Long-read Whole-genome Sequencing Research Articles

Expansion of GGC Repeat in GIPC1 Is Associated with Oculopharyngodistal Myopathy

Oculopharyngodistal myopathy (OPDM) is an adult-onset inherited neuromuscular disorder characterized by progressive ptosis, external ophthalmoplegia, and weakness of the masseter, facial, pharyngeal, and distal limb muscles. The myopathological features are presence of rimmed vacuoles (RVs) in the muscle fibers and myopathic changes of differing severity. Inheritance is variable, with either putative autosomal-dominant or autosomal-recessive pattern. Here, using a comprehensive strategy combining whole-genome sequencing (WGS), long-read whole-genome sequencing (LRS), linkage analysis, repeat-primed polymerase chain reaction (RP-PCR), and fluorescence amplicon length analysis polymerase chain reaction (AL-PCR), we identified an abnormal GGC repeat expansion in the 5' UTR of GIPC1 in one out of four families and three sporadic case subjects from a Chinese OPDM cohort. Expanded GGC repeats were further confirmed as the cause of OPDM in an additional 2 out of 4 families and 6 out of 13 sporadic Chinese individuals with OPDM, as well as 7 out of 194 unrelated Japanese individuals with OPDM. Methylation, qRT-PCR, and western blot analysis indicated that GIPC1 mRNA levels were increased while protein levels were unaltered in OPDM-affected individuals. RNA sequencing indicated p53 signaling, vascular smooth muscle contraction, ubiquitin-mediated proteolysis, and ribosome pathways were involved in the pathogenic mechanisms of OPDM-affected individuals with GGC repeat expansion in GIPC1. This study provides further evidence that OPDM is associated with GGC repeat expansions in distinct genes and highly suggests that expanded GGC repeat units are essential in the pathogenesis of OPDM, regardless of the genes in which the expanded repeats are located.

Multispecies Outbreak of Verona Integron-Encoded Metallo-ß-Lactamase-Producing Multidrug Resistant Bacteria Driven by a Promiscuous Incompatibility Group A/C2 Plasmid.

Antibiotic resistance is often spread through bacterial populations via conjugative plasmids. However, plasmid transfer is not well recognized in clinical settings because of technical limitations, and health care-associated infections are usually caused by clonal transmission of a single pathogen. In 2015, multiple species of carbapenem-resistant Enterobacteriaceae (CRE), all producing a rare carbapenemase, were identified among patients in an intensive care unit. This observation suggested a large, previously unrecognized plasmid transmission chain and prompted our investigation. Electronic medical record reviews, infection control observations, and environmental sampling completed the epidemiologic outbreak investigation. A laboratory analysis, conducted on patient and environmental isolates, included long-read whole-genome sequencing to fully elucidate plasmid DNA structures. Bioinformatics analyses were applied to infer plasmid transmission chains and results were subsequently confirmed using plasmid conjugation experiments. We identified 14 Verona integron-encoded metallo-ß-lactamase (VIM)-producing CRE in 12 patients, and 1 additional isolate was obtained from a patient room sink drain. Whole-genome sequencing identified the horizontal transfer of blaVIM-1, a rare carbapenem resistance mechanism in the United States, via a promiscuous incompatibility group A/C2 plasmid that spread among 5 bacterial species isolated from patients and the environment. This investigation represents the largest known outbreak of VIM-producing CRE in the United States to date, which comprises numerous bacterial species and strains. We present evidence of in-hospital plasmid transmission, as well as environmental contamination. Our findings demonstrate the potential for 2 types of hospital-acquired infection outbreaks: those due to clonal expansion and those due to the spread of conjugative plasmids encoding antibiotic resistance across species.

Natural Selection Shapes Variation in Genome-wide Recombination Rate in Drosophila pseudoobscura.

