Haplotyping Using Long-Range PCR and Nanopore Sequencing to Phase Variants: Lessons Learned From the ABCA4 Locus.

Short-read next-generation sequencing has revolutionized our ability to identify variants underlying inherited diseases; however, it does not allow the phasing of variants to clarify their diagnostic interpretation. The advent of widespread, increasingly accurate long-read sequencing has opened up new applications not currently available through short-read next-generation sequencing. One such use is the ability to phase variants to clarify their diagnostic interpretation and to investigate the increasingly prevalent role of cis-acting variants in the pathogenesis of the inherited disease, so-called complex alleles. Complex alleles are becoming an increasingly prevalent part of the study of genes associated with inherited diseases, for example, in ABCA4-related diseases. We sought to establish a cost-effective method to phase contiguous segments of the 130-kb ABCA4 locus by long-read sequencing of overlapping amplification products. Using the comprehensively characterized CEPH sample, NA12878, we verified the accuracy and robustness of our assay. However, in-field assessment of its utility using clinical test cases was hampered by the paucity and distribution of identified variants and by PCR chimerism, particularly where the number of PCR cycles was high. Despite this, we were able to construct robust phase blocks of up to 94.9 kb, representing 73% of the ABCA4 locus. We conclude that, although haplotype analysis of variants located within discrete amplification products was robust and informative, the stitching together of larger phase blocks using overlapping single-molecule reads remained practically challenging.

Inherited CD19 Deficiency Does Not Impair Plasma Cell Formation or Response to CXCL12.

BACKGROUND: The human CD19 antigen is expressed throughout B cell ontogeny with the exception of neoplastic plasma cells and a subset of normal plasma cells. CD19 plays a role in propagating signals from the B cell receptor and other receptors such as CXCR4 in mature B cells. Studies of CD19-deficient patients have confirmed its function during the initial stages of B cell activation and the production of memory B cells; however, its role in the later stages of B cell differentiation is unclear. OBJECTIVE: Using B cells from a newly identified CD19-deficient individual, we investigated the role of CD19 in the generation and function of plasma cells using an in vitro differentiation model. METHODS: Flow cytometry and long-read nanopore sequencing using locus-specific long-range amplification products were used to screen a patient with suspected primary immunodeficiency. Purified B cells from the patient and healthy controls were activated with CD40L, IL-21, IL-2, and anti-Ig, then transferred to different cytokine conditions to induce plasma cell differentiation. Subsequently, the cells were stimulated with CXCL12 to induce signalling through CXCR4. Phosphorylation of key downstream proteins including ERK and AKT was assessed by Western blotting. RNA-seq was also performed on in vitro differentiating cells. RESULTS: Long-read nanopore sequencing identified the homozygous pathogenic mutation c.622del (p.Ser208Profs*19) which was corroborated by the lack of CD19 cell surface staining. CD19-deficient B cells that are predominantly naïve generate phenotypically normal plasma cells with expected patterns of differentiation-associated genes and normal levels of CXCR4. Differentiated CD19-deficient cells were capable of responding to CXCL12; however, plasma cells derived from naïve B cells, both CD19-deficient and sufficient, had relatively diminished signaling compared to those generated from total B cells. Additionally, CD19 ligation on normal plasma cells results in AKT phosphorylation. CONCLUSION: CD19 is not required for generation of antibody-secreting cells or the responses of these populations to CXCL12, but may alter the response other ligands that require CD19 potentially affecting localization, proliferation, or survival. The observed hypogammaglobulinemia in CD19-deficient individuals is therefore likely attributable to the lack of memory B cells.

Long-read sequencing to resolve the parent of origin of a de novo pathogenic UBE3A variant.

