Liquid biopsies are increasingly being used as a cancer detection modality. Epigenetic characterization of cell-free DNA (cfDNA), specifically DNA methylation, is an emerging approach for sensitive detection and quantification of tumor burden. Detection of early-stage cancers remains an ongoing challenge due to lower amounts of tumor-specific DNA molecules found in cfDNA. In the context of breast cancer detection, existing cfDNA analysis methods suffer from poor sensitivity especially at earlier stages of malignancy. Solving this challenge, we developed a novel approach for single-molecule methylation analysis of cfDNA from cancer patients. Our study consists of a large cohort (~200 with stage I-II breast cancer and ~400 healthy controls). We successfully distinguished breast cancer cfDNA samples from healthy controls based on separate validation datasets. Overall, our study demonstrates that our nanopore-based cfDNA sequencing method can be an effective and scalable method for the non-invasive detection of breast cancer from blood.

Standard clinical genetic testing for individuals with a suspected imprinting disorder, such as Prader Willi syndrome (PWS) or Angelman syndrome (AS), typically involves multiple assays performed sequentially and can take months to years to complete. Because long-read sequencing (LRS) data can be analyzed for copy number variation, single nucleotide and indel variants, structural variants, and differences in methylation, we hypothesized that it would have high concordance to standard testing. Anonymized samples with previous clinical results for PWS or AS were selected for whole genome LRS on an Oxford Nanopore Technologies PromethION device. Reports with copy number state, methylation pattern, and pattern of single nucleotide variants for the relevant region of chromosome 15 were independently reviewed. The genetic defect was correctly identified in all 15 samples. LRS shows potential future utility as an efficient diagnostic tool for suspected imprinting disorders.

Telomeres are the protective, nucleoprotein structure at the ends of linear eukaryotic chromosomes. The accurate measurement of both telomeric length and composition of individual telomeres in mammalian cells has been challenged by the length and repetitive nature of telomeres. With nanopore sequencing, it is now technically possible to sequence entire telomeres and map them to individual chromosome arms. Here, we report a reliable method to enrich, sequence and analyze human telomeres using nanopore sequencing. To enrich for telomeric sequences, we combine the ligation of adapters complementary to the telomeric G-overhang with restriction enzyme digestion to sequence the telomeric C-strand and part of the adjacent subtelomere. The subtelomeric information is harvested to map individual telomeric reads to specific chromosome arms. We have measured bulk and chromosome-arm specific telomere length dynamics during cellular aging of cultured primary cells and in a patient-derived aging cohort. To address the impact of the telomere maintenance mechanism on telomere length and composition, we have sequenced five well-established telomerase- and ALT-positive cancer cell lines. Our results suggest that, based on nanopore telomere long-read sequencing, ALT-positive cells can be easily discriminated from normal and telomerase-positive cancer cells. In summary, nanopore sequencing of telomeres grants a deeper understanding of individual telomere composition through telomere length measurement and mapping to specific chromosome arms. As such, telomere sequencing using our nanopore-based enrichment method is a valuable tool to study telomere biology in aging and cancer.

The year is 2043. Following the first human landing on Mars in 2040, NASA has just embarked on the first sustained crewed mission. On the Martian surface, a prefabricated habitat with advanced and sustainable life support systems, shelf-stable food, power and communication infrastructure, and a suite of scientific hardware welcomes the crew. Within the science suite is the MinION Mk1X, which will prove invaluable for crew health monitoring, plant cultivation and agriculture, microbial surveillance within the environmental control and life support systems, and, possibly, the most transformative moment in history — the discovery of life beyond Earth. A question that has been left unanswered by previous sample return and robotic missions. On the brink of potentially redefining our understanding of life, a reflection on the scientific advancements that led us here is key. 2016 brought the dawn of the molecular space age with the success of DNA sequencing onboard the International Space Station. In the years that followed, pivotal experiments reduced Earth-dependence for complex sample analysis. Space-based genomics moved at a rapid pace throughout the early 2020s, validating off-Earth microbial identifications, full genome analysis, transcriptomics, epigenomics, targeted biomarker tracking, and data analytics. The MinION-based Microbial Monitoring System used onboard Gateway was expanded for planetary protection to create microbiome baselines as an alert for forward and backward contamination. The lunar surface provided the paving ground to define suit and vehicle leak rates, decontamination strategies, and microbial dispersion and transport. The culmination of these breakthroughs resulted in the MinION’s path to Mars.

