PacBio vs Oxford Nanopore: Which Long-Read Sequencing Technology is Right for Your Research

In recent years, the cycle time for second-generation sequencing library construction has seen remarkable improvements. Thesecond-generation short-read sequencingtechnology continues to maintain a dominant position in the sequencing market. However, since its inception in 2008, third-generation sequencing technology has been advancing at an impressive rate. With its unique advantage of long-read capabilities and PCR-free sequencing process, it has enabled the individual sequencing of each DNA molecule. This technology is now widely applied in various areas such as genome assembly, pathogen research, and mutation identification.

In the realm of genomics, Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) have emerged as the leading pioneers inlong-read sequencing. They are actively pushing the boundaries of complex genome analysis, structural variation detection, and real-time biological research. PacBio's Single Molecule Real-Time (SMRT) sequencing technology and Nanopore's nanopore-based electrical signal detection capitalize on their unique principles to overcome the limitations of short-read technologies. These have become essential tools in precision medicine, pathogen monitoring, and evolutionary biology research.

Development of Long-read Sequencing Technology (2008-2012).

Comparison of PacBio and Nanopore Technologies

The core differences between PacBio and Nanopore technologies are reflected in sequencing principles, read length and accuracy, as well as throughput and cost. PacBio employsSMRT sequencingtechnology to record DNA synthesis through fluorescent signals, generating highly accurate HiFi reads. In contrast,Nanopore sequencingdetects DNA sequences using nanopore electrical current, enabling real-time data streaming and ultra-long reads. In terms of throughput and cost, PacBio's Sequel IIe is well-suited for large-scale, high-precision projects, while Nanopore's PromethION is renowned for its flexibility and lower entry cost. Below, a detailed comparative analysis is offered from dimensions of principles, read length and accuracy, and throughput and cost, along with a summarized tabular representation.

PacBio: Detection of Structural Variations and Precision Transcriptome Analysis

PacBio's HiFi reads technology stands out in the analysis of complex genomes, particularly beneficial in the following applications:

1. Structural Variation Detection: HiFi reads accurately identify structural variations such as insertions, deletions, inversions, and translocations, supplying critical data for cancer genomics and rare disease research.
2. Precision Transcriptome Analysis: HiFi reads enable full-length transcript sequencing, capturing RNA isoforms directly and revealing gene expression regulatory mechanisms.
3. Epigenetic Research: PacBio can directly detect DNA methylation, such as 5mC, and other base modifications, providing high-resolution data for epigenetic regulatory network studies.

Nanopore: Real-Time Monitoring and Portability in Various Scenarios

Nanopore's real-time sequencing capacity and portability make it highly advantageous for monitoring dynamic biological processes and in-field applications:

1. Real-Time Pathogen Monitoring: Nanopore supports real-time data streaming, allowing for pathogen genome sequencing and analysis within hours, suitable for rapid response to infectious disease outbreaks.
   Portable Sequencing Scenarios: The lightweight and portable MinION device is suited for genome sequencing in field, polar, and even space environments.
2. Clinical Point-Of-Care Testing (POCT): Nanopore technology enables bedside testing for rapid pathogen diagnostics or patient genome variant analysis.
3. Direct RNA Sequencing: Nanopore technology reads RNA sequences and modifications directly without reverse transcription, offering new perspectives for functional genomics research.

Principle Differences: SMRT Sequencing (Enzyme-Driven) vs. Nanopore Electrical Signal (Electrochemical Detection)

PacBio's SMRT technology leverages zero-mode waveguides (ZMW) and DNA polymerase, where fluorescently labeled dNTPs are excited by a laser to record the base synthesis in real-time. Its primary advantage lies in the production of high-fidelity HiFi reads, achieving single-molecule accuracy levels exceeding >99.9% through cyclic consensus sequencing (CCS) to correct random errors.

Flowchart of HiFi sequence read generation and downstream applications. (Hon, T.,et al., 2020)

Conversely, Nanopore technology operates on electrochemical detection principles, where single-stranded DNA traversing a protein nanopore induces specific current changes, directly translating into sequence information. Nanopore technology requires no amplification or labeling, supporting real-time data flow and direct detection of epigenetic modifications such as methylation. The latest R10 chip, with its dual-reader head design, significantly enhances accuracy in homopolymeric regions.

A schematic diagram of the mechanism of Oxford Nanopore Technologies (ONT) sequencing. (Beckett, Angela H., et al., 2021)

Read Length and Accuracy: PacBio's High-Fidelity Model vs. Nanopore's Real-Time Sequencing Characteristics

PacBio's Sequel IIe platform achieves an average enzyme read length exceeding 70 kb, with HiFi reads reaching 1020 kb, and post-error correction accuracy surpassing 99.9%, ideal for high-accuracy assembly and structural variation detection. Nanopore's PromethION provides single-molecule read lengths up to megabase levels, with an N50 of approximately 35 kb. The R10 chip's initial read accuracy has been improved to 93.8%, and consensus sequences (at 50X coverage) reach Q44 (99.996%).

Throughput and Cost: Comparative Platform Throughput (e.g., PromethION vs. Sequel IIe)

PacBio's Sequel IIe generates 120 Gb of HiFi data per run, fitting medium to large-scale projects, though it entails higher equipment costs and complex sample preparation. Nanopore's PromethION offers a throughput of up to 1.9 Tb per run, supports flexible scaling, and features lightweight equipment (e.g., MinION), catering to budget-constrained or field-based applications.

Through this comparative analysis and the summary table, it is evident that PacBio excels in accuracy and the detection of epigenetic modifications, whereas Nanopore stands out in real-time capability, portability, and ultra-long reads. The choice of technology should be carefully balanced against research objectives (such as assembly integrity and real-time requirements) and budget considerations.

Application Scenarios of PacBio and ONT

PacBio and Nanopore each offer unique advantages across various research domains, with their specific technological characteristics rendering them indispensable in certain settings. PacBio excels in structural variation analysis and transcriptome research due to its high-precision HiFi reads and capacity for epigenetic modification detection. On the other hand, Nanopore's real-time capability and portability provide unmatched utility in field monitoring and on-the-spot clinical diagnostics. Below is a detailed analysis of their applications in specific scenarios.