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Third-generation sequencing

Third-generation sequencing (TGS), also known as long-read sequencing, refers to advanced DNA and RNA sequencing technologies that analyze individual nucleic acid molecules in real time without prior PCR amplification, generating reads ranging from thousands to millions of base pairs in length. Introduced in the early 2010s, TGS emerged as a response to the limitations of second-generation sequencing (SGS), such as short read lengths (typically 150–300 bp) that hinder accurate assembly of repetitive genomic regions, detection of structural variants, and phasing of haplotypes. The primary platforms driving TGS are (PacBio) and (ONT). PacBio employs single-molecule (SMRT) sequencing, where incorporates fluorescently labeled in zero-mode waveguides, allowing continuous, long-read sequencing with average lengths of 15–20 kb and accuracy up to 99.95% using circular consensus methods. In contrast, ONT uses nanopore-based sequencing, passing DNA or RNA through protein nanopores to measure changes in ionic current, enabling ultra-long reads up to 4 Mb, analysis, and direct detection of base modifications like without conversion. Notable devices include PacBio's Revio system, which achieves up to 360 Gb per day, and ONT's portable (up to 50 Gb) and high-throughput PromethION (up to 13.3 Tb). TGS offers significant advantages over SGS, including improved de novo genome assembly, resolution of complex structural variations (e.g., insertions, deletions, inversions), and comprehensive transcriptomics through full-length isoform identification. It also facilitates epigenomic studies by directly sequencing modified bases and supports applications in , cancer , and rapid pathogen detection, such as during the and outbreaks. Despite these benefits, challenges persist, including higher per-base error rates (5–15% before correction) and the need for sophisticated computational tools to handle long reads. Ongoing advancements in TGS, such as improved base-calling algorithms and approaches combining TGS with SGS, promise to reduce costs and enhance accuracy, with recent developments including PacBio's SPRQ-Nx chemistry and ONT's PromethION Plus flow cells further advancing throughput and multiomic capabilities, positioning it as a for precision medicine and large-scale genomic projects.

Overview

Definition and key characteristics

Third-generation sequencing (TGS), also referred to as long-read sequencing, encompasses a suite of DNA and RNA sequencing technologies designed to generate extended sequence reads ranging from 10 kilobases (kb) to over 1 megabase (Mb) in length, facilitating the interrogation of complex genomic regions such as repetitive sequences and structural variants that challenge shorter-read methods. These platforms achieve this by directly sequencing native nucleic acid molecules, bypassing the need for fragmentation or amplification, which minimizes biases associated with polymerase chain reaction (PCR) processes. Key characteristics of TGS include single-molecule resolution, where individual DNA or RNA strands are sequenced without clonal amplification, enabling the detection of rare variants and heterogeneity at the molecular level. Real-time data acquisition is another hallmark, allowing immediate base calling during the sequencing process rather than post-sequencing assembly. Additionally, TGS supports the native detection of epigenetic modifications, such as DNA methylation, by analyzing kinetic signatures or ionic current changes inherent to the unmodified molecule, providing insights into gene regulation without separate bisulfite conversion steps. In comparison to earlier generations, first-generation produces short reads of approximately 800–1,000 base pairs () with low throughput, suitable for targeted validation but inefficient for large-scale . Second-generation next-generation sequencing (NGS) methods, such as Illumina platforms, yield high-throughput short reads of 100–300 but rely on amplification, introducing biases and complicating the resolution of repetitive or low-complexity regions. TGS shifts the paradigm by prioritizing read length and structural fidelity over per-base accuracy (which has improved to >99% in recent iterations), though it initially traded higher error rates for these benefits. TGS technologies first emerged in the late 2000s, with launching its single-molecule real-time (SMRT) platform in 2010, and by 2025, ultra-long reads exceeding 4 Mb have been achieved, particularly with nanopore-based systems.