Background The ever-increasing capacity of short-read sequencing instruments is driving the adoption of whole genome sequencing (WGS) as a universal approach to the diagnosis of rare genetic disorders. However, many challenging genomic regions remain, for which alternative technologies must be deployed in order to address the clinical question satisfactorily. Methods Here we report the use of long-read sequencing to resolve ambiguity over a suspected diagnosis of Angelman syndrome. Results Despite a normal chromosomal microarray result and methylation studies at the imprinted 15q11q13 locus, the continued clinical suspicion of Angelman Syndrome prompted trio WGS of the proband and his parents. A de novo heterozygous frameshift variant, c.2370_2373del (NM_130838.2) p.(Asp790Glufs*7), in UBE3A was identified. To determine the parental allele on which this variant arose, long-read sequencing of the flanking genomic region was performed. Comparison of the resulting haplotypes allowed us to determine that the pathogenic frameshift variant arose on the maternal allele, confirming a diagnosis of Angelman syndrome in this case. Conclusion Long-read nanopore sequencing provides significant clinical utility when assessing the parental origin of de novo variants.

Identification of a novel MAGT1 mutation supports a diagnosis of XMEN disease.

XMEN (X-linked immunodeficiency with magnesium defect) is caused by loss-of-function mutations in MAGT1 which is encoded on the X chromosome. The disorder is characterised by CD4 lymphopenia, severe chronic viral infections and defective T-lymphocyte activation. XMEN patients are susceptible to Epstein-Barr virus infections and persistently low levels of intracellular Mg2+. Here we describe a patient that presented with multiple recurrent infections and a subsequent diffuse B-cell lymphoma. Molecular genetic analysis by exome sequencing identified a novel hemizygous MAGT1 nonsense mutation c.1005T>A (NM_032121.5) p.(Cys335*), confirming a diagnosis of XMEN deficiency. Follow-up immunophenotyping was performed by antibody staining and flow cytometry; proliferation was determined by 3H-thymidine uptake after activation by PHA and anti-CD3. Cytotoxic natural killer cell activity was assessed with K562 target cells using the NKTESTTM assay. While lymphocyte populations were superficially intact, B cells were largely naive with a reduced memory cell compartment. Translated NKG2D was absent on both NK and T cells in the proband, and normally expressed in the carrier mother. In vitro NK cell activity was intact in both the proband and his mother. This report adds to the growing number of identified XMEN cases, raising awareness of a, still rare, X-linked immunodeficiency.

Long-read nanopore DNA sequencing can resolve complex intragenic duplication/deletion variants, providing information to enable preimplantation genetic diagnosis.

BACKGROUND: The adoption of massively parallel short-read DNA sequencing methods has greatly expanded the scope and availability of genetic testing for inherited diseases. Indeed, the power of these methods has encouraged the integration of whole genome sequencing, the most comprehensive single approach to genomic analysis, into clinical practice. Despite these advances, diagnostic techniques that incompletely resolve the precise molecular boundaries of pathogenic sequence variants continue to be routinely deployed. This can present a barrier for certain prenatal diagnostic approaches. For example, the pre-referral workup for couples seeking preimplantation genetic diagnosis requires intragenic dosage variants to be characterised at nucleotide resolution. OBJECTIVE: We sought to assess the use of long-read nanopore sequencing to rapidly characterise an apparent heterozygous RB1 exon 23 deletion that was initially identified by multiplex ligation-dependent probe amplification (MLPA), in a patient with bilateral retinoblastoma. METHODS: Target enrichment was performed by long-range polymerase chain reaction (PCR) amplification prior to Flongle sequencing on a MinION long-read sequencer. RESULTS: Characterisation of the deletion breakpoint included an unexpected 85-bp insertion which duplicated RB1 exon 24 (and was undetected by MLPA). The long-read sequence permitted design of a multiplex PCR assay, which confirmed that the mutation arose de novo. CONCLUSION: Our experience demonstrates the diagnostic utility of long-read technology for the precise characterisation of structural variants, and highlights how this technology can be efficiently deployed to enable onward referral to reproductive medicine services.

Assessing the utility of long-read nanopore sequencing for rapid and efficient characterization of mobile element insertions.

