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Li X, Huang Q, Zhong F, Liu Y, Chen Z, Lin J, Fan Z, Lan F, Wang Z. Preimplantation genetic testing for inborn errors of metabolism: observations from a reproductive genetic laboratory in China. J Hum Genet 2025; 70:113-119. [PMID: 39582021 DOI: 10.1038/s10038-024-01307-9] [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: 08/02/2024] [Revised: 11/03/2024] [Accepted: 11/11/2024] [Indexed: 11/26/2024]
Abstract
In this study, we aimed to apply preimplantation genetic testing for monogenic disorders (PGT-M) based on mutated allele revealed by sequencing with aneuploidy and linkage analyses (MARSALA) to block the transmission of inborn errors of metabolism (IEMs). After the disease-causing variants were identified through genetic testing, four carrier couples having children affected with IEMs, including methylmalonic aciduria, glutaric acidemia type 1, beta-ketothiolase deficiency, and ornithine transcarbamylase deficiency, sought PGT-M. A series of PGT procedures involving intracytoplasmic sperm injection, blastocyst culture, biopsy of trophectoderm cells, and next-generation sequencing (NGS)-based MARSALA, was performed to provide comprehensive chromosome screening and variant gene analysis. Finally, embryos were selected for transfer, and prenatal diagnosis was conducted to confirm the PGT-M results. All four carrier couples obtained transferrable embryos after PGT. The results of the prenatal diagnosis were consistent with the PGT results, and all couples gave birth to healthy babies free of IEMs. The results of this study confirm that NGS-based MARSALA is an effective approach for families with IEMs to prevent the subsequent transmission of pathological genetic variants to the next generation.
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Affiliation(s)
- Xiaoli Li
- Department of Clinical Laboratory Medicine, Dongfang Hospital of Xiamen University, School of Medicine, Xiamen University, Fuzhou, Fujian, China
| | - Qiuxiang Huang
- Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Dongfang Hospital of Xiamen University, School of Medicine, Xiamen University, Fuzhou, Fujian, China
| | - Fuchun Zhong
- Laboratory of Basic Medicine, Fujian Provincial Key Laboratory of Transplant Biology, Dongfang Hospital of Xiamen University, School of Medicine, Xiamen University, Fuzhou, Fujian, China
| | - Yun Liu
- Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Dongfang Hospital of Xiamen University, School of Medicine, Xiamen University, Fuzhou, Fujian, China
| | - Zhibiao Chen
- Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Dongfang Hospital of Xiamen University, School of Medicine, Xiamen University, Fuzhou, Fujian, China
| | - Juan Lin
- Laboratory of Basic Medicine, Fujian Provincial Key Laboratory of Transplant Biology, Dongfang Hospital of Xiamen University, School of Medicine, Xiamen University, Fuzhou, Fujian, China
| | - Zhongli Fan
- Laboratory of Basic Medicine, Fujian Provincial Key Laboratory of Transplant Biology, Dongfang Hospital of Xiamen University, School of Medicine, Xiamen University, Fuzhou, Fujian, China
| | - Fenghua Lan
- Laboratory of Basic Medicine, Fujian Provincial Key Laboratory of Transplant Biology, Dongfang Hospital of Xiamen University, School of Medicine, Xiamen University, Fuzhou, Fujian, China
| | - Zhihong Wang
- Laboratory of Basic Medicine, Fujian Provincial Key Laboratory of Transplant Biology, Dongfang Hospital of Xiamen University, School of Medicine, Xiamen University, Fuzhou, Fujian, China.
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2
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Muñoz E, Bronet F, Lledo B, Palacios-Verdú G, Martinez-Rocca L, Altmäe S, Pla J. To transfer or not to transfer: the dilemma of mosaic embryos - a narrative review. Reprod Biomed Online 2024; 48:103664. [PMID: 38408811 DOI: 10.1016/j.rbmo.2023.103664] [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: 06/06/2023] [Revised: 10/19/2023] [Accepted: 10/30/2023] [Indexed: 02/28/2024]
Abstract
A frequent finding after preimplantation genetic diagnostic testing for aneuploidies using next-generation sequencing is an embryo that is putatively mosaic. The prevalence of this outcome remains unclear and varies with technical and external factors. Mosaic embryos can be classified by the percentage of cells affected, type of chromosome involvement (whole or segmental), number of affected chromosomes or affected cell type (inner mass cell, trophectoderm or both). The origin of mosaicism seems to be intrinsic as a post-zygotic mitotic error, but some external factors can play a role. As experience has increased with the transfer of mosaic embryos, clinical practice has gradually become more flexible in recent years. Nevertheless, clinical results show lower implantation, pregnancy and clinical pregnancy rates and higher miscarriage rates with mosaic embryo transfer when compared with the transfer of euploid embryos. Prenatal diagnosis is highly recommended after the transfer of mosaic embryos. This narrative review is intended to serve as reference material for practitioners in reproductive medicine who must manage a mosaic embryo result after preimplantation genetic testing for aneuploidies.
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Affiliation(s)
- Elkin Muñoz
- Reproductive Medicine, IVIRMA Vigo, Vigo, Spain; Department of Obstetrics and Gynecology, University of Cauca, Popayan, Colombia
| | | | | | - Gabriela Palacios-Verdú
- Unit of Genomic Medicine, Department of Obstetrics, Gynecology and Reproductive Medicine, Institut Universitari Quirón Dexeus, Barcelona, Spain
| | | | - Signe Altmäe
- Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Granada, Granada, Spain; Instituto de Investigación Biosanitaria ibs, Granada, Granada, Spain; Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden
| | - Josep Pla
- Reproductive Genetics Unit, IVIRMA Global, Barcelona, Spain.
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Giuliano R, Maione A, Vallefuoco A, Sorrentino U, Zuccarello D. Preimplantation Genetic Testing for Genetic Diseases: Limits and Review of Current Literature. Genes (Basel) 2023; 14:2095. [PMID: 38003038 PMCID: PMC10671162 DOI: 10.3390/genes14112095] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2023] [Revised: 10/26/2023] [Accepted: 11/16/2023] [Indexed: 11/26/2023] Open
Abstract
Preimplantation genetic testing (PGT) has emerged as a revolutionary technique in the field of reproductive medicine, allowing for the selection and transfer of healthy embryos, thus reducing the risk of transmitting genetic diseases. However, despite remarkable advancements, the implementation of PGT faces a series of limitations and challenges that require careful consideration. This review aims to foster a comprehensive reflection on the constraints of preimplantation genetic diagnosis, encouraging a broader discussion about its utility and implications. The objective is to inform and guide medical professionals, patients, and society overall in the conscious and responsible adoption of this innovative technology, taking into account its potential benefits and the ethical and practical challenges that it presents.
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Affiliation(s)
- Roberta Giuliano
- Preimplantation Genetic Diagnosis, Department of Women’s and Children’s Health, University of Padova, 35128 Padova, Italy
| | - Anna Maione
- Fertility Unit, Maternal-Child Department, AOU Federico II, 80131 Naples, Italy;
| | - Angela Vallefuoco
- Department of Clinical Medicine and Surgery, University of Naples Federico II, 80138 Naples, Italy;
| | - Ugo Sorrentino
- Clinical Genetics and Epidemiology Unit, University Hospital of Padova, Via Giustiniani 3, 35128 Padova, Italy; (U.S.); (D.Z.)
| | - Daniela Zuccarello
- Clinical Genetics and Epidemiology Unit, University Hospital of Padova, Via Giustiniani 3, 35128 Padova, Italy; (U.S.); (D.Z.)
