Background: although short-read whole-genome sequencing has become the clinical standard, important genomic regions including structural variants, tandem repeat expansions, segmental duplications, and complex pharmacogenomic loci remain difficult to resolve. PacBio HiFi sequencing offers highly accurate long reads capable of simultaneously detecting small variants, structural rearrangements, repeat expansions, methylation, and haplotype phase within a single assay.Objective: this review evaluates current evidence supporting PacBio HiFi whole-genome sequencing for clinical implementation and discusses its potential role in Southeast Asia, where high genetic diversity, founder mutations, and pharmacogenomic variation create unique diagnostic challenges.Evidence Summary: published clinical studies consistently demonstrate higher diagnostic yield compared with conventional short-read sequencing, particularly for rare diseases, repeat expansion disorders, structural variation, and pharmacogenomics. Recent advances in sequencing chemistry and bioinformatics further support clinical scalability.Conclusion: PacBio HiFi WGS represents a promising next-generation clinical sequencing platform capable of improving diagnostic accuracy and precision medicine implementation across Southeast Asia.
1. Introduction
Clinical genomics has moved through successive technology epochs. Illumina short-read whole-exome and whole-genome sequencing (WGS), widespread from ~2008, accelerated Mendelian gene discovery and became a first- or second-tier diagnostic test. Yet diagnostic yield plateaued at roughly 30–50% across rare-disease cohorts, leaving many multi-year diagnostic odysseys unresolved because short reads cannot reliably resolve repetitive regions, detect structural variants (SVs), or phase alleles across long distances. Long-read single-molecule platforms addressed this gap: Pacific Biosciences' single-molecule real-time (SMRT) sequencing reads DNA synthesis by a polymerase within a zero-mode waveguide
[11][12]
The decisive advance was circular consensus sequencing (CCS): a polymerase passes repeatedly over a circularised template, and the noisy subreads collapse into a single "HiFi" read of 10–25 kb at Q30+ (~99.9%) accuracy. Wenger et al.
[4]
Three findings motivate genome-wide HiFi. First, SVs are pervasive and clinically significant: long-read sequencing of 3,622 Icelanders found a median ~22,600 SVs per genome, 4–5× the short-read estimate, including disease-relevant SVs (e.g. affecting PCSK9, ACAN) miscalled by short reads
[3][1][8][7][5][20]
This review appraises HiFi WGS for clinical use, emphasizing the disease epidemiology, founder-mutation landscape, and pharmacogenomic profile of Southeast Asia (SEA), while critically noting real obstacles including cost, throughput, interpretation bottlenecks, and ancestry-specific reference gaps.
This is a narrative non-systematic review synthesising peer-reviewed primary literature, reference-consortium releases (Telomere-to-Telomere, HPRC, GenomeAsia 100K, SG10K_Health), and published clinical-cohort reports on PacBio HiFi WGS. No search protocol with predefined date limits was registered, sources reflect landmark and representative evidence. Manufacturer specifications are reported as declared performance, distinct from independent clinical validation. Diagnostic-yield figures are given as published, without meta-analysis or formal risk-of-bias scoring.
2. Results & discussion
PacBio Single-Molecule Real-Time (SMRT) sequencing is based on three core innovations originally described by Eid et al. First, DNA synthesis is monitored within a zero-mode waveguide (ZMW), a nanoscale reaction chamber that enables real-time observation of a single DNA polymerase molecule. Second, phospholinked fluorescent nucleotides are incorporated during DNA synthesis, with the fluorescent label released as part of the natural polymerization process, leaving the newly synthesized DNA unmodified. Third, the platform records the kinetics of nucleotide incorporation, including interpulse duration (IPD) and pulse width, which vary in the presence of DNA base modifications such as 5-methylcytosine (5mC) and N6-methyladenine (6mA). This unique capability enables the direct detection of DNA methylation from native DNA without bisulfite conversion or antibody-based enrichment.