Historical development and generational context

The development of DNA sequencing technologies traces back to the first generation, which emerged in the 1970s with Frederick Sanger's chain-termination method introduced in 1977. This technique, relying on dideoxynucleotides and , produced reads of less than 1 kb and was pivotal for the , where it enabled the sequencing of the 3 billion base pair over 13 years from 1990 to 2003. The second generation of sequencing, launched in the mid-2000s, shifted to high-throughput platforms that amplified and sequenced millions of short DNA fragments in parallel, drastically reducing costs and time. Key early systems included 454 Life Sciences' pyrosequencing instrument in 2005, which generated 400–500 bp reads, and Illumina's Genome Analyzer in 2006, utilizing reversible terminator chemistry for even higher output. These technologies dominated the 2000s and 2010s, facilitating large-scale genomic studies but struggling with repetitive sequences and structural variants due to short read lengths. Early single-molecule sequencing without amplification began in 2008 with Helicos BioSciences' true single-molecule sequencing platform, but long-read TGS technologies started in 2010 with ' launch of its PacBio RS system, introducing single-molecule (SMRT) sequencing for continuous long-read generation. entered the field in 2014 with , a USB-powered device that allowed portable, sequencing of ultra-long strands. Subsequent key events in the and focused on enhancing TGS accuracy and utility. In 2015, Oxford Nanopore released for widespread early access, emphasizing its portability for field applications. The brought major accuracy boosts, including ' 2019 introduction of HiFi reads through circular consensus sequencing, yielding >99% accuracy for 15–20 kb reads, and Oxford Nanopore's 2020 rollout of adaptive sampling for real-time targeted enrichment without library modifications. TGS represents a generational shift by overcoming second-generation limitations in assembling complex genomes, particularly in repetitive regions and structural variant detection, through reads often exceeding 10 . By 2025, TGS adoption has accelerated via hybrid workflows combining short- and long-read data, with the market valued at approximately USD 881 million in 2025 and exhibiting a of over 20% through 2032, reflecting its integration into routine . From 2023 to 2025, advancements emphasized precision, such as Oxford Nanopore's R10.4 pores paired with Q20+ chemistry, achieving >99% raw read accuracy for , further refined by AI-driven error correction in the Dorado basecaller for consistent high-quality outputs. In October 2025, Oxford Nanopore announced the PromethION Plus flow cell, which significantly increases output for large-scale genomic studies.

Technologies

Pacific Biosciences SMRT sequencing

Pacific Biosciences' Single Molecule, Real-Time (SMRT) sequencing technology enables the direct observation of by individual molecules, providing long-read sequencing data with high accuracy through consensus generation. Introduced commercially in 2010 following foundational research published in 2009, SMRT sequencing has evolved to support scalable genomic applications, including the 2022 launch of the Revio system, which facilitates sequencing of human genomes at 30x coverage in approximately 24 hours using a single SMRT Cell. Recent 2025 updates include the announcement of SPRQ-Nx chemistry for enhanced throughput on Revio, with beta testing beginning in November 2025. The core mechanism relies on zero-mode waveguides (ZMWs), nanoscale wells etched into a fused silica that confine excitation light to a volume of about 20 nm in depth, allowing real-time optical detection of incorporation without illuminating the entire reaction volume. A highly processive , such as a modified phi29, incorporates fluorescently labeled —each with a distinct attached to the terminal —into a growing DNA strand complementary to the template. As incorporation occurs, the fluorophore is cleaved and diffuses away, producing a characteristic light pulse captured by high-speed cameras; the sequence of pulses corresponds to the DNA template sequence. To enable multiple observations of the same molecule, the template is prepared as a circular SMRTbell , where the repeatedly traverses the insert region, generating subreads that can be aligned for consensus. The workflow begins with sample preparation to create SMRTbell libraries: high-molecular-weight DNA is sheared to desired fragment sizes (typically 10-20 kb for HiFi applications), ends are repaired and A-tailed, and adapters are ligated to form the double-stranded circular template. Libraries are then bound to and loaded onto SMRT Cells containing millions of ZMWs (8 million on IIe, 25 million on Revio), where sequencing occurs in . Raw data produce continuous long reads (CLR) from single passes or high-fidelity (HiFi) reads via circular consensus sequencing (), where multiple subreads (typically 10-30 passes) are computationally aligned to generate a . SMRT sequencing specifications vary by instrument and chemistry. On the , CLR reads exceed 20 , while HiFi reads average 15-20 with >99% accuracy (Q30 or better). The Revio system enhances throughput, yielding up to 120 of HiFi data per SMRT Cell in a 24-hour run at 15-20 read lengths, supporting two phased 20x human genomes per cell. HiFi accuracy derives from over multiple passes, approximated by the error rate formula: \text{error rate} \approx \frac{\text{initial error}}{\sqrt{n_{\text{passes}}}} where n_{\text{passes}} is the number of cycles (10-30 typical), reducing random errors from the ~13-15% raw rate to >99.9% at 10 passes.