Our laboratory has been developing testing and analysis tools focused on next generation sequencing (NGS) to improve bacterial disease public health work for the last 10 years. Nanopore sequencing has enhanced and improved our work in many ways. We have utilized long-read nanopore sequencing to investigate the relatedness of plasmids in antibiotic resistant bacteria and elucidate antimicrobial resistance mechanisms. Our targeted NGS approaches have benefited from nanopore sequencing which has provided rapid, accurate and cost-effective testing to identify drug-resistant Mycobacterium tuberculosis from patient specimens Additionally, testing to identify unknown bacteria to the species level by 16S and other conserved targets is being utilized. Future work will include exploring direct specimen NGS and continuing to optimize NGS testing approaches for public health action.

Genome sequencing can better identify disease-causing variants in inherited retinal diseases (IRDs), however, all known IRD genomic loci constitute less than 3% of total DNA in the human genome. Targeted enrichment utilizing adaptive sampling on the Oxford Nanopore Technologies platform avoids wasting sequencing bandwidth on uninformative reads to allow much deeper coverage from the same sequencing effort. In this talk we will discuss how real-time analysis can be performed to allow target enrichment by directly rejecting or accepting DNA molecules without specialized sample preparation to generate phased data sets for narrowing of disease-causing variants from the proband alone and reveal candidate non-coding variants in cases of missing heritability.

Pediatric diffuse midline gliomas (pDMG) are universally fatal childhood brain tumors with a median survival rate of only 9-12 months. Frequent radiographic monitoring (via MRI/CT) is desirable to track response to therapy but remains difficult due to the diffuse nature of these tumors and the potential for radiation induced swelling to masquerade as tumor progression (pseudo-progression). Recent work has shown that quantification of plasma cell-free tumor DNA (cf-tDNA) in pDMGs via digital droplet PCR (ddPCR) might supplement radiographic monitoring, and enable more quantitative, convenient, and frequent monitoring as long as results can be returned within a few days. ddPCR is the current gold standard but is expensive and requires burdensome assay design and validation. Plasma is an ideal biofluid, but due to the selective permeability of the blood brain barrier, plasma cf-tDNA signal is very low (typically <0.5% allele frequency) preventing analysis via native, un-error-corrected nanopore sequencing in most contexts. We combine rapid loop-mediated isothermal amplification (LAMP) of narrow cf-DNA fragments with concatemeric error correction to improve nanopore sequencing accuracy. When applied to serial timepoint samples collected during treatment of three patients (n = 5,5,8 respectively), we show that error corrected nanopore results correlate with ddPCR and that concatemeric error correction is important to reduce native error. In two patients, small increases in cfDNA measured by our approach correlated with clinical progression. To the best of our knowledge, this is the first demonstration of plasma-based serial time-point monitoring performed by any sequencing technology on central nervous system tumors.

Using genomic data to monitor the spread of current and emerging pathogens or understanding how to treat and prevent infection requires high-quality pathogen genomes. The recent global increase in fungal infections caused by Candida species has brought attention to the emergence of antimicrobial resistance among fungal pathogens, the rise in incidence of invasive non-albicans Candida (iNAC) species, and the increasing burden this brings to the healthcare system. Many fungal genomes contain long repetitive sequences and GC-rich regions, making assembly challenging when relying solely on short-read sequencing platforms. However, long-read sequencing platforms generate sequence reads that can span these problematic regions and enable the assembly of complete and accurate fungal genomes. Here, nanopore sequencing was utilized in de novo genome assembly of a Candida parapsilosis (one of the leading causes of iNAC infection) isolate derived from the blood specimen of a neonate with sepsis. While the mother of the neonate was asymptomatic, we also generated an additional C. parapsilosis genome derived from placental tissue that upheld a previous microscopy finding of candidiasis in formalin-fixed paraffin-embedded tissue. The generation of high-quality genome assemblies using long-read sequencing enabled comparative genomic analyses of two cases of infection by the same iNAC species and provided invaluable feedback to physicians on a complex case of maternal-fetal transmission at Texas Children’s Hospital.