Genomic DNA is sheared to ~15–25 kb fragments and ligated to hairpin SMRTbell adaptors to create a topologically closed, single-stranded circular template. A strand-displacing polymerase traverses the template multiple times during the SMRT cell run. The resulting subreads for each an imperfect ~10–15% raw-error copy, are then computationally collapsed into a single high-quality CCS / HiFi consensus read. Typical HiFi yields are 15–25 kb reads at Q30+ (~99.9% per-base accuracy), with current Revio output ~360 Gb per SMRT cell (≥4–5 whole human genomes at 30× equivalent depth per 24-hour run).
Because HiFi sequencing requires no PCR amplification, base modifications present on native genomic DNA are preserved through library preparation and read out via polymerase kinetics. The current Revio primary analysis pipeline calls 5-methylcytosine at CpG sites with sensitivity comparable to whole-genome bisulfite sequencing (WGBS) and concordance with EPIC/450K arrays generally >95%
[6]
Read-length distribution is shaped by library shearing (Megaruptor, gTUBE) and SMRTbell size selection (BluePippin, AMPure). Typical clinical HiFi WGS libraries yield N50 read length 15–20 kb, with reads >25 kb common; this exceeds the size of >99% of typical pathogenic structural variants, the vast majority of mobile-element insertions, and the unexpanded alleles of all known disease-associated tandem repeats. Read length is the single most important parameter determining the resolution of complex segmental duplications (e.g., PMS2/PMS2CL, SMN1/SMN2, CYP2D6/CYP2D7, PKD1/PKD1P), since at least some HiFi reads must uniquely span the region of divergence between paralogues.
In direct head-to-head benchmarking on the Genome in a Bottle reference samples (HG002 trio):
1.
2.
3.
[4][13][14]
Each platform has legitimate clinical niches. HiFi WGS currently provides the most balanced performance across accuracy, throughput, epigenetic profiling, and comprehensive variant detection, making it the most suitable option for routine clinical genomics. In contrast, ONT remains particularly valuable for ultra-long-read applications such as telomere-to-telomere assembly, resolving very large structural variants, and rapid or field-based sequencing workflows. For routine clinical WGS, rare disease diagnostics, pharmacogenomics, and methylation-aware genome analysis, current evidence strongly supports HiFi WGS as the most mature single-assay solution. Conversely, ultra-long-range assembly, telomere-to-telomere reconstruction, and certain highly repetitive structural rearrangements remain areas in which ONT retains important advantages.
Coverage requirements differ by application:
1.
2.
[5]
3.
The Revio platform (launched in 2023), serves as the high-throughput foundation for population-scale HiFi sequencing, enabling simultaneous operation of up to four SMRT Cells and generating approximately 1,300 Gb of data per run. In contrast, the Vega benchtop system, launched in 2025, is designed for single SMRT Cell sequencing and is intended for clinical laboratories, regional reference centres, and academic institutions that do not require the throughput capacity of Revio. For most clinical genomics initiatives in Southeast Asia, a hub-and-spoke deployment model is likely to be both operationally efficient and cost-effective, with Revio systems centralized at regional sequencing hubs and Vega instruments deployed at tertiary academic hospitals to support rapid-turnaround clinical genome sequencing.