Short-read next generation sequencing (NGS) has become the predominant first-line technique used to diagnose patients with rare genetic conditions. Inherent limitations of short-read technology, notably for the detection and characterization of complex insertion-containing variants, are offset by the ability to concurrently screen many disease genes. "Third-generation" long-read sequencers are increasingly being deployed as an orthogonal adjunct technology, but their full potential for molecular genetic diagnosis has yet to be exploited. Here, we describe three diagnostic cases in which pathogenic mobile element insertions were refractory to characterization by short-read sequencing. To validate the accuracy of the long-read technology, we first used Sanger sequencing to confirm the integration sites and derive curated benchmark sequences of the variant-containing alleles. Long-read nanopore sequencing was then performed on locus-specific amplicons. Pairwise comparison between these data and the previously determined benchmark alleles revealed 100% identity of the variant-containing sequences. We demonstrate a number of technical advantages over existing wet-laboratory approaches, including in silico size selection of a mixed pool of amplification products, and the relative ease with which an automated informatics workflow can be established. Our findings add to a growing body of literature describing the diagnostic utility of long-read sequencing.

Biallelic Mutations in LRRC56, Encoding a Protein Associated with Intraflagellar Transport, Cause Mucociliary Clearance and Laterality Defects.

Primary defects in motile cilia result in dysfunction of the apparatus responsible for generating fluid flows. Defects in these mechanisms underlie disorders characterized by poor mucus clearance, resulting in susceptibility to chronic recurrent respiratory infections, often associated with infertility; laterality defects occur in about 50% of such individuals. Here we report biallelic variants in LRRC56 (known as oda8 in Chlamydomonas) identified in three unrelated families. The phenotype comprises laterality defects and chronic pulmonary infections. High-speed video microscopy of cultured epithelial cells from an affected individual showed severely dyskinetic cilia but no obvious ultra-structural abnormalities on routine transmission electron microscopy (TEM). Further investigation revealed that LRRC56 interacts with the intraflagellar transport (IFT) protein IFT88. The link with IFT was interrogated in Trypanosoma brucei. In this protist, LRRC56 is recruited to the cilium during axoneme construction, where it co-localizes with IFT trains and is required for the addition of dynein arms to the distal end of the flagellum. In T. brucei carrying LRRC56-null mutations, or a variant resulting in the p.Leu259Pro substitution corresponding to the p.Leu140Pro variant seen in one of the affected families, we observed abnormal ciliary beat patterns and an absence of outer dynein arms restricted to the distal portion of the axoneme. Together, our findings confirm that deleterious variants in LRRC56 result in a human disease and suggest that this protein has a likely role in dynein transport during cilia assembly that is evolutionarily important for cilia motility.

Cas9-based enrichment and single-molecule sequencing for precise characterization of genomic duplications.

The widespread use of genome-wide diagnostic screening methods has greatly increased the frequency with which incidental (but possibly pathogenic) copy number changes affecting single genes are detected. These findings require validation to allow appropriate clinical management. Deletion variants can usually be readily validated using a range of short-read next-generation sequencing (NGS) strategies, but the characterization of duplication variants at nucleotide resolution remains challenging. This presents diagnostic problems, since pathogenicity cannot generally be assessed without knowing the structure of the variant. We have used a novel Cas9 enrichment strategy, in combination with long-read single-molecule nanopore sequencing, to address this need. We describe the nucleotide-level resolution of two problematic cases, both of whom presented with neurodevelopmental problems and were initially investigated by array CGH. In the first case, an incidental 1.7-kb imbalance involving a partial duplication of VHL exon 3 was detected. This variant was inherited from the patient's father, who had a history of renal cancer at 38 years. In the second case, an incidental ~200-kb de novo duplication that included DMD exons 30-44 was resolved. In both cases, the long-read data yielded sufficient information to enable Sanger sequencing to define the rearrangement breakpoints, and creation of breakpoint-spanning PCR assays suitable for testing of relatives. Our Cas9 enrichment and nanopore sequencing approach can be readily adopted by molecular diagnostic laboratories for cost-effective and rapid characterization of challenging duplication-containing alleles. We also anticipate that in future this method may prove useful for characterizing acquired translocations in tumor cells, and for precisely identifying transgene integration sites in mouse models.