Oxford Nanopore sequencing

Oxford Nanopore sequencing, developed by Oxford Nanopore Technologies (ONT), utilizes a protein nanopore embedded in a membrane to detect nucleic acid sequences through changes in ionic current. In this method, DNA or RNA molecules are driven through the nanopore by an applied voltage, while a motor protein, such as a helicase, controls the translocation speed to approximately 450 bases per second, allowing sufficient time for signal detection. As the nucleotide sequence passes through the nanopore—typically variants like R9 or R10—the partial blockage of the pore by each base or k-mer (short sequence motif) generates distinct disruptions in the ionic current flowing across the membrane. These current variations are recorded as a time-series signal, which is then analyzed to infer the underlying sequence. The workflow begins with library preparation, where native barcoding is ligated to DNA or RNA fragments to enable multiplexing of up to 96 samples without amplification, preserving epigenetic modifications. Prepared libraries are loaded into flow cells containing thousands of nanopores, and sequencing occurs on portable devices like the MinION or high-throughput platforms such as the PromethION. During or post-sequencing, raw current signals undergo real-time basecalling using AI-driven models like Dorado, which employs neural networks to translate signals into FASTQ-formatted sequences with integrated modification calling. This process supports both single-end and duplex modes, where complementary strands are sequenced for consensus accuracy. Key specifications include ultra-long read lengths exceeding 1 megabase, enabling assembly of complex genomes without fragmentation. Raw single-read accuracy typically ranges from 90-95%, but advances in chemistry, such as the Q20+ kits introduced post-2023, achieve over 99% consensus accuracy through duplex basecalling. Throughput varies by device: , launched in 2014 as a USB-powered, palm-sized sequencer for field use, yields up to 48 gigabases per flow cell, while a single PromethION flow cell delivers up to 290 gigabases in 72 hours. Recent 2024-2025 updates incorporate adaptive sampling, a software feature that enables real-time targeted enrichment by selectively slowing or rejecting off-target reads during sequencing, and the introduction of PromethION Plus Flow Cells in limited release in Q4 2025 for higher output. A distinctive feature is the direct detection of base modifications like 5-methylcytosine (5mC) and N6-methyladenine (6mA) by modeling characteristic current signal perturbations, eliminating the need for bisulfite conversion or other harsh treatments that can damage DNA. This native approach integrates modification calling during basecalling, providing simultaneous sequence and epigenome data at single-molecule resolution with accuracies exceeding 90% for 5mC in high-coverage regions.

Emerging and alternative platforms

While and Oxford Nanopore dominate third-generation sequencing (TGS), several emerging platforms explore alternative single-molecule approaches to address limitations in throughput, accuracy, and cost, with recent 2025 updates including enhancements to established systems like PacBio's SPRQ-Nx chemistry and ONT's PromethION Plus. These technologies often integrate solid-state or detection methods but remain in developmental stages with limited adoption as of 2025. One influential early platform was Helicos BioSciences' HeliScope, launched in 2009 as the first true single-molecule sequencing (tSMS) system. This technology immobilized DNA strands on a flow cell surface and used fluorescently labeled reversible terminators for direct imaging of individual molecules, bypassing amplification biases common in earlier generations. Although Helicos filed for bankruptcy in 2012, its demonstration of unbiased, high-throughput single-molecule readout paved the way for subsequent TGS innovations by proving the feasibility of direct nucleic acid interrogation without cloning. Quantapore represents a solid-state nanopore alternative, leveraging silicon nanopores integrated with CMOS chips for massively parallel detection. The platform threads single DNA molecules through nanopores while using optical readout to monitor ionic current changes and base-specific signals, aiming for long reads and reduced costs through scalable chip fabrication. As of 2025, Quantapore remains focused on research prototypes without widespread commercial availability, emphasizing hybrid integration for proteomics and genomics applications. Stratos Genomics' sequencing by expansion (SBX) technology, acquired by Roche in 2020, introduces a novel amplification-free method using Xpandomers—synthetic polymers that biochemically convert and expand DNA templates by over 50-fold to encode sequence information with high signal-to-noise ratios. These expanded molecules are then sequenced via nanopore sensing of reporter codes, combining short-read accuracy with long-range phasing potential. Unveiled by Roche in February 2025, SBX is in development with demonstrated F1 scores >99.8% for single nucleotide variants (SNVs) and >99.7% for insertions/deletions (InDels) in whole genome samples, along with collaborations for clinical validation. In October 2025, Roche announced major advances, including a Guinness World Record for sequencing a human genome in under 4 hours, highlighting its speed and reliability, though it holds no significant market share yet and prioritizes cost-effective scalability.