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Ordóñez CD, Mayoral-Campos C, Egas C, Redrejo-Rodríguez M. A primer-independent DNA polymerase-based method for competent whole-genome amplification of intermediate to high GC sequences. NAR Genom Bioinform 2023; 5:lqad073. [PMID: 37608803 PMCID: PMC10440786 DOI: 10.1093/nargab/lqad073] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2023] [Revised: 07/03/2023] [Accepted: 08/09/2023] [Indexed: 08/24/2023] Open
Abstract
Multiple displacement amplification (MDA) has proven to be a useful technique for obtaining large amounts of DNA from tiny samples in genomics and metagenomics. However, MDA has limitations, such as amplification artifacts and biases that can interfere with subsequent quantitative analysis. To overcome these challenges, alternative methods and engineered DNA polymerase variants have been developed. Here, we present new MDA protocols based on the primer-independent DNA polymerase (piPolB), a replicative-like DNA polymerase endowed with DNA priming and proofreading capacities. These new methods were tested on a genomes mixture containing diverse sequences with high-GC content, followed by deep sequencing. Protocols relying on piPolB as a single enzyme cannot achieve competent amplification due to its limited processivity and the presence of ab initio DNA synthesis. However, an alternative method called piMDA, which combines piPolB with Φ29 DNA polymerase, allows proficient and faithful amplification of the genomes. In addition, the prior denaturation step commonly performed in MDA protocols is dispensable, resulting in a more straightforward protocol. In summary, piMDA outperforms commercial methods in the amplification of genomes and metagenomes containing high GC sequences and exhibits similar profiling, error rate and variant determination as the non-amplified samples.
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Affiliation(s)
- Carlos D Ordóñez
- Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain
| | - Carmen Mayoral-Campos
- Departamento de Bioquímica, Universidad Autónoma de Madrid (UAM) and Instituto de Investigaciones Biomédicas Sols-Morreale (CSIC-UAM), Madrid, Spain
| | - Conceição Egas
- Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal
- Biocant, Transfer Technology Association, Cantanhede, Portugal
| | - Modesto Redrejo-Rodríguez
- Departamento de Bioquímica, Universidad Autónoma de Madrid (UAM) and Instituto de Investigaciones Biomédicas Sols-Morreale (CSIC-UAM), Madrid, Spain
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Kim J, Kim S, Yeom H, Song SW, Shin K, Bae S, Ryu HS, Kim JY, Choi A, Lee S, Ryu T, Choi Y, Kim H, Kim O, Jung Y, Kim N, Han W, Lee HB, Lee AC, Kwon S. Barcoded multiple displacement amplification for high coverage sequencing in spatial genomics. Nat Commun 2023; 14:5261. [PMID: 37644058 PMCID: PMC10465490 DOI: 10.1038/s41467-023-41019-w] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2023] [Accepted: 08/21/2023] [Indexed: 08/31/2023] Open
Abstract
Determining mutational landscapes in a spatial context is essential for understanding genetically heterogeneous cell microniches. Current approaches, such as Multiple Displacement Amplification (MDA), offer high genome coverage but limited multiplexing, which hinders large-scale spatial genomic studies. Here, we introduce barcoded MDA (bMDA), a technique that achieves high-coverage genomic analysis of low-input DNA while enhancing the multiplexing capabilities. By incorporating cell barcodes during MDA, bMDA streamlines library preparation in one pot, thereby overcoming a key bottleneck in spatial genomics. We apply bMDA to the integrative spatial analysis of triple-negative breast cancer tissues by examining copy number alterations, single nucleotide variations, structural variations, and kataegis signatures for each spatial microniche. This enables the assessment of subclonal evolutionary relationships within a spatial context. Therefore, bMDA has emerged as a scalable technology with the potential to advance the field of spatial genomics significantly.
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Affiliation(s)
- Jinhyun Kim
- Department of Electrical and Computer Engineering, Seoul National University, Seoul, 08826, Republic of Korea
| | - Sungsik Kim
- Interdisciplinary Program in Bioengineering, Seoul National University, Seoul, 08826, Republic of Korea
| | - Huiran Yeom
- Division of Data Science, College of Information and Communication Technology, The University of Suwon, Hwaseong, 18323, Republic of Korea
| | - Seo Woo Song
- Basic Science and Engineering Initiative, Children's Heart Center, Stanford University, Stanford, CA, USA
| | - Kyoungseob Shin
- Department of Electrical and Computer Engineering, Seoul National University, Seoul, 08826, Republic of Korea
| | - Sangwook Bae
- Renal Division and Division of Engineering in Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA
| | - Han Suk Ryu
- Cancer Research Institute, Seoul National University, Seoul, 03080, Republic of Korea
- Department of Pathology, Seoul National University College of Medicine, Seoul, 03080, Republic of Korea
| | - Ji Young Kim
- Biomedical Research Institute, Seoul National University Hospital, Seoul, 03080, Republic of Korea
| | - Ahyoun Choi
- Interdisciplinary Program in Bioengineering, Seoul National University, Seoul, 08826, Republic of Korea
| | - Sumin Lee
- Department of Electrical and Computer Engineering, Seoul National University, Seoul, 08826, Republic of Korea
- Meteor Biotech, Co. Ltd., Seoul, 08826, Republic of Korea
| | - Taehoon Ryu
- ATG Lifetech Inc., Seoul, 08507, Republic of Korea
| | - Yeongjae Choi
- School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, 61005, Republic of Korea
| | - Hamin Kim
- Interdisciplinary Program in Bioengineering, Seoul National University, Seoul, 08826, Republic of Korea
| | - Okju Kim
- ATG Lifetech Inc., Seoul, 08507, Republic of Korea
| | - Yushin Jung
- ATG Lifetech Inc., Seoul, 08507, Republic of Korea
| | - Namphil Kim
- Department of Electrical and Computer Engineering, Seoul National University, Seoul, 08826, Republic of Korea
| | - Wonshik Han
- Cancer Research Institute, Seoul National University, Seoul, 03080, Republic of Korea
- Biomedical Research Institute, Seoul National University Hospital, Seoul, 03080, Republic of Korea
- Department of Surgery, Seoul National University College of Medicine, Seoul, 03080, Republic of Korea
| | - Han-Byoel Lee
- Cancer Research Institute, Seoul National University, Seoul, 03080, Republic of Korea.
- Biomedical Research Institute, Seoul National University Hospital, Seoul, 03080, Republic of Korea.
- Department of Surgery, Seoul National University College of Medicine, Seoul, 03080, Republic of Korea.
| | - Amos C Lee
- Bio-MAX Institute, Seoul National University, Seoul, 08826, Republic of Korea.
- Meteor Biotech, Co. Ltd., Seoul, 08826, Republic of Korea.
| | - Sunghoon Kwon
- Department of Electrical and Computer Engineering, Seoul National University, Seoul, 08826, Republic of Korea.
- Interdisciplinary Program in Bioengineering, Seoul National University, Seoul, 08826, Republic of Korea.
- Biomedical Research Institute, Seoul National University Hospital, Seoul, 03080, Republic of Korea.
- Bio-MAX Institute, Seoul National University, Seoul, 08826, Republic of Korea.
- Inter-University Semiconductor Research Center, Seoul National University, Seoul, 08826, Republic of Korea.
- Institutes of Entrepreneurial BioConvergence, Seoul National University, Seoul, 08826, Republic of Korea.