Avian influenza virus (AIV) is a significant infectious agent worldwide. This became particularly evident during the ongoing Highly Pathogenic Avian Influenza (HPAI) outbreak. In this context of an outbreak response, rapid and accurate diagnosis is essential. The current diagnostic workflow relies on a combination of qPCR and genome sequencing, which can take days to weeks and is too slow for outbreak control. Nanopore sequencing offers many features which make it suitable for improving genome sequencing in veterinary diagnostics. However, working directly from clinical samples is challenging due to the overabundance of host nucleic acid, compromising the assay’s sensitivity. In this study, we aim to optimize the nanopore sequencing workflow for AIV identification and characterization from clinical samples by comparing different enrichment strategies. We tested host nucleic acid depletion, target-specific and target-independent enrichment approaches during the pre-processing steps. Host nucleic acid depletion treatment involved DNA and rRNA depletion. Treated and untreated RNA extracts were amplified via sequence-independent, single-primer amplification (SISPA) to increase viral reads. Enrichment and depletion strategies effectively reduced non-target DNA and RNA concentration from the samples, while SISPA increased viral detection. Additionally, target-specific enrichment, using Influenza A Universal Primers, will be used to specifically amplify our AIV target sequences. This amplicon-based approach will be compared to SISPA enrichment, in its sensitivity and genome coverage results. The final goal of this study is to generate a complete workflow for AIV diagnosis using nanopore sequencing, reducing the time for diagnostic, while keeping sensitivity and accuracy. Consequently, this will optimize the extraction of genetic information from clinical samples.

Long-read sequencing allows for the quantification of many complex aspects of RNA and so it can be used to profile biological causes of complex problems such as cancer drug resistance. We developed FLAIR, a tool for detecting alternative splicing of genes from long reads, as well as the haplotype of the reads supporting each isoform. This allows us to detect isoform-level allele bias, particularly of clinically relevantmutations. To further examine the mutational state of the transcriptome, we also developed FLAIR-fusion, which identifies the expressed isoforms of gene fusions, which are prevalent in many cancer types. We used long-read single cell sequencing and these tools to investigate the total mutational state of expressed transcripts in a cancer cell line both before and after drug treatment. We identified known and novel gene fusions and somatic mutations as well as distinct subpopulations with changes in splicing of KRAS, an important cancer driver.

Antimicrobial resistance (AMR) remains a critical public health concern, particularly among bacteria such as Citrobacter freundii and Acinetobacter baumannii. Addressing this issue necessitates identifying genes associated with AMR, which in turn aids in understanding and mitigating outbreaks. However, conventional short-read sequencing technologies often yield truncated gene sequences and limited plasmid identification capabilities. To assess the potential of nanopore sequencing for AMR gene surveillance, we conducted a comprehensive analysis of 178 bacterial isolates from 12 distinct organisms within a public health context. Our findings demonstrate that nanopore sequencing exhibits a higher likelihood of completing partial gene matches and offers a more comprehensive depiction of plasmid content within the isolates. These promising results underscore the utility of nanopore sequencing as a valuable tool for advancing AMR surveillance and management strategies.

The phase-out of community testing programs and minimal reporting of at-home test results have resulted in a slow decline in the availability of genome surveillance for SARS-CoV-2. Environmental surveillance can fill the gaps left by community testing programs with the added benefit of being able to collect anonymous data from multiple individuals at once. In addition, environmental surveillance can provide information about other potential pathogens in a community such as respiratory syncytial virus (RSV) or influenza viruses. As a proof of concept, we used environmental air sampling in combination with unbiased metagenomic sequencing to study a household outbreak of SARS-CoV-2 and to detect diverse human viruses in community settings. This community level data can be especially useful for monitoring populations where virus outbreaks are of especially high consequence such as in long-term care facilities or to identify emerging threats in transportation hubs such as airports.

APHL Global Health leverages next-generation sequencing (NGS), bioinformatics, and genomic epidemiology to enhance public health decision-making in 78 countries. NGS emerged as a crucial component of the COVID-19 pandemic response, with internal and external investments catalyzing advancements in global pathogen genomic capabilities. We emphasize establishing regional capacities in a post/peri-pandemic setting, enabling real-time technical support. A fundamental principle is prioritizing a south-to-south global approach, fostering collaboration among nations with similar challenges. Crucially, we aim to assess the costs of pathogen genomics initiatives, ensuring their long-term sustainability. Exploring diverse use cases demonstrates the broad impact of pathogen genomics on public health outcomes, attracting funding from various entities. While the COVID-19 pandemic brought enduring challenges, it accelerated global pathogen genomics by a decade. APHL seeks to harness this momentum by showcasing the transformative potential of NGS technology, bioinformatics, genomic epidemiology, and data visualization, fostering better health outcomes at individual and population levels.