Advantages

Extended read lengths and structural resolution

Third-generation sequencing (TGS) technologies, such as those from and Oxford Nanopore, produce reads typically exceeding 10 kilobases (kb) in length, enabling them to span repetitive genomic regions that fragment short-read next-generation sequencing (NGS) assemblies. This capability resolves ambiguities in complex structures like segmental duplications and tandem repeats, which often exceed the 100-300 limits of NGS reads. For instance, the telomere-to-telomere (T2T) assembly of the CHM13 in 2022 achieved a fully contiguous 3.055 billion by leveraging high-fidelity long reads to bridge previously unresolvable gaps in centromeres and acrocentric regions. These extended read lengths facilitate superior haplotype phasing. In diverse human populations, TGS has enabled phasing of a high proportion of structural variants across haplotypes, revealing allele-specific configurations in repetitive loci that NGS struggles to disambiguate. Additionally, TGS excels at detecting large insertions and deletions (indels) greater than 50 base pairs, capturing full event spans that short reads misalign or overlook, thus improving resolution of structural variants contributing to phenotypic . In bacterial , TGS routinely assembles over 99% of closed circular chromosomes into a single contig, contrasting with NGS's typical fragmentation into dozens of contigs due to repeats and . Recent 2025 analyses of eukaryotic assemblies demonstrate improvements in gap closure rates compared to hybrid NGS approaches, particularly in polyploid crops and model organisms where long reads scaffold heterochromatic regions without additional mate-pair data. This inherent scaffolding power of long reads eliminates the need for paired-end libraries, streamlining workflows for high-resolution structural annotation.

Native detection of molecular modifications

Third-generation sequencing technologies, such as Pacific Biosciences SMRT and Oxford Nanopore, enable the direct detection of epigenetic modifications like 5-methylcytosine (5mC) and N6-methyladenine (6mA) in native DNA molecules, avoiding the need for bisulfite conversion or other chemical treatments that can introduce artifacts and DNA damage. In these platforms, modifications are identified through disruptions in sequencing signals: Oxford Nanopore detects them as shifts in ionic current blockade as the modified base passes through the nanopore, while PacBio SMRT sequencing observes variations in polymerase kinetics, such as altered inter-pulse durations (IPD) and pulse widths during nucleotide incorporation. This native approach preserves the integrity of the DNA and allows simultaneous sequencing and modification profiling at single-molecule resolution. Specialized software tools facilitate the analysis of these signals for accurate modification calling. For Oxford Nanopore data, Nanopolish employs a (HMM) trained on signal patterns to distinguish modified from unmodified bases, computing a modification probability score as the log-likelihood ratio of the modified versus unmodified models: \text{Score} = \log \left( \frac{P(\text{data} \mid \text{modified})}{P(\text{data} \mid \text{unmodified})} \right) A positive score above a (typically ) indicates evidence of modification, enabling site-specific calling of 5mC and 6mA. Similarly, PacBio's SMRT-Link software performs base modification detection by modeling kinetic signatures, identifying motifs associated with 4mC, 5mC, and 6mA through IPD ratio analysis and Phred-scaled quality scores. These tools support comprehensive methylome profiling without amplification biases. By 2025, these methods have achieved high accuracy for 6mA detection in prokaryotic genomes, with PacBio SMRT-Link reaching an F1 score of 0.958 (equivalent to >95% balanced accuracy) in wild-type Pseudomonas syringae, demonstrating low false positive rates at the site level. This precision enables single-molecule epigenome mapping, revealing heterogeneous modification patterns across individual DNA strands and facilitating studies of bacterial restriction-modification systems. Such capabilities extend to broader epigenetic applications, including the integration with structural variant detection for comprehensive genome annotation.

Real-time processing and portability

Third-generation sequencing (TGS) platforms, particularly those from Oxford Nanopore Technologies (ONT), enable real-time basecalling, where raw electrical signals from nanopores are converted into nucleotide sequences as the DNA or RNA strand transits the pore, allowing immediate data analysis without post-sequencing delays. This capability supports adaptive sampling, a process in which the sequencer dynamically rejects off-target DNA molecules and directs the process toward regions of interest, such as specific genes, enabling users to stop sequencing upon reaching target coverage and optimize resource use. For instance, basecalling occurs in real time via software like Guppy, which processes signals on-the-fly to inform decisions during the run. The portability of TGS devices further enhances their utility in field settings, exemplified by ONT's , a compact sequencer weighing approximately 87 grams and powered via USB connection to a or , facilitating deployment in remote or resource-limited environments. This design supports on-site sequencing without reliance on laboratory infrastructure, making it ideal for rapid response scenarios. Real-world applications include the 2015 Ebola outbreak in , where devices enabled portable sequencing of virus samples, yielding results in less than 24 hours from sample receipt, with the sequencing itself taking 15–60 minutes. More recently, in 2025, a nanopore-based platform was used for near real-time of wheat stem rust lineages in agricultural fields, allowing on-site detection of fungicide sensitivity to inform immediate crop management decisions. To facilitate real-time workflows, ONT's EPI2ME integrates cloud-based or local pipelines that process during sequencing runs, supporting applications like adaptive enrichment and preliminary variant calling. For short reads, basecalling latency is minimal, often enabling decisions within seconds to minutes, which is critical for time-sensitive tasks such as pathogen identification. Streaming basecalling, enhanced by GPU acceleration, further improves efficiency; for example, neural network-based tools like those in achieve up to 4-fold speedups on a single GPU compared to CPU-only processing, with scalability to multi-GPU setups yielding even greater performance gains for live runs.

