive partnerships, diverse technologies from groups like NanoString and Olink are also seamlessly integrated into Illumina's sequencing platforms.

The most recent advancement to Illumina’s portfolio is the release of their NovaSeq X and X Plus. These devices are their highest throughput sequencers yet and come equipped with the DRAGEN Bio-IT Platform for immediate data processing. Illumina has also begun using their XLEAP-SBS chemistry on newer instruments, which boasts faster and more accurate enzymes. The introduction of the Illumina Complete Long Reads (ICLR) technology is another development that allows users to generate long-read information on existing short-read instruments. Additionally, continual updates to the NextSeq 1000/2000 series instruments further increase the capabilities of their medium-throughput devices and increase the range of various applications.

Newer Entrants

Element Biosciences

Since its inception, Element Biosciences has been a notable innovator with a mission to democratize access to genomic information. Their flagship instrument, the AVITI system, is a powerful, short-read benchtop sequencer that has seen many technical advancements since its initial release. The device runs on their proprietary avidity sequencing chemistry, which improves the stability of the sequencing complex and increases accuracy through a multivalent approach, using nucleotide analogs called avidites. Additionally, avidity sequencing reduces reagent consumption and enhances data accuracy by minimizing sequencing signal noise. Element recently announced plans for the first-ever “integrated biology platform” known as AVITI24. The new instrument is designed to perform the detection of RNA, proteins, morphology, and DNA data for a comprehensive, multiomic analysis, which was termed In Vitro 3D ABC Sequencing.

Ultima Genomics

The latest entrant into the field of sequencing instruments is Ultima Genomics. Their high-throughput sequencer, the UG 100, was recently given a full release to the public. Boasting the ability to process up to 20,000 human genomes per year, the UG 100 was highlighted for its output and 24/7 run capabilities. Additionally, the system comes with a low price of approximately $1 per gigabase (Gb), translating to around $100 for a complete human genome. This makes it especially appealing to large sequencing labs that can supply the necessary volume of samples. Recent advances in Ultima Genomics' technology emphasize improvements in handling complex genomic elements like homopolymers, leading to enhanced indel accuracy. Furthermore, the introduction of Ultima's novel sequencing method, PPMseq, represents the ability to achieve high accuracy without the need for over-sequencing or unique molecular indices.

Singular Genomics

Singular Genomics is making significant strides in the field of genomics. The company's G4 Sequencing System was designed with input from scientists and clinicians to address the limitations of existing sequencing tools. This benchtop platform distinguishes itself through its unique flow cell design, scalability of runs, and customizable sequencing options. Singular Genomics has continuously advanced the G4's capabilities through improvements in quality, flow cells, and throughput. Looking ahead, Singular released their G4X Spatial Sequencer, which aims to combine in situ spatial multiomics and NGS in one device. The G4X Spatial Sequencer employs two sequencing modes to target specific RNA sequences and gene expression, as well as incorporates protein imaging and fluorescent H&E staining for tissue analysis. Current G4 sequencer users can anticipate having the G4X as an available upgrade by the end of the year.