A precise genetic diagnosis is vital for clinical management and informed care decisions. While clinical genetic testing has advanced with sequencing, it has also revealed many rare genetic disorders. However, over half of the individuals with suspected disorders remain undiagnosed after thorough evaluation. Therefore, there is a pressing need for new tools and techniques to improve the diagnostic rate. In the GREGoR consortium, our objective is to unravel the genetic causes of complex Mendelian diseases that have eluded traditional methods (approximately 100 unsolved cases per year). We leveraged nanopore sequencing, enabling accurate haplotyping and variant calling, including SNVs, indels, SVs, and methylation events. By utilizing PRINCESS pipelines, we identified variants in 40 samples from affected individuals with various disorders, such as Aicardi-Goutières syndrome, craniofacial microsomia, Charge syndrome, and Multifocal lymphanioendotheliomatosis (12 trios and 3 single samples). Parental genomes aided in detecting de novo mutations and distinguishing inherited variants from disease-associated mutations. Our framework for variant calling and interpretation incorporated diverse functional genomics and population annotations as well as in silico prediction tools. Through the investigation of multiple trios, we will obtain a better understanding of these disorders and explore the potential of nanopore sequencing in uncovering the variants linked to the diseases. Preliminary analysis has yielded promising results, revealing several candidate genes and variants that warrant further investigation to discern their functional consequences. This research paves the way for improved diagnostics, prognostics, and interventions in the realm of genetic medicine.

Beginning in melanin-producing cells (melanocytes), melanoma is the most serious form of skin cancer. While it is highly curable if diagnosed and treated early, most advanced cases can be fatal. Pan-cancer analysis on exome and RNA sequencing data from The Cancer Genome Atlas and other cohorts shows that melanoma is one of the most heterogeneous cancers. A better understanding of the key genomic and epigenomic events that characterize the diverse subclonal populations in melanoma may reveal key insights into what drives its progression and therapeutic resistance. In this study, 24 distinct clonal sublines were derived in vitro from single cells of mouse B2905 melanoma cell line culture, and the genetically homogeneous population from each subline was sequenced using PromethION R10 flowcells. Empowered by the possibility to directly call 5mC base modifications and to perform haplotype phasing with long nanopore sequencing reads, we integrate insights in detected CpG methylation, small variants, and structural variants in our study of melanoma evolution. Preliminary results show that the tumor phylogeny constructed using phased CpG methylation profiles of sublines derived from long-read data corroborates phylogeny based on bulk exome/transcriptome data in a prior work. By associating structural variations and small variants with the constructed phylogeny, our analysis offers a better characterization of the epigenetic mechanism of subclonal evolution in melanoma. In particular, structural variation analysis revealed dozens of clonal and subclonal somatic events, ranging from indels to mosaic chromosomal translocations. These results inform an on-going study on subclone-specific responses to immune checkpoint blockade therapy on a preclinical melanoma model.

RNA splicing factors are recurrently mutated in blood diseases, but their impact on hematopoiesis remains unclear. The study of splicing factor mutations has been technologically challenging. To overcome such limitations, we integrated genotyping of transcriptomes (GoT) with long-read single-cell transcriptomics and proteo-genomics for single-cell profiling of transcriptomes, surface proteins, somatic mutations, and RNA splicing (GoT-Splice). We applied GoT-Splice to hematopoietic progenitors from myelodysplastic syndrome (MDS) patients with mutations in the core splicing factor SF3B1 as well as clonal hematopoiesis samples (CH). In both conditions, we observed an erythroid bias and cell-type-specific cryptic 3′ splice site usage in SF3B1 mutant cells. Collectively, our work demonstrated the conserved mechanisms of mutational impact profiled directly in human samples.