Challenges

Error rates and correction strategies

Third-generation sequencing (TGS) technologies are characterized by higher raw rates than short-read methods, primarily due to the challenges of single-molecule detection without . In (PacBio) single-molecule real-time (SMRT) sequencing, continuous long reads (CLR) exhibit error rates of approximately 8-15%, stemming from polymerase kinetics and signal noise during single-pass traversal of the DNA molecule. These errors are predominantly substitutions and insertions/deletions (indels), with random distribution but occasional systematic biases in repetitive regions. In contrast, (ONT) sequencing shows raw error rates of 5-15%, with systematic homopolymer errors being particularly prominent; these arise from imprecise ionic current measurements in stretches of identical bases, often leading to over- or underestimation of repeat lengths by 5-10%. Post-correction strategies can reduce ONT errors to 1-2% at level, while PacBio's high-fidelity (HiFi) reads, derived from multiple passes, achieve error rates below 0.1%. Correction strategies for TGS errors leverage both platform-specific consensus mechanisms and external data integration. PacBio's circular consensus sequencing (CCS) generates HiFi reads by repeatedly sequencing the same molecule (typically 10-20 passes), yielding accuracy that follows the binomial model for error reduction: \text{Consensus accuracy} = 1 - (1 - p)^n where p represents single-pass accuracy (around 85%) and n is the coverage depth or number of passes. This approach corrects random errors effectively but requires sufficient subread coverage to resolve systematic issues. Hybrid polishing combines TGS assemblies with high-accuracy short-read next-generation sequencing (NGS) data; for instance, NextPolish aligns Illumina-like short reads to long-read contigs and iteratively corrects single-nucleotide variants (SNVs) and indels, achieving up to 99% polishing accuracy in microbial genomes. Advancements in have further refined error correction, especially for ONT. Deep learning basecallers like , an open-source PyTorch-based model, process raw signal data to predict bases with reduced indel rates, outperforming earlier (RNN) models by 2-5% in accuracy on R9 pores. By 2025, ONT's Q20+ kits, incorporating R10.4 pore chemistry, have lowered raw error rates to under 1% (Q20+ modal accuracy >99%), while enhancements on R10 pores enable up to 99.75% single-read accuracy through optimized convolutional architectures. These strategies collectively address TGS error profiles, enabling reliable downstream applications like assembly with minimal residual bias.

Throughput, cost, and scalability issues

Third-generation sequencing (TGS) platforms, such as those from (PacBio) and (ONT), offer significant advantages in read length but face notable limitations in throughput compared to second-generation sequencing (NGS) systems like Illumina's NovaSeq X. The PacBio Revio system achieves up to 480 Gb of high-fidelity (HiFi) reads per day, enabling the equivalent of approximately 2,500 human whole genomes annually when accounting for typical coverage requirements. Similarly, a single ONT PromethION flow cell generates up to 200 Gb of output, though full PromethION 24 systems can scale to 2.4–4.8 Tb across 24 flow cells in a 72-hour run. In contrast, NGS platforms like the NovaSeq X Plus deliver up to 16 Tb per dual-flow-cell run, often achieving terabase-scale output per day in high-throughput configurations, making TGS generally 10-100 times lower in daily volume for large-scale projects. This disparity arises from TGS's reliance on single-molecule detection, which prioritizes length over parallelization density, limiting its suitability for population-scale studies without extensive . Cost remains a key barrier for TGS adoption, though prices have declined steadily into 2025. ONT sequencing costs approximately $345 per as of 2025, with PromethION flow cells priced around $900–$1,000 for 100–200 Gb outputs, translating to roughly $4.50–$9 per Gb depending on library efficiency and run conditions. PacBio's Revio offers HiFi genomes at approximately $250–300 each as of late 2025, with SMRT cells yielding up to 480 Gb and costs reduced through new SPRQ chemistry, or approximately $2–$3 per Gb. Initial setup costs are higher for portable systems, such as ONT's device at around $1,000-5,000, compared to NGS where per-Gb rates can fall below $1 through bulk runs. Despite these reductions—driven by improved chemistries and —TGS remains 5-10 times more expensive than NGS for routine high-volume tasks like whole-genome resequencing, though it provides better value for applications requiring long reads. Scalability challenges in TGS stem from hardware constraints and limited options, though 2025 innovations have mitigated some issues. Flow is possible with ONT's protocols, which remove prior libraries and refresh nanopores, enabling multiple runs per and contributing to cost savings of up to 50% in sequential experiments by avoiding full replacements. However, each reduces active pores, capping practical at 3-6 cycles with diminishing yields. PacBio SMRT s are largely single-use, though the Revio's supports higher parallelism. Recent advances, such as PacBio's barcoding for up to 48 samples per Revio SMRT and ONT's adaptive sampling for 96+ barcodes, have boosted per-run capacity, allowing efficient scaling for mid-sized cohorts without proportional cost increases. The 2025 introduction of ONT's PromethION Plus flow , in limited release as of , further enhances scalability by delivering significantly increased outputs optimized for 15–30 kb fragments without , reducing the per-30x cost below $345 through built-in efficiencies. These developments position TGS for hybrid workflows, where it complements NGS for structural insights, but full scalability for terabase projects still requires multi-instrument deployments.