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6
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Bi Q, Huang S, Wang H, Gao X, Ma M, Han M, Lu S, Kang D, Nourbakhsh A, Yan D, Blanton S, Liu X, Yuan Y, Yao Y, Dai P. Preimplantation genetic testing for hereditary hearing loss in Chinese population. J Assist Reprod Genet 2023:10.1007/s10815-023-02753-8. [PMID: 37017887 PMCID: PMC10352472 DOI: 10.1007/s10815-023-02753-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2022] [Accepted: 02/13/2023] [Indexed: 04/06/2023] Open
Abstract
PURPOSE To evaluate the clinical validity of preimplantation genetic testing (PGT) to prevent hereditary hearing loss (HL) in Chinese population. METHODS A PGT procedure combining multiple annealing and looping-based amplification cycles (MALBAC) and single-nucleotide polymorphisms (SNPs) linkage analyses with a single low-depth next-generation sequencing run was implemented. Forty-three couples carried pathogenic variants in autosomal recessive non-syndromic HL genes, GJB2 and SLC26A4, and four couples carried pathogenic variants in rare HL genes: KCNQ4, PTPN11, PAX3, and USH2A were enrolled. RESULTS Fifty-four in vitro fertilization (IVF) cycles were implemented, 340 blastocysts were cultured, and 303 (89.1%) of these received a definite diagnosis of a disease-causing variant testing, linkage analysis and chromosome screening. A clinical pregnancy of 38 implanted was achieved, and 34 babies were born with normal hearing. The live birth rate was 61.1%. CONCLUSIONS AND RELEVANCE In both the HL population and in hearing individuals at risk of giving birth to offspring with HL in China, there is a practical need for PGT. The whole genome amplification combined with NGS can simplify the PGT process, and the efficiency of PGT process can be improved by establishing a universal SNP bank of common disease-causing gene in particular regions and nationalities. This PGT procedure was demonstrated to be effective and lead to satisfactory clinical outcomes.
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Affiliation(s)
- Qingling Bi
- College of Otolaryngology Head and Neck Surgery, Chinese PLA General Hospital, Chinese PLA Medical School, National Clinical Research Center for Otolaryngologic Diseases, Key Lab of Hearing Impairment Science of Ministry of Education, Key Lab of Hearing Impairment Prevention and Treatment of Beijing, #28 Fuxing Road, Beijing, 100853, China
- Departments of Otolaryngology Head & Neck Surgery, China-Japan Friendship Hospital, 2#Yinghua Road, Beijing, 100029, China
| | - Shasha Huang
- College of Otolaryngology Head and Neck Surgery, Chinese PLA General Hospital, Chinese PLA Medical School, National Clinical Research Center for Otolaryngologic Diseases, Key Lab of Hearing Impairment Science of Ministry of Education, Key Lab of Hearing Impairment Prevention and Treatment of Beijing, #28 Fuxing Road, Beijing, 100853, China
| | - Hui Wang
- Reproductive Center, Chinese PLA General Hospital, 28#Fuxing Road, Beijing, 100853, China
| | - Xue Gao
- Department of Otolaryngology, PLA Rocket Force Characteristic Medical Center, 16# XinWai Da Jie, Beijing, 100088, China
| | - Minyue Ma
- Reproductive Center, Chinese PLA General Hospital, 28#Fuxing Road, Beijing, 100853, China
| | - Mingyu Han
- College of Otolaryngology Head and Neck Surgery, Chinese PLA General Hospital, Chinese PLA Medical School, National Clinical Research Center for Otolaryngologic Diseases, Key Lab of Hearing Impairment Science of Ministry of Education, Key Lab of Hearing Impairment Prevention and Treatment of Beijing, #28 Fuxing Road, Beijing, 100853, China
| | - Sijia Lu
- Department of Clinical Research, Yikon Genomics, 1698 Wangyuan Road, Fengxian District Shanghai, 201400, China
| | - Dongyang Kang
- College of Otolaryngology Head and Neck Surgery, Chinese PLA General Hospital, Chinese PLA Medical School, National Clinical Research Center for Otolaryngologic Diseases, Key Lab of Hearing Impairment Science of Ministry of Education, Key Lab of Hearing Impairment Prevention and Treatment of Beijing, #28 Fuxing Road, Beijing, 100853, China
| | - Aida Nourbakhsh
- Department of Otolaryngology, University of Miami Miller School of Medicine, Miami, FL, 33136, USA
| | - Denise Yan
- Department of Otolaryngology, University of Miami Miller School of Medicine, Miami, FL, 33136, USA
| | - Susan Blanton
- Department of Otolaryngology, University of Miami Miller School of Medicine, Miami, FL, 33136, USA
| | - Xuezhong Liu
- Department of Otolaryngology, University of Miami Miller School of Medicine, Miami, FL, 33136, USA
| | - Yongyi Yuan
- College of Otolaryngology Head and Neck Surgery, Chinese PLA General Hospital, Chinese PLA Medical School, National Clinical Research Center for Otolaryngologic Diseases, Key Lab of Hearing Impairment Science of Ministry of Education, Key Lab of Hearing Impairment Prevention and Treatment of Beijing, #28 Fuxing Road, Beijing, 100853, China.
| | - Yuanqing Yao
- Reproductive Center, Chinese PLA General Hospital, 28#Fuxing Road, Beijing, 100853, China.
| | - Pu Dai
- College of Otolaryngology Head and Neck Surgery, Chinese PLA General Hospital, Chinese PLA Medical School, National Clinical Research Center for Otolaryngologic Diseases, Key Lab of Hearing Impairment Science of Ministry of Education, Key Lab of Hearing Impairment Prevention and Treatment of Beijing, #28 Fuxing Road, Beijing, 100853, China.
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7
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Volozonoka L, Miskova A, Gailite L. Whole Genome Amplification in Preimplantation Genetic Testing in the Era of Massively Parallel Sequencing. Int J Mol Sci 2022; 23:4819. [PMID: 35563216 PMCID: PMC9102663 DOI: 10.3390/ijms23094819] [Citation(s) in RCA: 23] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2022] [Revised: 04/24/2022] [Accepted: 04/25/2022] [Indexed: 12/16/2022] Open
Abstract
Successful whole genome amplification (WGA) is a cornerstone of contemporary preimplantation genetic testing (PGT). Choosing the most suitable WGA technique for PGT can be particularly challenging because each WGA technique performs differently in combination with different downstream processing and detection methods. The aim of this review is to provide insight into the performance and drawbacks of DOP-PCR, MDA and MALBAC, as well as the hybrid WGA techniques most widely used in PGT. As the field of PGT is moving towards a wide adaptation of comprehensive massively parallel sequencing (MPS)-based approaches, we especially focus our review on MPS parameters and detection opportunities of WGA-amplified material, i.e., mappability of reads, uniformity of coverage and its influence on copy number variation analysis, and genomic coverage and its influence on single nucleotide variation calling. The ability of MDA-based WGA solutions to better cover the targeted genome and the ability of PCR-based solutions to provide better uniformity of coverage are highlighted. While numerous comprehensive PGT solutions exploiting different WGA types and adjusted bioinformatic pipelines to detect copy number and single nucleotide changes are available, the ones exploiting MDA appear more advantageous. The opportunity to fully analyse the targeted genome is influenced by the MPS parameters themselves rather than the solely chosen WGA.
