Hard-to-Sequence Genes | Native Long-Read Sequencing

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Hard-to-sequence genes and complex genomic regions are defined by features that are difficult to characterize with short-read sequencing, including high homology genes, repetitive sequences, and structural variation.
Because short-read sequencing fragments DNA into short segments that often lack sufficient context to resolve these features, conventional approaches frequently produce incomplete or ambiguous results.
Long-read sequencing overcomes these limitations by capturing extended genomic context within single molecules, enabling accurate variant detection, haplotype resolution, and structural interpretation.

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Flexible, Scalable Long-Read Resolution

Renew integrates native long-read sequencing with both whole-genome and targeted strategies to overcome these limitations while maintaining resolution and scalability.

This Approach Enables

Accurate resolution of homologous and repetitive genomic regions

Full haplotype resolution across clinically relevant genomic loci

De novo assembly of complex
and structurally diverse genomes

Which Is Supported With

Cohesive interpretation of sequence and structural genomic context

Flexible study design across whole-genome and targeted approaches

End-to-end bioinformatics support for analysis and interpretation

Applications

Our applications span pharmacogenomics, telomere biology, and other complex genomic analyses, where standard approaches fall short due to structural complexity. In each case, long-read sequencing enables precise, decision-relevant insight beyond what conventional methods can provide.

Where allele structure directly impacts clinical interpretation, long-read sequencing provides critical resolution. Clinical PGx value today is concentrated in a defined set of genes with well-established roles in drug metabolism and response.

While many of these genes can be adequately resolved with short-read sequencing, structurally complex loci, such as CYP2D6, HLA, UGT1A1, and select DPYD/TPMT contexts, require long-read approaches to accurately capture duplications, hybrid alleles, and phased variants that determine functional output.

Table comparing expected and called CYP2D6 genotypes for various cell lines with replicates and barcodes.

Figure 1. Long-read sequencing enables decision-grade CYP2D6 haplotype and structural variant resolution. Per-sample CYP2D6 star-allele calls, including copy number and hybrid structures (e.g., *1/*4x2, *36+*10), were compared to expected haplotypes across truth-set samples (GIAB, Corielle; ≥30x). Despite minor discrepancies in biologically complex regions, high concordance was observed, demonstrating the ability of long-read sequencing to accurately resolve PGx haplotypes and structural variation in clinically relevant genes.

Legacy telomere methods force trade-offs between accessibility and resolution. Techniques such as qPCR, Flow-FISH, and TRF provide global estimates of telomere length but rely on averaged signals across chromosomes, obscuring arm-specific variation, subtelomeric structure, and genomic context.

This lack of resolution limits both research and clinical insight. For researchers, it masks locus-specific mechanisms driving genomic instability, aging biology, and disease progression. For clinicians, it can obscure high-risk profiles, where critically short telomeres on specific chromosome arms remain hidden within a “normal” global average, limiting accurate risk assessment and patient stratification. Long-read sequencing overcomes these limitations by enabling direct, chromosome-specific measurement of native telomere length while preserving subtelomeric context.

Box plots and scatter plots show mean recalling times for HARD and EASY materials in a study.

Violin plot showing FAR data distribution for June and July, with July data more spread and higher values.

Graph comparing vertical launch stress on pilots from a shuttle rocket and a fairing capsule.

Violin plots comparing task durations by gender showing women generally take longer than men.

Figure 2. ONT long-read sequencing resolves telomere arm-length differences masked by global averages. Per-arm telomere length distributions were compared between healthy and CLL PBMC donors using long-read sequencing. Despite modest global shifts, multiple chromosome arms showed significant median differences (~2.5–5.5 kb).

Clinically-relevant genes with highly homologous pseudogenes represent a major blind spot for short-read sequencing, where high sequence similarity leads to misalignment, off-target mapping, and unreliable variant calls. This challenge is exemplified by PKD1 (Polycystic Kidney Disease 1), which shares >97% homology with multiple pseudogenes, making accurate variant detection difficult with short reads.

Long-read sequencing overcomes this limitation by spanning homologous regions within single molecules, enabling accurate mapping and variant resolution without reliance on complex PCR strategies. In targeted nanopore workflows, this also allows simultaneous interrogation of sequence variation and methylation within structurally complex loci.

Box plots comparing read percentages for target and pseudogene/hemolog genes by DNA type on the X chromosome.

Figure 3. ONT long-read sequencing enables accurate alignment in highly homologous PKD1 pseudogenes. Read alignments across PKD1 and its pseudogenes (>97% homology) demonstrate accurate mapping to the functional gene, with significantly higher reads per kilobase compared to homologous loci. Across 634 target regions, SNV detection achieved 98.5% precision and 99.5% recall with >120× mean coverage and >1,600x enrichment.