Data management and computational requirements

Third-generation sequencing (TGS) platforms produce exceptionally large datasets, with a single run on high-throughput devices like the Oxford Nanopore PromethION generating over 100 GB of raw data in POD5 format, necessitating robust storage infrastructure for initial handling. This volume arises from the capture of detailed electrical current signals ("squiggles") during sequencing, which are significantly bulkier than the basecalled outputs; for instance, raw POD5 files from Oxford Nanopore technologies (ONT) can occupy approximately 10 times the storage space per gigabase sequenced compared to next-generation sequencing (NGS) FASTQ files, due to the uncompressed signal traces essential for downstream analyses. Basecalled FASTQ files require about 0.65 GB per gigabase, while aligned BAM files demand around 1.4 GB per gigabase, further amplifying storage needs for processed data. Computational demands escalate during alignment and assembly, where tools like minimap2 for mapping long reads to reference genomes—such as the —may require 64 GB or more of RAM for high-coverage datasets with long reads, though standard alignments typically use 30–60 GB. Basecalling, a prerequisite step, relies on neural network-based software such as or the high-performance from ONT, which convert raw signals to sequences but still demand substantial GPU resources for or . Assembly tools like Hifiasm address these challenges by leveraging the accuracy of high-fidelity reads (e.g., from PacBio) to produce haplotype-resolved genomes efficiently, often completing human-sized assemblies in hours on multi-core systems with hundreds of GB of memory. Recent advancements in 2025, including AI-driven optimizations in basecalling and pipelines, have reduced overall compute requirements by up to 40% through techniques like accelerated neural networks and efficient data compression, enabling faster processing on standard . Cloud-based solutions, such as AWS HealthOmics, further mitigate these burdens by providing scalable and analysis, achieving up to 72% cost reductions in TGS workflows through optimized and . A distinctive computational aspect of TGS involves signal-level for epigenetic modifications, where pipelines like Nanocompore perform comparative modeling of raw current traces to detect RNA modifications (e.g., m6A) without basecalling, employing Gaussian mixture models that require specialized scripting and moderate CPU/GPU resources for across large signal datasets.