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Affiliation(s)
- Ludmila Volozonoka
- Scientific Laboratory of Molecular Genetics, Riga Stradins University, LV-1007 Riga, Latvia;
| | - Anna Miskova
- Department of Obstetrics and Gynaecology, Riga Stradins University, LV-1007 Riga, Latvia;
| | - Linda Gailite
- Scientific Laboratory of Molecular Genetics, Riga Stradins University, LV-1007 Riga, Latvia;
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8
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O'Grady CJ, Dhandapani V, Colbourne JK, Frisch D. Refining the evolutionary time machine: An assessment of whole genome amplification using single historical Daphnia eggs. Mol Ecol Resour 2021; 22:946-961. [PMID: 34672105 DOI: 10.1111/1755-0998.13524] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2021] [Revised: 09/03/2021] [Accepted: 09/07/2021] [Indexed: 12/14/2022]
Abstract
Whole genome sequencing is instrumental for the study of genome variation in natural populations, delivering important knowledge on genomic modifications and potential targets of natural selection at the population level. Large dormant eggbanks of aquatic invertebrates such as the keystone herbivore Daphnia, a microcrustacean widespread in freshwater ecosystems, provide detailed sedimentary archives to study genomic processes over centuries. To overcome the problem of limited DNA amounts in single Daphnia dormant eggs, we developed an optimized workflow for whole genome amplification (WGA), yielding sufficient amounts of DNA for downstream whole genome sequencing of individual historical eggs, including polyploid lineages. We compare two WGA kits, applied to recently produced Daphnia magna dormant eggs from laboratory cultures, and to historical dormant eggs of Daphnia pulicaria collected from Arctic lake sediment between 10 and 300 years old. Resulting genome coverage breadth in most samples was ~70%, including those from >100-year-old isolates. Sequence read distribution was highly correlated among samples amplified with the same kit, but less correlated between kits. Despite this, a high percentage of genomic positions with single nucleotide polymorphisms in one or more samples (maximum of 74% between kits, and 97% within kits) were recovered at a depth required for genotyping. As a by-product of sequencing we obtained 100% coverage of the mitochondrial genomes even from the oldest isolates (~300 years). The mitochondrial DNA provides an additional source for evolutionary studies of these populations. We provide an optimized workflow for WGA followed by whole genome sequencing including steps to minimize exogenous DNA.
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Affiliation(s)
- Christopher James O'Grady
- School of Life Sciences, University of Warwick, Coventry, UK.,Cell and Gene Therapy Catapult, London, UK.,School of Biosciences, University of Birmingham, Birmingham, UK
| | | | | | - Dagmar Frisch
- School of Biosciences, University of Birmingham, Birmingham, UK.,Leibniz Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin, Germany
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9
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Rogers A, Menezes M, Kane SC, Zander-Fox D, Hardy T. Preimplantation Genetic Testing for Monogenic Conditions: Is Cell-Free DNA Testing the Next Step? Mol Diagn Ther 2021; 25:683-690. [PMID: 34495483 DOI: 10.1007/s40291-021-00556-0] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/24/2021] [Indexed: 11/25/2022]
Abstract
Genetic assessment of an embryo via preimplantation genetic testing (PGT) represents an important reproductive option for couples wanting to try and improve success rates from in vitro fertilisation (IVF) cycles, as well as reduce their risk of having a child born with a genetic condition. Currently, biopsy of the developing embryo prior to transfer allows genetic assessment of an embryo for either chromosome copy number (aneuploidy [PGT-A] or segmental rearrangement [PGT-SR]) or to avoid the transmission of a single gene condition (monogenic conditions [PGT-M]). However, this technology is invasive and commands considerable resources. Non-invasive PGT (niPGT) offers a potential alternate mode of embryonic analysis. Whilst the utility of niPGT-A has been recently explored, there has been limited consideration of niPGT-M as an option for couples at risk of passing on a single gene or chromosomal condition. This review examines the historical and current clinical context of preimplantation embryonic analysis for monogenic conditions, in addition to important considerations surrounding the origin and analysis of cell-free deoxyribose nucleic acid (cfDNA), whether it is sourced via blastocentesis or spent embryonic culture medium (SCM). Future capabilities of this testing modality will almost certainly be enhanced by integration of whole genome sequencing into everyday practice. In addition, the increased utilisation of reproductive carrier screening as part of standard reproductive healthcare will likely result in the identification of a larger high-risk population. As a result, stratification of limited and highly specialised reproductive genetic resources will be required. Prospective parents should continue to be made aware of the limitations of this technology, with prenatal confirmatory testing remaining an essential part of antenatal care in these patients. However, niPGT-M poses an important alternate testing modality for high-risk couples, particularly in the setting of embryos that cannot be biopsied for traditional PGT-M and as demand for this treatment continues to grow.
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Affiliation(s)
- Alice Rogers
- Genetics, Repromed, Monash IVF, 180 Fullarton Road, Dulwich, SA, 5065, Australia
| | - Melody Menezes
- Monash Ultrasound for Women, Richmond, VIC, Australia
- Department of Paediatrics, The University of Melbourne, Parkville, VIC, Australia
| | - Stefan C Kane
- Monash Ultrasound for Women, Richmond, VIC, Australia
- Department of Obstetrics and Gynaecology, The University of Melbourne, Parkville, VIC, Australia
- Department of Maternal Fetal Medicine, The Royal Women's Hospital, Parkville, VIC, Australia
| | - Deirdre Zander-Fox
- Monash IVF Group, Clayton, VIC, Australia
- Monash University, Clayton, VIC, Australia
- The University of Adelaide, Adelaide, SA, Australia
- University of South Australia, Adelaide, SA, Australia
| | - Tristan Hardy
- Genetics, Repromed, Monash IVF, 180 Fullarton Road, Dulwich, SA, 5065, Australia.
- Genetics and Molecular Pathology, SA Pathology, Adelaide, Australia.
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10
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Lyu R, Tsui V, McCarthy DJ, Crismani W. Personalized genome structure via single gamete sequencing. Genome Biol 2021; 22:112. [PMID: 33874978 PMCID: PMC8054432 DOI: 10.1186/s13059-021-02327-w] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2020] [Accepted: 03/25/2021] [Indexed: 12/13/2022] Open
Abstract
Genetic maps have been fundamental to building our understanding of disease genetics and evolutionary processes. The gametes of an individual contain all of the information required to perform a de novo chromosome-scale assembly of an individual's genome, which historically has been performed with populations and pedigrees. Here, we discuss how single-cell gamete sequencing offers the potential to merge the advantages of short-read sequencing with the ability to build personalized genetic maps and open up an entirely new space in personalized genetics.
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Affiliation(s)
- Ruqian Lyu
- Bioinformatics and Cellular Genomics, St. Vincent's Institute of Medical Research, Melbourne, Australia
- Melbourne Integrative Genomics, Faculty of Science, The University of Melbourne, Melbourne, Australia
| | - Vanessa Tsui
- DNA Repair and Recombination Laboratory, St. Vincent's Institute of Medical Research, Melbourne, Australia
- The Faculty of Medicine, Dentistry and Health Science, The University of Melbourne, Melbourne, Australia
| | - Davis J McCarthy
- Bioinformatics and Cellular Genomics, St. Vincent's Institute of Medical Research, Melbourne, Australia.
- Melbourne Integrative Genomics, Faculty of Science, The University of Melbourne, Melbourne, Australia.
| | - Wayne Crismani
- DNA Repair and Recombination Laboratory, St. Vincent's Institute of Medical Research, Melbourne, Australia.
- The Faculty of Medicine, Dentistry and Health Science, The University of Melbourne, Melbourne, Australia.
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11
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Pregnancy and Neonatal Outcomes after Transfer of Mosaic Embryos: A Review. J Clin Med 2021; 10:jcm10071369. [PMID: 33801687 PMCID: PMC8037456 DOI: 10.3390/jcm10071369] [Citation(s) in RCA: 33] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Revised: 03/17/2021] [Accepted: 03/25/2021] [Indexed: 12/18/2022] Open
Abstract
Preimplantation genetic testing for aneuploidy (PGT-A) seeks to identify embryos with a normal chromosome complement during in vitro fertilization (IVF). Transfer of one euploid embryo at a time maximizes the chance of implantation while minimizing the risk of multiple pregnancy. The emergence of new technologies including next generation sequencing (NGS) has led to increased diagnosis of embryonic mosaicism, suggesting the presence of karyotypically distinct cells within a single trophectoderm (TE). Clinical implications of embryonic mosaicism are important in both naturally conceived and IVF pregnancies. Although information regarding outcomes after mosaic embryo transfer (MET) is limited, more than 100 live births have now been documented with rather reassuring outcomes with no abnormal phenotype. Here, we aim to provide a summary of recent data regarding clinical and neonatal outcomes after transfer of mosaic embryos in IVF/PGT-A cycles.