While recombination is widely recognized to be a key modulator of numerous evolutionary phenomena, we have a poor understanding of how recombination rate itself varies and evolves within a species. Here, we performed a comprehensive study of recombination rate (rate of meiotic crossing over) in two natural populations of Drosophila pseudoobscura from Utah and Arizona, USA. We used an amplicon sequencing approach to obtain high-quality genotypes in approximately 8,000 individual backcrossed offspring (17 mapping populations with roughly 530 individuals each), for which we then quantified crossovers. Interestingly, variation in recombination rate within and between populations largely manifested as differences in genome-wide recombination rate rather than remodeling of the local recombination landscape. Comparing populations, we discovered individuals from the Utah population displayed on average 8% higher crossover rates than the Arizona population, a statistically significant difference. Using a QST-FST analysis, we found that this difference in crossover rate was dramatically higher than expected under neutrality, indicating that this difference may have been driven by natural selection. Finally, using a combination of short- and long-read whole-genome sequencing, we found no significant association between crossover rate and structural variation at the 200-400 kb scale. Our results demonstrate that (1) there is abundant variation in genome-wide crossover rate in natural populations, (2) at the 200-400 kb scale, recombination rate appears to vary largely genome-wide, rather than in specific intervals, and (3) interpopulation differences in recombination rate may be the result of local adaptation.

New genomic features of the polled intersex syndrome variant in goats unraveled by long-read whole-genome sequencing.

In domestic goats, the polled intersex syndrome (PIS) refers to XX female-to-male sex reversal associated with the absence of horn growth (polled). The causal variant was previously reported as a 11.7kb deletion at approximately 129Mb on chromosome 1 that affects the transcription of both FOXL2 and several long non-coding RNAs. In the meantime the presence of different versions of the PIS deletion was postulated and trials to establish genetic testing with the existing molecular genetic information failed. Therefore, we revisited this variant by long-read whole-genome sequencing of two genetically female (XX) goats, a PIS-affected and a horned control. This revealed the presence of a more complex structural variant consisting of a deletion with a total length of 10159bp and an inversely inserted approximately 480kb-sized duplicated segment of a region located approximately 21Mb further downstream on chromosome 1 containing two genes, KCNJ15 and ERG. Publicly available short-read whole-genome sequencing data, Sanger sequencing of the breakpoints and FISH using BAC clones corresponding to both involved genome regions confirmed this structural variant. A diagnostic PCR was developed for simultaneous genotyping of carriers for this variant and determination of their genetic sex. We showed that the variant allele was present in all 334 genotyped polled goats of diverse breeds and that all analyzed 15 PIS-affected XX goats were homozygous. Our findings enable for the first time a precise genetic diagnosis for polledness and PIS in goats and add a further genomic feature to the complexity of the PIS phenomenon.

Elucidation of de novo small insertion/deletion biology with parent-of-origin phasing.

The mechanisms underlying de novo insertion/deletion (indel) genesis, such as polymerase slippage, have been hypothesized but not well characterized in the human genome. We implemented two methodological improvements, which were leveraged to dissect indel mutagenesis. We assigned de novo variants to parent-of-origin (i.e., phasing) with low-coverage long-read whole-genome sequencing, achieving better phasing compared to short-read sequencing (medians of 84% and 23%, respectively). We then wrote an application programming interface to classify indels into three subtypes according to sequence context. Across three cohorts with different phasing methods (Ntrios = 540, all cohorts), we observed that one de novo indel subtype, change in copy count (CCC), was significantly correlated with father's (p = 7.1 × 10-4 ) but not mother's (p = .45) age at conception. We replicated this effect in three cohorts without de novo phasing (ppaternal = 1.9 × 10-9 , pmaternal = .61; Ntrios = 3,391, all cohorts). Although this is consistent with polymerase slippage during spermatogenesis, the percentage of variance explained by paternal age was low, and we did not observe an association with replication timing. These results suggest that spermatogenesis-specific events have a minor role in CCC indel mutagenesis, one not observed for other indel subtypes nor for maternal age in general. These results have implications for indel modeling in evolution and disease.