Genomic applications

and genome

Third-generation sequencing (TGS) has transformed genome assembly by enabling the construction of complete genomes from raw reads without a reference, primarily through overlap-layout-consensus (OLC) paradigms that leverage long reads to bridge repetitive regions. In OLC approaches, reads are first aligned based on overlaps, then organized into a layout graph, and finally consensus sequences are generated to correct errors; this method excels with TGS data due to read lengths often exceeding 10 kb, allowing resolution of repeats larger than 10 kb that fragment short-read assemblies. For instance, the Canu assembler, designed for noisy long reads from PacBio platforms, employs adaptive k-mer weighting to separate repeats and has successfully produced near-complete assemblies for microbial and eukaryotic genomes, such as a assembly with contig N50 exceeding 20 Mb. Hybrid assembly strategies further enhance contiguity by integrating TGS long reads to scaffold or extend contigs from next-generation sequencing (NGS) short reads, combining the high accuracy of short reads with the structural spanning power of long reads. Tools like use a "mega-reads" approach, where short reads are paired into longer composites aligned to TGS reads, yielding assemblies with dramatically improved metrics; for example, in the model plant , achieved a contig N50 of up to 9.15 Mb, a substantial improvement over NGS-only assemblies. Such hybrid methods typically boost N50 lengths by 10- to 100-fold across diverse taxa, reducing fragmentation in complex regions like centromeres. A landmark application occurred in 2022 when the Telomere-to-Telomere (T2T) Consortium utilized ultralong (ONT) reads for scaffolding alongside high-fidelity PacBio reads to assemble the first complete (T2T-CHM13), resolving all gaps including repetitive and achieving a contig N50 of over 100 Mb. By 2025, automated tools like Verkko have streamlined this process, integrating phased assembly graphs with long-read data to produce telomere-to-telomere human assemblies with minimal manual intervention, as demonstrated in diploid samples with chromosome-level contigs. The contig N50 metric quantifies assembly quality, defined as the smallest contig length such that 50% of the total genome length is contained in contigs of that length or longer; TGS routinely achieves N50 values >10 Mb, compared to ~100 kb for typical NGS de novo assemblies, underscoring the former's superiority in capturing large-scale structure.

Structural variant detection and epigenetic analysis

Third-generation sequencing (TGS) technologies, such as (PacBio) HiFi and (ONT), enhance structural variant () detection by producing long reads that span repetitive genomic regions and complex rearrangements, allowing alignment to reference genomes to identify variants like deletions, insertions, inversions, and translocations. Tools like align these reads and detect SVs by analyzing discordant alignments, split reads, and coverage depth, achieving high precision (up to 94%) for deletions and duplications. Split-read alignment, where reads are partially mapped on either side of an SV breakpoint, enables precise localization of variant boundaries, particularly for insertions and deletions in long-read data. For large SVs exceeding 1 kb, such as inversions and translocations, TGS demonstrates superior resolution compared to short-read next-generation sequencing (NGS), with tools like Sniffles2 and cuteSV reporting rates above 90% in benchmarked datasets. In cancer genomes, 2025 studies using TGS have identified up to two to six times more SVs than NGS, uncovering hidden complexities in tumor rearrangements that short reads often miss due to fragmentation in repetitive elements. This improved sensitivity stems from the ability of long reads to bridge SV breakpoints directly, reducing false negatives in challenging regions like segmental duplications. In epigenetic analysis, TGS enables direct detection of DNA modifications without bisulfite conversion, leveraging signal changes in raw sequencing data to call (5mC) at single-molecule resolution. The F5C tool, an optimized version of Nanopolish, processes ONT event alignments to quantify 5mC probabilities, facilitating genome-wide profiling with base-level accuracy. Furthermore, TGS supports phasing of epigenetic modifications across haplotypes by integrating calls with variant phasing, as demonstrated in methods like MethPhaser, which resolve allele-specific patterns in diploid genomes. ONT sequencing has also advanced the mapping of N6-methyladenine (6mA) in bacterial genomes at the single-molecule level, detecting modification motifs through ionic current disruptions without amplification bias. Recent 2025 benchmarks confirm ONT's efficacy for 6mA calling, with tools achieving high concordance across replicates in diverse bacterial strains, aiding in the study of restriction-modification systems.

Advanced applications

Transcriptomic profiling and isoform resolution

Third-generation sequencing technologies enable comprehensive transcriptomic profiling by capturing full-length transcripts, allowing direct observation of and isoform diversity without the fragmentation required in short-read methods. PacBio's Iso-Seq sequences full-length cDNA molecules, typically poly-A selected transcripts exceeding 10 , providing complete representations of mature mRNAs from 5' to poly-A . Similarly, (ONT) supports direct sequencing of native strands, preserving modifications and enabling isoform quantification at the single-molecule level. These approaches facilitate the resolution of complex splicing patterns, such as , mutually exclusive exons, and alternative polyadenylation sites, which are often ambiguous in short-read data due to computational assembly needs. Isoform resolution in third-generation sequencing distinguishes splice variants by sequencing uninterrupted full-length transcripts, eliminating fragmentation-induced biases and enabling precise annotation of novel isoforms. Tools like SQANTI3 perform quality control and curation of long-read transcript models, classifying them against reference annotations to identify structural categories such as full-length non-chimeric reads and filtering artifacts for accurate isoform discovery. This full-length consensus approach supports the detection of previously unannotated transcripts, revealing hidden transcriptional complexity in genes with high alternative splicing rates, such as those involved in neuronal development. Recent applications of single-cell Iso-Seq have demonstrated its power in uncovering isoform diversity in tissues, with studies identifying over 30% novel isoforms during cortical through high-fidelity long-read sequencing. correction in these datasets is achieved using tools like FLAIR, which clusters noisy reads, corrects splice junctions with reference or short-read support, and generates high-confidence consensus sequences for isoform quantification and analysis. Such advancements highlight third-generation sequencing's role in transcriptomic profiling, particularly for discovering cell-type-specific isoforms that short-read methods overlook.