Comparison of clinical sequencing platforms for whole-genome analysis
| Feature | Illumina SR-WGS | ONT R10.4.1 | PacBio Revio HiFi | PacBio Vega HiFi |
| Read length | 150 bp | 10–100+ kb (ultra-long) | 10–25 kb | 10–25 kb |
| Per-base accuracy | ~Q30 (99.9%) | Q20–Q30 | Q30+ (99.9%) | Q30+ (99.9%) |
| SNV recall (GIAB) | ≥99.5% | 99.4–99.7% | ≥99.9% | ≥99.9% |
| SV recall (≥50 bp) | 30–50% | ~90% | ≥90% | ≥90% |
| Repeat expansions | Mostly invisible | Native detection | Native detection | Native detection |
| Native 5mC/5hmC/6mA | No | Yes | Yes (SPRQ-Nx) | Yes (SPRQ-Nx) |
| Throughput/run | Very high | Variable | ~4–5 genomes/24h | 1 SMRT cell |
| Best clinical fit | Population panels, oncology panels | Ultra-long phasing, rapid in-field | Population WGS hub | Tertiary clinical site |
Low-pass HiFi (1–5×) supports cfDNA/NIPT with preserved fragmentomic and methylation signatures
[15][6]
– SNVs/indels via DeepVariant at ≥99.9% precision/recall
[16]
– SVs via sawfish/Sniffles2 at ≥90% recall/precision across deletions, insertions, duplications, and inversions
[13]
– mobile-element insertions (Alu, L1, SVA) recovered;
– tandem repeats genotyped by TRGT with 99.56% Mendelian concordance across ~937,000 loci [
1819
In pediatric rare disease, 50–70% of well-phenotyped patients remain undiagnosed after exome or short-read WGS, largely because of SV-, repeat-, and methylation-class variants specifically structural variants, mobile-element insertions, tandem-repeat expansions, complex rearrangements, variants within segmental duplications, and deep-intronic or "dark"-region variants. HiFi WGS captures SNVs, indels, SVs, repeats, phase, and methylation together; the Radboudumc cohort reached 93% sensitivity against 2–6 assays
[5][6][21][22]
Recurrent CNV syndromes (DiGeorge, Williams, Prader–Willi, Angelman) gain single-nucleotide breakpoint resolution and parent-of-origin assignment via imprinted-locus methylation
[3][7][18][23][24][6]
In neurodegeneration, HiFi distinguishes GBA1 from its pseudogene GBAP1 and detects SNCA multiplications in Parkinson disease, sizes C9orf72 in ALS/FTD (with SOD1, FUS, TARDBP), resolves the PMP22 duplication of CMT1A, and interrogates APP, APOE/TOMM40, PRNP codon-129 phase, and the "dark" CR1 locus in Alzheimer disease
[1][25][26][64]
Pharmacogenomics historically needed many assays because CYP2D6, UGT1A1, SLCO1B1, NUDT15, TPMT, HLA, and DPYD are afflicted by structural variation and paralogues. StarPhase
[27][28][13][29][30]
Southeast Asia comprises eleven countries (Brunei, Cambodia, Indonesia, Laos, Malaysia, Myanmar, Philippines, Singapore, Thailand, Timor-Leste, Vietnam) with a combined population of 680 million. The region carries some of the world's highest prevalence of certain monogenic disorders and pharmacogenetic risk variants, yet is dramatically underrepresented in major reference resources (gnomAD, 1000 Genomes Project, UK Biobank, All of Us). This combination with high genetic disease burden plus poor reference coverage makes SEA both an urgent priority for HiFi WGS deployment and a region where the technology's advantages are most acute.
Thalassaemia and hemoglobinopathies are by far the most prevalent monogenic disorders in SEA, with α-thalassaemia carrier frequencies of 30–40% in Northern Thailand and Laos, β-thalassaemia 1–9% across most of the region, and HbE gene frequencies of 50–60% at the Laos–Cambodia–Thailand junction. The most common deletional α-thalassaemia allele for the Southeast Asian (SEA) deletion together with the –α^3.7 and –α^4.2 deletions, Hb Constant Spring, and the HbE β-globin variant, defines the regional clinical profile.
Traditional diagnostics rely on PCR-RDB, gap-PCR, and MLPA, each blind to rare or novel variants. Multiple groups have demonstrated that PacBio HiFi long-molecule sequencing accurately detects all common α- and β-thalassaemia deletions plus rare and novel SNVs and SVs missed by conventional assays, including newly described −α^3.7 subtype III, ααα^anti3.7 triplications, and 7–14 kb δβ-fusion deletions
[31][32][33][34][35]
1.
[36][37][38][39]
2.
[40]
3.
[41]
4.