First report of the mcr-1 colistin resistance gene identified in two Escherichia coli isolates from clinical samples, Philippines, 2018

ObjectivesThe first report of a plasmid-borne colistin resistance gene (mcr-1) detected in an Escherichia coli isolate from China heralded the emergence of pandrug-resistant bacteria. Since then, the mcr-1 gene has been detected worldwide, but to date it has not been reported in the Philippines. MethodsIn this study, 123 antimicrobial-resistant isolates collected from January–June 2018 from patients admitted to a tertiary hospital in Manila, Philippines, were characterised. Biochemical identification and antimicrobial susceptibility testing were performed using a BD Phoenix™ M50 system with NMIC/ID-95 panel. Conventional PCR was performed to detect the genes mcr-1 to mcr-5, and short- and long-read whole-genome sequencing was performed. ResultsTwo mcr-1-positive E. coli clinical isolates from separate patients harboured mcr-1 on an IncI2 plasmid. One isolate was shown to carry 12 antimicrobial resistance genes (ARGs) in addition to mcr-1, including the extended-spectrum β-lactamase blaCTX-M-55, whilst the other E. coli isolate carried 6 ARGs in addition to mcr-1. ConclusionBoth patients had no prior colistin treatment recorded in their medical history and no travel history outside of the country within the past 6 months from the date of hospital admission, indicating local transmission and acquisition of the colistin-resistant strain from either community or hospital settings within the Philippines. This report should serve as a signal to local public-health officials of the need to intensify surveillance efforts and to increase vigilance and implementation of antimicrobial stewardship programmes to contain and slow the spread of antimicrobial resistance.

Combinations of exonic deletions and rare mutations lead to misdiagnosis of propionic acidemia

Propionic acidemia (PA) is an inborn metabolic error characterized by the accumulation of propionic acid due to deficiency of propionyl-CoA carboxylase (PCC). In this study, we present an intractable case with PCC activity defects. Although next-generation sequencing was applied twice to test genetic defects of the patients, no pathogenic mutations of a metabolic disease gene were identified. Mutations related to the disease were screened in prenatal diagnosis, but the mother still gave birth to an unhealthy neonate. We analyzed the second sequencing data and found that a novel synonymous PCCA mutation c.1746 G>C (p.S582S), which leads to an exon 19 skip, was screened out. Furthermore, a deletion mutation covering exon 3 and exon 4 of the PCCA gene was identified using q-PCR and DNA breakpoint test. Both of these can result in the loss of PCCA protein function. The finding expands the mutation spectrum of the PCCA gene and indicates that another technology such as cDNA analysis, multiplex ligation-dependent probe amplification (MLPA), or long-read whole-genome sequencing should be considered to improve the detection rates of special cases.

A Cluster of Colistin- and Carbapenem-Resistant Klebsiella pneumoniae Carrying blaNDM-1 and mcr-8.2.

Klebsiella pneumoniae resistant to both carbapenems and colistin imposes severe challenges for management. In this study, we report a cluster of 5 carbapenem-resistant K pneumoniae clinical strains belonging to ST1 and K57 types, 4 of which were also resistant to colistin, from 2 hospitals. The 5 strains were subjected to whole-genome sequencing (WGS) using the short-read Illumina HiSeq platform, and 2 strains were also selected for long-read WGS using MinION. Clonal relatedness of the 5 strains was determined based on single-nucleotide polymorphisms (SNPs). Conjugation experiments were performed to obtain self-transmissible plasmids. All 5 strains carried the carbapenemase-encoding gene blaNDM-1, whereas the 4 colistin-resistant strains also harbored a new variant of the mcr-8 colistin resistance gene, namely, mcr-8.2. MCR-8.2 differs from MCR-8.1 by four amino acid substitutions (A51V, A232S, N365Y, and N480K). mcr-8.2 was located on a large, hybrid, nonself-transmissible plasmid containing IncQ, IncR, and IncFII replicons, whereas blaNDM-1 was carried by self-transmissible IncX3 plasmids. Phylogenetic analysis based on SNPs revealed that the 5 strains were likely to have a common origin. Both the intra- and interhospital transfer of strains carrying mcr-8 and blaNDM-1 were identified, which represents an emerging threat for clinical management and infection control.

Expansion of GGC repeat in the human-specific NOTCH2NLC gene is associated with essential tremor.