Novel and non-model genomes often present challenges that limit accurate assembly with short-read sequencing, including high repetitive content, structural complexity, and degraded or low-quality input DNA. These constraints frequently result in fragmented assemblies, misaligned contigs, and incomplete representation of functionally important regions.

Long-read sequencing overcomes these limitations by spanning repetitive elements and preserving long-range genomic context, enabling more contiguous and complete assemblies. Wasatch BioLabs applies optimized extraction, high-molecular-weight DNA workflows, and long-read nanopore sequencing to generate high-quality genome assemblies from challenging samples, supporting downstream functional, evolutionary, and translational research.

Bar chart showing species sequencing difficulty with T. Ignis highlighted as fifth hardest to sequence.

Figure 4. Long-read sequencing enables high-quality, functionally-complete de novo genome assembly. BUSCO analysis of the Tedania ignis genome assembly demonstrates high completeness (86.4%), indicating strong representation of conserved orthologs relative to reference species. RNA-seq validation showed >82% alignment to the assembled genome, supporting structural accuracy and functional integrity, and highlighting the effectiveness of long-read sequencing for complex, non-model genome assembly.

Accurate microbial community profiling requires resolving species-level composition without distortion from amplification bias or incomplete genomic context. Both conventional and long-read targeted 16S approaches rely on PCR and other enrichment methods, introducing bias and artifacts while excluding broader genomic and epigenetic information.

Long-read, whole-genome sequencing overcomes these limitations by directly profiling native DNA, enabling species- and strain-level resolution with integrated methylation detection. Wasatch BioLabs’ Direct Whole Methylome Sequencing (dWMS) platform delivers a PCR-free, scalable workflow that preserves native genomic and epigenetic context, supporting high-fidelity microbial profiling even at reduced sequencing depth.

Bar chart comparing frequency of first and repeated COVID-19 vaccine doses by age group in 2023.

Figure 5. dWMS accurately recapitulates microbial community composition and outperforms targeted 16S methods. Whole-genome dWMS sequencing of a 10-species microbial standard achieved accurate species identification and relative abundance estimates, with an average deviation of 5.7% from theoretical composition. Compared to both Illumina short-read and ONT long-read 16S workflows, dWMS demonstrated improved species-level accuracy and reduced bias, maintaining performance under downsampled conditions and highlighting the value of long-read, PCR-free profiling.

Questions & Answers

What types of genomic regions are considered hard to sequence?

Genomic regions that are difficult to resolve with traditional short-read sequencing are considered hard to sequence. These often include regions with high sequence homology, repetitive elements, structural variation, copy number changes, or complex haplotypes that are difficult to resolve from short read fragments alone.

How does long-read sequencing improve analysis of complex genomic regions?

Long reads span more of a complex genomic region and its surrounding context, enabling more accurate assembly and resolution than short-read sequencing.

What types of variants can Renew resolve in hard-to-sequence genes?

Depending on the locus and study design, Renew can support analysis of SNVs, indels, structural variants, copy number changes, repeat expansions, hybrid genes, and phased haplotypes within complex genomic regions.

Can Renew analyze clinically relevant complex genes such as CYP2D6 or HLA?

Yes. Renew supports long-read analysis of structurally complex human loci including CYP2D6, HLA, UGT1A1, and select DPYD and TPMT genes where duplications, hybrid alleles, or phased variants may affect clinical interpretation and decision making.

Does Renew offer both whole-genome and targeted approaches for complex regions?

Yes. Renew offers both whole-genome and targeted long-read sequencing approaches tailored to research objectives, from broad genomic characterization to deeper, scalable analysis of specific genes and regions.

What sample types and input requirements are compatible with these studies?

Specific sample requirements depend on the services selected and the study objectives. Renew works with each team to define DNA input, quality, and preparation requirements based on the locus, sequencing strategy, and study objectives.

How does Renew support custom analysis for hard-to-sequence regions?

Renew provides study design, sequencing strategy, bioinformatics, and interpretation support tailored to complex loci, including haplotype resolution, structural variant analysis, de novo assembly, and other project-specific endpoints.

Renew combines platform flexibility, nanopore expertise, and operational excellence to resolve areas of the genome that standard approaches cannot.

Hard-to-Sequence Genes & Complex Regions

Flexible, Scalable Long-Read Resolution

Applications

Questions & Answers

What types of genomic regions are considered hard to sequence?

How does long-read sequencing improve analysis of complex genomic regions?

What types of variants can Renew resolve in hard-to-sequence genes?

Can Renew analyze clinically relevant complex genes such as CYP2D6 or HLA?

Does Renew offer both whole-genome and targeted approaches for complex regions?

What sample types and input requirements are compatible with these studies?

How does Renew support custom analysis for hard-to-sequence regions?