[30]
SCA3 (Machado–Joseph disease) is markedly enriched in Han Chinese, Taiwanese, and Singaporean Chinese populations. SCA1, SCA2, SCA6, SCA12, and DRPLA show population-specific distributions across SEA. NIID (
[23][24]
[9]
SG10K_Health. The Singapore National Precision Medicine (NPM) program sequenced ~10,000 Singaporeans across Chinese (58.4%), Malay (21.8%), and Indian backgrounds at ~13.7× coverage in the pilot
[42][10][43][44]
Indonesian Genome Diversity Project, Thai Reference Genome, Vietnamese Genome Project. Emerging short-read population resources establishing baseline allele frequencies but underpowered for SVs, tandem repeats, and methylation.
HUGO Pan-Asian SNP Initiative and Singapore Genome Variation Project. Historical genotype-array resources are still cited for ancestry-specific allele frequencies.
SEA harbors numerous founder mutations and population-specific variants that simultaneously:
Inflate the population-allele-frequency–weighted estimate of "rare disease–causing variants" in clinical settings, since some variants are common in SEA but absent or extremely rare in gnomAD.
Underlie ACMG/AMP variant-classification difficulties [
45
Are systematically misclassified by clinical interpretation pipelines built on Caucasian-dominated reference data.
GnomAD v4 contains ~807,000 individuals globally but <1% are from SEA, dramatically inflating variant-of-uncertain-significance (VUS) rates in regional patients. Population-specific HiFi WGS reference panels analogous to SG10K but with HiFi data are essential to convert VUSs to actionable calls.
Clinical genomics adoption across SEA is heterogeneous:
– Singapore (mature, NPM-led precision-medicine program);
– Malaysia and Thailand (tertiary academic centers with growing clinical exome capacity);
– Indonesia (emerging, mostly tertiary referral);
– Vietnam and the Philippines (rapidly developing);
– Cambodia, Laos, Myanmar, Brunei, Timor-Leste (limited tertiary capacity, dependent on referral).
The deployment opportunity is therefore neither uniform nor reducible to a single regional strategy; HiFi WGS adoption should be calibrated to local clinical infrastructure, regulatory frameworks, and reimbursement environments.
Selected clinically actionable variants of high relevance to Southeast Asian populations
| Gene/Locus | Variant/Allele | Clinical implication | SEA AF (%) | HiFi value |
| HBA1/HBA2 | –SEA, –α3.7, –α4.2 | α-thalassemia | 5–40 | Resolves segdup; SV native |
| HBB | Codon 41/42, IVS1-1, HbE | β-thalassemia, HbE | 1–9 (HbE up to 60) | Direct phasing |
| G6PD | Mahidol, Viangchan, Mediterranean | Hemolysis risk | 3–15 | Pan-allele detection |
| HLA-B | *15:02 | Carbamazepine SJS/TEN | 5–15 | StarPhase 4-field |
| HLA-B | *58:01 | Allopurinol SCAR | 5–15 | StarPhase 4-field |
| NUDT15 | *3 (c.415C>T) | Thiopurine toxicity | 5–10 | Direct call |
| CYP2C19 | *2, *3 | Clopidogrel, PPI | 10–35 | StarPhase diplotype |
| NAT2 | Slow acetylator | Isoniazid hepatotoxicity | 30–60 | Direct phasing |
| ATXN3 | CAG expansion | SCA3 (Machado–Joseph) | Variable | TRGT sizing |
| NOTCH2NLC | GGC expansion | NIID | Variable | GC-rich, HiFi essential |
| SAMD12 | TTTCA/TTTTA | BAFME-1 | Variable | Intronic, HiFi essential |
The most rigorous direct comparison to date is the Radboudumc 100-sample study
[5]
Several smaller series support similar conclusions:
1. Mantere, Kersten & Hoischen
[12]
2. Mahmoud et al.
[2]
3. Beyter et al.
[3]
4. Liao et al. (HPRC)
[7]
5. Nurk et al. (T2T-CHM13)
[8]
6. Aganezov et al.
[46]
7. Logsdon, Vollger & Eichler
[47]
Cheung et al.
[6]
Capper et al.
[28]
Although formal health-economic evaluations of HiFi WGS are still limited, early implementation studies suggest that the technology has the potential to improve the cost-effectiveness of rare disease diagnostics. In the Radboudumc setting, replacing 2–6 orthogonal short-read–based assays per patient with a single HiFi WGS demonstrably reduces total diagnostic cost per case while shortening turnaround time. The economic value proposition strengthens further as HiFi reagent costs decrease with SPRQ-Nx chemistry and Vega platform deployment, and as automated library preparation (24–96 samples per run at Radboudumc) drives operational efficiency.
The cost calculation must include downstream value: a single HiFi WGS provides not only the immediate diagnostic answer but also lifelong pharmacogenomic, carrier-screening, and future-reanalysis utility from a single sample—a property no orthogonal assay stack offers.
SMRT Link is the PacBio-supplied primary analysis software, performing CCS read generation from raw subreads, demultiplexing, and basic QC. On Revio, primary analysis (including CpG methylation calling) executes on-instrument; SPRQ chemistry runs incorporate further sensitivity gains and 6mA calling.
pbmm2 is the PacBio-tuned wrapper around minimap2
[48][49]
DeepVariant
[16][50]
sawfish (PacBio-supplied SV caller) and Sniffles2
[13]
TRGT (Tandem Repeat Genotyping Tool)
[18][19]
Paraphase
[26]
StarPhase
[27]
CpG methylation is called natively during primary analysis on Revio (and via secondary tools on Sequel IIe data). Downstream analysis tools are available in the MethBat tools collection, and with modbamtools for visualization. The Cheung et al.
[6]
The HPRC v1.0 draft pangenome
[7][51][49]
Variant prioritization for clinical reporting integrates ACMG/AMP classification
[45][52][53]
A 30× HiFi WGS BAM is typically 50 GB (including methylation); compute requirements for full HiFi WGS analysis (alignment, small-variant, SV, repeat, methylation, PGx) are typically 100–500 CPU-hours per sample on standard cloud or HPC infrastructure. For SEA deployment, regional cloud or government-hosted compute capacity (Singapore's NSCC; Malaysian and Thai national HPC) supports clinical-scale operation; data residency and privacy regulations vary by country.
Key bioinformatics tools for clinical HiFi WGS analysis
| Task | Tool | Function | Reference |
| Alignment | pbmm2 / minimap2 | HiFi-tuned long-read alignment | [48] |
| SNV/Indel calling | DeepVariant (HiFi model) | Deep-learning small-variant caller | [16] |
| Phasing | HiPhase | Read-based haplotype phasing | PacBio |
| SV calling | Sniffles2 / pbsv | Structural variant detection (incl. mosaic) | [13] |
| Tandem repeats | TRGT / TRGT-denovo | Repeat sizing + methylation + de novo | [18] |
| Paralogues | Paraphase | Segmental duplication resolution | [26] |
| Pharmacogenomics | StarPhase / Pangu / PharmCAT | PGx diplotypes + HLA + CYP2D6 | [27] |
| Methylation | modkit / methbat | 5mC/6mA calling and analysis | PacBio/community |
| Pangenome calling | vg giraffe / PanGenie / Minigraph-Cactus | Pangenome-aware variant calling | [51] |
| Variant interpretation | Exomiser / LIRICAL / AMELIE | HPO-driven prioritization | [52] |
Methylation, a free by-product of every run, will become a routine reporting layer within 3–5 years as pathogenic catalogues grow
[6][28][125]
3. Limitations
Despite its substantial advantages, HiFi WGS is not free of meaningful limitations. Honest critical appraisal is essential to calibrate clinical expectations and roadmap deployment. HiFi reagents remain ~2–4× short-read cost favourable per diagnosis against the multi-assay stack it replaces, but still a barrier without reimbursement in lower-middle-income SEA countries. Throughput (~4-5 genomes/24 h on Revio) limits population scale outside Singapore, though higher-throughput long-read platforms are emerging. Library preparation needs high-molecular-weight DNA (SPRQ has lowered input to ≤500 ng), limiting FFPE/archival use. A 30× genome yields ~4-5 million variants, and classification frameworks remain weaker for long-read-resolved classes (complex SVs in segmental duplications, non-canonical repeats, methylation signals), whereas difficulty amplifies for under-represented SEA genomes. Ancestry-specific reference gaps (limited SEA in gnomAD and the HPRC pangenome) inflate VUS and "novel"-SV rates.
ELSI frameworks (consent, return of secondary findings, data sovereignty), EHR/HPO integration, regulatory/IVD pathways (long-read sequencing is recognised by the FDA and EMA in research contexts and CLIA validation exists, but routine IVD adoption and national SEA approvals remain heterogeneous), and clinical-genomics workforce remain immature across much of SEA, and structured phenotyping (HPO) and longitudinal reanalysis pipelines are variably implemented. Residual technical gaps persist where acrocentric/rDNA/centromeric arrays still need complementary ONT ultra-long reads, mosaicism below ~1% VAF needs deep or targeted coverage, and some centromeric/telomeric balanced rearrangements need optical mapping or Hi-C. These residual gaps are areas of active development and are progressively narrowing.
4. Conclusion
As of 2026, PacBio HiFi whole-genome sequencing represents the most comprehensive single-assay clinical genomic test currently available. By producing 10–25 kb reads at Q30+ accuracy with simultaneous native methylation calling, HiFi WGS resolves the structural variants, tandem repeats, mobile-element insertions, paralogue-pseudogene complexes, and methylation events that collectively a substantial fraction of clinically meaningful but short-read–invisible disease. The Radboudumc 100-sample cohort demonstrated 93% pathogenic-variant sensitivity against a stack of orthogonal assays, and the GA4K methylation study established native HiFi methylation as a clinically deployable analytical layer. The bioinformatics ecosystem such as DeepVariant, Sniffles2, sawfish, TRGT, Paraphase, StarPhase, HiPhase, and the HPRC pangenome has matured to clinical-grade specifications.
For Southeast Asia, the case for HiFi WGS is unusually compelling. The regional disease burden for thalassaemias, hemoglobinopathies, G6PD deficiency, congenital adrenal hyperplasia, SMA, repeat-expansion ataxias, NIID, and others overwhelmingly involves variant classes for which HiFi WGS provides decisive advantages. The pharmacogenomic risk profile such as HLA-B*15:02, HLA-B*58:01, NUDT15, CYP2C19, NAT2 is exactly the class of high-impact variation for which StarPhase from a single HiFi run delivers pan-pharmacogenome typing. Population genomic under-representation in gnomAD, HPRC, and other Caucasian-dominated resources further amplifies the per-sample diagnostic value of HiFi WGS in SEA patients.
Real obstacles remain including cost, throughput, workforce, regulatory frameworks, and SEA-specific reference resources should be honestly acknowledged rather than minimized. Yet none is intrinsically intractable on current technology trajectories. The scientific framework supporting HiFi WGS as a first-tier clinical test is, in our judgment, now established beyond reasonable contest. The remaining question is one of implementation: how rapidly the SEA region can build the population reference data, regulatory pathways, reimbursement architectures, and clinical workforce required to deliver this technology equitably to the patients who stand to benefit most.
The additional file for this article can be found as follows:
Further description of analytic pipeline and patient demographic information. DOI:
YSDS gratefully acknowledges the global scientific community whose published work forms the evidence base of this Review, including investigators at Radboud University Medical Center, Children’s Mercy Kansas City, the Human Pangenome Reference Consortium, the Telomere-to-Telomere Consortium, GenomeAsia 100K, SG10K_Health, Pacific Biosciences, and the many SEA national genomic medicine initiatives.