Essential tremor is one of the most common movement disorders. Despite its high prevalence and heritability, the genetic aetiology of essential tremor remains elusive. Up to now, only a few genes/loci have been identified, but these genes have not been replicated in other essential tremor families or cohorts. Here we report a genetic study in a cohort of 197 Chinese pedigrees clinically diagnosed with essential tremor. Using a comprehensive strategy combining linkage analysis, whole-exome sequencing, long-read whole-genome sequencing, repeat-primed polymerase chain reaction and GC-rich polymerase chain reaction, we identified an abnormal GGC repeat expansion in the 5' region of the NOTCH2NLC gene that co-segregated with disease in 11 essential tremor families (5.58%) from our cohort. Clinically, probands that had an abnormal GGC repeat expansion were found to have more severe tremor phenotypes, lower activities of daily living ability. Obvious genetic anticipation was also detected in these 11 essential tremor-positive families. These results indicate that abnormal GGC repeat expansion in the 5' region of NOTCH2NLC gene is associated with essential tremor, and provide strong evidence that essential tremor is a family of diseases with high clinical and genetic heterogeneities.

A Complete Genome Screening Program of Clinical Methicillin-Resistant Staphylococcus aureus Isolates Identifies the Origin and Progression of a Neonatal Intensive Care Unit Outbreak.

Whole-genome sequencing (WGS) of Staphylococcus aureus is increasingly used as part of infection prevention practices. In this study, we established a long-read technology-based WGS screening program of all first-episode methicillin-resistant Staphylococcus aureus (MRSA) blood infections at a major urban hospital. A survey of 132 MRSA genomes assembled from long reads enabled detailed characterization of an outbreak lasting several months of a CC5/ST105/USA100 clone among 18 infants in a neonatal intensive care unit (NICU). Available hospital-wide genome surveillance data traced the origins of the outbreak to three patients admitted to adult wards during a 4-month period preceding the NICU outbreak. The pattern of changes among complete outbreak genomes provided full spatiotemporal resolution of its progression, which was characterized by multiple subtransmissions and likely precipitated by equipment sharing between adults and infants. Compared to other hospital strains, the outbreak strain carried distinct mutations and accessory genetic elements that impacted genes with roles in metabolism, resistance, and persistence. This included a DNA recognition domain recombination in the hsdS gene of a type I restriction modification system that altered DNA methylation. Transcriptome sequencing (RNA-Seq) profiling showed that the (epi)genetic changes in the outbreak clone attenuated agr gene expression and upregulated genes involved in stress response and biofilm formation. Overall, our findings demonstrate the utility of long-read sequencing for hospital surveillance and for characterizing accessory genomic elements that may impact MRSA virulence and persistence.

Complete nontuberculous mycobacteria whole genomes using an optimized DNA extraction protocol for long-read sequencing

BackgroundNontuberculous mycobacteria (NTM) are a major cause of pulmonary and systemic disease in at-risk populations. Gaps in knowledge about transmission patterns, evolution, and pathogenicity during infection have prompted a recent surge in genomic NTM research. Increased availability and affordability of whole genome sequencing (WGS) techniques provide new opportunities to sequence and construct complete bacterial genomes faster and at a lower cost. However, extracting large quantities of pure genomic DNA is particularly challenging with NTM due to its slow growth and recalcitrant cell wall. Here we report a DNA extraction protocol that is optimized for long-read WGS of NTM, yielding large quantities of highly pure DNA with no additional clean-up steps.ResultsOur DNA extraction method was compared to 6 other methods with variations in timing of mechanical disruption and enzymatic digestion of the cell wall, quantity of matrix material, and reagents used in extraction and precipitation. We tested our optimized method on 38 clinical isolates from the M. avium and M. abscessus complexes, which yielded optimal quality and quantity measurements for Oxford Nanopore Technologies sequencing. We also present the efficient completion of circularized M. avium subspecies hominissuis genomes using our extraction technique and the long-read sequencing MinION platform, including the identification of a novel plasmid.ConclusionsOur optimized extraction protocol and assembly pipeline was both sufficient and efficient for genome closure. We expect that our finely-tuned extraction method will prove to be a valuable tool in long-read sequencing and completion of mycobacterial genomes going forward. Utilization of comprehensive, long-read based approaches will advance the understanding evolution and pathogenicity of NTM infections.

Plant evolution and environmental adaptation unveiled by long-read whole-genome sequencing of Spirodela

Aquatic plants have to adapt to the environments distinct from where land plants grow. A critical aspect of adaptation is the dynamics of sequence repeats, not resolved in older sequencing platforms due to incomplete and fragmented genome assemblies from short reads. Therefore, we used PacBio long-read sequencing of the Spirodela polyrhiza genome, reaching a 44-fold increase of contiguity with an N50 (a median of contig lengths) of 831 kb and filling 95.4% of gaps left from the previous version. Reconstruction of repeat regions indicates that sequentially nested long terminal repeat (LTR) retrotranspositions occur early in monocot evolution, featured with both prokaryote-like gene-rich regions and eukaryotic repeat islands. Protein-coding genes are reduced to 18,708 gene models supported by 492,435 high-quality full-length PacBio complementary DNA (cDNA) sequences. Different from land plants, the primitive architecture of Spirodela's adventitious roots and lack of lateral roots and root hairs are consistent with dispensable functions of nutrient absorption. Disease-resistant genes encoding antimicrobial peptides and dirigent proteins are expanded by tandem duplications. Remarkably, disease-resistant genes are not only amplified, but also highly expressed, consistent with low levels of 24-nucleotide (nt) small interfering RNA (siRNA) that silence the immune system of land plants, thereby protecting Spirodela against a wide spectrum of pathogens and pests. The long-read sequence information not only sheds light on plant evolution and adaptation to the environment, but also facilitates applications in bioenergy and phytoremediation.

Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak.

The global spread of antibiotic resistance among Enterobacteriaceae is largely due to multidrug resistance plasmids that can transfer between different bacterial strains and species. Horizontal gene transfer of resistance plasmids can complicate hospital outbreaks and cause problems in epidemiological tracing, since tracing is usually based on bacterial clonality. We have developed a method, based on optical DNA mapping combined with Cas9-assisted identification of resistance genes, which is used here to characterize plasmids during an extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae outbreak at a Swedish neonatal intensive care unit. The outbreak included 17 neonates initially colonized with ESBL-producing Klebsiella pneumoniae (ESBL-KP), some of which were found to carry additional ESBL-producing Escherichia coli (ESBL-EC) in follow-up samples. We demonstrate that all ESBL-KP isolates contained two plasmids with the blaCTX-M-15 gene located on the smaller one (~80 kbp). The same ESBL-KP clone was present in follow-up samples for up to 2 years in some patients, and the plasmid carrying the blaCTX-M-15 gene was stable throughout this time period. However, extensive genetic rearrangements within the second plasmid were observed in the optical DNA maps for several of the ESBL-KP isolates. Optical mapping also demonstrated that even though other bacterial clones and species carrying blaCTX-M group 1 genes were found in some neonates, no transfer of resistance plasmids had occurred. The data instead pointed toward unrelated acquisition of ESBL-producing Enterobacteriaceae (EPE). In addition to revealing important information about the specific outbreak, the method presented is a promising tool for surveillance and infection control in clinical settings.IMPORTANCE This study presents how a novel method, based on visualizing single plasmids using sequence-specific fluorescent labeling, could be used to analyze the genetic dynamics of an outbreak of resistant bacteria in a neonatal intensive care unit at a Swedish hospital. Plasmids are a central reason for the rapid global spread of bacterial resistance to antibiotics. In a single experimental procedure, this method replaces many traditional plasmid analysis techniques that together provide limited details and are slow to perform. The method is much faster than long-read whole-genome sequencing and offers direct genetic comparison of patient samples. We could conclude that no transfer of resistance plasmids had occurred between different bacteria during the outbreak and that secondary cases of ESBL-producing Enterobacteriaceae carriage were instead likely due to influx of new strains. We believe that the method offers potential in improving surveillance and infection control of resistant bacteria in hospitals.

Tying up Loose Ends in the Malaria Antigenic Variation Story

A recent paper (Zhang et al., PLoS Biol., 2019) shines remarkable new light onto the malaria antigenic variation story. Using CRISPR/Cas9-targeted chromosome breaks and long-read whole-genome sequencing, they followed the fate of detached subtelomeric PfEMP1/var genes and demonstrated that these initiate cascades of recombination at sites far from the original break.

Long-read sequencing unveils IGH-DUX4 translocation into the silenced IGH allele in B-cell acute lymphoblastic leukemia

IGH@ proto-oncogene translocation is a common oncogenic event in lymphoid lineage cancers such as B-ALL, lymphoma and multiple myeloma. Here, to investigate the interplay between IGH@ proto-oncogene translocation and IGH allelic exclusion, we perform long-read whole-genome and transcriptome sequencing along with epigenetic and 3D genome profiling of Nalm6, an IGH-DUX4 positive B-ALL cell line. We detect significant allelic imbalance on the wild-type over the IGH-DUX4 haplotype in expression and epigenetic data, showing IGH-DUX4 translocation occurs on the silenced IGH allele. In vitro, this reduces the oncogenic stress of DUX4 high-level expression. Moreover, patient samples of IGH-DUX4 B-ALL have similar expression profile and IGH breakpoints as Nalm6, suggesting a common mechanism to allow optimal dosage of non-toxic DUX4 expression.

Transient multifocal genomic crisis creating chromothriptic and non-chromothriptic rearrangements in prezygotic testicular germ cells

BackgroundThe co-occurrence of multiple de novo copy number variations (CNVs) is a rare phenomenon in the human genome. Recently, an “organismal CNV mutator phenotype” has been reported to result in transient genomic instability introducing multiple de novo CNVs in primary oocytes and early-stage zygotes. These findings opened a new area of human genome research.MethodsWe performed genome-wide copy number analysis for ~ 2100 individuals with various congenital defects. Furthermore, extensive molecular analyses, including synthetic long-read whole-genome sequencing and haplotype-phasing, were carried out for an individual with multiple de novo CNVs.ResultsA boy was found to have de novo rearrangements on five chromosomes. The rearrangements comprised simple duplication and inversion as well as chaotic changes, all of which affected paternally derived chromosomes. Postzygotic genomic instability was ruled out. The duplicated regions on 6q and 13q contained both diallelic and triallelic loci, indicating that the genomic rearrangements were initially created during premeiotic mitosis and subsequently modified by physiological cross-over during meiosis I. Breakpoints of the rearrangements were indicative of non-homologous end joining, replication-based errors, and/or chromothripsis. The mutagenic event was independent of specific local DNA motifs or de novo point mutations, but may be driven by spermatogenesis-specific factors.ConclusionsThese results indicate that during spermatogenesis, a transient multifocal genomic crisis can introduce several chromothriptic and non-chromothriptic changes into the genome. These findings broaden the concept of the “organismal CNV mutator phenotype”. This study provides insights into mechanisms for altering the global chromosomal architecture of human embryos.

Rapid antigen diversification through mitotic recombination in the human malaria parasite Plasmodium falciparum

Malaria parasites possess the remarkable ability to maintain chronic infections that fail to elicit a protective immune response, characteristics that have stymied vaccine development and cause people living in endemic regions to remain at risk of malaria despite previous exposure to the disease. These traits stem from the tremendous antigenic diversity displayed by parasites circulating in the field. For Plasmodium falciparum, the most virulent of the human malaria parasites, this diversity is exemplified by the variant gene family called var, which encodes the major surface antigen displayed on infected red blood cells (RBCs). This gene family exhibits virtually limitless diversity when var gene repertoires from different parasite isolates are compared. Previous studies indicated that this remarkable genome plasticity results from extensive ectopic recombination between var genes during mitotic replication; however, the molecular mechanisms that direct this process to antigen-encoding loci while the rest of the genome remains relatively stable were not determined. Using targeted DNA double-strand breaks (DSBs) and long-read whole-genome sequencing, we show that a single break within an antigen-encoding region of the genome can result in a cascade of recombination events leading to the generation of multiple chimeric var genes, a process that can greatly accelerate the generation of diversity within this family. We also found that recombinations did not occur randomly, but rather high-probability, specific recombination products were observed repeatedly. These results provide a molecular basis for previously described structured rearrangements that drive diversification of this highly polymorphic gene family.

Detecting a long insertion variant in SAMD12 by SMRT sequencing: implications of long-read whole-genome sequencing for repeat expansion diseases.

Long-read sequencing technology is now capable of reading single-molecule DNA with an average read length of more than 10 kb, fully enabling the coverage of large structural variations (SVs). This advantage may pave the way for the detection of unprecedented SVs as well as repeat expansions. Pathogenic SVs of only known genes used to be selectively analyzed based on prior knowledge of target DNA sequence. The unbiased application of long-read whole-genome sequencing (WGS) for the detection of pathogenic SVs has just begun. Here, we apply PacBio SMRT sequencing in a Japanese family with benign adult familial myoclonus epilepsy (BAFME). Our SV selection of low-coverage WGS data (7×) narrowed down the candidates to only six SVs in a 7.16-Mb region of the BAFME1 locus and correctly determined an approximately 4.6-kb SAMD12 intronic repeat insertion, which is causal of BAFME1. These results indicate that long-read WGS is potentially useful for evaluating all of the known SVs in a genome and identifying new disease-causing SVs in combination with other genetic methods to resolve the genetic causes of currently unexplained diseases.

Complex structural variants in Mendelian disorders: identification and breakpoint resolution using short- and long-read genome sequencing

BackgroundStudies have shown that complex structural variants (cxSVs) contribute to human genomic variation and can cause Mendelian disease. We aimed to identify cxSVs relevant to Mendelian disease using short-read whole-genome sequencing (WGS), resolve the precise variant configuration and investigate possible mechanisms of cxSV formation.MethodsWe performed short-read WGS and analysis of breakpoint junctions to identify cxSVs in a cohort of 1324 undiagnosed rare disease patients. Long-read WGS and gene expression analysis were used to resolve one case.ResultsWe identified three pathogenic cxSVs: a de novo duplication-inversion-inversion-deletion affecting ARID1B, a de novo deletion-inversion-duplication affecting HNRNPU and a homozygous deletion-inversion-deletion affecting CEP78. Additionally, a de novo duplication-inversion-duplication overlapping CDKL5 was resolved by long-read WGS demonstrating the presence of both a disrupted and an intact copy of CDKL5 on the same allele, and gene expression analysis showed both parental alleles of CDKL5 were expressed. Breakpoint analysis in all the cxSVs revealed both microhomology and longer repetitive elements.ConclusionsOur results corroborate that cxSVs cause Mendelian disease, and we recommend their consideration during clinical investigations. We show that resolution of breakpoints can be critical to interpret pathogenicity and present evidence of replication-based mechanisms in cxSV formation.

Long-read sequencing identified a causal structural variant in an exome-negative case and enabled preimplantation genetic diagnosis

BackgroundFor a proportion of individuals judged clinically to have a recessive Mendelian disease, only one heterozygous pathogenic variant can be found from clinical whole exome sequencing (WES), posing a challenge to genetic diagnosis and genetic counseling. One possible reason is the limited ability to detect disease causal structural variants (SVs) from short reads sequencing technologies. Long reads sequencing can produce longer reads (typically 1000 bp or longer), therefore offering greatly improved ability to detect SVs that may be missed by short-read sequencing.ResultsHere we describe a case study, where WES identified only one heterozygous pathogenic variant for an individual suspected to have glycogen storage disease type Ia (GSD-Ia), which is an autosomal recessive disease caused by bi-allelic mutations in the G6PC gene. Through Nanopore long-read whole-genome sequencing, we identified a 7.1 kb deletion covering two exons on the other allele, suggesting that complex structural variants (SVs) may explain a fraction of cases when the second pathogenic allele is missing from WES on recessive diseases. Both breakpoints of the deletion are within Alu elements, and we designed Sanger sequencing and quantitative PCR assays based on the breakpoints for preimplantation genetic diagnosis (PGD) for the family planning on another child. Four embryos were obtained after in vitro fertilization (IVF), and an embryo without deletion in G6PC was transplanted after PGD and was confirmed by prenatal diagnosis, postnatal diagnosis, and subsequent lack of disease symptoms after birth.ConclusionsIn summary, we present one of the first examples of using long-read sequencing to identify causal yet complex SVs in exome-negative patients, which subsequently enabled successful personalized PGD.