Long-Read Sequencing in CKD Diagnostics: Breaking Genomic Barriers and Expanding Global Inclusion

Abstract

Chronic kidney disease (CKD) is a major global health burden, and genetic testing is increasingly used to clarify diagnosis and guide management, particularly in inherited and early-onset disease. Short-read sequencing (SRS), including targeted gene panels, exome sequencing, and genome sequencing, provides diagnostic-grade detection of many exonic single nucleotide variants and small insertions or deletions, but important diagnostic gaps remain in genomic regions that are difficult to resolve with short reads. Long-read sequencing (LRS) addresses several of these limitations by enabling improved detection of structural variants, repeat expansions, and complex or highly homologous loci, and by providing long-range haplotype context.

This review focuses on the current and emerging clinical applications of LRS in CKD diagnostics, highlighting scenarios where LRS is most likely to add value beyond SRS, including unresolved suspected monogenic kidney disease and technically challenging genes such as PKD1 and MUC1. We also discuss how improved representation of structural variation and ancestry-specific reference data may reduce uncertainty in variant interpretation for patients who are poorly represented in existing databases.

Although routine clinical implementation of LRS is still evolving, ongoing advances in laboratory workflows, bioinformatics pipelines, and reference resources are accelerating translation into nephrology practice.

CKD remains a major global health challenge, imposing substantial economic burdens.1, 2, 3 However, recent advancements in genomic technologies have improved our broad understanding of the genetic causes of CKD, offering opportunities for early diagnosis and targeted interventions.^4,5

Genetics in CKD exists on a continuum, ranging from monogenic kidney diseases caused by high-penetrance variants to common CKD, where genetic variants act as risk modifiers alongside environmental factors,6, 7, 8 including socio-economic status and lifestyle choices.9, 10, 11 In clinical practice, monogenic kidney diseases are often suspected in conditions such as autosomal dominant polycystic kidney disease (ADPKD), most commonly caused by variants in PKD1 or PKD2, although additional genes including GANAB, DNAJB11, ALG8, and ALG9 are now recognized and routinely included in clinical gene panels,^12,13 Alport syndrome associated with pathogenic variants in COL4A3–COL4A5,¹⁴ or nephronophthisis caused by variants in NPHP genes.⁷ In contrast, common forms of CKD, such as diabetic kidney disease¹⁵ or hypertensive nephropathy,¹⁶ typically reflect polygenic risk combined with environmental exposures rather than a single causal variant. Genetic testing is therefore most frequently pursued in patients with suspected inherited or early-onset disease, where a single pathogenic variant is more likely to explain the phenotype.^17,18 In this diagnostic context, particularly when SRS fails to resolve complex or structurally challenging loci, LRS may offer additional diagnostic value.19, 20, 21

LRS, also known as third-generation sequencing, offers transformative potential in CKD genetics by enabling the precise detection of structural variants, deep intronic variants , and other complex genomic alterations that are often missed by SRS methods (Table 1).22, 23, 24 However, the successful implementation of LRS necessitates tailored methodological workflows for DNA extraction, library preparation, and sequencing, as well as suitable computational sources, robust bioinformatics pipelines, and reference databases for variant interpretation.25, 26, 27, 28 Ongoing developments in the field and multidisciplinary efforts are progressively addressing these challenges, enhancing the clinical applicability of LRS.^29,30 The higher up-front cost of LRS reflects sequencing consumable costs, lower multiplexing capacity, and greater analytical complexity compared with short-read methods.³¹ As the technology becomes more accessible and cost-effective in relation to its diagnostic yield and clinical impact, its adoption is expected to expand, offering promising avenues for early diagnosis and personalized treatment strategies in CKD.³²

Table 1.

Technical characteristics of short-read sequencing (SRS) and long-read sequencing (LRS) relevant to clinical genomics

Performance metrics	SRS	LRS	References
SNV/indel performance (clinical)	F1 ≈ 0.98–0.99 (Illumina)	F1 ≈ 0.95–0.98 (PacBio HiFi); 0.90–0.96 (ONT, model-dependent)	Performance metrics derived from Genome in a Bottle benchmarking datasets49,134,135
Per-read base accuracy	High ∼99.8–99.9%	Lower ∼ 99.8% (PacBio HiFi); ∼95–98% (ONT, chemistry/model-dependent)	Wenger et al.49
Cost-effectiveness (clinical context dependent)	Lower cost; cost-efficient for routine SNV detection	Higher upfront cost; may reduce downstream testing in structurally complex or unresolved cases	Groopman et al.44
Throughput	High (hundreds–thousands of samples/run)	Medium (lower sample multiplexing; improving)	Logsdon et al.22 and Goodwin et al.136
Time efficiency	Moderate (batch-based workflows, delayed results)	High (real-time sequencing and analysis possible)	Logsdon et al.22, Quick et al.137, and De Coster et al.138
SVs detection	Some (mostly CNVs; breakpoint resolution poor)	Yes (balanced SVs, repeat-associated SVs, complex rearrangements) simultaneous with sequencing.	Chaisson et al.50 and Sedlazeck et al.139
Epigenetic modifications detection	Indirect (separate assays required)	Direct (simultaneous with sequencing)	Simpson et al.140 and Liu et al.141
Maximum read length	∼150–300 bp	10–25 kb (PacBio HiFi); > 100 kb (ONT)	Logsdon et al.22, Amarasinghe et al.27, Wenger et al.49, and Jain et al.142