An alternative to array-based diagnostics: a prospectively recruited cohort, comparing arrayCGH to next-generation sequencing to evaluate foetal structural abnormalities.

Molecular diagnostic investigations, following the identification of foetal abnormalities, are routinely performed using array comparative genomic hybridisation (aCGH). Despite the utility of this technique, contemporary approaches for the detection of copy number variation are typically based on next-generation sequencing (NGS). We sought to compare an in-house NGS-based workflow (CNVseq) with aCGH, for invasively obtained foetal samples from pregnancies complicated by foetal structural abnormality. DNA from 40 foetuses was screened using both 8 × 60 K aCGH oligoarrays and low-coverage whole genome sequencing. Sequencer-compatible libraries were combined in a ten-sample multiplex and sequenced using an Illumina HiSeq2500. The mean resolution of CNVseq was 29 kb, compared to 60 kb for aCGH analyses. Four clinically significant, concordant, copy number imbalances were detected using both techniques, however, genomic breakpoints were more precisely defined by CNVseq. This data indicates CNVseq is a robust and sensitive alternative to aCGH, for the prenatal investigation of foetuses with structural abnormalities. Impact statement What is already known about this subject? Copy number variant analysis using next-generation sequencing has been successfully applied to investigations of tumour specimens and patients with developmental delays. The application of our approach, to a prospective prenatal diagnosis cohort, has not hitherto been assessed. What do the results of this study add? Next-generation sequencing has a comparable turnaround time and assay sensitivity to copy number variant analysis performed using array CGH. We demonstrate that having established a next-generation sequencing facility, high-throughput CNVseq sample processing and analysis can be undertaken within the framework of a regional diagnostic service. What are the implications of these findings for clinical practice and/or further research? Array CGH is a legacy technology which is likely to be superseded by low-coverage whole genome sequencing, for the detection of copy number variants, in the prenatal diagnosis of structural abnormalities.

Deficiency of the myogenic factor MyoD causes a perinatally lethal fetal akinesia.

BACKGROUND: Lethal fetal akinesia deformation sequence (FADS) describes a clinically and genetically heterogeneous phenotype that includes fetal akinesia, intrauterine growth retardation, arthrogryposis and developmental anomalies. Affected babies die as a result of pulmonary hypoplasia. We aimed to identify the underlying genetic cause of this disorder in a family in which there were three affected individuals from two sibships. METHODS: Autosomal-recessive inheritance was suggested by a family history of consanguinity and by recurrence of the phenotype between the two sibships. We performed exome sequencing of the affected individuals and their unaffected mother, followed by autozygosity mapping and variant filtering to identify the causative gene. RESULTS: Five autozygous regions were identified, spanning 31.7 Mb of genomic sequence and including 211 genes. Using standard variant filtering criteria, we excluded all variants as being the likely pathogenic cause, apart from a single novel nonsense mutation, c.188C>A p.(Ser63*) (NM_002478.4), in MYOD1. This gene encodes an extensively studied transcription factor involved in muscle development, which has nonetheless not hitherto been associated with a hereditary human disease phenotype. CONCLUSIONS: We provide the first description of a human phenotype that appears to result from MYOD1 mutation. The presentation with FADS is consistent with a large body of data demonstrating that in the mouse, MyoD is a major controller of precursor cell commitment to the myogenic differentiation programme.

GeneTIER: prioritization of candidate disease genes using tissue-specific gene expression profiles.

MOTIVATION: In attempts to determine the genetic causes of human disease, researchers are often faced with a large number of candidate genes. Linkage studies can point to a genomic region containing hundreds of genes, while the high-throughput sequencing approach will often identify a great number of non-synonymous genetic variants. Since systematic experimental verification of each such candidate gene is not feasible, a method is needed to decide which genes are worth investigating further. Computational gene prioritization presents itself as a solution to this problem, systematically analyzing and sorting each gene from the most to least likely to be the disease-causing gene, in a fraction of the time it would take a researcher to perform such queries manually. RESULTS: Here, we present Gene TIssue Expression Ranker (GeneTIER), a new web-based application for candidate gene prioritization. GeneTIER replaces knowledge-based inference traditionally used in candidate disease gene prioritization applications with experimental data from tissue-specific gene expression datasets and thus largely overcomes the bias toward the better characterized genes/diseases that commonly afflict other methods. We show that our approach is capable of accurate candidate gene prioritization and illustrate its strengths and weaknesses using case study examples. AVAILABILITY AND IMPLEMENTATION: Freely available on the web at http://dna.leeds.ac.uk/GeneTIER/. CONTACT: umaan@leeds.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Enhanced diagnostic yield in Meckel-Gruber and Joubert syndrome through exome sequencing supplemented with split-read mapping.

BACKGROUND: The widespread adoption of high-throughput sequencing technologies by genetic diagnostic laboratories has enabled significant expansion of their testing portfolios. Rare autosomal recessive conditions have been a particular focus of many new services. Here we report a cohort of 26 patients referred for genetic analysis of Joubert (JBTS) and Meckel-Gruber (MKS) syndromes, two clinically and genetically heterogeneous neurodevelopmental conditions that define a phenotypic spectrum, with MKS at the severe end. METHODS: Exome sequencing was performed for all cases, using Agilent SureSelect v5 reagents and Illumina paired-end sequencing. For two cases medium-coverage (9×) whole genome sequencing was subsequently undertaken. RESULTS: Using a standard analysis pipeline for the detection of single nucleotide and small insertion or deletion variants, molecular diagnoses were confirmed in 12 cases (4%). Seeking to determine whether our cohort harboured pathogenic copy number variants (CNV), in JBTS- or MKS-associated genes, targeted comparative read-depth analysis was performed using FishingCNV. These analyses identified a putative intragenic AHI1 deletion that included three exons spanning at least 3.4 kb and an intergenic MPP4 to TMEM237 deletion that included exons spanning at least 21.5 kb. Whole genome sequencing enabled confirmation of the deletion-containing alleles and precise characterisation of the mutation breakpoints at nucleotide resolution. These data were validated following development of PCR-based assays that could be subsequently used for "cascade" screening and/or prenatal diagnosis. CONCLUSIONS: Our investigations expand the AHI1 and TMEM237 mutation spectrum and highlight the importance of performing CNV screening of disease-associated genes. We demonstrate a robust increasingly cost-effective CNV detection workflow that is applicable to all MKS/JBTS referrals.

Rapid Detection of Rare Deleterious Variants by Next Generation Sequencing with Optional Microarray SNP Genotype Data.

Autozygosity mapping is a powerful technique for the identification of rare, autosomal recessive, disease-causing genes. The ease with which this category of disease gene can be identified has greatly increased through the availability of genome-wide SNP genotyping microarrays and subsequently of exome sequencing. Although these methods have simplified the generation of experimental data, its analysis, particularly when disparate data types must be integrated, remains time consuming. Moreover, the huge volume of sequence variant data generated from next generation sequencing experiments opens up the possibility of using these data instead of microarray genotype data to identify disease loci. To allow these two types of data to be used in an integrated fashion, we have developed AgileVCFMapper, a program that performs both the mapping of disease loci by SNP genotyping and the analysis of potentially deleterious variants using exome sequence variant data, in a single step. This method does not require microarray SNP genotype data, although analysis with a combination of microarray and exome genotype data enables more precise delineation of disease loci, due to superior marker density and distribution.

Robust diagnostic genetic testing using solution capture enrichment and a novel variant-filtering interface.