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12
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Jamal R, Li X, Weidhaas J. Template length, concentration and guanidine and cytosine content influence on multiple displacement amplification efficiency. J Microbiol Methods 2021; 181:106146. [PMID: 33493489 DOI: 10.1016/j.mimet.2021.106146] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2020] [Revised: 01/12/2021] [Accepted: 01/14/2021] [Indexed: 01/09/2023]
Abstract
Detection of low abundance human health pathogens in environmental samples is a challenge for water monitoring. This limitation can be overcome by the introduction of multiple displacement amplification (MDA) where a minute amount of genetic material can be amplified using a phi-29 DNA polymerase. However, the genetic makeup and the concentration of the polynucleotides might influence the amplification process due to inherent assay bias. Herein, a series of experiments were designed to demonstrate the effect of genome length, guanidine and cytosine content, and template concentration on the efficiency of MDA. Quantitative polymerase chain reaction (qPCR) was performed to quantify pre- and post-MDA concentrations of selected genes. Linear regression between pre- and post-MDA log gene copies L-1 of both environmental and lab-grown samples showed a positive correlation (F = 77.59, P < 0.001, R2 = 0.7, slope = 1.01). Correlation between relative polynucleotide increase after MDA and target organism length and gene target guanidine and cytosine (G + C) content (F = 4.3, P = 0.02) shows that lower G + C and higher genome length is favored in the MDA process. The MDA process was shown to favor a longer genome over a shorter genome (1.19 and 1.04 change in log gene copy L-1, respectively) and a lower G + C content over a higher G + C content (1.11 and 0.61 change in log gene copy L-1, respectively). There was no MDA bias observed when polynucleotides had the same G + C and genome length but different initial concentrations. This study highlights the need for increased caution when interpreting relative abundance of organisms amplified by MDA such as in next generation sequencing.
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Affiliation(s)
- Rubayat Jamal
- Civil and Environmental Engineering, University of Utah, 110 Central Campus Drive, Salt Lake City, UT 84112, USA.
| | - Xiang Li
- Southern University of Science and Technology, School of Environmental Science and Engineering, Shenzhen, China
| | - Jennifer Weidhaas
- Civil and Environmental Engineering, University of Utah, 110 Central Campus Drive, Salt Lake City, UT 84112, USA.
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13
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Long N, Qiao Y, Xu Z, Tu J, Lu Z. Recent advances and application in whole-genome multiple displacement amplification. QUANTITATIVE BIOLOGY 2020. [DOI: 10.1007/s40484-020-0217-2] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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14
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Popovic M, Dhaenens L, Boel A, Menten B, Heindryckx B. Chromosomal mosaicism in human blastocysts: the ultimate diagnostic dilemma. Hum Reprod Update 2020; 26:313-334. [DOI: 10.1093/humupd/dmz050] [Citation(s) in RCA: 62] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2019] [Revised: 11/29/2019] [Indexed: 12/30/2022] Open
Abstract
Abstract
BACKGROUND
Trophectoderm (TE) biopsy and next generation sequencing (NGS) are currently the preferred techniques for preimplantation genetic testing for aneuploidies (PGT-A). Although this approach delivered important improvements over previous testing strategies, increased sensitivity has also prompted a rise in diagnoses of uncertain clinical significance. This includes reports of chromosomal mosaicism, suggesting the presence of karyotypically distinct cells within a single TE biopsy. Given that PGT-A relies on the chromosomal constitution of the biopsied cells being representative of the entire embryo, the prevalence and clinical implications of blastocyst mosaicism continue to generate considerable controversy.
OBJECTIVE AND RATIONALE
The objective of this review was to evaluate existing scientific evidence regarding the prevalence and impact of chromosomal mosaicism in human blastocysts. We discuss insights from a biological, technical and clinical perspective to examine the implications of this diagnostic dilemma for PGT-A.
SEARCH METHODS
The PubMed and Google Scholar databases were used to search peer-reviewed publications using the following terms: ‘chromosomal mosaicism’, ‘human’, ‘embryo’, ‘blastocyst’, ‘implantation’, ‘next generation sequencing’ and ‘clinical management’ in combination with other keywords related to the subject area. Relevant articles in the English language, published until October 2019 were critically discussed.
OUTCOMES
Chromosomal mosaicism predominately results from errors in mitosis following fertilization. Although it appears to be less pervasive at later developmental stages, establishing the true prevalence of mosaicism in human blastocysts remains exceedingly challenging. In a clinical context, blastocyst mosaicism can only be reported based on a single TE biopsy and has been ascribed to 2–13% of embryos tested using NGS. Conversely, data from NGS studies disaggregating whole embryos suggests that mosaicism may be present in up to ~50% of blastocysts. However, differences in testing and reporting strategies, analysis platforms and the number of cells sampled inherently overshadow current data, while added uncertainties emanate from technical artefacts. Moreover, laboratory factors and aspects of in vitro culture generate further variability. Outcome data following the transfer of blastocysts diagnosed as mosaic remain limited. Current studies suggest that the transfer of putative mosaic embryos may lead to healthy live births, but also results in significantly reduced ongoing pregnancy rates compared to the transfer of euploid blastocysts. Observations that a subset of mosaic blastocysts has the capacity to develop normally have sparked discussions regarding the ability of embryos to self-correct. However, there is currently no direct evidence to support this assumption. Nevertheless, the exclusion of mosaic blastocysts results in fewer embryos available for transfer, which may inevitably compromise treatment outcomes.
WIDER IMPLICATIONS
Chromosomal mosaicism in human blastocysts remains a perpetual diagnostic and clinical dilemma in the context of PGT-A. This review offers an important scientific resource, informing about the challenges, risks and value of diagnosing mosaicism. Elucidating these uncertainties will ultimately pave the way towards improved clinical and patient management.
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Affiliation(s)
- Mina Popovic
- Ghent-Fertility and Stem Cell Team (G-FAST), Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium
| | - Lien Dhaenens
- Ghent-Fertility and Stem Cell Team (G-FAST), Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium
| | - Annekatrien Boel
- Ghent-Fertility and Stem Cell Team (G-FAST), Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium
| | - Björn Menten
- Center for Medical Genetics, Ghent University Hospital, 9000 Ghent, Belgium
| | - Björn Heindryckx
- Ghent-Fertility and Stem Cell Team (G-FAST), Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium
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15
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Hardy T. The role of prenatal diagnosis following preimplantation genetic testing for single-gene conditions: A historical overview of evolving technologies and clinical practice. Prenat Diagn 2020; 40:647-651. [PMID: 32037566 DOI: 10.1002/pd.5662] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2019] [Revised: 12/11/2019] [Accepted: 01/26/2020] [Indexed: 11/12/2022]
Abstract
Preimplantation genetic testing for monogenic conditions (PGT-M) has become a valued reproductive option for couples at risk of having a child with a single gene condition. In line with developments in molecular genetics, there has been an overall trend toward laboratory techniques with higher accuracy in comparison to earlier PGT-M techniques. The recommendation for confirmatory prenatal diagnostic testing has remained a standard component of PGT-M counseling, reflecting the inherent difficulties of testing the limited number of cells obtained from embryo biopsy, as well as recognition of the biological and human factors that may lead to misdiagnosis in a PGT-M cycle. Reported misdiagnosis rates are less than 1 in 200 pregnancies following PGT-M, although updated data regarding newer methods of PGT-M are required. There is limited evidence available regarding clinician and patient behavior in pregnancies resulting from PGT-M cycles. It remains essential that clinicians involved in the care of patients undergoing PGT-M provide appropriate counseling regarding the risks of misdiagnosis and the importance of confirmatory prenatal diagnosis. The nature of PGT-M test design lends itself to cell-free DNA-based noninvasive prenatal testing for monogenic conditions (NIPT-M), which is likely to become a popular method in the near future.