bp, base pairs; CNV, copy number variant; F1, F1 score (harmonic mean of precision and recall); indel, insertion/deletion; ONT, Oxford Nanopore Technologies; SNV, single nucleotide variant; SV, structural variant.

Performance metrics are derived from published benchmarking studies and reflect variant-type–specific capabilities. SRS remains highly accurate for SNVs and small insertions/deletions (indels) in well-characterized genomic regions, whereas LRS provides advantages for SV detection, repeat expansions, complex or highly homologous loci, and haplotype resolution. Epigenetic information can be obtained using SRS through dedicated assays requiring separate workflows, whereas certain LRS platforms enable direct detection of base modifications from native DNA within a single sequencing run.^49,81,136, 143, 144

CKD arises from multiple subtypes influenced by over 600 identified genes, rendering it a multifaceted disorder shaped by the intricate interplay between genetic predispositions and environmental stressors, including epigenetic modifications like DNA methylation that modulate gene expression.33, 34, 35, 36 In patients with suspected inherited or monogenic forms of CKD, early genomic diagnosis can prevent costly and high-risk invasive procedures such as hemodialysis and kidney transplant, whereas also enabling family screening and early delivery of targeted treatments.³⁷ The 2022 Kidney Disease: Improving Global Outcomes Controversies Conference highlighted the substantial influence of genetics in the assessment of CKD cases, stressing the need for thorough family history evaluations, consideration of the age of CKD onset, extrarenal symptoms, and the inclusion of genetic testing in the diagnostic process.³⁶

This review examines the current and emerging role of LRS in the diagnostic evaluation of CKD, with emphasis on scenarios in which LRS provides added value beyond conventional SRS. We focus on its application in suspected monogenic kidney disease, particularly in resolving structurally complex loci and repeat-associated variants, and in cases remaining unsolved after standard testing. By synthesizing recent primary studies, we aim to clarify the clinical positioning of LRS as a complementary strategy within nephrogenetic practice.

Current Genetic Testing in CKD and Limitations

Although conventional diagnostic approaches such a biochemical test, imaging, and biopsies remain central to the evaluation of nephropathy, they often fail to establish an underlying diagnosis in a subset of patients.^38,39 An important proportion of patients frequently experience uncertainty during the diagnostic journey and express a desire for earlier and clearer information, particularly in the absence of a known family history.⁴⁰ Patient and caregiver studies in inherited kidney disease, including ADPKD, highlight the perceived value of early genetic information and the emotional burden associated with diagnostic uncertainty and unclear prognosis.41, 42, 43 These observations highlight the clinical consequences of delayed or uncertain etiological diagnosis and underscore the potential value of earlier genetic evaluation in selected patients with CKD, particularly when conventional clinical assessment fails to establish a clear cause.

Current genetic testing options for CKD include targeted gene panels, exome sequencing, and genome sequencing which are routinely performed using next-generation SRS platforms and enable parallel interrogation of multiple kidney disease genes.^44,45 Sanger sequencing remains a cost-effective and accurate method for confirmatory testing or targeted analysis of known familial variants but is not used for broad, first-line genetic evaluation.⁴⁶ Copy number variants are now typically detected through SRS-based approaches rather than chromosomal microarray analysis.⁴⁷ These approaches provide diagnostic-grade detection of single nucleotide variants and small insertions or deletions, and have become the standard of care for many suspected inherited kidney diseases. However, the short-read lengths (typically ∼150 base pairs) limits accurate variant detection in certain genomic contexts. Structural variants (SVs), repeat expansions, guanine-cytosine (GC)-rich regions, and highly homologous or duplicated loci remain challenging to resolve, contributing to unresolved variants in a subset of Mendelian disorders. These limitations provide the clinical rationale for exploring complementary sequencing approaches, such as LRS, in selected patients with CKD, where standard testing has failed to establish a molecular diagnosis.48, 49, 50

A relevant example illustrating this analytical difficulty is the clinically important gene PKD1. PKD1 represents a technically challenging locus because of the presence of 6 highly homologous pseudogenes spanning exons 1 to 33, which share approximately 97% sequence identity with the functional gene. Although clinical testing of PKD1 using SRS is widely implemented and supported by specialized laboratory workflows, these approaches remain technically complex and may fail to resolve certain intronic, structural, or splice-altering variants. Recent studies incorporating targeted LRS have demonstrated improved resolution of PKD1 variants, including detection of atypical splice variants and more accurate differentiation from pseudogene sequences, supporting an integrative role for LRS in selected unresolved or complex cases.^23,51 Our understanding of CKD mechanisms and causes has advanced in tandem with emerging technologies, from the first genome-wide association studies in 2007 to the adoption of LRS in 2020, highlighting significant progress in unravelling this complex disease and offering hope for more precise diagnostics and deeper insight in the years ahead (Figure 1).48, 49, 50

Figure 1.