Targeted hybridization enrichment prior to next-generation sequencing is a widespread method for characterizing sequence variation in a research setting, and is being adopted by diagnostic laboratories. However, the number of variants identified can overwhelm clinical laboratories with strict time constraints, the final interpretation of likely pathogenicity being a particular bottleneck. To address this, we have developed an approach in which, after automatic variant calling on a standard unix pipeline, subsequent variant filtering is performed interactively, using AgileExomeFilter and AgilePindelFilter (http://dna.leeds.ac.uk/agile), tools designed for clinical scientists with standard desktop computers. To demonstrate the method's diagnostic efficacy, we tested 128 patients using (1) a targeted capture of 36 cancer-predisposing genes or (2) whole-exome capture for diagnosis of the genetically heterogeneous disorder primary ciliary dyskinesia (PCD). In the cancer cohort, complete concordance with previous diagnostic data was achieved across 793 variant genotypes. A high yield (42%) was also achieved for exome-based PCD diagnosis, underscoring the scalability of our method. Simple adjustments to the variant filtering parameters further allowed the identification of a homozygous truncating mutation in a presumptive new PCD gene, DNAH8. These tools should allow diagnostic laboratories to expand their testing portfolios flexibly, using a standard set of reagents and techniques.

Detection of somatic mutations in tumors using unaligned clonal sequencing data.

Most cancers arise and evolve as a consequence of somatic mutations. These mutations influence tumor behavior and clinical outcome. Consequently, there is considerable interest in identifying somatic variants within specific genes (such as BRAF, KRAS and EGFR) so that chemotherapy can be tailored to the patient's tumor genotype rather than using a generic treatment based on histological diagnosis alone. Owing to the heterogeneous nature of tumors, a somatic mutation may be present in only a subset of cells, necessitating the use of quantitative techniques to detect rare variants. The highly quantitative nature of next-generation sequencing (NGS), together with the ability to multiplex numerous samples, makes NGS an attractive choice with which to screen for somatic variants. However, the large volumes of sequence data present significant difficulties when applying NGS for the detection of somatic mutations. To alleviate this, we have developed methodologies including a set of data analysis programs, which allow the rapid screening of multiple formalin-fixed, paraffin-embedded samples for the presence of specified somatic variants using unaligned Illumina NGS data.

Diagnostic whole genome sequencing and split-read mapping for nucleotide resolution breakpoint identification in CNTNAP2 deficiency syndrome.

Whole genome sequencing (WGS) has the potential to report on all types of genetic abnormality, thus converging diagnostic testing on a single methodology. Although WGS at sufficient depth for robust detection of point mutations is still some way from being affordable for diagnostic purposes, low-coverage WGS is already an excellent method for detecting copy number variants ("CNVseq"). We report on a family in which individuals presented with a presumed autosomal recessive syndrome of severe intellectual disability and epilepsy. Array comparative genomic hybridization (CGH) analysis had revealed a homozygous deletion apparently lying within intron 3 of CNTNAP2. Since this was too small for confirmation by FISH, CNVseq was used, refining the extent of this mutation to approximately 76.8 kb, encompassing CNTNAP2 exon 3 (an out-of-frame deletion). To characterize the precise breakpoints and provide a rapid molecular diagnostic test, we resequenced the CNVseq library at medium coverage and performed split read mapping. This yielded information for a multiplex polymerase chain reaction (PCR) assay, used for cascade screening and/or prenatal diagnosis in this family. This example demonstrates a rapid, low-cost approach to converting molecular cytogenetic findings into robust PCR-based tests.

Long-Read Nanopore Sequencing of RPGR ORF15 is Enhanced Following DNase I Treatment of MinION Flow Cells.

INTRODUCTION: RPGR ORF15 is an exon present almost exclusively in the retinal transcript of RPGR. It is purine-rich, repetitive and notoriously hard to sequence, but is a hotspot for mutations causing X-linked retinitis pigmentosa. METHODS: Long-read nanopore sequencing on MinION and Flongle flow cells was used to sequence RPGR ORF15 in genomic DNA from patients with inherited retinal dystrophy. A flow cell wash kit was used on a MinION flow cell to increase yield. Findings were confirmed by PacBio SMRT long-read sequencing. RESULTS: We showed that long-read nanopore sequencing successfully reads through a 2 kb PCR-amplified fragment containing ORF15. We generated reads of sufficient quality and cumulative read-depth to detect pathogenic RP-causing variants. However, we observed that this G-rich, repetitive DNA segment rapidly blocks the available pores, resulting in sequence yields less than 5% of the expected output. This limited the extent to which samples could be pooled, increasing cost. We tested the utility of a MinION wash kit containing DNase I to digest DNA fragments remaining on the flow cell, regenerating the pores. Use of the DNase I treatment allowed repeated re-loading, increasing the sequence reads obtained. Our customised workflow was used to screen pooled amplification products from previously unsolved inherited retinal disease (IRD) in patients, identifying two new cases with pathogenic ORF15 variants. DISCUSSION: We report the novel finding that long-read nanopore sequencing can read through RPGR-ORF15, a DNA sequence not captured by short-read next-generation sequencing (NGS), but with a more reduced yield. Use of a flow cell wash kit containing DNase I unblocks the pores, allowing reloading of further library aliquots over a 72-h period, increasing yield. The workflow we describe provides a novel solution to the need for a rapid, robust, scalable, cost-effective ORF15 screening protocol.

Targeted nanopore sequencing enables complete characterisation of structural deletions initially identified using exon-based short-read sequencing strategies.

BACKGROUND: The widespread adoption of exome sequencing has greatly increased the rate of genetic diagnosis for inherited conditions. However, the detection and validation of large deletions remains challenging. While numerous bioinformatics approaches have been developed to detect deletions from whole - exome sequencing and targeted panels, further work is typically required to define the physical breakpoints or integration sites. Accurate characterisation requires either expensive follow - up whole - genome sequencing or the time - consuming, laborious process of PCR walking, both of which are challenging when dealing with the repeat sequences which frequently intersect deletion breakpoints. The aim of this study was to develop a cost-effective, long-range sequencing method to characterise deletions. METHODS: Genomic DNA was amplified with primers spanning the deletion using long-range PCR and the products purified. Sequencing was performed on MinION flongle flowcells. The resulting fast5 files were basecalled using Guppy, trimmed using Porechop and aligned using Minimap2. Filtering was performed using NanoFilt. Nanopore sequencing results were verified by Sanger sequencing. RESULTS: Four cases with deletions detected following comparative read-depth analysis of targeted short-read sequencing were analysed. Nanopore sequencing defined breakpoints at the molecular level in all cases including homozygous breakpoints in EYS, CNGA1 and CNGB1 and a heterozygous deletion in PRPF31. All breakpoints were verified by Sanger sequencing. CONCLUSIONS: In this study, a quick, accurate and cost - effective method is described to characterise deletions identified from exome, and similar data, using nanopore sequencing.

Long-read nanopore sequencing enables accurate confirmation of a recurrent PMS2 insertion-deletion variant located in a region of complex genomic architecture.

Targeted next generation sequencing (NGS) is the predominant methodology for the molecular genetic diagnosis of inherited conditions. In many laboratories, NGS-identified variants are routinely validated using a different method, to minimize the risk of a false-positive diagnosis. This can be particularly important when pathogenic variants are located in complex genomic regions. In this situation, new long-read sequencing technologies have potential advantages over existing alternatives. However, practical examples of their utility for diagnostic purposes remain scant. Here, we report the use of nanopore sequencing to validate a PMS2 mutation refractory to conventional methods. In a patient who presented with colorectal cancer and loss of PMS2 immunostaining, short-read NGS of Lynch syndrome-associated genes identified the recurrent PMS2 insertion-deletion variant, c.736_741delinsTGTGTGTGAAG (p.Pro246Cysfs*3). Confirmation of this variant using bidirectional Sanger sequencing was impeded by an upstream intron 6 poly(T) tract. Using a locus-specific amplicon template, we undertook nanopore long-read sequencing in order to assess the diagnostic accuracy of this platform. Pairwise comparison between a curated benchmark allele (derived from short-read NGS and unidirectional Sanger sequencing) and the consensus nanopore dataset revealed 100% sequence identity. Our experience provides insight into the robustness and ease of deployment of "third-generation" sequencing for accurate characterisation of pathogenic variants.