Osteosarcoma (OS) is an aggressive bone malignancy in children and young adults. Implementation and optimization of neoadjuvant chemotherapy led to significant improvements in outcome, but patients with recurrence or metastases at diagnosis continue to have poor outcomes. Whole genome sequencing (WGS) revealed that a hallmark of OS is a highly rearranged genome with complex copy number aberrations. In many respects, canine and human OS are similar. A significant difference is that initial surgery in canine OS allowed us to explore tumor evolution unperturbed by treatment. Here we performed bulk WGS of multiple tumor and normal sites in two canines to analyze the genetic and epigenetic evolution of osteosarcoma. In both canines, structural variation analysis using Severus revealed chromothripsis-like rearrangements in chr11 leading to the deletion of Cdkn2a, a known cell cycle regulator and tumor suppressor. We further detected multi-break rearrangements in chr5, chr8, and chr10. The multi-site analysis identified subclonal structural alterations in TP53 and ATRX genes. Direct phasing of heterozygous germline variants revealed copy-neutral loss-of-heterozygosity events and long identical-by-descent blocks. Haplotype-specific copy-number analysis showed different aneuploidy patterns in both canines, with primarily tetraploid karyotype and partial chromosome arm losses. Our analysis allowed us to identify clonal and subclonal genetic and epigenetic factors involved in tumor progression and evolution in canine OS,which can potentially translate to human disease.

Short-read WGS has been reliably used as a single technique simultaneously detecting all genetic variants, making the diagnostic process both comprehensive and efficient. Nevertheless, there are several shortcomings that cannot be effectively addressed by short-read sequencing, such as determination of the precise size and sequence of STR expansions, phasing of potentially compound recessive variants, resolution of some structural variants and exact determination of their boundaries. In some cases variants can only be tentatively detected by short-read sequencing and require orthogonal confirmation for clinical reporting or regulatory purposes. At Variantyx we have developed, validated, and implemented an integrated clinical genetic testing approach, augmenting short-read WGS variant detection with long nanopore sequencing reads, for orthogonal confirmation of all types of variants with additional benefit of improved identification of exact size and position of the detected aberrations. Currently Variantyx is developing an enhanced approach to variant detection, where short-read WGS results are combined with structural variant detection, STR length and sequence elucidation, and methylation utilizing long-reads. Our results demonstrate an advantage of such approach, especially when the pathogenicity is defined not only by the variant length but also by a specific sequence or methylation, taking genetic testing to a whole new level.

More than half of individuals with a suspected Mendelian condition remain without a precise molecular diagnosis after comprehensive clinical evaluation, limiting their ability to benefit from precision therapies or N-of-1 trials. One major obstacle is that traditional sequencing technologies (i.e. short-read genome sequencing [srGS]) cannot reliably identify and map structural variants (SVs; insertions, deletions, inversions, or repeat expansions larger than 50 bp). Consequently, there is a lack of understanding about the diversity of SVs in the human genome, making it difficult to filter for and prioritize candidate disease-causing variants. To address these limitations, the 1000 Genomes ONT Sequencing Consortium, building upon the 1000 Genomes Project, is using Oxford Nanopore Technologies’ long-read sequencing (LRS) platform to sequence 750 reportedly healthy individuals in an attempt to characterize normal genome-wide SV patterns and identify variants in challenging regions that are not effectively evaluated using srGS. Preliminary data from the first 250 genomes, sequenced to 30x coverage with an average read of 50 kbp, demonstrate the potential of this approach. Here we use both alignment and de novo assembly-based methods, resulting in contig N50 values exceeding 30 Mbp. With this data, we performed phased variant calling and SV calling, enabling us to filter and prioritize SVs in unresolved clinical cases. The generated dataset will be publicly available, encouraging collaborative efforts within the human genetics community to identify disease-causing variants among individuals sequenced with nanopore sequencing.

The assignment of variants across haplotypes, a process called phasing, is crucial for predicting the consequences, interaction, and inheritance of mutations and is a critical step in improving our understanding of phenotype and disease. While there are three main phasing methods, only one (read-based phasing) can comprehensively provide information also about de novo single nucleotide variants (SNVs) and their origin. But this is often limited by the read size and length of homozygous regions in the genome. To address this, we developed MethPhaser, the first method that uses haplotype-specific methylation signals from nanopore sequencing reads to extend SNV-based phasing. MethPhaser operates on a set of already-phased SNVs to extend or merge individual phased regions together, often by extending the phase blocks into homozygous regions that contain haplotype-specific methylation signatures. Benchmarking using trio-based phasing data showed that MethPhaser is able to extend the genome-wide phasing by 1.6 to 2.5-fold, while only marginally increasing the phasing error rate from 0.03% to 0.05%. We further evaluated its performance on various human populations (HG01109, HG02080, and HG03098), as well as across blood samples from a cohort of patients with cardiovascular disease. In each case, MethPhaser is able to improve phaseblock N50 with methylation information. MethPhaser represents a novel approach that uses easily accessible nanopore methylation data to improve phasing and thus the interpretation of variant interactions across many medically important genes. MethPhaser is open-source and available at https://github.com/treangenlab/methphaser.