Timeline of major genomic advancements in chronic kidney disease (CKD) research and clinical diagnostics. The timeline illustrates the progression from early genome-wide association studies (GWAS) to the adoption of short-read sequencing (SRS)-based target gene panels and exome sequencing of Mendelian CKD, and more recently to long-read (LRS) technologies. Key milestones highlight the evolving contributions of genomics to understanding CKD mechanisms, improving diagnostic yield, and enabling more precise molecular characterization. CRISPR, clustered regularly interspaced short palindromic repeats; ES, exome sequencing; GS, genome sequencing; LRS, long-read sequencing; PRS, polygenic risk score; SRS, short-read sequencing^52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62 Created with BioRender (BioRender.com).

Reported diagnostic yields for short-read genomic sequencing in established CKD cohorts range from approximately 10% to 30% in adult populations. In this context, diagnostic yield refers to the proportion of individuals in whom sequencing identifies a pathogenic or likely pathogenic variant that explains the patient’s clinical phenotype. Primary studies applying exome sequencing in adult CKD cohorts have reported diagnostic rates between approximately 9% and 24%, with higher yields observed in selected groups such as early-onset disease, positive family history, or syndromic presentations.^44,63,64 In some cases, genomic diagnosis leads to reclassification of the clinical diagnosis and influences management decisions, including avoidance of inappropriate immunosuppression or unnecessary invasive investigations. Although whole genome sequencing (WGS) can improve variant detection compared with exome sequencing in technically challenging regions, many diagnostic gaps persist, reflecting the intrinsic limitations of short-read technologies rather than gene content alone.^65,66

LRS overcomes many of these constraints by generating extended reads that span complex genomic regions, enabling improved alignment, variant phasing, and structural variant detection. The key technical differences between SRS and LRS that underpin these diagnostic advantages are illustrated in Figure 2. LRS has shown several applications in the clinical discovery of recently identified pathogenic gene variants in human diseases, investigating genetic disorders with previously unknown genetic cause or strongly suspected disease loci.^67,68

Figure 2.

Advantages of long-read sequencing (LRS) compared with short-read sequencing (SRS) in complex genetic regions. Short reads often fail to capture the extended genetic context, which complicates accurate alignment. In contrast, LRS generates extended reads, enabling precise placement and improved resolution of such regions. (a) LRS demonstrates enhanced capacity to identify structural variants (SVs) relative to SRS, particularly in difficult genomic areas like repetitive DNA sequences (depicted in blue), which frequently contribute to SV formation. (b) LRS facilitates superior sequencing and alignment of short tandem repeat (STR) regions (shown in green) compared with SRS, allowing precise identification of STR expansions. Notable genes with STR expansions linked to disorders include PKD1 (autosomal dominant polycystic kidney disease),⁶⁹NPHP1 (nephronophthisis),⁷⁰ and COL4A5 (Alport syndrome).⁷¹ (c) LRS improves the mapping and coverage of genomic regions with high sequence homology (illustrated in red), which pose challenges for SRS. This capability supports the distinction between genes and their pseudogenes, enhancing the detection of variations in these complex regions. Examples of disease-related genes in such homologous regions include PKD2 (polycystic kidney disease),⁷²CFHR5 (complement factor H-related 5 glomerulopathy),⁷³ and MYH9.⁷⁴ (d) The ability to sequence native DNA strands using LRS allows simultaneous evaluation of base modifications, such as distinguishing methylated cytosines (marked in red) from unmethylated cytosines (marked in blue) within cytosine-guanine dinucleotides (CpGs). This can be paired with phasing data to explore imprinted regions exhibiting parent-specific DNA methylation. Examples of imprinted genes with methylation patterns linked to CKD include WT1 (altered methylation in kidney development defects)⁷⁵ and HNF1B (methylation changes in renal cysts and diabetes syndrome).⁷⁶ (e) LRS enables direct haplotype phasing, allowing determination of whether multiple variants occur on the same chromosome (in cis) or on opposite chromosomes (in trans). This information is critical for interpreting variant interactions that modify disease risk. For example, at the APOL1 locus, the p.N264K (M1) variant has been shown to confer a strong protective effect against G2-associated kidney disease when present on the same haplotype, highlighting the importance of accurate phasing when evaluating APOL1 risk configurations in CKD genetics.^77,78 CKD, chronic kidney disease. Created with BioRender (BioRender.com).