Metagenomics and pathogen identification

Third-generation sequencing technologies, with their ability to generate long reads spanning repetitive regions and structural variations, have significantly advanced by facilitating the assembly of genomes from unculturable microorganisms that dominate microbial ecosystems. Unlike short-read approaches, which often fragment assemblies due to incomplete coverage of complex genomic elements, long reads enable the reconstruction of near-complete metagenome-assembled genomes (MAGs) from diverse, low-abundance taxa in environmental samples. This capability is particularly valuable in studying microbial communities where cultivation-independent methods are essential, as unculturable account for over 99% of microbial . A key tool in this domain is metaFlye, an assembler designed specifically for long-read metagenomic data, which uses repeat graphs to handle uneven community composition and intra-species heterogeneity. By resolving strain-level variants—such as single-nucleotide polymorphisms and insertions/deletions within closely related populations—metaFlye improves the recovery of high-quality MAGs, achieving contiguity scores that surpass traditional short-read assemblers in simulated and real datasets. This strain resolution is crucial for understanding microbial evolution, functional redundancy, and interactions in complex consortia, such as those in or microbiomes. Taxonomic binning in long-read metagenomics further enhances analysis by grouping reads or contigs based on compositional features, including k-mer frequencies that capture species-specific signatures over extended sequences. Methods employing coverage and k-mer profiles allow reference-free classification of long reads, outperforming short-read binning in accuracy for low-abundance taxa and reducing chimerism in diverse samples. This approach supports precise community profiling without relying on marker genes, enabling deeper insights into phylogenetic diversity. In pathogen identification, real-time sequencing via (ONT) platforms enables rapid metagenomic surveillance during outbreaks, allowing identification of causative agents directly from clinical or environmental samples within hours. The portability of devices like supports on-site deployment in remote settings, such as field investigations, where immediate data generation informs containment strategies. For viral pathogens, ONT delivers consensus genomes with modal accuracies exceeding 97%, sufficient for epidemiological tracking and variant detection in real-time workflows. A notable application occurred in the Canadian High Arctic, where sequencing was performed on-site in 2018 to analyze metagenomes from cryoconites, yielding high-quality MAGs from uncultured and in extreme environments. Such portable TGS efforts have since facilitated the detection of novel resistance genes in polar soils, revealing reservoirs of resistance to clinically relevant antibiotics like beta-lactams and aminoglycosides, with implications for global amid climate-driven microbial dispersal.

Clinical diagnostics and personalized medicine

Third-generation sequencing (TGS) has significantly advanced clinical diagnostics by enabling the precise characterization of repeat expansions, which are challenging for short-read technologies due to their length and repetitive nature. For instance, in , long-read sequencing accurately sizes CAG repeats in the HTT gene, detecting somatic expansions exceeding 150 repeats that drive selective neuronal degeneration in striatal projection neurons. This approach has facilitated rapid and comprehensive diagnostic methods for repeat expansion disorders, improving diagnostic yield in neurological conditions where traditional methods fall short. In Mendelian disorders, TGS enhances structural variant (SV) detection with high , particularly for complex rearrangements in repetitive genomic regions often missed by short-read sequencing. Long-read methods have identified causal SVs in , contributing to diagnostic rates of up to 20-30% in reanalysis of undiagnosed patients by resolving variants in genes associated with genetic diseases. This capability is crucial for conditions involving copy number variations or inversions, where TGS provides complete phasing and . For , TGS supports phasing of pharmacogenes, such as and HLA loci, enabling diplotyping that predicts individual drug responses more accurately than alone. Real-time tumor sequencing with platforms like (ONT) allows intra-operative profiling to guide , identifying neoantigens and tumor-specific alterations for tailored treatments like checkpoint inhibitors. In diverse populations, TGS resolves up to 40% more nucleotide-resolved deletions compared to short-read approaches, addressing biases in variant calling and improving equity in genomic diagnostics across ancestries. Portable TGS devices, such as the ONT , extend these benefits to low-resource settings by enabling on-site prenatal screening and rapid pathogen identification for , achieving results from blood cultures in under 2 hours through analysis. This portability facilitates in remote areas, supporting timely interventions for conditions like structural anomalies in fetuses without relying on centralized labs.

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