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Affiliation(s)
- Tristan Hardy
- Repromed, Adelaide, Australia.,Genetics and Molecular Pathology, SA Pathology, Adelaide, Australia
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16
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Zhao M, Lian M, Cheah FSH, Tan ASC, Agarwal A, Chong SS. Identification of Novel Microsatellite Markers Flanking the SMN1 and SMN2 Duplicated Region and Inclusion Into a Single-Tube Tridecaplex Panel for Haplotype-Based Preimplantation Genetic Testing of Spinal Muscular Atrophy. Front Genet 2019; 10:1105. [PMID: 31781167 PMCID: PMC6851269 DOI: 10.3389/fgene.2019.01105] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2019] [Accepted: 10/11/2019] [Indexed: 12/12/2022] Open
Abstract
Preimplantation genetic testing for the monogenic disorder (PGT-M) spinal muscular atrophy (SMA) is significantly improved by supplementation of SMN1 deletion detection with marker-based linkage analysis. To expand the availability of informative markers for PGT-M of SMA, we identified novel non-duplicated and highly polymorphic microsatellite markers closely flanking the SMN1 and SMN2 duplicated region. Six of the novel markers within 0.5 Mb of the 1.7 Mb duplicated region containing SMN1 and SMN2 (SMA6863, SMA6873, SMA6877, SMA7093, SMA7115, and SMA7120) and seven established markers (D5S1417, D5S1413, D5S1370, D5S1408, D5S610, D5S1999, and D5S637), all with predicted high heterozygosity values, were selected and optimized in a tridecaplex PCR panel, and their polymorphism indices were determined in two populations. Observed marker heterozygosities in the Chinese and Caucasian populations ranged from 0.54 to 0.86, and 98.4% of genotyped individuals (185 of 188) were heterozygous for ≥2 markers on either side of SMN1. The marker panel was evaluated for disease haplotype phasing using single cells from two parent–child trios after whole-genome amplification, and applied to a clinical IVF (in vitro fertilization) PGT-M cycle in an at-risk couple, in parallel with SMN1 deletion detection. Both direct and indirect test methods determined that none of five tested embryos were at risk for SMA, with haplotype analysis further identifying one embryo as unaffected and four as carriers. Fresh transfer of the unaffected embryo did not lead to implantation, but subsequent frozen-thaw transfer of a carrier embryo produced a pregnancy, with fetal genotype confirmed by amniocentesis, and a live birth at term.
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Affiliation(s)
- Mingjue Zhao
- Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
| | - Mulias Lian
- Preimplantation Genetic Diagnosis Center, Khoo Teck Puat-National University Children's Medical Institute, National University Health System, Singapore, Singapore
| | - Felicia S H Cheah
- Preimplantation Genetic Diagnosis Center, Khoo Teck Puat-National University Children's Medical Institute, National University Health System, Singapore, Singapore
| | - Arnold S C Tan
- Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.,Preimplantation Genetic Diagnosis Center, Khoo Teck Puat-National University Children's Medical Institute, National University Health System, Singapore, Singapore
| | - Anupriya Agarwal
- Clinic for Human Reproduction, Department of Obstetrics and Gynecology, National University Hospital, Singapore, Singapore
| | - Samuel S Chong
- Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.,Preimplantation Genetic Diagnosis Center, Khoo Teck Puat-National University Children's Medical Institute, National University Health System, Singapore, Singapore.,Molecular Diagnosis Center and Clinical Cytogenetics Service, Department of Laboratory Medicine, National University Hospital, Singapore, Singapore
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17
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Fu Y, Shen X, Chen D, Wang Z, Zhou C. Multiple displacement amplification as the first step can increase the diagnostic efficiency of preimplantation genetic testing for monogenic disease for β-thalassemia. J Obstet Gynaecol Res 2019; 45:1515-1521. [PMID: 31141845 DOI: 10.1111/jog.14003] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2019] [Accepted: 04/30/2019] [Indexed: 12/30/2022]
Abstract
AIM To evaluate whether using multiple displacement amplification (MDA) as the first step can increase the diagnostic efficiency of preimplantation genetic testing for monogenic disease (PGT-M) for β-thalassemia. METHODS This is a retrospective cohort study. All included patients underwent PGT-M cycles (n = 307) for β-thalassemia in our center from January 2014 to February 2018. We divided the patients into two groups based on two different detection methods. For the polymerase chain reaction (PCR) group (n = 115), multiplex nested PCR+ reverse dot blot analysis was performed directly after cell lysis. For the MDA group (n = 192), the whole genomes of single cells were directly amplified using MDA and then examined by singleplex PCR + reverse dot blot for β-thalassemia. RESULTS A total of 2315 embryos were tested. The overall diagnostic efficiency of the MDA group was significantly higher than that of the PCR group (96.99% vs 88.15%, P < 0.001). The percentage of embryos available for transfer was significantly higher in the MDA group than in the PCR group (74.28% vs 64.98%, P < 0.001). Furthermore, the carrier embryo rate of the MDA group was significantly higher than that of the PCR group (50.11% vs 35.95%, P < 0.001). CONCLUSION This study indicates that MDA, as the first step in PGT-M for β-thalassemia, can increase diagnostic efficiency.
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Affiliation(s)
- Yu Fu
- Reproductive Medicine Center, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.,Guangdong Provincial Key Laboratory of Reproductive Medicine, Guangzhou, China
| | - Xiaoting Shen
- Reproductive Medicine Center, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.,Guangdong Provincial Key Laboratory of Reproductive Medicine, Guangzhou, China
| | - Dongjia Chen
- Reproductive Medicine Center, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.,Guangdong Provincial Key Laboratory of Reproductive Medicine, Guangzhou, China
| | - Zengyan Wang
- Reproductive Medicine Center, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.,Guangdong Provincial Key Laboratory of Reproductive Medicine, Guangzhou, China
| | - Canquan Zhou
- Reproductive Medicine Center, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.,Guangdong Provincial Key Laboratory of Reproductive Medicine, Guangzhou, China
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18
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Karyomapping in Preimplantation Genetic Testing of Patients with Beta-thalassemia and Sickle Cell Anemia. ANADOLU KLINIĞI TIP BILIMLERI DERGISI 2019. [DOI: 10.21673/anadoluklin.438117] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
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19
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Nakhuda G, Jing C, Butler R, Guimond C, Hitkari J, Taylor E, Tallon N, Yuzpe A. Frequencies of chromosome-specific mosaicisms in trophoectoderm biopsies detected by next-generation sequencing. Fertil Steril 2018; 109:857-865. [PMID: 29778385 DOI: 10.1016/j.fertnstert.2018.01.011] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2017] [Revised: 01/02/2018] [Accepted: 01/08/2018] [Indexed: 11/30/2022]
Abstract
OBJECTIVE To examine the chromosome-specific frequencies of mosaicism detected by next-generation sequencing (NGS) compared with constitutional aneuploidy. DESIGN Retrospective cross-sectional review of NGS results from trophectoderm biopsies analyzed by per-chromosome prevalence of mosaicism and constitutional aneuploidy. SETTING Private fertility clinic. PATIENT(S) A total of 378 patients who underwent preimplantation genetic screening by NGS for routine clinical indications from February 2016 to April 2017. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Aneuploidies and mosaicisms were tabulated per chromosome, and whole-chromosome and segmental mosaicisms were also analyzed. RESULT(S) NGS results were analyzed from 1,547 blastocysts. Mosaicism was detected as the sole abnormality in 17.5% (n = 270) of samples but were also found in 196/634 aneuploid embryos, so the overall incidence of mosaicism per biopsy was 30.1%. Mosaicism did not statistically vary when stratified by maternal age. The mean rate of overall mosaicism per chromosome was 2.46%. When whole chromosome and segmental mosaicisms were compared, unequal frequencies were found in several chromosomes. Trisomy was more frequently detected as whole-chromosome mosaicism, although monosomy was more frequently seen in segmental mosaicism. Aneuploidy and mosaicism displayed different patterns of distribution in various chromosomes. CONCLUSION(S) Mosaicism is unequally detected in various chromosomes and appears distinct from the distribution pattern of constitutional aneuploidy. Whole chromosome and segmental mosaicisms are also differentially detected. These results contribute to the study of mosaicism, illuminating a differential pattern of detection across the genome.