Clinical Applications of LRS in Kidney Disease

There are currently 2 major LRS technologies: single molecule real-time and nanopore-based sequencing platforms. Single molecule real-time sequencing, developed by Pacific Biosciences (PacBio, Menlo Park, CA) was the first widely adopted LRS technology. It facilitates the direct observation of DNA polymerase activity as it synthesizes new DNA. Fluorescently labelled bases incorporated by the polymerase, allow for real-time detection through zero-mode waveguides. PacBio LRS platforms currently include the Sequel IIe, Revio, and Vega sequencers.²²

Nanopore-based sequencing, developed by Oxford Nanopore Technologies (ONT; Oxford, UK), has grown significantly since its first commercial release in 2014. This method uses a membrane with embedded nanopores to measure ionic current changes as DNA or RNA passes through. During library preparation, motor protein-tagged adapters are attached to guide the polynucleotides into the pores. As the template strand translocates through the nanopore, unique disruptions in current flow correspond to each nucleotide, enabling real-time sequencing. ONT platforms include MinION, GridION, and PromethION, which are applicable in both basic and clinical research.^79,80

LRS can significantly enhance the detection and characterization of SVs, improve haplotype resolution, accurately differentiate pseudogenes, and sequence tandem repeat expansions (Figure 2). Thus, LRS holds the potential to increase the diagnostic yield of genetic testing and facilitate the discovery of novel disease-associated genes.⁶⁷

A pilot study conducted across All of Us research program sequencing centers applied LRS to a set of control samples to evaluate platform performance and reproducibility. This study demonstrated that LRS substantially improved genome assembly quality and structural variant detection compared with SRS, with PacBio HiFi reads achieving the highest single-nucleotide variant accuracy and ONT sequencing excelling in structural variant identification across medically relevant genomic regions.⁸¹

To date, published applications of LRS in kidney disease can be broadly categorized into the following 3 domains: (i) resolution of technically complex loci, (ii) detection of repeat-associated pathogenic variants (including variable number of tandem repeats [VNTRs] and mobile element insertions), and (iii) improved structural variant detection and haplotype phasing in unresolved or ambiguous cases.²²

ADPKD (PKD1/PKD2)

A recent study investigated families with typical ADPKD, who lacked a genetic diagnosis after standard testing. By using a combination of short-read and long-read genome sequencing, the authors identified an atypical splice variant impacting PKD1 in most previously unresolved families, with multiple pathogenic changes located in noncoding regions that are not captured by standard workflows and/or are difficult to interpret without transcript or splicing assessment.⁴ More recently, targeted long-read approaches have been evaluated as clinically implementable assays for ADPKD, demonstrating improved resolution of PKD1/PKD2 variant detection (including difficult PKD1 calls) and supporting the role for LRS in complex or unresolved cases.^23,72 These studies align with the well-known technical constraint that PKD1 pseudogene homology can complicate alignment and variant attribution using short reads, motivating long-read, or long-range polymerase chain reaction (PCR)–based solutions in selected cases⁵¹ (Figure 2c).

Autosomal Dominant Tubulointerstitial Kidney Disease (MUC1)

In a Japanese cohort of suspected patients with autosomal dominant tubulointerstitial kidney disease (ADTKD), LRS identified frameshift variants in the GC-rich MUC1 known as VNTR that were missed or incompletely resolved by short-read approaches (Figure 2b), supporting the role of LRS for this repeat-associated diagnostic blind spot.⁸² The VNTR region, a GC-rich (> 80%), repetitive 60-nucleotide sequence, leads to frameshift variants (e.g., 27dupC) that produce a toxic MUC1fs protein, evading standard SRS because of alignment issues.⁸³ Consistent with this, targeted single molecule real-time LRS has been shown to enable complete VNTR assembly and precise positioning of causative MUC1 variants, providing an alternative to specialized non-SRS assays in ADTKD-MUC1.⁸⁴

APOL1-Associated Nephropathy (Phasing/cis–trans Interpretation)

The long contiguous reads generated by LRS enable direct haplotype phasing of multiple variants within the same gene, allowing determination of whether pathogenic variants are in cis or trans (Figure 2e). This information can materially change interpretation when variant combinations modify disease risk.^22,49 At the APOL1 locus, a large human genetics study reported a strong protective effect of the p.N264K (M1) variant against G2-associated kidney disease, emphasizing the importance of accurately resolving haplotypes when interpreting APOL1 risk configurations.⁸⁵ Because long reads can phase across the locus directly on single DNA molecules, LRS provides a practical route to establish whether protective and risk alleles occur on the same haplotype in an individual, something that can be challenging with short reads depending on distance/coverage.^77,78

Structural Variants and Complex Rearrangements in Inherited Kidney Disease

Structural variants (SVs) are a major, underdetected contributor to genetic kidney disease, particularly when breakpoints fall in repeat-rich regions, segmental duplications, or when rearrangements are complex (Figure 2a). In a nephronophthisis-related ciliopathy (Senior–Løken syndrome),²⁴ long-read technologies were used to resolve an otherwise hidden mobile element insertion with downstream splice consequences, illustrating the value of long reads for “cryptic” structural variation missed by routine testing.⁸⁶ For kidney tubulopathies, LRS has also been used to uncover previously undetected intronic/splice-altering variants in suspected Gitelman syndrome, demonstrating how LRS can recover missing pathogenic variation after standard approaches.⁸⁷

Epigenetic Profiling and Regulatory Variation in CKD

LRS generates millions of reads in real-time and can be performed without PCR amplification, thereby reducing amplification-related biases such as GC-content bias, preferential amplification of shorter fragments, allelic imbalance, and dropout of repetitive or structurally complex genomic regions.³² Because DNA is sequenced in its native state, LRS also enables the direct detection of base modifications including cytosine-guanine dinucleotides methylation^88,89 (Figure 2d). The use of LRS for epigenetic profiling facilitates the understanding of the regulatory mechanisms governing cellular gene expression and integrates the impacts of genetic variation and environmental influences. Heritability studies have shown that methylation explains a larger share of kidney disease heritability than gene expression.^35,90,91 As the application of LRS technology expands, it is expected to further our understanding of molecular mechanisms underlying kidney dysfunction.

Detectability Versus Diagnostic Yield

Despite these clear technical advantages, it is important to distinguish improved variant detectability from overall diagnostic yield. Although LRS offers clear technical advantages in variant detectability and genomic resolution, improved technical detection does not necessarily translate into substantially higher diagnostic yields across all CKD cohorts. In unselected adult CKD populations, diagnostic yields achieved with targeted gene panels, exome sequencing, and short-read WGS are often comparable, reflecting the fact that many genetic kidney diseases are caused by exonic variants in a relatively limited number of well-characterized genes that are already detectable using short-read technologies.

Consequently, the additional variants uniquely detectable by LRS, such as deep intronic variants, complex structural rearrangements, or repeat-associated variants are expected to account for a smaller proportion of diagnoses and may be rare at the population level. The clinical value of LRS therefore lies less in increasing overall diagnostic yield in broad CKD cohorts, and more in its ability to resolve diagnostically challenging cases, clarify ambiguous findings, and provide definitive molecular diagnoses in patients who remain undiagnosed after standard testing. Current evidence supports LRS primarily as a complementary or reflex strategy, particularly when a monogenic etiology remains strongly suspected in SRS-negative diagnostic pathways.¹⁹

To date, published data on LRS in kidney disease derive primarily from selected patient groups, those with unresolved suspected monogenic disease or technically challenging loci, rather than unselected CKD populations. No published study has applied genome-wide LRS to a consecutively enrolled or unselected CKD cohort, and truly blinded head-to-head comparisons of SRS and LRS diagnostic yield in kidney disease remain lacking. Del Gobbo and Boycott¹⁹ performed one of the most systematic comparisons of LRS versus SRS in rare disease broadly, demonstrating incremental diagnostic yield in SRS-negative cases; however, their cohort was not kidney-specific.

LRS Limitations

A recognized limitation of LRS is its comparatively lower per-base coverage in whole-genome applications relative to targeted gene panels or exome sequencing, which may limit sensitivity for detecting low-level mosaic variants. Detection of mosaic variants is inherently challenging because they often occur at low variant allele fractions, and most existing mosaic variant detection algorithms were originally developed for SRS, with dedicated tools for long-read data only recently emerging.⁹² Mosaic pathogenic variants have been identified in a proportion of patients with ADPKD who had negative standard genetic testing, highlighting the clinical relevance of mosaicism in CKD context.⁹³

Detection of variants generally requires high read depth, which, currently is more readily achieved with short-read targeted approaches. Accordingly, LRS should be viewed as complementary rather than universally optimal, with its use guided by the specific clinical question and previous testing results.

Strategies for LRS in CKD Diagnostics

In clinical nephrogenetics, the choice between WGS-LRS and targeted long-read approaches is guided by the underlying clinical question.⁹⁴ WGS, whether performed using short-read or long-read platforms, provides comprehensive genome-wide analysis and enables hypothesis-free variant discovery.95, 96, 97 However, because sequencing resources are distributed across the entire genome, WGS typically achieves lower per-locus depth compared with targeted enrichment approaches, which concentrate sequencing coverage on selected regions.⁹⁵ Consequently, targeted strategies may offer higher sensitivity for specific loci at lower cost, whereas WGS provides broader discovery potential at the expense of increased data volume, analytical complexity, and overall resource requirements.⁹⁸

Owing to higher cost and historically higher per-read error rates associated with LRS relative to SRS, target enrichment techniques have gained prominence for enhancing the capture of disease-relevant genomic regions while addressing the issue of error rates, for example, those associated with ONT platforms.⁹⁹ In a study, targeted LRS was applied to 10 individuals with suspected recessive or X-linked Mendelian diseases whose molecular diagnosis was incomplete after previous testing. Targeted LRS improved diagnostic yield by identifying structural, intronic, and complex variants that had been missed by earlier methods.¹⁰⁰ Common enrichment strategies used alongside LRS include the following: adaptive sampling (AS), hybridization-based capture, amplicon-based enrichment via long-range PCR (LR-PCR), and clustered regularly interspaced short palindromic repeats (CRISPR)-guided techniques, each with distinct advantages, limitations, and applications in CKD research.

Real-Time Enrichment – AS

Specifically developed for ONT sequencers, a real-time, molecule-by-molecule selective sequencing approach, also known as the “Read Until” technique, was introduced as an alternative to conventional enrichment approaches.¹⁰¹ During sequencing, DNA strands translocate through nanopores, generating real-time electrical current signals that are decoded into nucleotide sequences through basecalling and subsequently aligned to a reference genome, with files specifying the genomic coordinates of target regions. The system provides real-time feedback to the sequencing pore, allowing fragments of interest to continue sequencing, whereas noninformative DNA is selectively ejected, freeing the pore for other molecules.¹⁰¹ Advantages of AS include cost efficiency, as it enables background depletion and target enrichment in a single run without additional hardware, and flexibility for real-time adjustments, which is ideal for low-input samples without the need for amplification.¹⁰²

In CKD research, AS has shown promise for real-time enrichment of nephrogenetic regions, as demonstrated in a 2025 study that implemented a customized AS approach, referred to as Cornetto, to specifically enrich and assemble the MUC1 locus in VNTR region.¹⁰³ However, AS is not error-free; a 2022 study highlighted that AS could suffer from inaccurate strand ejection because of mapping errors, leading to false negative rejections (where target molecules are ejected, though observed at low occurrence), off-target sequencing, and reduced enrichment efficiency, particularly for short DNA molecules or large reference databases. Additionally, its reliance on high-performance computing, often requiring costly graphics processing units, can limit throughput in resource-constrained settings.¹⁰⁴

Hybridization-Based Capture

Hybridization-based capture commonly is used with both ONT and PacBio platforms. This method excels in covering larger genomic regions (e.g., up to megabases) with high specificity and uniformity, making it suitable for comprehensive variant detection in complex loci which makes the method suitable for clinical application.³⁰ However, this method also brings important disadvantages, such as higher costs for probe design and more time-consuming, error-prone library preparation. An extended and often complicated library construction protocol is needed, which involves multiple PCR amplification steps that limits the length of DNA captured and can introduce biases in repetitive or GC-rich areas. The coverage drops significantly in extreme GC-content regions, performing worse than SRS, next-generation WGS in regions of extreme GC content.⁹⁵

A 2021 study applied hybridization capture with PacBio LRS to mismatch repair genes, achieving 98% on-target reads and superior coverage in repetitive loci.⁶⁷ In patients with ADPKD, hybridization-based capture combined with PacBio LRS has been used to resolve PKD1 variants, achieving higher diagnostic yields by accurately distinguishing true genetic variants from pseudogene artifacts.²³

In CKD diagnostics, hybridization-based capture is most useful for known disease loci where increased coverage can resolve complex or duplicated regions.

Enzyme-Guided Enrichment – CRISPR-Cas Approaches

Because of its high programmability and specificity, the CRISPR-Cas9 system has been used in a wide variety of biotechnological applications that involve validate CKD-associated variant,⁵² genome editing, gene therapy, and targeted sequencing.^105,106 CRISPR-guided enrichment uses Cas9 or similar enzymes to cleave and isolate target regions, enabling precise enrichment of native DNA without amplification.¹⁰⁷ Although in CKD context CRISPR-based enrichment has not been attempted, studies in other fields have shown that it has strong potential.

The CRISPR-guided enrichment process necessitates meticulous optimization of guide RNAs and entails risks of incomplete cleavage in complex samples, requiring substantial technical expertise and financial investment for synthesis. These factors currently constrain the routine application of this approach in clinical settings, including its potential use in CKD diagnostics.¹⁰⁸

Amplicon-Based Enrichment – LR-PCR

LR-PCR amplifies large DNA targets using specialized polymerases, followed by LRS to generate error-corrected consensus reads. Its advantages include procedural simplicity and compatibility with PacBio circular consensus sequencing, whereas limitations involve PCR bias, artifacts in homologous regions, and failure to detect certain SVs.⁵¹ In CKD diagnostics, LR-PCR has been used to detect MUC1 frameshift variants in ADTKD; notably, a 2022 study enhanced diagnostic accuracy by combining LR-PCR with ONT sequencing in clinically diagnosed patients with ADTKD.⁸²

Selecting the optimal enrichment strategy requires consideration of target complexity, sample quality, and sequencing platform. For CKD, hybridization and LR-PCR are widely adopted and used for their robustness in duplicated regions, whereas CRISPR-based enrichment could further improve cost-effectiveness and precision for larger panels. Together, these enrichment strategies highlight that targeted LRS approaches are complementary tools in CKD diagnostics, best applied in selected clinical contexts rather than as universal replacements for WGS.^19,23

Reference Bias, SV Representation, and Underrepresented Population

Genomic research plays an indirect but critical role in improving clinical care by expanding variant discovery and reference databases, particularly through the inclusion of diverse and historically underrepresented populations.¹⁰⁹ Publicly available genetic variation datasets, like the widely used Genome Aggregation Database, lack population-specific data for many indigenous and First Nations groups.¹¹⁰ Large-scale research sequencing efforts enable the identification of population-specific single nucleotide variants and structural variants, refine allele frequency estimates, and reduce uncertainly in variant interpretation, especially for patients whose ancestry is poorly represented in existing databases.^109,111,112 However, the translation of research genomics into routine clinical management remains an evolving process and is not yet fully optimized, particularly for complex disorders such as CKD. The practical expansion of genomic access in remote and underserved communities is more likely to be achieved in the near term through centralized SRS workflows, including sample batching and transport to core sequencing facilities, than through point-of-care LRS. Portable ONT devices such as the MinION are well suited to targeted or surveillance applications, but their use for large-scale population genomics in remote areas remains logistically constrained. The more specific contribution of LRS in indigenous and underrepresented populations is in structural variant discovery and epigenetic characterization, capabilities that are not accessible with short-read methods. These advancements represent incremental but meaningful progress in CKD research and clinical care, with LRS contributing complementary capabilities that are not achievable with SRS methods alone.

It should be noted that the advantage of LRS in SV detection is not specific to any population. However, in populations with limited SRS-based reference data, such as many indigenous and First Nations groups, LRS de novo assembly provides a reference-independent approach to identify structural variation that would be missed by SRS alignment pipelines reliant on existing reference genomes. This has been demonstrated empirically — Reis et al.,¹¹³ using Nanopore LRS, identified over 160,000 SVs unique to Aboriginal Australians not present in the Genome Aggregation Database, illustrating the complementary contribution of LRS to population genomics in this context. The primary solution to database underrepresentation, however, remains broader inclusion of diverse populations in well-funded SRS programs.

By expanding structurally resolved genomic datasets across ancestries, LRS may enhance the accuracy of variant interpretation and contribute to reducing diagnostic uncertainty, thereby potentially mitigating health inequities in indigenous communities globally.

Clinical Integration and Implementation Considerations

A diagnostic workflow using LRS technology relies on close collaboration across multiple fields, including healthcare, molecular biology, biotechnology, bioinformatics, and genetics. The need for interdisciplinary collaboration is crucial, and the integration of genetic testing into CKD care faces multiple systemic and practical challenges.

Economic Aspects and Access

Genetic testing enables timely and accurate diagnoses in nephrology, informing treatment decisions and potentially reducing costs by minimizing prolonged diagnostic investigations and ineffective treatments. However, its upfront costs limit widespread adoption, highlighting the need to improve access to this valuable technology. Disparities persist because of limited access to care in marginalized communities, exacerbating health inequities.^10,114,115 A study reviewed CKD-related health policies in 160 countries, finding that fewer than 40% have dedicated strategies to solve access issues. It highlights major global disparities in CKD prevention, care, and workforce capacity. Low-income countries are particularly underserved. Authors call for urgent global action to integrate CKD into national health agendas.¹¹⁶ As the access to genetic screening technology remains a challenge in many parts of the world, disparities exist based on socioeconomic status, geographic location, and healthcare infrastructure, which can limit access to laboratories, technology improvements, and specialist care, for certain populations.¹¹⁷

However, since 2019, when SRS averaged ∼$942 and LRS with PacBio’s Sequel II cost ∼$1,500, genomic sequencing costs have substantially decreased; in 2025, ONT have currently reduced the LRS costs of the human genome at 30 times depth to about $850 per individual.¹¹⁸ As sequencing technologies advance, further upfront cost reductions are anticipated, fostering optimism for enhanced global accessibility and equitable integration into clinical practice.

Ethical Aspects

Legal and psychological challenges associated with genetic data play a significant role in hindering progress and limiting the adoption of genetic screening.⁴⁸ Public health policies increasingly address ethical issues regarding genetic testing, such as privacy, consent, and potential discrimination based on genetic information.¹¹⁹ Concerns have emerged over the accessibility of genetic testing, which may lead to information overload and potential misuse of data related to nonmedical traits.¹²⁰

Technical Aspects

Accurate interpretation of structural and intronic variants remains challenging across genomic medicine. In CKD, where SVs and deep intronic variants contribute to unresolved diagnoses in a subset of monogenic conditions, integration of LRS-derived data into shared population databases (such as Genome Aggregation Database and ClinVar) would directly support clinical variant classification.¹²¹ Furthermore, some initiatives propose integrating LRS with artificial intelligence-based pipelines and cloud-based data-sharing frameworks to create dynamic, learning databases, but these remain in early stages.¹²²

There is also a growing need for tailored analytical workflows integrating advanced bioinformatics tools to interpret the vast amount of data these techniques generate and translate it into meaningful insights and conclusions. The integration of these tools into clinical practice relies heavily on bioinformatics expertise and the development of standardized protocols.123, 124, 125 Moreover, significant gaps remain in physicians’ knowledge of genetics, further complicating its clinical application.¹²⁶ To fully realize the clinical potential of genomics in CKD, it is essential to invest in multidisciplinary collaboration among molecular biologists, bioinformaticians, and geneticists, and develop user-friendly infrastructures that support both clinical workflows and family-based screening.¹⁸

Conclusions and Future Research Directions

Beyond its current role in resolving diagnostically challenging inherited kidney diseases, LRS may contribute to selected advances in CKD research and precision medicine, particularly in technically challenging diagnostic scenarios, though its broader impact will depend on continued reductions in cost and improvements in analytical pipelines. By enabling more comprehensive characterization of genetic, epigenetic, and regulatory variation, and by facilitating integration with multiomics and computational approaches, LRS may offer incremental diagnostic improvements beyond SRS in selected clinical scenarios. Its contribution to the understanding of CKD pathogenesis and informing therapeutic strategies will require further validation at scale before broader clinical translation can be achieved. Understanding the functional consequences of genetic variation is a key step toward translating genomic discoveries into clinical insight. Recent large-scale functional genomics studies in kidney tissue have begun to bridge this gap by linking genetic variants to gene expression and cellular pathways relevant to CKD. A comprehensive expression Quantitative Trait Locus mapping from 659 kidney samples identified over 9000 cell-type-specific genes influencing traits like estimated glomerular filtration rate and blood pressure, prioritizing proximal tubules for kidney function, and nominating drug-targetable genes like ACE for CKD therapies.¹²⁷ In parallel, another key advancement in CKD research has been the integration of multiomics approaches.^128,129 By combining genomics, transcriptomics, epigenomics, and proteomics, we are gaining a more nuanced understanding of the complex biological processes that contribute to disease progression. Multiomics not only fills gaps left by traditional genome-wide association studies, but also helps explain the so-called “missing heritability” of CKD. On the computational side, advanced machine learning algorithms are facilitating more accurate variant calling, phenotype classification. Tools such as DeepVariant and Clair3,¹³⁰ specifically optimized for LRS data, enhance the detection of rare or complex genetic variants.

Within multiomics frameworks, the integration of genomics, proteomics, metabolomics, and transcriptomics is reshaping disease classification and prognosis. Artificial intelligence-driven models show promise for CKD risk prediction and personalized treatment planning by integrating multibiomarker panels with clinical data.¹²⁹ However, challenges remain in biomarker standardization, large-scale validation, and clinical implementation.

Continued expansion of genomic research to include diverse and historically underrepresented populations, supported by both SRS and LRS technologies, has the potential to improve variant interpretation and reduce diagnostic uncertainty across all CKD patient groups.¹³¹

Future research should focus on refining LRS technologies for clinical use, working to reduce its cost, identifying population-specific genetic variants to discover novel CKD markers, and enhancing bioinformatics tools to translate large-scale data into actionable clinical outcomes.^132,133 Emerging multiomics and artificial intelligence, which incorporate LRS, must address challenges in clinical validation, biomarker standardization, and the development of practical strategies for seamless incorporation into routine clinical practice, underscoring the need for continued research.¹²⁹

Addressing social and ethical considerations is crucial, requiring strategies to build community trust, ensure privacy, promote equitable access, and implement transparent benefit-sharing, alongside public awareness campaigns and genetic counselling to empower underserved communities for informed decision-making and improved CKD prevention and care.

Disclosure

AJM declares being a member of advisory boards for Natera, GSK, and PYC, with travel support from Otsuka, and an unpaid Board member of the Australia and New Zealand Society of Nephrology. The other authors declared that they have no competing interests.