Short tandem repeats (STR) are used in forensic studies to identify individuals. To harness the power of the MinION device, we developed STRspy 2.0 which can produce accurate forensic STR profiles from third-generation sequencing data. STRspy 2.0 is an improved version of STRspy, working as the same streamlined method capable of producing length and sequence-based allele designations from long-read data. Additionally, a new method has been implemented in STRspy 2.0, which automatically creates the autosome (auSTRs) and chrY-specific (Y-STRs) database based on the STRSeq NCBI BioProject. This expanded database (v2.0) represents the most comprehensive collection of validated sequence-based STR alleles available, enabling simultaneous profiling of auSTR (24 loci) and Y-STRs (26 loci) targeted in different next generation sequencing amplification kits. STRspy 2.0 was evaluated using four DNA controls (NIST A, B, C, and Promega 2800M) amplified and sequenced on the MinION device. The three male samples were profiled across both auSTRs and Y-STRs, whereas only auSTRs were analyzed for the female (NIST A). In auSTRs, normalized read counts were used to predict locus homozygosity or heterozygosity (reporting the top two alleles) using a threshold of 0.4. For Y-STR haplotype predictions, only the top allele was considered. The study reported a 100% concordance of each STR (auSTR and Y-STR) present in the control samples. STRspy 2.0, is the first and only third-generation sequencing platform-specific method that successfully profiles the entire autosomal STRs and Y-Chr amplified by a commercially available multiplex.

Autism Spectrum Disorder (ASD) is the most common childhood developmental disability, affecting 1 in 58 Canadian school-aged children. Despite extensive study, causal variants and molecular diagnosis remain elusive due to the heterogeneity of both the phenotype, as well as the genetic landscape associated with phenotype, which includes both inherited and de novo mutations. Currently, diagnosis is complex and behaviourally based, oftentimes occurring years after the ideal 1-2 years age. Structural variants are large and complex genomic variants which likely have underrepresented contribution to ASD. Methylation patterns associated with imprinting regions and ASD genes remain largely unexplored for their contribution to ASD. Long-read sequencing (LRS) is optimal for the comprehensive interrogation of each affected individuals’ genome for variants of clinical importance, particularly of structural variants, tandem repeats, and methylation variability. This project uses LRS integrated with phenotypic data for earlier, individualized diagnosis and treatment of ASD.

The mangroves of the Colombian Pacific represent a biodiverse and highly productive region, inhabited by a population that largely relies on economic activities such as fishing and ecotourism, with limited access to energy. This ecosystem plays a pivotal role in maintaining equilibrium within coastal and benthic marine environments. However, as a region that is widely exposed to marine vehicle traffic due to the presence of ports, it is susceptible to a looming threat of hydrocarbon exploitation, as well as other anthropogenic disturbances and the effects of climate change. We used a novel approach—field amplicon sequencing—to explore the diverse microorganisms inhabiting mangrove-associated environments. This approach overcomes challenges posed by extreme weather, transportation limitations, and energy scarcity, enabling us to illuminate the intricate microbial network within mangrove ecosystems. Leveraging state-of-the-art portable, next-generation nanopore sequencing and other compact and lightweight instrumentation, we could establish field laboratories even in remote areas. Field sequencing offers unprecedented insights into the intricate microbial diversity within mangrove ecosystems along the western Colombian Pacific coast. This pioneering study describes the microbial communities associated with the Colombian mangroves using nanopore sequencing.

Carrier screening (CS) aims to identify couples at risk for having a child with a severe genetic disorder. Although NGS is a widely used method, it fails to resolve many problematic genes, such as those with tandem repeats, copy number variation, pseudogenes, and structural variation. These genes require specialized techniques and only cover a fraction of carrier risk. We combined three innovations into a single workflow to address these shortcomings: novel short and long-range PCR enrichment, scalable any-length nanopore sequencing, and bioinformatic analysis software. The prototype assay panel interrogates 11 genes critical for CS representing ~70% of all pathogenic variants for a severe inherited disorder in at risk couples compared to gene panels at least 15 times larger. Results on over 450 samples demonstrate that both challenging and conventional genes can be analyzed in an efficient, scalable single workflow with the potential to enable CS in any laboratory.