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Affiliation(s)
- Gary Nakhuda
- Olive Fertility Centre, Vancouver, British Columbia, Canada; University of British Columbia, Vancouver, British Columbia, Canada.
| | - Chen Jing
- Olive Fertility Centre, Vancouver, British Columbia, Canada
| | - Rachel Butler
- Olive Fertility Centre, Vancouver, British Columbia, Canada; University of British Columbia, Vancouver, British Columbia, Canada
| | - Colleen Guimond
- Olive Fertility Centre, Vancouver, British Columbia, Canada; University of British Columbia, Vancouver, British Columbia, Canada
| | - Jason Hitkari
- Olive Fertility Centre, Vancouver, British Columbia, Canada; University of British Columbia, Vancouver, British Columbia, Canada
| | - Elizabeth Taylor
- Olive Fertility Centre, Vancouver, British Columbia, Canada; University of British Columbia, Vancouver, British Columbia, Canada
| | - Niamh Tallon
- Olive Fertility Centre, Vancouver, British Columbia, Canada; University of British Columbia, Vancouver, British Columbia, Canada
| | - Albert Yuzpe
- Olive Fertility Centre, Vancouver, British Columbia, Canada; University of British Columbia, Vancouver, British Columbia, Canada
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20
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Cruaud A, Groussier G, Genson G, Sauné L, Polaszek A, Rasplus JY. Pushing the limits of whole genome amplification: successful sequencing of RADseq library from a single microhymenopteran (Chalcidoidea, Trichogramma). PeerJ 2018; 6:e5640. [PMID: 30356952 PMCID: PMC6195110 DOI: 10.7717/peerj.5640] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2018] [Accepted: 08/27/2018] [Indexed: 11/20/2022] Open
Abstract
A major obstacle to high-throughput genotyping of microhymenoptera is their small size. As species are difficult to discriminate, and because complexes may exist, the sequencing of a pool of specimens is hazardous. Thus, one should be able to sequence pangenomic markers (e.g., RADtags) from a single specimen. To date, whole genome amplification (WGA) prior to library construction is still a necessity as at most 10 ng of DNA can be obtained from single specimens (sometimes less). However, this amount of DNA is not compatible with manufacturer's requirements for commercial kits. Here we test the accuracy of the GenomiPhi kit V2 on Trichogramma wasps by comparing RAD libraries obtained from the WGA of single specimens (F0 and F1 generation, about1 ng input DNA for the WGA (0.17-2.9 ng)) and a biological amplification of genomic material (the pool of the progeny of the F1 generation). Globally, we found that 99% of the examined loci (up to 48,189 for one of the crosses, 109 bp each) were compatible with the mode of reproduction of the studied model (haplodiploidy) and Mendelian inheritance of alleles. The remaining 1% (0.01% of the analysed nucleotides) could represent WGA bias or other experimental/analytical bias. This study shows that the multiple displacement amplification method on which the GenomiPhi kit relies, could also be of great help for the high-throughput genotyping of microhymenoptera used for biological control, or other organisms from which only a very small amount of DNA can be extracted, such as human disease vectors (e.g., sandflies, fleas, ticks etc.).
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Affiliation(s)
- Astrid Cruaud
- CBGP, INRA, CIRAD, IRD, Montpellier SupAgro, Univ Montpellier, Montpellier, France
| | - Géraldine Groussier
- Institut Sophia Agrobiotech, INRA, CNRS, Université Côte d’Azur, Sophia Antipolis, France
| | - Guenaëlle Genson
- CBGP, INRA, CIRAD, IRD, Montpellier SupAgro, Univ Montpellier, Montpellier, France
| | - Laure Sauné
- CBGP, INRA, CIRAD, IRD, Montpellier SupAgro, Univ Montpellier, Montpellier, France
| | - Andrew Polaszek
- Department of Life Sciences, Natural History Museum, London, United Kingdom
| | - Jean-Yves Rasplus
- CBGP, INRA, CIRAD, IRD, Montpellier SupAgro, Univ Montpellier, Montpellier, France
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21
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Volozonoka L, Perminov D, Korņejeva L, Alkšere B, Novikova N, Pīmane EJ, Blumberga A, Kempa I, Miskova A, Gailīte L, Fodina V. Performance comparison of two whole genome amplification techniques in frame of multifactor preimplantation genetic testing. J Assist Reprod Genet 2018; 35:1457-1472. [PMID: 29687370 PMCID: PMC6086788 DOI: 10.1007/s10815-018-1187-4] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2017] [Accepted: 04/12/2018] [Indexed: 11/29/2022] Open
Abstract
PURPOSE To compare multiple displacement amplification and OmniPlex whole genome amplification technique performance during array comparative genome hybridization (aCGH), Sanger sequencing, SNaPshot and fragment size analysis downstream applications in frame of multifactor embryo preimplantation genetic testing. METHODS Preclinical workup included linked short tandem repeat (STR) marker selection and primer design for loci of interest. It was followed by a family haplotyping, after which an in vitro fertilization preimplantation genetic testing (IVF-PGT) cycle was carried out. A total of 62 embryos were retrieved from nine couples with a confirmed single gene disorder being transmitted in their family with various inheritance traits-autosomal dominant (genes-ACTA2, HTT, KRT14), autosomal recessive (genes-ALOX12B, TPP1, GLB1) and X-linked (genes-MTM1, DMD). Whole genome amplification (WGA) for the day 5 embryo trophectoderm single biopsies was carried out by multiple displacement amplification (MDA) or polymerase chain reaction (PCR)-based technology OmniPlex and was used for direct (Sanger sequencing, fragment size analysis, SNaPshot) and indirect mutation assessment (STR marker haplotyping), and embryo aneuploidy testing by array comparative genome hybridization (aCGH). RESULTS Family haplotyping revealed informative/semi-informative microsatellite markers for all clinical cases for all types of inheritance. Indirect testing gave a persuasive conclusion for all embryos assessed, which was confirmed through direct testing. The overall allele dropout (ADO) rate was higher for PCR-based WGA, and MDA shows a better genomic recovery scale. Five euploid embryos were subjected to elective single embryo transfer (eSET), which resulted in four clinical pregnancies and birth of two healthy children, which proved free of disease causative variants running in the family postnataly. CONCLUSIONS A developed multifactor PGT protocol can be adapted and applied to virtually any genetic condition and is capable of improving single gene disorder preimplantation genetic testing in a patient-tailored manner thus increasing pregnancy rates, saving costs and increasing patient reliability.
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Affiliation(s)
- Ludmila Volozonoka
- Scientific Laboratory of Molecular Genetics, Riga Stradins University, Dzirciema street 16, Riga, LV-1007, Latvia.
- Centre of Genetics, "IVF Riga" Reproductive Genetics Clinic, Riga, LV-1010, Latvia.
| | - Dmitry Perminov
- Centre of Genetics, "IVF Riga" Reproductive Genetics Clinic, Riga, LV-1010, Latvia
- Department of Molecular Biology, "E. Gulbja Laboratory", Riga, LV-1006, Latvia
| | - Liene Korņejeva
- Centre of Genetics, "IVF Riga" Reproductive Genetics Clinic, Riga, LV-1010, Latvia
| | - Baiba Alkšere
- Centre of Genetics, "IVF Riga" Reproductive Genetics Clinic, Riga, LV-1010, Latvia
| | - Natālija Novikova
- Centre of Genetics, "IVF Riga" Reproductive Genetics Clinic, Riga, LV-1010, Latvia
- Faculty of Medicine, University of Latvia, Riga, LV-1586, Latvia
| | - Evija Jokste Pīmane
- Centre of Genetics, "IVF Riga" Reproductive Genetics Clinic, Riga, LV-1010, Latvia
| | - Arita Blumberga
- Centre of Genetics, "IVF Riga" Reproductive Genetics Clinic, Riga, LV-1010, Latvia
| | - Inga Kempa
- Scientific Laboratory of Molecular Genetics, Riga Stradins University, Dzirciema street 16, Riga, LV-1007, Latvia
| | - Anna Miskova
- Department of Obstetrics and Gynecology, Riga Stradins University, Riga, LV-1007, Latvia
| | - Linda Gailīte
- Scientific Laboratory of Molecular Genetics, Riga Stradins University, Dzirciema street 16, Riga, LV-1007, Latvia
| | - Violeta Fodina
- Centre of Genetics, "IVF Riga" Reproductive Genetics Clinic, Riga, LV-1010, Latvia
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22
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Handyside AH. ‘Designer babies’ almost thirty years on. Reproduction 2018; 156:F75-F79. [DOI: 10.1530/rep-18-0157] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2018] [Accepted: 06/12/2018] [Indexed: 11/08/2022]
Abstract
The first pregnancies and live births following in vitro fertilisation (IVF) and preimplantation genetic testing (PGT), formerly known as preimplantation genetic diagnosis, were reported in 1990, almost 30 years ago, in several couples at risk of X-linked inherited conditions, which typically only affect boys inheriting the X chromosome with the affected gene from their carrier mothers. At that time, it was only possible to identify the sex of the embryo by amplifying a Y-linked repeat sequence in single cells biopsied at cleavage stages and avoid the transfer of males, half of which would be affected. The extensive publicity surrounding these cases and the perceived risk of using IVF and PGT for desirable characteristics not related to health, such as sex selection, led to the epithet of ‘designer babies’ which continues to resonate to this day. Here, I briefly reflect on how the technology of PGT has evolved over the decades and whether it deserves this reputation. With efficient methods for whole genome amplification and the genomic revolution, we now have highly accurate universal tests that combine marker-based diagnosis of almost any monogenic disorder with the detection of aneuploidy. PGT is now clinically well established and is likely to remain a valuable alternative for couples at risk of having affected children.
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Abstract
PURPOSE OF REVIEW Aneuploidy is a leading cause of pregnancy failure. Although initial attempts to perform preimplantation genetic screening did not improve outcomes, validated techniques were developed to safely and effectively increase pregnancy rates. Still, many embryos designated as euploid do not implant. Current approaches are being refined to provide additional biologic insight into why this is the case. At present, the diagnosis and clinical relevance of segmental aneuploidy and mosaicism are amongst the more heavily investigated. RECENT FINDINGS Class I data have proven the safety of trophectoderm biopsy and validation studies have shown single nucleotide polymorphism array and quantitative PCR can accurately detect whole chromosome aneuploidy. Similar studies to validate next generation sequencing are underway. Although randomized control trials have demonstrated the clinical utility of preimplantation genetic screening, recent data on the impact of mosaicism and segmental aneuploidy require clarification. SUMMARY Several well powered randomized control trials have shown preimplantation genetic screening improves implantation rate. Plausible explanations for euploid failures include undetected mosaicism and segmental aneuploidy. However, the true incidence and dispersion of mosaicism within the embryo is unknown. Likewise, the resolution of detection and clinical significance of segmental aneuploidy is unclear. Further research to validate proposed detection algorithms and class I data to determine if detection impacts outcomes is needed.
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A Total-variation Constrained Permutation Model for Revealing Common Copy Number Patterns. Sci Rep 2017; 7:9666. [PMID: 28851906 PMCID: PMC5575355 DOI: 10.1038/s41598-017-09139-8] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2016] [Accepted: 07/24/2017] [Indexed: 01/20/2023] Open
Abstract
Variations in DNA copy number carry important information on genome evolution and regulation of DNA replication in cancer cells. The rapid development of single-cell sequencing technology enables exploration of gene-expression heterogeneity among single cells, providing important information on cell evolution. Evolutionary relationships in accumulated sequence data can be visualized by adjacent positioning of similar cells so that similar copy-number profiles are shown by block patterns. However, single-cell DNA sequencing data usually have low amount of starting genome, which requires an extra step of amplification to accumulate sufficient samples, introducing noise and making regular pattern-finding challenging. In this paper, we will propose to tackle this issue of recovering the hidden blocks within single-cell DNA-sequencing data through continuous sample permutations such that similar samples are positioned adjacently. The permutation is guided by the total variational norm of the recovered copy number profiles, and is continued until the total variational norm is minimized when similar samples are stacked together to reveal block patterns. An efficient numerical scheme for finding this permutation is designed, tailored from the alternating direction method of multipliers. Application of this method to both simulated and real data demonstrates its ability to recover the hidden structures of single-cell DNA sequences.
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Abstract
INTRODUCTION Preimplantation genetic diagnosis and screening (PGD/PGS) has been applied clinically for >25 years however inherent drawbacks include the necessity to tailor each case to the trait in question, and that technology to detect monogenic and chromosomal disorders respectively is fundamentally different. Areas covered: The area of preimplantation genetics has evolved over the last 25 years, adapting to changes in technology and the need for more efficient, streamlined diagnoses. Karyomapping allows the determination of inheritance from the (grand)parental haplobocks through assembly of inherited chromosomal segments. The output displays homologous chromosomes, crossovers and the genetic status of the embryos by linkage comparison, as well as chromosomal disorders. It also allows for determination of heterozygous SNP calls, avoiding the risks of allele dropout, a common problem with other PGD techniques. Manuscripts documenting the evolution of preimplantation genetics, especially those investigating technologies that would simultaneously detect monogenic and chromosomal disorders, were selected for review. Expert commentary: Karyomapping is currently available for detection of single gene disorders; ~1000 clinics worldwide offer it (via ~20 diagnostic laboratories) and ~2500 cases have been performed. Due an inability to detect post-zygotic trisomy reliably however and confounding problems of embryo mosaicism, karyomapping has yet to be applied clinically for detection of chromosome disorders.
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Affiliation(s)
- Rebecca L Gould
- a The Bridge Centre , London , UK.,b School of Biological Sciences , University of Kent , Canterbury , UK
| | - Darren K Griffin
- b School of Biological Sciences , University of Kent , Canterbury , UK
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Sanders KD, Griffin DK. