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Vaisbourd E, Bren A, Alon U, Glass DS. Preventing Multimer Formation in Commonly Used Synthetic Biology Plasmids. ACS Synth Biol 2025; 14:1309-1315. [PMID: 40101192 PMCID: PMC12012879 DOI: 10.1021/acssynbio.4c00508] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2024] [Revised: 10/08/2024] [Accepted: 03/03/2025] [Indexed: 03/20/2025]
Abstract
Plasmids are an essential tool for basic research and biotechnology applications. To optimize plasmid-based circuits, it is crucial to control plasmid integrity, including the formation of plasmid multimers. Multimers are tandem repeats of entire plasmids formed by failed dimer resolution during replication. Multimers can affect the behavior of synthetic circuits, especially ones that include DNA-editing enzymes. However, occurrence of multimers is not commonly assayed. Here we survey four commonly used plasmid backbones for occurrence of multimers in cloning (JM109) and wild-type (MG1655) strains of Escherichia coli. We find that multimers occur appreciably only in MG1655, with the fraction of plasmids existing as multimers increasing with both plasmid copy number and culture passaging. In contrast, transforming multimers into JM109 can yield strains that contain no singlet plasmids. We present an MG1655 ΔrecA single-locus knockout that avoids multimer production. These results can aid synthetic biologists in improving design and reliability of plasmid-based circuits.
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Affiliation(s)
- Elizabeth Vaisbourd
- Department
of Molecular Cell Biology, Weizmann Institute
of Science, Rehovot, Israel 76100
| | - Anat Bren
- Department
of Molecular Cell Biology, Weizmann Institute
of Science, Rehovot, Israel 76100
| | - Uri Alon
- Department
of Molecular Cell Biology, Weizmann Institute
of Science, Rehovot, Israel 76100
| | - David S. Glass
- Department
of Molecular Cell Biology, Weizmann Institute
of Science, Rehovot, Israel 76100
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2
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Gürkan H, Satkın NB. The Importance of Genetic Diagnosis in Rare Diseases. Balkan Med J 2025; 42:92-93. [PMID: 40033553 PMCID: PMC11881545 DOI: 10.4274/balkanmedj.galenos.2025.2025-270125] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/05/2025] Open
Affiliation(s)
- Hakan Gürkan
- Department of Medical Genetics, Trakya University Faculty of Medicine, Edirne, Türkiye
| | - Nihan Bilge Satkın
- Department of Medical Genetics, Trakya University Faculty of Medicine, Edirne, Türkiye
- Department of Genetics and Bioengineering, Trakya University Faculty of Engineering, Edirne, Türkiye
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Natsuga K. Advanced phasing techniques in congenital skin diseases. J Dermatol 2025; 52:392-399. [PMID: 39723554 PMCID: PMC11883850 DOI: 10.1111/1346-8138.17597] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2024] [Revised: 11/26/2024] [Accepted: 12/10/2024] [Indexed: 12/28/2024]
Abstract
Phasing, the process of determining which alleles at different loci on homologous chromosomes belong together on the same chromosome, is crucial in the diagnosis and management of autosomal recessive diseases. Advances in long-read sequencing technologies have significantly enhanced our ability to accurately determine haplotypes. This review discusses the application of low-coverage long-read sequencing, nanopore Cas9-guided long-read sequencing, and adaptive sampling in phasing, highlighting their utility in complex clinical scenarios. Through clinical vignettes, we explore the importance of phasing in gene therapy design for recessive dystrophic epidermolysis bullosa and the role of revertant mosaicism in therapeutic epidermal autografts. Despite its promise, phasing with long-read sequencing faces challenges, including low efficiency in enriching target regions and the inherent error rate of nanopore sequencing. Future developments in long-read sequencing technologies will be critical in overcoming these limitations and expanding the applicability of phasing across various clinical settings.
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Affiliation(s)
- Ken Natsuga
- Department of Dermatology, Faculty of Medicine and Graduate School of MedicineHokkaido UniversitySapporoJapan
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4
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Muthaffar OY, Alazhary NW, Alyazidi AS, Alsubaie MA, Bahowarth SY, Odeh NB, Bamaga AK. Clinical description and evaluation of 30 pediatric patients with ultra-rare diseases: A multicenter study with real-world data from Saudi Arabia. PLoS One 2024; 19:e0307454. [PMID: 39024300 PMCID: PMC11257271 DOI: 10.1371/journal.pone.0307454] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2024] [Accepted: 07/04/2024] [Indexed: 07/20/2024] Open
Abstract
BACKGROUND With the advancement of next-generation sequencing, clinicians are now able to detect ultra-rare mutations that are barely encountered by the majority of physicians. Ultra-rare and rare diseases cumulatively acquire a prevalence equivalent to type 2 diabetes with 80% being genetic in origin and more prevalent among high consanguinity communities including Saudi Arabia. The challenge of these diseases is the ability to predict their prevalence and define clear phenotypic features. METHODS This is a non-interventional retrospective multicenter study. We included pediatric patients with a pathogenic variant designated as ultra-rare according to the National Institute for Clinical Excellence's criteria. Demographic, clinical, laboratory, and radiological data of all patients were collected and analyzed using multinomial regression models. RESULTS We included 30 patients. Their mean age of diagnosis was 16.77 months (range 3-96 months) and their current age was 8.83 years (range = 2-15 years). Eleven patients were females and 19 were males. The majority were of Arab ethnicity (96.77%). Twelve patients were West-Saudis and 8 patients were South-Saudis. SCN1A mutation was reported among 19 patients. Other mutations included SZT2, ROGDI, PRF1, ATP1A3, and SHANK3. The heterozygous mutation was reported among 67.86%. Twenty-nine patients experienced seizures with GTC being the most frequently reported semiology. The mean response to ASMs was 45.50% (range 0-100%). CONCLUSION The results suggest that ultra-rare diseases must be viewed as a distinct category from rare diseases with potential demographic and clinical hallmarks. Additional objective and descriptive criteria to detect such cases are needed.
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Affiliation(s)
- Osama Y Muthaffar
- Department of Pediatric, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
| | - Noura W Alazhary
- Department of General Pediatric, Dr. Soliman Fakeeh Hospital, Jeddah, Saudi Arabia
| | - Anas S Alyazidi
- Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
| | | | - Sarah Y Bahowarth
- Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
| | - Nour B Odeh
- College of Medicine, Alfaisal University, Riyadh, Saudi Arabia
| | - Ahmed K Bamaga
- Department of Pediatric, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
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Shelton WJ, Zandpazandi S, Nix JS, Gokden M, Bauer M, Ryan KR, Wardell CP, Vaske OM, Rodriguez A. Long-read sequencing for brain tumors. Front Oncol 2024; 14:1395985. [PMID: 38915364 PMCID: PMC11194609 DOI: 10.3389/fonc.2024.1395985] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2024] [Accepted: 05/27/2024] [Indexed: 06/26/2024] Open
Abstract
Brain tumors and genomics have a long-standing history given that glioblastoma was the first cancer studied by the cancer genome atlas. The numerous and continuous advances through the decades in sequencing technologies have aided in the advanced molecular characterization of brain tumors for diagnosis, prognosis, and treatment. Since the implementation of molecular biomarkers by the WHO CNS in 2016, the genomics of brain tumors has been integrated into diagnostic criteria. Long-read sequencing, also known as third generation sequencing, is an emerging technique that allows for the sequencing of longer DNA segments leading to improved detection of structural variants and epigenetics. These capabilities are opening a way for better characterization of brain tumors. Here, we present a comprehensive summary of the state of the art of third-generation sequencing in the application for brain tumor diagnosis, prognosis, and treatment. We discuss the advantages and potential new implementations of long-read sequencing into clinical paradigms for neuro-oncology patients.
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Affiliation(s)
- William J Shelton
- Department of Neurosurgery, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, United States
| | - Sara Zandpazandi
- Department of Neurosurgery, Medical University of South Carolina, Charleston, SC, United States
| | - J Stephen Nix
- Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR, United States
| | - Murat Gokden
- Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR, United States
| | - Michael Bauer
- Department of Biomedical Informatics, University of Arkansas for Medical Sciences, Little Rock, AR, United States
| | - Katie Rose Ryan
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, United States
| | - Christopher P Wardell
- Department of Biomedical Informatics, University of Arkansas for Medical Sciences, Little Rock, AR, United States
| | - Olena Morozova Vaske
- Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, CA, United States
| | - Analiz Rodriguez
- Department of Neurosurgery, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, United States
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Wang N, Jiao K, He J, Zhu B, Cheng N, Sun J, Chen L, Chen W, Gong L, Qiao K, Xi J, Wu Q, Zhao C, Zhu W. Diagnosis of Challenging Spinal Muscular Atrophy Cases with Long-Read Sequencing. J Mol Diagn 2024; 26:364-373. [PMID: 38490302 DOI: 10.1016/j.jmoldx.2024.02.004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2023] [Revised: 01/17/2024] [Accepted: 02/07/2024] [Indexed: 03/17/2024] Open
Abstract
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder primarily caused by the deletion or mutation of the survival motor neuron 1 (SMN1) gene. This study assesses the diagnostic potential of long-read sequencing (LRS) in three patients with SMA. For Patient 1, who has a heterozygous SMN1 deletion, LRS unveiled a missense mutation in SMN1 exon 5. In Patient 2, an Alu/Alu-mediated rearrangement covering the SMN1 promoter and exon 1 was identified through a blend of multiplex ligation-dependent probe amplification, LRS, and PCR across the breakpoint. The third patient, born to a consanguineous family, bore four copies of hybrid SMN genes. LRS determined the genomic structures, indicating two distinct hybrids of SMN2 exon 7 and SMN1 exon 8. However, a discrepancy was found between the SMN1/SMN2 ratio interpretations by LRS (0:2) and multiplex ligation-dependent probe amplification (0:4), which suggested a limitation of LRS in SMA diagnosis. In conclusion, this newly adapted long PCR-based third-generation sequencing introduces an additional avenue for SMA diagnosis.
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Affiliation(s)
- Ningning Wang
- Department of Neurology, Huashan Hospital, Fudan University, Shanghai, China; National Center for Neurological Disorders, Shanghai, China; Huashan Rare Disease Center, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China
| | - Kexin Jiao
- Department of Neurology, Huashan Hospital, Fudan University, Shanghai, China; National Center for Neurological Disorders, Shanghai, China; Huashan Rare Disease Center, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China
| | - Jin He
- Department of Neurology and Institute of Neurology of First Affiliated Hospital, Institute of Neuroscience, and Fujian Key Laboratory of Molecular Neurology, Fujian Medical University, Fuzhou, China
| | - Bochen Zhu
- Department of Neurology, Huashan Hospital, Fudan University, Shanghai, China; National Center for Neurological Disorders, Shanghai, China; Huashan Rare Disease Center, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China
| | - Nachuan Cheng
- Department of Neurology, Huashan Hospital, Fudan University, Shanghai, China; National Center for Neurological Disorders, Shanghai, China; Huashan Rare Disease Center, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China
| | - Jian Sun
- Department of Neurology, Huashan Hospital, Fudan University, Shanghai, China; National Center for Neurological Disorders, Shanghai, China; Huashan Rare Disease Center, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China
| | - Lan Chen
- Department of Neurology, Nantong First People's Hospital, Nantong, China
| | - Wanjin Chen
- Department of Neurology and Institute of Neurology of First Affiliated Hospital, Institute of Neuroscience, and Fujian Key Laboratory of Molecular Neurology, Fujian Medical University, Fuzhou, China
| | - Lingyun Gong
- Department of Neurology, Huashan Hospital, Fudan University, Shanghai, China; National Center for Neurological Disorders, Shanghai, China; Huashan Rare Disease Center, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China
| | - Kai Qiao
- Department of Neurology, Huashan Hospital, Fudan University, Shanghai, China; National Center for Neurological Disorders, Shanghai, China; Huashan Rare Disease Center, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China
| | - Jianying Xi
- Department of Neurology, Huashan Hospital, Fudan University, Shanghai, China; National Center for Neurological Disorders, Shanghai, China; Huashan Rare Disease Center, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China
| | - Qihan Wu
- Shanghai Ministry of Science and Technology Key Laboratory of Health and Disease Genomics, National Health Commission Key Lab of Reproduction Regulation, Shanghai Institute for Biomedical and Pharmaceutical Technologies, Shanghai, China
| | - Chongbo Zhao
- Department of Neurology, Huashan Hospital, Fudan University, Shanghai, China; National Center for Neurological Disorders, Shanghai, China; Huashan Rare Disease Center, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China
| | - Wenhua Zhu
- Department of Neurology, Huashan Hospital, Fudan University, Shanghai, China; National Center for Neurological Disorders, Shanghai, China; Huashan Rare Disease Center, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
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Olivucci G, Iovino E, Innella G, Turchetti D, Pippucci T, Magini P. Long read sequencing on its way to the routine diagnostics of genetic diseases. Front Genet 2024; 15:1374860. [PMID: 38510277 PMCID: PMC10951082 DOI: 10.3389/fgene.2024.1374860] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2024] [Accepted: 02/26/2024] [Indexed: 03/22/2024] Open
Abstract
The clinical application of technological progress in the identification of DNA alterations has always led to improvements of diagnostic yields in genetic medicine. At chromosome side, from cytogenetic techniques evaluating number and gross structural defects to genomic microarrays detecting cryptic copy number variants, and at molecular level, from Sanger method studying the nucleotide sequence of single genes to the high-throughput next-generation sequencing (NGS) technologies, resolution and sensitivity progressively increased expanding considerably the range of detectable DNA anomalies and alongside of Mendelian disorders with known genetic causes. However, particular genomic regions (i.e., repetitive and GC-rich sequences) are inefficiently analyzed by standard genetic tests, still relying on laborious, time-consuming and low-sensitive approaches (i.e., southern-blot for repeat expansion or long-PCR for genes with highly homologous pseudogenes), accounting for at least part of the patients with undiagnosed genetic disorders. Third generation sequencing, generating long reads with improved mappability, is more suitable for the detection of structural alterations and defects in hardly accessible genomic regions. Although recently implemented and not yet clinically available, long read sequencing (LRS) technologies have already shown their potential in genetic medicine research that might greatly impact on diagnostic yield and reporting times, through their translation to clinical settings. The main investigated LRS application concerns the identification of structural variants and repeat expansions, probably because techniques for their detection have not evolved as rapidly as those dedicated to single nucleotide variants (SNV) identification: gold standard analyses are karyotyping and microarrays for balanced and unbalanced chromosome rearrangements, respectively, and southern blot and repeat-primed PCR for the amplification and sizing of expanded alleles, impaired by limited resolution and sensitivity that have not been significantly improved by the advent of NGS. Nevertheless, more recently, with the increased accuracy provided by the latest product releases, LRS has been tested also for SNV detection, especially in genes with highly homologous pseudogenes and for haplotype reconstruction to assess the parental origin of alleles with de novo pathogenic variants. We provide a review of relevant recent scientific papers exploring LRS potential in the diagnosis of genetic diseases and its potential future applications in routine genetic testing.
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Affiliation(s)
- Giulia Olivucci
- IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy
- Department of Surgical and Oncological Sciences, University of Palermo, Palermo, Italy
| | - Emanuela Iovino
- IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy
| | - Giovanni Innella
- Department of Medical and Surgical Sciences (DIMEC), University of Bologna, Bologna, Italy
- Medical Genetics Unit, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy
| | - Daniela Turchetti
- Department of Medical and Surgical Sciences (DIMEC), University of Bologna, Bologna, Italy
- Medical Genetics Unit, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy
| | - Tommaso Pippucci
- IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy
| | - Pamela Magini
- Medical Genetics Unit, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy
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Damaraju N, Miller AL, Miller DE. Long-Read DNA and RNA Sequencing to Streamline Clinical Genetic Testing and Reduce Barriers to Comprehensive Genetic Testing. J Appl Lab Med 2024; 9:138-150. [PMID: 38167773 DOI: 10.1093/jalm/jfad107] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2023] [Accepted: 10/24/2023] [Indexed: 01/05/2024]
Abstract
BACKGROUND Obtaining a precise molecular diagnosis through clinical genetic testing provides information about disease prognosis or progression, allows accurate counseling about recurrence risk, and empowers individuals to benefit from precision therapies or take part in N-of-1 trials. Unfortunately, more than half of individuals with a suspected Mendelian condition remain undiagnosed after a comprehensive clinical evaluation, and the results of any individual clinical genetic test ordered during a typical evaluation may take weeks or months to return. Furthermore, commonly used technologies, such as short-read sequencing, are limited in the types of disease-causing variation they can identify. New technologies, such as long-read sequencing (LRS), are poised to solve these problems. CONTENT Recent technical advances have improved accuracy, increased throughput, and decreased the costs of commercially available LRS technologies. This has resolved many historical concerns about the use of LRS in the clinical environment and opened the door to widespread clinical adoption of LRS. Here, we review LRS technology, how it has been used in the research setting to clarify complex variants or identify disease-causing variation missed by prior clinical testing, and how it may be used clinically in the near future. SUMMARY LRS is unique in that, as a single data source, it has the potential to replace nearly every other clinical genetic test offered today. When analyzed in a stepwise fashion, LRS will simplify laboratory processes, reduce barriers to comprehensive genetic testing, increase the rate of genetic diagnoses, and shorten the amount of time required to make a molecular diagnosis.
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Affiliation(s)
- Nikhita Damaraju
- Institute for Public Health Genetics, University of Washington, Seattle, WA 98195, United States
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA 98195, United States
| | - Angela L Miller
- Department of Pediatrics, University of Washington, Seattle, WA 98195, United States
| | - Danny E Miller
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA 98195, United States
- Department of Pediatrics, University of Washington, Seattle, WA 98195, United States
- Brotman Baty Institute for Precision Medicine, University of Washington, Seattle, WA 98195, United States
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Yu SY, Xi YL, Xu FQ, Zhang J, Liu YS. Application of long read sequencing in rare diseases: The longer, the better? Eur J Med Genet 2023; 66:104871. [PMID: 38832911 DOI: 10.1016/j.ejmg.2023.104871] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2023] [Revised: 10/11/2023] [Accepted: 10/22/2023] [Indexed: 06/06/2024]
Abstract
Rare diseases encompass a diverse group of genetic disorders that affect a small proportion of the population. Identifying the underlying genetic causes of these conditions presents significant challenges due to their genetic heterogeneity and complexity. Conventional short-read sequencing (SRS) techniques have been widely used in diagnosing and investigating of rare diseases, with limitations due to the nature of short-read lengths. In recent years, long read sequencing (LRS) technologies have emerged as a valuable tool in overcoming these limitations. This minireview provides a concise overview of the applications of LRS in rare disease research and diagnosis, including the identification of disease-causing tandem repeat expansions, structural variations, and comprehensive analysis of pathogenic variants with LRS.
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Affiliation(s)
- Si-Yan Yu
- Department of Pediatric Laboratory, Affiliated Children's Hospital of Jiangnan University (Wuxi Children's Hospital), Wuxi, Jiangsu, China; The First School of Clinical Medicine, Nanjing Medical University, Nanjing, Jiangsu, China
| | - Yu-Lin Xi
- Wuxi School of Medicine, Jiangnan University, Wuxi, Jiangsu, China
| | - Fu-Qiang Xu
- Department of Gynecology, Beijing Youan Hospital, Capital Medical University, Beijing, China
| | - Jian Zhang
- Department of Medical Laboratory, Affiliated Children's Hospital of Jiangnan University (Wuxi Children's Hospital), Wuxi, Jiangsu, China.
| | - Yan-Shan Liu
- Department of Pediatric Laboratory, Affiliated Children's Hospital of Jiangnan University (Wuxi Children's Hospital), Wuxi, Jiangsu, China; Wuxi School of Medicine, Jiangnan University, Wuxi, Jiangsu, China.
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Xu P, Wang L, Li J, Huang S, Gao M, Kang R, Li J, Xie H, Liu X, Yan J, Gao X, Gao Y. OGM and WES identifies translocation breakpoints in PKD1 gene in an polycystic kidney patient and healthy baby delivered using PGT. BMC Med Genomics 2023; 16:285. [PMID: 37953234 PMCID: PMC10642002 DOI: 10.1186/s12920-023-01725-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2023] [Accepted: 11/02/2023] [Indexed: 11/14/2023] Open
Abstract
BACKGROUND Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common autosomal dominant genetic diseases. Whole exome sequencing (WES) is a routine tool for diagnostic confirmation of genetic diseases, and it is usually performed to confirm the clinical diagnosis in ADPKD. Reciprocal translocation is the most common chromosomal structural abnormalities and most of its carriers have normal phenotypes until they are encountered infertility problems in adulthood. However, for the polycystic kidney disease caused by abnormal chromosome structure, WES is difficult to achieve the purpose of gene diagnosis. METHODS ADPKD-related genes were detected by WES; Chromosomal karyotyping and Optical Genome Mapping (OGM) were used to detect structural variant; The genomic break-point locations and the abnormal splicing were detected by reverse transcription-PCR and Sanger sequencing; The karyomapping gene chip and Next-Generation Sequencing (NGS) were performed to screen aneuploidy and to distinguish the non-carrier embryos from the carrier embryos. RESULTS No pathogenic variant was found after the first round of WES analysis. Karyotyping data showed 46, XX, t (16; 17) (p13.3; q21.3). With the help of OGM, the translocation breakpoint on chromosome 16 was located within the PKD1 gene. With re-analysis of WES raw data, the breakpoint of translocation was verified to be located at the c.10618 + 3 of PKD1 gene. Based on this molecular diagnosis, a non-carrier embryo was selected out from three blastocysts. With preimplantation genetic testing (PGT) after in vitro fertilization (IVF), it was then transferred into uterus. With confirmation by prenatal and postnatal testing, the pedigree delivered a healthy baby. CONCLUSION We identified a case of ADPKD caused by balanced translocation and assisted the patient to have a healthy child. When the phenotype was closely related with a monogenic disease and the WES analysis was negative, chromosomal structural analysis would be recommended for further genetic diagnosis. Based on the precision diagnosis, preventing the recurrence of hereditary diseases in offspring would be reachable.
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Affiliation(s)
- Peiwen Xu
- Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong University, Jinan, 250012, Shandong, China
- Shandong Key Laboratory of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
- National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, 250012, Shandong, China
- Key laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, 250012, Shandong, China
- Shandong Provincial Clinical Medicine Research Center for Reproductive Health, Shandong University, Jinan, 250012, Shandong, China
| | - Lijuan Wang
- Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong University, Jinan, 250012, Shandong, China
- Shandong Key Laboratory of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
- National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, 250012, Shandong, China
- Key laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, 250012, Shandong, China
- Shandong Provincial Clinical Medicine Research Center for Reproductive Health, Shandong University, Jinan, 250012, Shandong, China
| | - Jing Li
- Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong University, Jinan, 250012, Shandong, China
- Shandong Key Laboratory of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
- National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, 250012, Shandong, China
- Key laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, 250012, Shandong, China
- Shandong Provincial Clinical Medicine Research Center for Reproductive Health, Shandong University, Jinan, 250012, Shandong, China
| | - Sexin Huang
- Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong University, Jinan, 250012, Shandong, China
- Shandong Key Laboratory of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
- National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, 250012, Shandong, China
- Key laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, 250012, Shandong, China
- Shandong Provincial Clinical Medicine Research Center for Reproductive Health, Shandong University, Jinan, 250012, Shandong, China
| | - Ming Gao
- Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong University, Jinan, 250012, Shandong, China
- Shandong Key Laboratory of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
- National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, 250012, Shandong, China
- Key laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, 250012, Shandong, China
- Shandong Provincial Clinical Medicine Research Center for Reproductive Health, Shandong University, Jinan, 250012, Shandong, China
| | - Ranran Kang
- Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong University, Jinan, 250012, Shandong, China
- Shandong Key Laboratory of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
- National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, 250012, Shandong, China
- Key laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, 250012, Shandong, China
- Shandong Provincial Clinical Medicine Research Center for Reproductive Health, Shandong University, Jinan, 250012, Shandong, China
| | - Jie Li
- Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong University, Jinan, 250012, Shandong, China
- Shandong Key Laboratory of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
- National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, 250012, Shandong, China
- Key laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, 250012, Shandong, China
- Shandong Provincial Clinical Medicine Research Center for Reproductive Health, Shandong University, Jinan, 250012, Shandong, China
| | - Hongqiang Xie
- Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong University, Jinan, 250012, Shandong, China
- Shandong Key Laboratory of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
- National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, 250012, Shandong, China
- Key laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, 250012, Shandong, China
- Shandong Provincial Clinical Medicine Research Center for Reproductive Health, Shandong University, Jinan, 250012, Shandong, China
| | - Xiaowei Liu
- Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong University, Jinan, 250012, Shandong, China
- Shandong Key Laboratory of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
- National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, 250012, Shandong, China
- Key laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, 250012, Shandong, China
- Shandong Provincial Clinical Medicine Research Center for Reproductive Health, Shandong University, Jinan, 250012, Shandong, China
| | - Junhao Yan
- Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong University, Jinan, 250012, Shandong, China
- Shandong Key Laboratory of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
- National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, 250012, Shandong, China
- Key laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, 250012, Shandong, China
- Shandong Provincial Clinical Medicine Research Center for Reproductive Health, Shandong University, Jinan, 250012, Shandong, China
| | - Xuan Gao
- Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong University, Jinan, 250012, Shandong, China
- Shandong Key Laboratory of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
- National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, 250012, Shandong, China
- Key laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, 250012, Shandong, China
- Shandong Provincial Clinical Medicine Research Center for Reproductive Health, Shandong University, Jinan, 250012, Shandong, China
| | - Yuan Gao
- Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong University, Jinan, 250012, Shandong, China.
- Shandong Key Laboratory of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China.
- National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, 250012, Shandong, China.
- Key laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, 250012, Shandong, China.
- Shandong Provincial Clinical Medicine Research Center for Reproductive Health, Shandong University, Jinan, 250012, Shandong, China.
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11
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Abstract
DNA sequencing has revolutionized medicine over recent decades. However, analysis of large structural variation and repetitive DNA, a hallmark of human genomes, has been limited by short-read technology, with read lengths of 100-300 bp. Long-read sequencing (LRS) permits routine sequencing of human DNA fragments tens to hundreds of kilobase pairs in size, using both real-time sequencing by synthesis and nanopore-based direct electronic sequencing. LRS permits analysis of large structural variation and haplotypic phasing in human genomes and has enabled the discovery and characterization of rare pathogenic structural variants and repeat expansions. It has also recently enabled the assembly of a complete, gapless human genome that includes previously intractable regions, such as highly repetitive centromeres and homologous acrocentric short arms. With the addition of protocols for targeted enrichment, direct epigenetic DNA modification detection, and long-range chromatin profiling, LRS promises to launch a new era of understanding of genetic diversity and pathogenic mutations in human populations.
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Affiliation(s)
- Peter E Warburton
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA; ,
- Center for Advanced Genomics Technology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Robert P Sebra
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA; ,
- Center for Advanced Genomics Technology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Icahn Genomics Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
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12
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Ohori S, Miyauchi A, Osaka H, Lourenco CM, Arakaki N, Sengoku T, Ogata K, Honjo RS, Kim CA, Mitsuhashi S, Frith MC, Seyama R, Tsuchida N, Uchiyama Y, Koshimizu E, Hamanaka K, Misawa K, Miyatake S, Mizuguchi T, Saito K, Fujita A, Matsumoto N. Biallelic structural variations within FGF12 detected by long-read sequencing in epilepsy. Life Sci Alliance 2023; 6:e202302025. [PMID: 37286232 PMCID: PMC10248215 DOI: 10.26508/lsa.202302025] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2023] [Revised: 05/22/2023] [Accepted: 05/23/2023] [Indexed: 06/09/2023] Open
Abstract
We discovered biallelic intragenic structural variations (SVs) in FGF12 by applying long-read whole genome sequencing to an exome-negative patient with developmental and epileptic encephalopathy (DEE). We also found another DEE patient carrying a biallelic (homozygous) single-nucleotide variant (SNV) in FGF12 that was detected by exome sequencing. FGF12 heterozygous recurrent missense variants with gain-of-function or heterozygous entire duplication of FGF12 are known causes of epilepsy, but biallelic SNVs/SVs have never been described. FGF12 encodes intracellular proteins interacting with the C-terminal domain of the alpha subunit of voltage-gated sodium channels 1.2, 1.5, and 1.6, promoting excitability by delaying fast inactivation of the channels. To validate the molecular pathomechanisms of these biallelic FGF12 SVs/SNV, highly sensitive gene expression analyses using lymphoblastoid cells from the patient with biallelic SVs, structural considerations, and Drosophila in vivo functional analysis of the SNV were performed, confirming loss-of-function. Our study highlights the importance of small SVs in Mendelian disorders, which may be overlooked by exome sequencing but can be detected efficiently by long-read whole genome sequencing, providing new insights into the pathomechanisms of human diseases.
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Affiliation(s)
- Sachiko Ohori
- Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan
- Department of Genetics, Kitasato University Hospital, Sagamihara, Japan
| | - Akihiko Miyauchi
- Department of Pediatrics, Jichi Medical School, Shimotsuke, Japan
| | - Hitoshi Osaka
- Department of Pediatrics, Jichi Medical School, Shimotsuke, Japan
| | - Charles Marques Lourenco
- Neurogenetics Department, Faculdade de Medicina de São José do Rio Preto, São Jose do Rio Preto, Brazil
- Personalized Medicine Department, Special Education Sector at DLE/Grupo Pardini, Belo Horizonte, Brazil
| | - Naohiro Arakaki
- Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Shizuoka, Japan
- Graduate Institute for Advanced Studies, SOKENDAI, Shizuoka, Japan
| | - Toru Sengoku
- Department of Biochemistry, Yokohama City University Graduate School of Medicine, Yokohama, Japan
| | - Kazuhiro Ogata
- Department of Biochemistry, Yokohama City University Graduate School of Medicine, Yokohama, Japan
| | - Rachel Sayuri Honjo
- Unidade de Genética Médica do Instituto da Criança, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, Brazil
| | - Chong Ae Kim
- Unidade de Genética Médica do Instituto da Criança, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, Brazil
| | - Satomi Mitsuhashi
- Department of Neurology, St. Marianna University School of Medicine, Kawasaki, Japan
| | - Martin C Frith
- Artificial Intelligence Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan
- Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Japan
- Computational Bio Big-Data Open Innovation Laboratory, AIST, Tokyo, Japan
| | - Rie Seyama
- Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan
- Department of Obstetrics and Gynecology, Juntendo University, Tokyo, Japan
| | - Naomi Tsuchida
- Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan
- Department of Rare Disease Genomics, Yokohama City University Hospital, Yokohama, Japan
| | - Yuri Uchiyama
- Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan
- Department of Rare Disease Genomics, Yokohama City University Hospital, Yokohama, Japan
| | - Eriko Koshimizu
- Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan
| | - Kohei Hamanaka
- Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan
| | - Kazuharu Misawa
- Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan
| | - Satoko Miyatake
- Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan
- Department of Clinical Genetics, Yokohama City University Hospital, Yokohama, Japan
| | - Takeshi Mizuguchi
- Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan
| | - Kuniaki Saito
- Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Shizuoka, Japan
- Graduate Institute for Advanced Studies, SOKENDAI, Shizuoka, Japan
| | - Atsushi Fujita
- Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan
| | - Naomichi Matsumoto
- Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan
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13
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Ahsan MU, Liu Q, Perdomo JE, Fang L, Wang K. A survey of algorithms for the detection of genomic structural variants from long-read sequencing data. Nat Methods 2023; 20:1143-1158. [PMID: 37386186 PMCID: PMC11208083 DOI: 10.1038/s41592-023-01932-w] [Citation(s) in RCA: 27] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2021] [Accepted: 05/31/2023] [Indexed: 07/01/2023]
Abstract
As long-read sequencing technologies are becoming increasingly popular, a number of methods have been developed for the discovery and analysis of structural variants (SVs) from long reads. Long reads enable detection of SVs that could not be previously detected from short-read sequencing, but computational methods must adapt to the unique challenges and opportunities presented by long-read sequencing. Here, we summarize over 50 long-read-based methods for SV detection, genotyping and visualization, and discuss how new telomere-to-telomere genome assemblies and pangenome efforts can improve the accuracy and drive the development of SV callers in the future.
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Affiliation(s)
- Mian Umair Ahsan
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA
| | - Qian Liu
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA
| | - Jonathan Elliot Perdomo
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA
- School of Biomedical Engineering, Drexel University, Philadelphia, PA, USA
| | - Li Fang
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA
- Department of Genetics and Biomedical Informatics, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China
| | - Kai Wang
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
- Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
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14
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黄 金, 张 碧, 刘 薇. [Clinical diagnostic techniques for rare genetic diseases in children: current status, advances, and thoughts]. ZHONGGUO DANG DAI ER KE ZA ZHI = CHINESE JOURNAL OF CONTEMPORARY PEDIATRICS 2023; 25:308-314. [PMID: 36946168 PMCID: PMC10032066 DOI: 10.7499/j.issn.1008-8830.2211010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Subscribe] [Scholar Register] [Received: 11/03/2022] [Accepted: 01/09/2023] [Indexed: 03/23/2023]
Abstract
Rare diseases refer to a group of single diseases with low incidence rates, complex pathogeneses, severe disease conditions, and rapid progression. Most rare diseases have a genetic background and may occur in childhood. Paying attention to the rare genetic diseases in children and performing early diagnosis and treatment can effectively delay the course of disease and improve the quality of life of children. Many rare diseases can be diagnosed with the help of various experimental techniques, but the diagnosis of rare diseases is still not widely understood. This article summarizes the laboratory diagnostic techniques currently used for rare genetic diseases in children, so as to provide clues for the diagnosis and treatment of such diseases and help to enhance the theoretical understanding and precise medical treatment of rare genetic diseases in children.
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15
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Xia Q, Li S, Ding T, Liu Z, Liu J, Li Y, Zhu H, Yao Z. Nanopore sequencing for detecting reciprocal translocation carrier status in preimplantation genetic testing. BMC Genomics 2023; 24:1. [PMID: 36593441 PMCID: PMC9809107 DOI: 10.1186/s12864-022-09103-5] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2022] [Accepted: 12/29/2022] [Indexed: 01/03/2023] Open
Abstract
BACKGROUND Balanced reciprocal translocation (BRT) is one of the most common chromosomal abnormalities that causes infertility, recurrent miscarriage, and birth defects. Preimplantation genetic testing (PGT) is widely used to select euploid embryos for BRT carriers to increase the chance of a healthy live birth. Several strategies can be used to distinguish reciprocal translocation carrier embryos from those with a normal karyotype; however, these techniques are time-consuming and difficult to implement in clinical laboratories. In this study, nanopore sequencing was performed in two reciprocal translocation carriers, and the results were validated using the next-generation sequencing-based method named, "Mapping Allele with Resolved Carrier Status" (MaReCs). RESULTS The translocation breakpoints in both reciprocal translocation carriers were accurately identified by nanopore sequencing and were in accordance with the results obtained using MaReCs. More than one euploid non-balanced translocation carrier embryo was identified in both patients. Amniocentesis results revealed normal karyotypes, consistent with the findings by MaReCs and nanopore sequencing. CONCLUSION Our results suggest that nanopore sequencing is a powerful strategy for accurately distinguishing non-translocation embryos from translocation carrier embryos and precisely localizing translocation breakpoints, which is essential for PGT and aids in reducing the propagation of reciprocal translocation in the population.
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Affiliation(s)
- Qiuping Xia
- grid.216417.70000 0001 0379 7164Reproductive Medicine Center, Xiangya Hospital, Central South University, 410008 Changsha, Hunan China
| | - Shenglan Li
- grid.216417.70000 0001 0379 7164Department of Gastroenterology, Xiangya Hospital, Central South University, 410008 Changsha, Hunan China
| | - Taoli Ding
- Yikon Genomics Co., Ltd, 215000 Suzhou, Jiangsu China
| | - Zhen Liu
- Yikon Genomics Co., Ltd, 215000 Suzhou, Jiangsu China
| | - Jiaqi Liu
- Yikon Genomics Co., Ltd, 215000 Suzhou, Jiangsu China
| | - Yanping Li
- grid.216417.70000 0001 0379 7164Reproductive Medicine Center, Xiangya Hospital, Central South University, 410008 Changsha, Hunan China
| | - Huimin Zhu
- grid.216417.70000 0001 0379 7164Center for Medical Genetics and Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, 410008 Changsha, Hunan China
| | - Zhongyuan Yao
- grid.216417.70000 0001 0379 7164Reproductive Medicine Center, Xiangya Hospital, Central South University, 410008 Changsha, Hunan China
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16
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Conlin LK, Aref-Eshghi E, McEldrew DA, Luo M, Rajagopalan R. Long-read sequencing for molecular diagnostics in constitutional genetic disorders. Hum Mutat 2022; 43:1531-1544. [PMID: 36086952 PMCID: PMC9561063 DOI: 10.1002/humu.24465] [Citation(s) in RCA: 36] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2022] [Revised: 09/03/2022] [Accepted: 09/06/2022] [Indexed: 11/11/2022]
Abstract
Long-read sequencing (LRS) has been around for more than a decade, but widespread adoption of the technology has been slow due to the perceived high error rates and high sequencing cost. This is changing due to the recent advancements to produce highly accurate sequences and the reducing costs. LRS promises significant improvement over short read sequencing in four major areas: (1) better detection of structural variation (2) better resolution of highly repetitive or nonunique regions (3) accurate long-range haplotype phasing and (4) the detection of base modifications natively from the sequencing data. Several successful applications of LRS have demonstrated its ability to resolve molecular diagnoses where short-read sequencing fails to identify a cause. However, the argument for increased diagnostic yield from LRS remains to be validated. Larger cohort studies may be required to establish the realistic boundaries of LRS's clinical utility and analytical validity, as well as the development of standards for clinical applications. We discuss the limitations of the current standard of care, and contrast with the applications and advantages of two major LRS platforms, PacBio and Oxford Nanopore, for molecular diagnostics of constitutional disorders, and present a critical argument about the potential of LRS in diagnostic settings.
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Affiliation(s)
- Laura K. Conlin
- Division of Genomic Diagnostics, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104
- Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104
| | - Erfan Aref-Eshghi
- Division of Genomic Diagnostics, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104
| | - Deborah A. McEldrew
- Division of Genomic Diagnostics, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104
| | - Minjie Luo
- Division of Genomic Diagnostics, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104
- Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104
| | - Ramakrishnan Rajagopalan
- Division of Genomic Diagnostics, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104
- Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104
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17
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Bai Y, Liu J, Xu J, Sun Y, Li J, Gao Y, Liu L, Jia C, Kong X, Wang L. Long-Read Sequencing Revealed Extragenic and Intragenic Duplications of Exons 56-61 in DMD in an Asymptomatic Male and a DMD Patient. Front Genet 2022; 13:878806. [PMID: 35615378 PMCID: PMC9125615 DOI: 10.3389/fgene.2022.878806] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2022] [Accepted: 03/31/2022] [Indexed: 11/13/2022] Open
Abstract
Expanded carrier screening (ECS) has become an increasingly common technique to assess the genetic risks of individuals in the prenatal or preconception period. Unexpected variants unrelated to referral are being increasingly detected in asymptomatic individuals through ECS. In this study, we reported an asymptomatic male with duplication of exons 56-61 in the DMD gene through ECS using whole-exome sequencing (WES), which was also detected in a male patient diagnosed with typical Duchenne muscular dystrophy (DMD). Breakpoint analysis was then performed to explore the potential mechanisms of phenotypic differences using long-read sequencing (LRS), PacBio single-molecule real-time (PacBio SMRT) target sequencing, and Sanger sequencing. Complex structural variations (SVs) on chromosome X were identified in the asymptomatic male, which revealed that the duplication occurred outside the DMD gene; whereas, the duplication in the patient with DMD was a tandem repeat. The phenotypic differences between the two men could be explained by the different breakpoint junctions. To the best of our knowledge, this is the first report of a breakpoint analysis of DMD duplication in two men with different phenotypes. Breakpoint analysis is necessary when the clinical phenotypes are inconsistent with genotypes, and it applies to prenatal testing.
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Affiliation(s)
- Ying Bai
- Genetic and Prenatal Diagnosis Center, Department of Obstetrics and Gynecology, First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Ju Liu
- Department of Neurology, First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Jinghan Xu
- Genetic and Prenatal Diagnosis Center, Department of Obstetrics and Gynecology, First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Yue Sun
- Genetic and Prenatal Diagnosis Center, Department of Obstetrics and Gynecology, First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Jingjing Li
- Genetic and Prenatal Diagnosis Center, Department of Obstetrics and Gynecology, First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Yong Gao
- GrandOmics Biosciences, Beijing, China
| | - Lina Liu
- Genetic and Prenatal Diagnosis Center, Department of Obstetrics and Gynecology, First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | | | - Xiangdong Kong
- Genetic and Prenatal Diagnosis Center, Department of Obstetrics and Gynecology, First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Li Wang
- Genetic and Prenatal Diagnosis Center, Department of Obstetrics and Gynecology, First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
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18
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Danis D, Jacobsen JOB, Balachandran P, Zhu Q, Yilmaz F, Reese J, Haimel M, Lyon GJ, Helbig I, Mungall CJ, Beck CR, Lee C, Smedley D, Robinson PN. SvAnna: efficient and accurate pathogenicity prediction of coding and regulatory structural variants in long-read genome sequencing. Genome Med 2022; 14:44. [PMID: 35484572 PMCID: PMC9047340 DOI: 10.1186/s13073-022-01046-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2021] [Accepted: 04/12/2022] [Indexed: 01/18/2023] Open
Abstract
Structural variants (SVs) are implicated in the etiology of Mendelian diseases but have been systematically underascertained owing to sequencing technology limitations. Long-read sequencing enables comprehensive detection of SVs, but approaches for prioritization of candidate SVs are needed. Structural variant Annotation and analysis (SvAnna) assesses all classes of SVs and their intersection with transcripts and regulatory sequences, relating predicted effects on gene function with clinical phenotype data. SvAnna places 87% of deleterious SVs in the top ten ranks. The interpretable prioritizations offered by SvAnna will facilitate the widespread adoption of long-read sequencing in diagnostic genomics. SvAnna is available at https://github.com/TheJacksonLaboratory/SvAnn a .
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Affiliation(s)
- Daniel Danis
- The Jackson Laboratory for Genomic Medicine, Farmington, CT, 06032, USA
| | - Julius O B Jacobsen
- William Harvey Research Institute, Charterhouse Square, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, EC1M 6BQ, UK
| | | | - Qihui Zhu
- The Jackson Laboratory for Genomic Medicine, Farmington, CT, 06032, USA
| | - Feyza Yilmaz
- The Jackson Laboratory for Genomic Medicine, Farmington, CT, 06032, USA
| | - Justin Reese
- Division of Environmental Genomics and Systems Biology, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA
| | - Matthias Haimel
- Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases, Vienna, Austria
- St. Anna Children's Cancer Research Institute, Vienna, Austria
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
- Present address: Global Computational Biology and Digital Sciences, Boehringer Ingelheim Regional Center Vienna GmbH & Co KG, 1120, Vienna, Austria
| | - Gholson J Lyon
- Department of Human Genetics, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York, USA
- Biology PhD Program, The Graduate Center, The City University of New York, New York, USA
| | - Ingo Helbig
- Division of Neurology, Children's Hospital of Philadelphia, Philadelphia, PA, USA
- The Epilepsy NeuroGenetics Initiative (ENGIN), Children's Hospital of Philadelphia, Philadelphia, PA, USA
- Department of Biomedical and Health Informatics (DBHi), Children's Hospital of Philadelphia, Philadelphia, PA, USA
- Department of Neurology, University of Pennsylvania, Philadelphia, PA, USA
| | - Christopher J Mungall
- Division of Environmental Genomics and Systems Biology, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA
| | - Christine R Beck
- The Jackson Laboratory for Genomic Medicine, Farmington, CT, 06032, USA
- Department of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, CT, 06032, USA
- Institute for Systems Genomics, University of Connecticut, Storrs, CT, 06269, USA
| | - Charles Lee
- The Jackson Laboratory for Genomic Medicine, Farmington, CT, 06032, USA
| | - Damian Smedley
- William Harvey Research Institute, Charterhouse Square, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, EC1M 6BQ, UK.
| | - Peter N Robinson
- The Jackson Laboratory for Genomic Medicine, Farmington, CT, 06032, USA.
- Department of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, CT, 06032, USA.
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19
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Marwaha S, Knowles JW, Ashley EA. A guide for the diagnosis of rare and undiagnosed disease: beyond the exome. Genome Med 2022; 14:23. [PMID: 35220969 PMCID: PMC8883622 DOI: 10.1186/s13073-022-01026-w] [Citation(s) in RCA: 167] [Impact Index Per Article: 55.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2021] [Accepted: 02/10/2022] [Indexed: 02/07/2023] Open
Abstract
Rare diseases affect 30 million people in the USA and more than 300-400 million worldwide, often causing chronic illness, disability, and premature death. Traditional diagnostic techniques rely heavily on heuristic approaches, coupling clinical experience from prior rare disease presentations with the medical literature. A large number of rare disease patients remain undiagnosed for years and many even die without an accurate diagnosis. In recent years, gene panels, microarrays, and exome sequencing have helped to identify the molecular cause of such rare and undiagnosed diseases. These technologies have allowed diagnoses for a sizable proportion (25-35%) of undiagnosed patients, often with actionable findings. However, a large proportion of these patients remain undiagnosed. In this review, we focus on technologies that can be adopted if exome sequencing is unrevealing. We discuss the benefits of sequencing the whole genome and the additional benefit that may be offered by long-read technology, pan-genome reference, transcriptomics, metabolomics, proteomics, and methyl profiling. We highlight computational methods to help identify regionally distant patients with similar phenotypes or similar genetic mutations. Finally, we describe approaches to automate and accelerate genomic analysis. The strategies discussed here are intended to serve as a guide for clinicians and researchers in the next steps when encountering patients with non-diagnostic exomes.
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Affiliation(s)
- Shruti Marwaha
- Department of Medicine, Division of Cardiovascular Medicine, School of Medicine, Stanford University, Stanford, CA, USA.
- Stanford Center for Undiagnosed Diseases, Stanford University, Stanford, CA, USA.
| | - Joshua W Knowles
- Department of Medicine, Division of Cardiovascular Medicine, School of Medicine, Stanford University, Stanford, CA, USA
- Department of Medicine, Diabetes Research Center, Cardiovascular Institute and Prevention Research Center, Stanford, CA, USA
| | - Euan A Ashley
- Department of Medicine, Division of Cardiovascular Medicine, School of Medicine, Stanford University, Stanford, CA, USA.
- Stanford Center for Undiagnosed Diseases, Stanford University, Stanford, CA, USA.
- Department of Genetics, School of Medicine, Stanford University, Stanford, CA, USA.
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20
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Pei Z, Deng K, Lei C, Du D, Yu G, Sun X, Xu C, Zhang S. Identifying Balanced Chromosomal Translocations in Human Embryos by Oxford Nanopore Sequencing and Breakpoints Region Analysis. Front Genet 2022; 12:810900. [PMID: 35116057 PMCID: PMC8804325 DOI: 10.3389/fgene.2021.810900] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2021] [Accepted: 12/13/2021] [Indexed: 01/02/2023] Open
Abstract
Background: Balanced chromosomal aberrations, especially balanced translocations, can cause infertility, recurrent miscarriage or having chromosomally defective offspring. Preimplantation genetic testing for structural rearrangement (PGT-SR) has been widely implemented to improve the clinical outcomes by selecting euploid embryos for transfer, whereas embryos with balanced translocation karyotype were difficult to be distinguished by routine genetic techniques from those with a normal karyotype. Method: In this present study, we developed a clinically applicable method for reciprocal translocation carriers to reduce the risk of pregnancy loss. In the preclinical phase, we identified reciprocal translocation breakpoints in blood of translocation carriers by long-read Oxford Nanopore sequencing, followed by junction-spanning polymerase chain reaction (PCR) and Sanger sequencing. In the clinical phase of embryo diagnosis, aneuploidies and unbalanced translocations were screened by comprehensive chromosomal screening (CCS) with single nucleotide polymorphism (SNP) microarray, carrier embryos were diagnosed by junction-spanning PCR and family haplotype linkage analysis of the breakpoints region. Amniocentesis and cytogenetic analysis of fetuses in the second trimester were performed after embryo transfer to conform the results diagnosed by the presented method. Results: All the accurate reciprocal translocation breakpoints were effectively identified by Nanopore sequencing and confirmed by Sanger sequencing. Twelve embryos were biopsied and detected, the results of junction-spanning PCR and haplotype linkage analysis were consistent. In total, 12 biopsied blastocysts diagnosed to be euploid, in which 6 were aneuploid or unbalanced, three blastocysts were identified to be balanced translocation carriers and three to be normal karyotypes. Two euploid embryos were subsequently transferred back to patients and late prenatal karyotype analysis of amniotic fluid cells was performed. The outcomes diagnosed by the current approach were totally consistent with the fetal karyotypes. Conclusions: In summary, these investigations in our study illustrated that chromosomal reciprocal translocations in embryos can be accurately diagnosed. Long-read Nanopore sequencing and breakpoint analysis contributes to precisely evaluate the genetic risk of disrupted genes, and provides a way of selecting embryos with normal karyotype, especially for couples those without a reference.
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Affiliation(s)
- Zhenle Pei
- Shanghai Ji Ai Genetics and IVF Institute, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China
| | - Ke Deng
- Shanghai Ji Ai Genetics and IVF Institute, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China
| | - Caixai Lei
- Shanghai Ji Ai Genetics and IVF Institute, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China
| | - Danfeng Du
- Shanghai Ji Ai Genetics and IVF Institute, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China
| | - Guoliang Yu
- Chigene (Beijing) Translational Medical Research Center Co. Ltd., Beijing, China
| | - Xiaoxi Sun
- Shanghai Ji Ai Genetics and IVF Institute, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China
| | - Congjian Xu
- Shanghai Ji Ai Genetics and IVF Institute, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China
- *Correspondence: Congjian Xu, ; Shuo Zhang,
| | - Shuo Zhang
- Shanghai Ji Ai Genetics and IVF Institute, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China
- *Correspondence: Congjian Xu, ; Shuo Zhang,
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21
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Wang Y, Zhao Y, Bollas A, Wang Y, Au KF. Nanopore sequencing technology, bioinformatics and applications. Nat Biotechnol 2021; 39:1348-1365. [PMID: 34750572 PMCID: PMC8988251 DOI: 10.1038/s41587-021-01108-x] [Citation(s) in RCA: 795] [Impact Index Per Article: 198.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2019] [Accepted: 09/22/2021] [Indexed: 12/13/2022]
Abstract
Rapid advances in nanopore technologies for sequencing single long DNA and RNA molecules have led to substantial improvements in accuracy, read length and throughput. These breakthroughs have required extensive development of experimental and bioinformatics methods to fully exploit nanopore long reads for investigations of genomes, transcriptomes, epigenomes and epitranscriptomes. Nanopore sequencing is being applied in genome assembly, full-length transcript detection and base modification detection and in more specialized areas, such as rapid clinical diagnoses and outbreak surveillance. Many opportunities remain for improving data quality and analytical approaches through the development of new nanopores, base-calling methods and experimental protocols tailored to particular applications.
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Affiliation(s)
- Yunhao Wang
- Department of Biomedical Informatics, The Ohio State University, Columbus, OH, USA
| | - Yue Zhao
- Department of Biomedical Informatics, The Ohio State University, Columbus, OH, USA
- Biomedical Informatics Shared Resources, The Ohio State University, Columbus, OH, USA
| | - Audrey Bollas
- Department of Biomedical Informatics, The Ohio State University, Columbus, OH, USA
| | - Yuru Wang
- Department of Biomedical Informatics, The Ohio State University, Columbus, OH, USA
| | - Kin Fai Au
- Department of Biomedical Informatics, The Ohio State University, Columbus, OH, USA.
- Biomedical Informatics Shared Resources, The Ohio State University, Columbus, OH, USA.
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22
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Plona KL, Eastman JF, Drumm ML. Classifying molecular phenotypes of G6PC variants for pathogenic properties and to guide therapeutic development. JIMD Rep 2021; 60:56-66. [PMID: 34258141 PMCID: PMC8260485 DOI: 10.1002/jmd2.12215] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/12/2020] [Revised: 03/02/2021] [Accepted: 03/19/2021] [Indexed: 12/02/2022] Open
Abstract
Due to advances in sequencing technologies, identification of genetic variants is rapid. However, the functional consequences of most genomic variants remain unknown. Consequently, variants of uncertain significance (VUS) that appear in clinical DNA diagnostic reports lack sufficient data for interpretation. Algorithms exist to aid prediction of a variant's likelihood of pathogenicity, but these predictions usually lack empiric evidence. To examine the feasibility of generating functional evidence in vitro for a given variant's role in disease, a panel of 29 coding sequence variants in the G6PC gene was assessed. G6PC encodes glucose-6 phosphatase enzyme, and reduction in its function causes the rare metabolic disease glycogen storage disease type 1a (GSD1a). Variants were heterologously expressed as fusion proteins in a hepatocyte-derived cell line and examined for effects on steady-state protein levels, biosynthetic processing, and intracellular distribution. The screen revealed variant effects on protein levels, N-linked glycosylation status, and cellular distribution. Of the eight VUS tested, seven behaved similar to wild-type protein while one VUS, p.Cys109Tyr, exhibited features consistent with pathogenicity for all molecular phenotypes assayed, including significantly reduced protein levels, alteration in protein glycosylation status, and abnormally diffuse protein localization pattern, and has recently been reported in a patient with GSD1a. Our results show that such a screen can add empiric evidence to existing databases to aid in diagnostics, and also provides further classification for molecular phenotypes that could be used in future therapeutic screening approaches for small molecule or gene editing strategies directed at specific variants.
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Affiliation(s)
- Kathleen L. Plona
- Genetics and Genome Sciences Department, School of MedicineCase Western Reserve UniversityClevelandOhioUSA
| | - Jean F. Eastman
- Genetics and Genome Sciences Department, School of MedicineCase Western Reserve UniversityClevelandOhioUSA
| | - Mitchell L. Drumm
- Genetics and Genome Sciences Department, School of MedicineCase Western Reserve UniversityClevelandOhioUSA
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23
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Study of complex structural variations of X-linked deafness-2 based on single-molecule sequencing. Biosci Rep 2021; 41:228372. [PMID: 33860785 PMCID: PMC8193640 DOI: 10.1042/bsr20203740] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2020] [Revised: 03/21/2021] [Accepted: 03/31/2021] [Indexed: 12/16/2022] Open
Abstract
X-linked deafness-2 (DFNX2) is cochlear incomplete partition type III (IP-III), one of inner ear malformations characterized by an abnormally wide opening in the bone separating the basal turn of the cochlea from the internal auditory canal, fixation of the stapes and cerebrospinal fluid (CSF) gusher upon stapedectomy or cochleostomy. The causative gene of DFNX2 was POU3F4. To investigate the genetic causes of DFNX2 and compare the efficiency of different sequencing methods, 12 unrelated patients were enrolled in the present study. Targeted next-generation sequencing (NGS) and long-read sequencing were used to analyze the genetic etiology of DFNX2. Six variants of POU3F4 were identified in this cohort by NGS. Three patients with a negative diagnosis based on NGS were enrolled in further long-read sequencing. Two of them were all found to carry structural variations (SVs) on chromosome X, consisting of an 870-kb deletion (DEL) at upstream of POU3F4 and an 8-Mb inversion (INV). The 870-kb DEL may have been due to non-homologous end joining (NHEJ), while non-allelic homologous recombination (NAHR) within a single chromatid may have accounted for the 8-Mb INV. Common POU3F4 mutations in DFNX2 included point mutations, small insertions and deletions (INDELs), and exon mutations, which can be detected by Sanger sequencing and NGS. Single-molecule long-read sequencing constitutes an additional and valuable method for accurate detection of pathogenic SVs in IP-III patients with negative NGS results.
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24
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Sun X, Song L, Yang W, Zhang L, Liu M, Li X, Tian G, Wang W. Nanopore Sequencing and Its Clinical Applications. Methods Mol Biol 2021; 2204:13-32. [PMID: 32710311 DOI: 10.1007/978-1-0716-0904-0_2] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Nanopore sequencing is a method for determining the order and modifications of DNA/RNA nucleotides by detecting the electric current variations when DNA/RNA oligonucleotides pass through the nanometer-sized hole (nanopore). Nanopore-based DNA analysis techniques have been commercialized by Oxford Nanopore Technologies, NabSys, and Sequenom, and widely used in scientific researches recently including human genomics, cancer, metagenomics, plant sciences, etc., moreover, it also has potential applications in the field of healthcare due to its fast turn-around time, portable and real-time data analysis. Those features make it a promising technology for the point-of-care testing (POCT) and its potential clinical applications are briefly discussed in this chapter.
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Affiliation(s)
- Xue Sun
- Geneis (Beijing) Co., Ltd., Beijing, People's Republic of China
| | - Lei Song
- Geneis (Beijing) Co., Ltd., Beijing, People's Republic of China
| | - Wenjuan Yang
- Geneis (Beijing) Co., Ltd., Beijing, People's Republic of China
| | - Lili Zhang
- Geneis (Beijing) Co., Ltd., Beijing, People's Republic of China
| | - Meng Liu
- Geneis (Beijing) Co., Ltd., Beijing, People's Republic of China
| | - Xiaoshuang Li
- Geneis (Beijing) Co., Ltd., Beijing, People's Republic of China
| | - Geng Tian
- Geneis (Beijing) Co., Ltd., Beijing, People's Republic of China
| | - Weiwei Wang
- Geneis (Beijing) Co., Ltd., Beijing, People's Republic of China.
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25
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Liu S, Wang H, Leigh D, Cram DS, Wang L, Yao Y. Third-generation sequencing: any future opportunities for PGT? J Assist Reprod Genet 2021; 38:357-364. [PMID: 33211225 PMCID: PMC7884560 DOI: 10.1007/s10815-020-02009-9] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2020] [Accepted: 11/08/2020] [Indexed: 12/15/2022] Open
Abstract
PURPOSE To investigate use of the third-generation sequencing (TGS) Oxford Nanopore system as a new approach for preimplantation genetic testing (PGT). METHODS Embryos with known structural variations underwent multiple displacement amplification to create fragments of DNA (average ~ 5 kb) suitable for sequencing on a nanopore. RESULTS High-depth sequencing identified the deletion interval for the relatively large HBA1/2--SEA alpha thalassemia deletion. In addition, STRs were able to be identified in the primary sequence data for potential use in conventional PGT-M linkage confirmation. Sequencing of amplified embryo DNA carrying a translocation enabled balanced embryos to be identified and gave the precise identification of translocation breakpoints, offering the opportunity to differentiate carriers from non-carrier embryos. Low-pass sequencing gave reproducible profiles suitable for simple identification of whole-chromosome and segmental aneuploidies. CONCLUSION TGS on the Oxford Nanopore is a possible alternative and versatile approach to PGT with potential for performing economical workups where the long read sequencing information can be used for assisting in a traditional PGT workup to design an accurate and reliable test. Additionally, application of TGS has the possibility of providing combined PGT-A/SR or in selected stand-alone PGT-M cases involving pathogenic deletions. Both of these applications offer the opportunity for simultaneous aneuploidy detection to select either balanced embryos for transfer or additional carrier identification. The low cost of the instrument offers new laboratories economical entry into onsite PGT.
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Affiliation(s)
- Sai Liu
- Department of Obstetrics and Gynecology, The First Medical Center of PLA General Hospital, Medical School of Chinese PLA, 28 Fuxing Road, Beijing, 100853, People's Republic of China
- Reproductive Medicine and Genetic Center, The First Hospital of Kunming Calmette Hospital, Kunming, People's Republic of China
| | - Hui Wang
- Department of Obstetrics and Gynecology, The First Medical Center of PLA General Hospital, Medical School of Chinese PLA, 28 Fuxing Road, Beijing, 100853, People's Republic of China
| | - Don Leigh
- Reproductive Medicine and Genetic Center, The First Hospital of Kunming Calmette Hospital, Kunming, People's Republic of China
| | - David S Cram
- Reproductive Medicine and Genetic Center, The First Hospital of Kunming Calmette Hospital, Kunming, People's Republic of China
| | - Li Wang
- Reproductive Medicine and Genetic Center, The First Hospital of Kunming Calmette Hospital, Kunming, People's Republic of China.
| | - Yuanqing Yao
- Department of Obstetrics and Gynecology, The First Medical Center of PLA General Hospital, Medical School of Chinese PLA, 28 Fuxing Road, Beijing, 100853, People's Republic of China.
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26
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Fatima N, Petri A, Gyllensten U, Feuk L, Ameur A. Evaluation of Single-Molecule Sequencing Technologies for Structural Variant Detection in Two Swedish Human Genomes. Genes (Basel) 2020; 11:E1444. [PMID: 33266238 PMCID: PMC7760597 DOI: 10.3390/genes11121444] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2020] [Revised: 11/24/2020] [Accepted: 11/26/2020] [Indexed: 01/23/2023] Open
Abstract
Long-read single molecule sequencing is increasingly used in human genomics research, as it allows to accurately detect large-scale DNA rearrangements such as structural variations (SVs) at high resolution. However, few studies have evaluated the performance of different single molecule sequencing platforms for SV detection in human samples. Here we performed Oxford Nanopore Technologies (ONT) whole-genome sequencing of two Swedish human samples (average 32× coverage) and compared the results to previously generated Pacific Biosciences (PacBio) data for the same individuals (average 66× coverage). Our analysis inferred an average of 17k and 23k SVs from the ONT and PacBio data, respectively, with a majority of them overlapping with an available multi-platform SV dataset. When comparing the SV calls in the two Swedish individuals, we find a higher concordance between ONT and PacBio SVs detected in the same individual as compared to SVs detected by the same technology in different individuals. Downsampling of PacBio reads, performed to obtain similar coverage levels for all datasets, resulted in 17k SVs per individual and improved overlap with the ONT SVs. Our results suggest that ONT and PacBio have a similar performance for SV detection in human whole genome sequencing data, and that both technologies are feasible for population-scale studies.
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Affiliation(s)
- Nazeefa Fatima
- Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, 752 36 Uppsala, Sweden; (N.F.); (A.P.); (U.G.); (L.F.)
| | - Anna Petri
- Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, 752 36 Uppsala, Sweden; (N.F.); (A.P.); (U.G.); (L.F.)
| | - Ulf Gyllensten
- Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, 752 36 Uppsala, Sweden; (N.F.); (A.P.); (U.G.); (L.F.)
| | - Lars Feuk
- Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, 752 36 Uppsala, Sweden; (N.F.); (A.P.); (U.G.); (L.F.)
| | - Adam Ameur
- Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, 752 36 Uppsala, Sweden; (N.F.); (A.P.); (U.G.); (L.F.)
- Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Clayton, VIC 3800, Australia
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27
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Logsdon GA, Vollger MR, Eichler EE. Long-read human genome sequencing and its applications. Nat Rev Genet 2020; 21:597-614. [PMID: 32504078 PMCID: PMC7877196 DOI: 10.1038/s41576-020-0236-x] [Citation(s) in RCA: 577] [Impact Index Per Article: 115.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/31/2020] [Indexed: 12/27/2022]
Abstract
Over the past decade, long-read, single-molecule DNA sequencing technologies have emerged as powerful players in genomics. With the ability to generate reads tens to thousands of kilobases in length with an accuracy approaching that of short-read sequencing technologies, these platforms have proven their ability to resolve some of the most challenging regions of the human genome, detect previously inaccessible structural variants and generate some of the first telomere-to-telomere assemblies of whole chromosomes. Long-read sequencing technologies will soon permit the routine assembly of diploid genomes, which will revolutionize genomics by revealing the full spectrum of human genetic variation, resolving some of the missing heritability and leading to the discovery of novel mechanisms of disease.
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Affiliation(s)
- Glennis A Logsdon
- Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA
| | - Mitchell R Vollger
- Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA
| | - Evan E Eichler
- Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA.
- Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA.
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28
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Xie Z, Sun C, Liu Y, Yu M, Zheng Y, Meng L, Wang G, Cornejo-Sanchez DM, Bharadwaj T, Yan J, Zhang L, Pineda-Trujillo N, Zhang W, Leal SM, Schrauwen I, Wang Z, Yuan Y. Practical approach to the genetic diagnosis of unsolved dystrophinopathies: a stepwise strategy in the genomic era. J Med Genet 2020; 58:743-751. [PMID: 32978268 DOI: 10.1136/jmedgenet-2020-107113] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2020] [Revised: 07/02/2020] [Accepted: 08/08/2020] [Indexed: 12/18/2022]
Abstract
OBJECTIVE To investigate the diagnostic value of implementing a stepwise genetic testing strategy (SGTS) in genetically unsolved cases with dystrophinopathies. METHODS After routine genetic testing in 872 male patients with highly suspected dystrophinopathies, we identified 715 patients with a pathogenic DMD variant. Of the 157 patients who had no pathogenic DMD variants and underwent a muscle biopsy, 142 patients were confirmed to have other myopathies, and 15 suspected dystrophinopathies remained genetically undiagnosed. These 15 patients underwent a more comprehensive evaluation as part of the SGTS pipeline, which included the stepwise analysis of dystrophin mRNA, short-read whole-gene DMD sequencing, long-read whole-gene DMD sequencing and in silico bioinformatic analyses. RESULTS SGTS successfully yielded a molecular diagnosis of dystrophinopathy in 11 of the 15 genetically unsolved cases. We identified 8 intronic and 2 complex structural variants (SVs) leading to aberrant splicing in 10 of 11 patients, of which 9 variants were novel. In one case, a molecular defect was detected on mRNA and protein level only. Aberrant splicing mechanisms included 6 pseudoexon inclusions and 4 alterations of splice sites and splicing regulatory elements. We showed for the first time the exonisation of a MER48 element as a novel pathogenic mechanism in dystrophinopathies. CONCLUSION Our study highlights the high diagnostic utility of implementing a SGTS pipeline in dystrophinopathies with intronic variants and complex SVs.
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Affiliation(s)
- Zhiying Xie
- Department of Neurology, Peking University First Hospital, Beijing, China
| | - Chengyue Sun
- Department of Neurology, Peking University First Hospital, Beijing, China
| | - Yilin Liu
- Department of Neurology, Peking University First Hospital, Beijing, China
| | - Meng Yu
- Department of Neurology, Peking University First Hospital, Beijing, China
| | - Yiming Zheng
- Department of Neurology, Peking University First Hospital, Beijing, China
| | - Lingchao Meng
- Department of Neurology, Peking University First Hospital, Beijing, China
| | - Gao Wang
- Center for Statistical Genetics, Sergievsky Center, Taub Institute for Alzheimer's Disease and the Aging Brain, and the Department of Neurology, Columbia University Medical Center, New York, New York, USA
| | - Diana M Cornejo-Sanchez
- Center for Statistical Genetics, Sergievsky Center, Taub Institute for Alzheimer's Disease and the Aging Brain, and the Department of Neurology, Columbia University Medical Center, New York, New York, USA
| | - Thashi Bharadwaj
- Center for Statistical Genetics, Sergievsky Center, Taub Institute for Alzheimer's Disease and the Aging Brain, and the Department of Neurology, Columbia University Medical Center, New York, New York, USA
| | - Jin Yan
- Science and Technology, Beijing Epigen Medical Technology Inc, Beijing, China
| | - Lingxiang Zhang
- Science and Technology, Beijing Epigen Medical Technology Inc, Beijing, China
| | - Nicolas Pineda-Trujillo
- Grupo Mapeo Genetico, Departamento de Pediatría, Facultad de Medicina, Universidad de Antioquia, Medellin, Colombia
| | - Wei Zhang
- Department of Neurology, Peking University First Hospital, Beijing, China
| | - Suzanne M Leal
- Center for Statistical Genetics, Sergievsky Center, Taub Institute for Alzheimer's Disease and the Aging Brain, and the Department of Neurology, Columbia University Medical Center, New York, New York, USA
| | - Isabelle Schrauwen
- Center for Statistical Genetics, Sergievsky Center, Taub Institute for Alzheimer's Disease and the Aging Brain, and the Department of Neurology, Columbia University Medical Center, New York, New York, USA
| | - Zhaoxia Wang
- Department of Neurology, Peking University First Hospital, Beijing, China
| | - Yun Yuan
- Department of Neurology, Peking University First Hospital, Beijing, China
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29
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Aganezov S, Goodwin S, Sherman RM, Sedlazeck FJ, Arun G, Bhatia S, Lee I, Kirsche M, Wappel R, Kramer M, Kostroff K, Spector DL, Timp W, McCombie WR, Schatz MC. Comprehensive analysis of structural variants in breast cancer genomes using single-molecule sequencing. Genome Res 2020; 30:1258-1273. [PMID: 32887686 PMCID: PMC7545150 DOI: 10.1101/gr.260497.119] [Citation(s) in RCA: 67] [Impact Index Per Article: 13.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2019] [Accepted: 08/07/2020] [Indexed: 12/14/2022]
Abstract
Improved identification of structural variants (SVs) in cancer can lead to more targeted and effective treatment options as well as advance our basic understanding of the disease and its progression. We performed whole-genome sequencing of the SKBR3 breast cancer cell line and patient-derived tumor and normal organoids from two breast cancer patients using Illumina/10x Genomics, Pacific Biosciences (PacBio), and Oxford Nanopore Technologies (ONT) sequencing. We then inferred SVs and large-scale allele-specific copy number variants (CNVs) using an ensemble of methods. Our findings show that long-read sequencing allows for substantially more accurate and sensitive SV detection, with between 90% and 95% of variants supported by each long-read technology also supported by the other. We also report high accuracy for long reads even at relatively low coverage (25×–30×). Furthermore, we integrated SV and CNV data into a unifying karyotype-graph structure to present a more accurate representation of the mutated cancer genomes. We find hundreds of variants within known cancer-related genes detectable only through long-read sequencing. These findings highlight the need for long-read sequencing of cancer genomes for the precise analysis of their genetic instability.
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Affiliation(s)
- Sergey Aganezov
- Department of Computer Science, Johns Hopkins University, Baltimore, Maryland 21211, USA
| | - Sara Goodwin
- Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA
| | - Rachel M Sherman
- Department of Computer Science, Johns Hopkins University, Baltimore, Maryland 21211, USA
| | - Fritz J Sedlazeck
- Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas 77030, USA
| | - Gayatri Arun
- Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA
| | - Sonam Bhatia
- Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA
| | - Isac Lee
- Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland 21211, USA
| | - Melanie Kirsche
- Department of Computer Science, Johns Hopkins University, Baltimore, Maryland 21211, USA
| | - Robert Wappel
- Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA
| | - Melissa Kramer
- Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA
| | | | - David L Spector
- Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA
| | - Winston Timp
- Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland 21211, USA
| | | | - Michael C Schatz
- Department of Computer Science, Johns Hopkins University, Baltimore, Maryland 21211, USA.,Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.,Department of Biology, Johns Hopkins University, Baltimore, Maryland 21211, USA
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30
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Lalonde E, Rentas S, Lin F, Dulik MC, Skraban CM, Spinner NB. Genomic Diagnosis for Pediatric Disorders: Revolution and Evolution. Front Pediatr 2020; 8:373. [PMID: 32733828 PMCID: PMC7360789 DOI: 10.3389/fped.2020.00373] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/26/2019] [Accepted: 06/02/2020] [Indexed: 12/14/2022] Open
Abstract
Powerful, recent advances in technologies to analyze the genome have had a profound impact on the practice of medical genetics, both in the laboratory and in the clinic. Increasing utilization of genome-wide testing such as chromosomal microarray analysis and exome sequencing have lead a shift toward a "genotype-first" approach. Numerous techniques are now available to diagnose a particular syndrome or phenotype, and while traditional techniques remain efficient tools in certain situations, higher-throughput technologies have become the de facto laboratory tool for diagnosis of most conditions. However, selecting the right assay or technology is challenging, and the wrong choice may lead to prolonged time to diagnosis, or even a missed diagnosis. In this review, we will discuss current core technologies for the diagnosis of classic genetic disorders to shed light on the benefits and disadvantages of these strategies, including diagnostic efficiency, variant interpretation, and secondary findings. Finally, we review upcoming technologies posed to impart further changes in the field of genetic diagnostics as we move toward "genome-first" practice.
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Affiliation(s)
- Emilie Lalonde
- Division of Genomic Diagnostics, Department of Pathology and Laboratory Medicine, School of Medicine, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA, United States
| | - Stefan Rentas
- Division of Genomic Diagnostics, Department of Pathology and Laboratory Medicine, School of Medicine, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA, United States
| | - Fumin Lin
- Division of Genomic Diagnostics, Department of Pathology and Laboratory Medicine, School of Medicine, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA, United States
| | - Matthew C. Dulik
- Division of Genomic Diagnostics, Department of Pathology and Laboratory Medicine, School of Medicine, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA, United States
| | - Cara M. Skraban
- Division of Human Genetics, Department of Pediatrics, School of Medicine, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA, United States
| | - Nancy B. Spinner
- Division of Genomic Diagnostics, Department of Pathology and Laboratory Medicine, School of Medicine, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA, United States
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31
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Bardet-Biedl syndrome and related disorders in Japan. J Hum Genet 2020; 65:847-853. [PMID: 32451492 DOI: 10.1038/s10038-020-0778-y] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2020] [Revised: 04/19/2020] [Accepted: 05/06/2020] [Indexed: 02/03/2023]
Abstract
Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder characterized by obesity, mental impairment, rod-cone dystrophy, polydactyly, male hypogonadism, and renal abnormalities. This disorder is caused by mutations in BBS1-21. Alström syndrome (AS), caused solely by mutations in ALMS1, is another genetic obesity syndrome clinically similar to BBS. We previously conducted the first nationwide survey of BBS in Japan and found four patients with genetically definite BBS. In this study, exome analyses were performed on new patients whose symptoms fulfilled the diagnostic criteria for BBS. We identified one reported heterozygous mutation in BBS1 (p.R429*) in one patient, two novel mutations (p.L493R and p.H719Y) in BBS20 in a second patient, and one novel mutation (p.Q920*) and one reported mutation (p.R2928*) in ALMS1 in a third patient, who was subsequently diagnosed with AS. The first patient with BBS was previously considered to have digenic heterozygous mutations in BBS1 and BBS4. RT-PCR and long-range genomic PCR analyses identified a new heterozygous mutation in BBS1, the deletion of exons 10 and 11. Thus, this patient was compound heterozygous for mutations in BBS1. Many studies have described digenic heterozygous mutations in BBS. However, undetected mutations might have existed in either one of the mutated genes.
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32
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Ross JP, Dion PA, Rouleau GA. Exome sequencing in genetic disease: recent advances and considerations. F1000Res 2020; 9:F1000 Faculty Rev-336. [PMID: 32431803 PMCID: PMC7205110 DOI: 10.12688/f1000research.19444.1] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 04/30/2020] [Indexed: 12/14/2022] Open
Abstract
Over the past decade, exome sequencing (ES) has allowed significant advancements to the field of disease research. By targeting the protein-coding regions of the genome, ES combines the depth of knowledge on protein-altering variants with high-throughput data generation and ease of analysis. New discoveries continue to be made using ES, and medical science has benefitted both theoretically and clinically from its continued use. In this review, we describe recent advances and successes of ES in disease research. Through selected examples of recent publications, we explore how ES continues to be a valuable tool to find variants that might explain disease etiology or provide insight into the biology underlying the disease. We then discuss shortcomings of ES in terms of variant discoveries made by other sequencing technologies that would be missed because of the scope and techniques of ES. We conclude with a brief outlook on the future of ES, suggesting that although newer and more thorough sequencing methods will soon supplant ES, its results will continue to be useful for disease research.
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Affiliation(s)
- Jay P. Ross
- Department of Human Genetics, McGill University, 3640 University, Montréal, QC, H3A 0C7, Canada
- Montreal Neurological Institute and Hospital, McGill University, 3801 University, Montréal, QC, H3A 2B4, Canada
| | - Patrick A. Dion
- Montreal Neurological Institute and Hospital, McGill University, 3801 University, Montréal, QC, H3A 2B4, Canada
- Department of Neurology and Neurosurgery, McGill University, 3801 University, Montréal, QC, H3A 2B4, Canada
| | - Guy A. Rouleau
- Department of Human Genetics, McGill University, 3640 University, Montréal, QC, H3A 0C7, Canada
- Montreal Neurological Institute and Hospital, McGill University, 3801 University, Montréal, QC, H3A 2B4, Canada
- Department of Neurology and Neurosurgery, McGill University, 3801 University, Montréal, QC, H3A 2B4, Canada
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33
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Xiao T, Zhou W. The third generation sequencing: the advanced approach to genetic diseases. Transl Pediatr 2020; 9:163-173. [PMID: 32477917 PMCID: PMC7237973 DOI: 10.21037/tp.2020.03.06] [Citation(s) in RCA: 71] [Impact Index Per Article: 14.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/29/2019] [Accepted: 02/05/2020] [Indexed: 01/05/2023] Open
Abstract
Genomic sequencing technologies have revolutionized mutation detection of the genetic diseases in the past few years. In recent years, the third generation sequencing (TGS) has been gaining insight into more genetic diseases owing to the single molecular and real time sequencing technology. This paper reviews the genomic sequencing revolutionary history first and then focuses on the genetic diseases discovered through the TGS and the clinical effects of the TGS, which is followed by the discussion of the improvement in the bioinformatic analysis for the TGS and its limitations. In summary, the TGS has been enhancing the diagnostic accuracy of genetic diseases in molecular level as well as paving a new way for basic researches and therapies.
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Affiliation(s)
- Tiantian Xiao
- Clinic of Neonatology, Children’s Hospital of Fudan University, Shanghai 201102, China
- Department of Neonatology, Chengdu Women’s and Children’s Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu 611731, China
| | - Wenhao Zhou
- Clinic of Neonatology, Children’s Hospital of Fudan University, Shanghai 201102, China
- Key Laboratory of Birth Defects, Children’s Hospital of Fudan University, Shanghai 201102, China
- Key Laboratory of Neonatal Diseases, Children’s Hospital of Fudan University, Shanghai 201102, China
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34
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Tham CY, Tirado-Magallanes R, Goh Y, Fullwood MJ, Koh BTH, Wang W, Ng CH, Chng WJ, Thiery A, Tenen DG, Benoukraf T. NanoVar: accurate characterization of patients' genomic structural variants using low-depth nanopore sequencing. Genome Biol 2020; 21:56. [PMID: 32127024 PMCID: PMC7055087 DOI: 10.1186/s13059-020-01968-7] [Citation(s) in RCA: 66] [Impact Index Per Article: 13.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2019] [Accepted: 02/21/2020] [Indexed: 12/19/2022] Open
Abstract
The recent advent of third-generation sequencing technologies brings promise for better characterization of genomic structural variants by virtue of having longer reads. However, long-read applications are still constrained by their high sequencing error rates and low sequencing throughput. Here, we present NanoVar, an optimized structural variant caller utilizing low-depth (8X) whole-genome sequencing data generated by Oxford Nanopore Technologies. NanoVar exhibits higher structural variant calling accuracy when benchmarked against current tools using low-depth simulated datasets. In patient samples, we successfully validate structural variants characterized by NanoVar and uncover normal alternative sequences or alleles which are present in healthy individuals.
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Affiliation(s)
- Cheng Yong Tham
- Cancer Science Institute of Singapore, National University of Singapore, Centre for Translational Medicine, 14 Medical Drive, #12-01, Singapore, 117599, Singapore
| | - Roberto Tirado-Magallanes
- Cancer Science Institute of Singapore, National University of Singapore, Centre for Translational Medicine, 14 Medical Drive, #12-01, Singapore, 117599, Singapore
| | - Yufen Goh
- Cancer Science Institute of Singapore, National University of Singapore, Centre for Translational Medicine, 14 Medical Drive, #12-01, Singapore, 117599, Singapore
| | - Melissa J Fullwood
- Cancer Science Institute of Singapore, National University of Singapore, Centre for Translational Medicine, 14 Medical Drive, #12-01, Singapore, 117599, Singapore.,School of Biological Sciences, Nanyang Technological University, Singapore, 637551, Singapore
| | - Bryan T H Koh
- Department of Orthopedic Surgery, National University Health Systems, Singapore, 119228, Singapore
| | - Wilson Wang
- Department of Orthopedic Surgery, National University Health Systems, Singapore, 119228, Singapore.,Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228, Singapore
| | - Chin Hin Ng
- Department of Hematology-Oncology, National University Cancer Institute of Singapore, National University Health System, Singapore, 119228, Singapore
| | - Wee Joo Chng
- Cancer Science Institute of Singapore, National University of Singapore, Centre for Translational Medicine, 14 Medical Drive, #12-01, Singapore, 117599, Singapore.,Department of Hematology-Oncology, National University Cancer Institute of Singapore, National University Health System, Singapore, 119228, Singapore.,Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228, Singapore
| | - Alexandre Thiery
- Department of Statistics and Applied Probability, National University of Singapore, Singapore, 117546, Singapore
| | - Daniel G Tenen
- Cancer Science Institute of Singapore, National University of Singapore, Centre for Translational Medicine, 14 Medical Drive, #12-01, Singapore, 117599, Singapore.,Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, 02115, USA
| | - Touati Benoukraf
- Cancer Science Institute of Singapore, National University of Singapore, Centre for Translational Medicine, 14 Medical Drive, #12-01, Singapore, 117599, Singapore. .,Discipline of Genetics, Faculty of Medicine, Memorial University of Newfoundland, St. John's, NL, A1B 3V6, Canada.
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35
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Combinations of exonic deletions and rare mutations lead to misdiagnosis of propionic acidemia. Clin Chim Acta 2020; 502:153-158. [DOI: 10.1016/j.cca.2019.12.021] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2019] [Revised: 12/17/2019] [Accepted: 12/25/2019] [Indexed: 12/23/2022]
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36
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Abstract
Identifying structural variation (SV) is essential for genome interpretation but has been historically difficult due to limitations inherent to available genome technologies. Detection methods that use ensemble algorithms and emerging sequencing technologies have enabled the discovery of thousands of SVs, uncovering information about their ubiquity, relationship to disease and possible effects on biological mechanisms. Given the variability in SV type and size, along with unique detection biases of emerging genomic platforms, multiplatform discovery is necessary to resolve the full spectrum of variation. Here, we review modern approaches for investigating SVs and proffer that, moving forwards, studies integrating biological information with detection will be necessary to comprehensively understand the impact of SV in the human genome.
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Affiliation(s)
- Steve S Ho
- Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA
| | - Alexander E Urban
- Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA, USA
- Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA
| | - Ryan E Mills
- Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA.
- Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA.
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37
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Simon R, Lischer HEL, Pieńkowska-Schelling A, Keller I, Häfliger IM, Letko A, Schelling C, Lühken G, Drögemüller C. New genomic features of the polled intersex syndrome variant in goats unraveled by long-read whole-genome sequencing. Anim Genet 2020; 51:439-448. [PMID: 32060960 DOI: 10.1111/age.12918] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2019] [Revised: 01/23/2020] [Accepted: 01/23/2020] [Indexed: 01/19/2023]
Abstract
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 11.7 kb deletion at approximately 129 Mb 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 10 159 bp and an inversely inserted approximately 480 kb-sized duplicated segment of a region located approximately 21 Mb 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.
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Affiliation(s)
- R Simon
- Institute of Animal Breeding and Genetics, Justus Liebig University, Giessen, 35390, Germany
| | - H E L Lischer
- Interfaculty Bioinformatics Unit, University of Bern, Bern, 3001, Switzerland.,Swiss Institute of Bioinformatics, Lausanne, 1015, Switzerland
| | - A Pieńkowska-Schelling
- Institute of Genetics, University of Bern, Bern, 3001, Switzerland.,Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zürich, Zürich, 8057, Switzerland
| | - I Keller
- Swiss Institute of Bioinformatics, Lausanne, 1015, Switzerland.,Department for BioMedical Research, University of Bern, Bern, 3001, Switzerland
| | - I M Häfliger
- Institute of Genetics, University of Bern, Bern, 3001, Switzerland
| | - A Letko
- Institute of Genetics, University of Bern, Bern, 3001, Switzerland
| | - C Schelling
- Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zürich, Zürich, 8057, Switzerland
| | - G Lühken
- Institute of Animal Breeding and Genetics, Justus Liebig University, Giessen, 35390, Germany
| | - C Drögemüller
- Institute of Genetics, University of Bern, Bern, 3001, Switzerland
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38
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Manor J, Lalani SR. Overgrowth Syndromes-Evaluation, Diagnosis, and Management. Front Pediatr 2020; 8:574857. [PMID: 33194904 PMCID: PMC7661798 DOI: 10.3389/fped.2020.574857] [Citation(s) in RCA: 35] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/21/2020] [Accepted: 09/29/2020] [Indexed: 12/14/2022] Open
Abstract
Abnormally excessive growth results from perturbation of a complex interplay of genetic, epigenetic, and hormonal factors that orchestrate human growth. Overgrowth syndromes generally present with inherent health concerns and, in some instances, an increased risk of tumor predisposition that necessitate prompt diagnosis and appropriate referral. In this review, we introduce some of the more common overgrowth syndromes, along with their molecular mechanisms, diagnostics, and medical complications for improved recognition and management of patients affected with these disorders.
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Affiliation(s)
- Joshua Manor
- Department of Molecular Genetics, Baylor College of Medicine, Houston, TX, United States
| | - Seema R Lalani
- Department of Molecular Genetics, Baylor College of Medicine, Houston, TX, United States
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39
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Zhou A, Lin T, Xing J. Evaluating nanopore sequencing data processing pipelines for structural variation identification. Genome Biol 2019; 20:237. [PMID: 31727126 PMCID: PMC6857234 DOI: 10.1186/s13059-019-1858-1] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2019] [Accepted: 10/10/2019] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Structural variations (SVs) account for about 1% of the differences among human genomes and play a significant role in phenotypic variation and disease susceptibility. The emerging nanopore sequencing technology can generate long sequence reads and can potentially provide accurate SV identification. However, the tools for aligning long-read data and detecting SVs have not been thoroughly evaluated. RESULTS Using four nanopore datasets, including both empirical and simulated reads, we evaluate four alignment tools and three SV detection tools. We also evaluate the impact of sequencing depth on SV detection. Finally, we develop a machine learning approach to integrate call sets from multiple pipelines. Overall SV callers' performance varies depending on the SV types. For an initial data assessment, we recommend using aligner minimap2 in combination with SV caller Sniffles because of their speed and relatively balanced performance. For detailed analysis, we recommend incorporating information from multiple call sets to improve the SV call performance. CONCLUSIONS We present a workflow for evaluating aligners and SV callers for nanopore sequencing data and approaches for integrating multiple call sets. Our results indicate that additional optimizations are needed to improve SV detection accuracy and sensitivity, and an integrated call set can provide enhanced performance. The nanopore technology is improving, and the sequencing community is likely to grow accordingly. In turn, better benchmark call sets will be available to more accurately assess the performance of available tools and facilitate further tool development.
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Affiliation(s)
- Anbo Zhou
- Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, NJ, 08854, USA
| | - Timothy Lin
- Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, NJ, 08854, USA
| | - Jinchuan Xing
- Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, NJ, 08854, USA.
- Human Genetics Institute of New Jersey, Rutgers, the State University of New Jersey, Piscataway, NJ, 08854, USA.
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40
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Long-read sequencing for rare human genetic diseases. J Hum Genet 2019; 65:11-19. [PMID: 31558760 DOI: 10.1038/s10038-019-0671-8] [Citation(s) in RCA: 58] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2019] [Revised: 08/28/2019] [Accepted: 09/03/2019] [Indexed: 12/19/2022]
Abstract
During the past decade, the search for pathogenic mutations in rare human genetic diseases has involved huge efforts to sequence coding regions, or the entire genome, using massively parallel short-read sequencers. However, the approximate current diagnostic rate is <50% using these approaches, and there remain many rare genetic diseases with unknown cause. There may be many reasons for this, but one plausible explanation is that the responsible mutations are in regions of the genome that are difficult to sequence using conventional technologies (e.g., tandem-repeat expansion or complex chromosomal structural aberrations). Despite the drawbacks of high cost and a shortage of standard analytical methods, several studies have analyzed pathogenic changes in the genome using long-read sequencers. The results of these studies provide hope that further application of long-read sequencers to identify the causative mutations in unsolved genetic diseases may expand our understanding of the human genome and diseases. Such approaches may also be applied to molecular diagnosis and therapeutic strategies for patients with genetic diseases in the future.
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41
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Sekiguchi F, Tsurusaki Y, Okamoto N, Teik KW, Mizuno S, Suzumura H, Isidor B, Ong WP, Haniffa M, White SM, Matsuo M, Saito K, Phadke S, Kosho T, Yap P, Goyal M, Clarke LA, Sachdev R, McGillivray G, Leventer RJ, Patel C, Yamagata T, Osaka H, Hisaeda Y, Ohashi H, Shimizu K, Nagasaki K, Hamada J, Dateki S, Sato T, Chinen Y, Awaya T, Kato T, Iwanaga K, Kawai M, Matsuoka T, Shimoji Y, Tan TY, Kapoor S, Gregersen N, Rossi M, Marie-Laure M, McGregor L, Oishi K, Mehta L, Gillies G, Lockhart PJ, Pope K, Shukla A, Girisha KM, Abdel-Salam GMH, Mowat D, Coman D, Kim OH, Cordier MP, Gibson K, Milunsky J, Liebelt J, Cox H, El Chehadeh S, Toutain A, Saida K, Aoi H, Minase G, Tsuchida N, Iwama K, Uchiyama Y, Suzuki T, Hamanaka K, Azuma Y, Fujita A, Imagawa E, Koshimizu E, Takata A, Mitsuhashi S, Miyatake S, Mizuguchi T, Miyake N, Matsumoto N. Genetic abnormalities in a large cohort of Coffin-Siris syndrome patients. J Hum Genet 2019; 64:1173-1186. [PMID: 31530938 DOI: 10.1038/s10038-019-0667-4] [Citation(s) in RCA: 50] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2019] [Revised: 08/13/2019] [Accepted: 08/25/2019] [Indexed: 01/15/2023]
Abstract
Coffin-Siris syndrome (CSS, MIM#135900) is a congenital disorder characterized by coarse facial features, intellectual disability, and hypoplasia of the fifth digit and nails. Pathogenic variants for CSS have been found in genes encoding proteins in the BAF (BRG1-associated factor) chromatin-remodeling complex. To date, more than 150 CSS patients with pathogenic variants in nine BAF-related genes have been reported. We previously reported 71 patients of whom 39 had pathogenic variants. Since then, we have recruited an additional 182 CSS-suspected patients. We performed comprehensive genetic analysis on these 182 patients and on the previously unresolved 32 patients, targeting pathogenic single nucleotide variants, short insertions/deletions and copy number variations (CNVs). We confirmed 78 pathogenic variations in 78 patients. Pathogenic variations in ARID1B, SMARCB1, SMARCA4, ARID1A, SOX11, SMARCE1, and PHF6 were identified in 48, 8, 7, 6, 4, 1, and 1 patients, respectively. In addition, we found three CNVs including SMARCA2. Of particular note, we found a partial deletion of SMARCB1 in one CSS patient and we thoroughly investigated the resulting abnormal transcripts.
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Affiliation(s)
- Futoshi Sekiguchi
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan
| | - Yoshinori Tsurusaki
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan.,Faculty of Nutritional Science, Sagami Women's University, Sagamihara, Kanagawa, Japan
| | - Nobuhiko Okamoto
- Department of Medical Genetics, Osaka Women's and Children's Hospital, Osaka, Japan
| | - Keng Wee Teik
- Department of Genetics, Hospital Kuala Lumpur, Kuala Lumpur, Malaysia
| | - Seiji Mizuno
- Department of Clinical Genetics, Central Hospital, Aichi Developmental Disability Center, Kasugai, Japan
| | - Hiroshi Suzumura
- Department of Pediatrics, Dokkyo Medical University, Tochigi, Japan
| | | | - Winnie Peitee Ong
- Department of Genetics, Hospital Kuala Lumpur, Kuala Lumpur, Malaysia
| | - Muzhirah Haniffa
- Department of Genetics, Hospital Kuala Lumpur, Kuala Lumpur, Malaysia
| | - Susan M White
- Victorian Clinical Genetics Services, Murdoch Children's Research Institute, Melbourne, Australia.,Department of Paediatrics, University of Melbourne, Melbourne, Australia
| | - Mari Matsuo
- Institute of Medical Genetics, Tokyo Women's Medical University, Tokyo, Japan
| | - Kayoko Saito
- Institute of Medical Genetics, Tokyo Women's Medical University, Tokyo, Japan
| | - Shubha Phadke
- Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India
| | - Tomoki Kosho
- Department of Medical Genetics, Shinshu University School of Medicine, Matsumoto, Japan
| | - Patrick Yap
- Genetic Health Service New Zealand, Auckland, New Zealand.,Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
| | - Manisha Goyal
- Rare Disease Clinic, J K Lone Hospital, SMS Medical College, Jaipur, Rajasthan, India
| | - Lorne A Clarke
- British Columbia Children's Hospital Research Institute, University of British Columbia, Vancouver, BC, Canada
| | - Rani Sachdev
- Centre for Clinical Genetics, Sydney Children's Hospital, Randwick, NSW, Australia
| | - George McGillivray
- Victorian Clinical Genetics Services, Murdoch Children's Research Institute, Melbourne, Australia
| | - Richard J Leventer
- Royal Children's Hospital Department of Neurology, Murdoch Children's Research Institute and University of Melbourne Department of Pediatrics, Parkville, 3052, Australia
| | - Chirag Patel
- Genetic Health Queensland, Royal Brisbane and Women's Hospital, Brisbane, QLD, Australia
| | | | - Hitoshi Osaka
- Department of Pediatrics, Jichi Medical University, Tochigi, Japan
| | - Yoshiya Hisaeda
- Department of Neonatology, Japanese Red Cross Medical Center, Tokyo, Japan
| | - Hirofumi Ohashi
- Division of Medical Genetics, Saitama Children's Medical Center, Saitama, Japan
| | - Kenji Shimizu
- Division of Medical Genetics, Saitama Children's Medical Center, Saitama, Japan
| | - Keisuke Nagasaki
- Department of Homeostatic Regulation and Development, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
| | - Junpei Hamada
- Department of Pediatrics, Ehime University Graduate School of Medicine, Ehime, Japan
| | - Sumito Dateki
- Department of Pediatrics, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
| | - Takashi Sato
- Asahikawa-Kosei General Hospital, Hokkaido, Japan
| | - Yasutsugu Chinen
- Department of Child Health and Welfare, Graduate School of Medicine, University of the Ryukyus, Nishihara, Japan
| | - Tomonari Awaya
- Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.,Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Takeo Kato
- Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Kougoro Iwanaga
- Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Masahiko Kawai
- Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Takashi Matsuoka
- Department of General Pediatrics, Okinawa Prefectural Nanbu Medical Center and Children's Medical Center, Okinawa, Japan
| | - Yoshikazu Shimoji
- Department of General Pediatrics, Okinawa Prefectural Nanbu Medical Center and Children's Medical Center, Okinawa, Japan
| | - Tiong Yang Tan
- Victorian Clinical Genetics Services, Murdoch Children's Research Institute, Melbourne, Australia.,Department of Paediatrics, University of Melbourne, Melbourne, Australia
| | - Seema Kapoor
- Division of Genetics, Department of Pediatrics, Maulana Azad Medical College, New Delhi, India
| | | | - Massimiliano Rossi
- Hospices Civils de Lyon, Service de Génétique, Centre de Référence Anomalies du Développement, and INSERM U1028, CNRS UMR5292, CRNL, GENDEV Team, UCBL1, Bron, France
| | - Mathieu Marie-Laure
- Hospices Civils de Lyon, Service de Génétique, Centre de Référence Anomalies du Développement, and INSERM U1028, CNRS UMR5292, CRNL, GENDEV Team, UCBL1, Bron, France
| | - Lesley McGregor
- South Australian Clinical Genetics Service, SA Pathology, Women's and Children's Hospital, Adelaide, Australia
| | - Kimihiko Oishi
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Lakshmi Mehta
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Greta Gillies
- Bruce Lefroy Centre for Genetic Health Research, Murdoch Children's Research Institute, Victoria, Australia
| | - Paul J Lockhart
- Bruce Lefroy Centre for Genetic Health Research, Murdoch Children's Research Institute, Victoria, Australia
| | - Kate Pope
- Bruce Lefroy Centre for Genetic Health Research, Murdoch Children's Research Institute, Victoria, Australia
| | - Anju Shukla
- Department of Medical Genetics, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, India
| | - Katta Mohan Girisha
- Department of Medical Genetics, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, India
| | - Ghada M H Abdel-Salam
- Department of Clinical Genetics, Human Genetics and Genome Research Division, National Research Centre, Cairo, Egypt
| | - David Mowat
- Department of Medical Genetics, Sydney Children's Hospital, Sydney, NSW, Australia
| | - David Coman
- Department of Paediatrics, The Wesley Hospital, Brisbane, QLD, Australia
| | - Ok Hwa Kim
- Department of Radiology, Ajou University Hospital, Suwon, Korea
| | | | - Kate Gibson
- Genetic Health Service New Zealand, Christchurch Hospital, Christchurch, New Zealand
| | | | - Jan Liebelt
- South Australian Clinical Genetics Services, Women's and Children's Hospital, North Adelaide, Australia
| | - Helen Cox
- West Midlands Regional Genetics Service, Birmingham Women's NHS Foundation Trust, Birmingham Women's Hospital, Edgbaston, Birmingham, B15 2TG, UK
| | - Salima El Chehadeh
- Service de Genetique Medicale, Hopital de Hautepierre, Strasbourg, France
| | | | - Ken Saida
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan
| | - Hiromi Aoi
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan.,Department of Obstetrics and Gynecology, Juntendo University Faculty of Medicine, Tokyo, Japan
| | - Gaku Minase
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan
| | - Naomi Tsuchida
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan
| | - Kazuhiro Iwama
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan
| | - Yuri Uchiyama
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan.,Department of Oncology, Yokohama City University Graduate School of Medicine, Yokohama, Japan.,Clinical Genetics Department, Yokohama City University Hospital, Yokohama, Kanagawa, Japan
| | - Toshifumi Suzuki
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan.,Department of Obstetrics and Gynecology, Juntendo University Faculty of Medicine, Tokyo, Japan
| | - Kohei Hamanaka
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan
| | - Yoshiteru Azuma
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan
| | - Atsushi Fujita
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan
| | - Eri Imagawa
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan.,Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Eriko Koshimizu
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan
| | - Atsushi Takata
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan
| | - Satomi Mitsuhashi
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan
| | - Satoko Miyatake
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan.,Clinical Genetics Department, Yokohama City University Hospital, Yokohama, Kanagawa, Japan
| | - Takeshi Mizuguchi
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan
| | - Noriko Miyake
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan
| | - Naomichi Matsumoto
- Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan.
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Zhang S, Liang F, Lei C, Wu J, Fu J, Yang Q, Luo X, Yu G, Wang D, Zhang Y, Lu D, Sun X, Liang Y, Xu C. Long-read sequencing and haplotype linkage analysis enabled preimplantation genetic testing for patients carrying pathogenic inversions. J Med Genet 2019; 56:741-749. [PMID: 31439719 PMCID: PMC6860410 DOI: 10.1136/jmedgenet-2018-105976] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2018] [Revised: 05/31/2019] [Accepted: 06/13/2019] [Indexed: 01/04/2023]
Abstract
Background Preimplantation genetic testing (PGT) has already been applied in patients known to carry chromosomal structural variants to improve the clinical outcome of assisted reproduction. However, conventional molecular techniques are not capable of reliably distinguishing embryos that carry balanced inversion from those with a normal karyotype. We aim to evaluate the use of long-read sequencing in combination with haplotype linkage analysis to address this challenge. Methods Long-read sequencing on Oxford Nanopore platform was employed to identify the precise positions of inversion break points in four patients. Comprehensive chromosomal screening and genome-wide haplotype linkage analysis were performed based on SNP microarray. The haplotypes, including the break point regions, the whole chromosomes involved in the inversion and the corresponding homologous chromosomes, were established using informative SNPs. Results All the inversion break points were successfully identified by long-read sequencing and validated by Sanger sequencing, and on average only 13 bp differences were observed between break points inferred by long-read sequencing and Sanger sequencing. Eighteen blastocysts were biopsied and tested, in which 10 were aneuploid or unbalanced and eight were diploid with normal or balanced inversion karyotypes. Diploid embryos were transferred back to patients, the predictive results of the current methodology were consistent with fetal karyotypes of amniotic fluid or cord blood. Conclusions Nanopore long-read sequencing is a powerful method to assay chromosomal inversions and identify exact break points. Identification of inversion break points combined with haplotype linkage analysis is an efficient strategy to distinguish embryos with normal or balanced inversion karyotypes, facilitating PGT applications.
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Affiliation(s)
- Shuo Zhang
- Shanghai Ji Ai Genetics & IVF Institute, Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China.,Collaborative Innovation Center for Genetics and Development, State Key Laboratory of Genetic Engineering, School of Life Science, Fudan University, Shanghai, China
| | - Fan Liang
- GrandOmics Biosciences, Beijing, China
| | - Caixia Lei
- Shanghai Ji Ai Genetics & IVF Institute, Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China
| | - Junping Wu
- Shanghai Ji Ai Genetics & IVF Institute, Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China
| | - Jing Fu
- Shanghai Ji Ai Genetics & IVF Institute, Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China
| | - Qi Yang
- GrandOmics Biosciences, Beijing, China
| | - Xiao Luo
- GrandOmics Biosciences, Beijing, China
| | | | | | - Yueping Zhang
- Shanghai Ji Ai Genetics & IVF Institute, Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China
| | - Daru Lu
- Collaborative Innovation Center for Genetics and Development, State Key Laboratory of Genetic Engineering, School of Life Science, Fudan University, Shanghai, China
| | - Xiaoxi Sun
- Shanghai Ji Ai Genetics & IVF Institute, Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China .,Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China
| | - Yu Liang
- GrandOmics Biosciences, Beijing, China
| | - Congjian Xu
- Shanghai Ji Ai Genetics & IVF Institute, Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China .,Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China
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43
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Mantere T, Kersten S, Hoischen A. Long-Read Sequencing Emerging in Medical Genetics. Front Genet 2019; 10:426. [PMID: 31134132 PMCID: PMC6514244 DOI: 10.3389/fgene.2019.00426] [Citation(s) in RCA: 251] [Impact Index Per Article: 41.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2018] [Accepted: 04/18/2019] [Indexed: 12/12/2022] Open
Abstract
The wide implementation of next-generation sequencing (NGS) technologies has revolutionized the field of medical genetics. However, the short read lengths of currently used sequencing approaches pose a limitation for the identification of structural variants, sequencing repetitive regions, phasing of alleles and distinguishing highly homologous genomic regions. These limitations may significantly contribute to the diagnostic gap in patients with genetic disorders who have undergone standard NGS, like whole exome or even genome sequencing. Now, the emerging long-read sequencing (LRS) technologies may offer improvements in the characterization of genetic variation and regions that are difficult to assess with the prevailing NGS approaches. LRS has so far mainly been used to investigate genetic disorders with previously known or strongly suspected disease loci. While these targeted approaches already show the potential of LRS, it remains to be seen whether LRS technologies can soon enable true whole genome sequencing routinely. Ultimately, this could allow the de novo assembly of individual whole genomes used as a generic test for genetic disorders. In this article, we summarize the current LRS-based research on human genetic disorders and discuss the potential of these technologies to facilitate the next major advancements in medical genetics.
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Affiliation(s)
- Tuomo Mantere
- Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands
- Laboratory of Cancer Genetics and Tumor Biology, Cancer and Translational Medicine Research Unit and Biocenter Oulu, University of Oulu, Oulu, Finland
| | - Simone Kersten
- Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands
- Department of Internal Medicine, Center for Infectious Diseases (RCI), Radboud University Medical Center, Nijmegen, Netherlands
- Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands
| | - Alexander Hoischen
- Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands
- Department of Internal Medicine, Center for Infectious Diseases (RCI), Radboud University Medical Center, Nijmegen, Netherlands
- Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands
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Maroilley T, Tarailo-Graovac M. Uncovering Missing Heritability in Rare Diseases. Genes (Basel) 2019; 10:E275. [PMID: 30987386 PMCID: PMC6523881 DOI: 10.3390/genes10040275] [Citation(s) in RCA: 34] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2019] [Revised: 03/29/2019] [Accepted: 04/01/2019] [Indexed: 12/14/2022] Open
Abstract
The problem of 'missing heritability' affects both common and rare diseases hindering: discovery, diagnosis, and patient care. The 'missing heritability' concept has been mainly associated with common and complex diseases where promising modern technological advances, like genome-wide association studies (GWAS), were unable to uncover the complete genetic mechanism of the disease/trait. Although rare diseases (RDs) have low prevalence individually, collectively they are common. Furthermore, multi-level genetic and phenotypic complexity when combined with the individual rarity of these conditions poses an important challenge in the quest to identify causative genetic changes in RD patients. In recent years, high throughput sequencing has accelerated discovery and diagnosis in RDs. However, despite the several-fold increase (from ~10% using traditional to ~40% using genome-wide genetic testing) in finding genetic causes of these diseases in RD patients, as is the case in common diseases-the majority of RDs are also facing the 'missing heritability' problem. This review outlines the key role of high throughput sequencing in uncovering genetics behind RDs, with a particular focus on genome sequencing. We review current advances and challenges of sequencing technologies, bioinformatics approaches, and resources.
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Affiliation(s)
- Tatiana Maroilley
- Departments of Biochemistry, Molecular Biology and Medical Genetics, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.
- Alberta Children's Hospital Research Institute, University of Calgary, Calgary, AB T2N 4N1, Canada.
| | - Maja Tarailo-Graovac
- Departments of Biochemistry, Molecular Biology and Medical Genetics, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.
- Alberta Children's Hospital Research Institute, University of Calgary, Calgary, AB T2N 4N1, Canada.
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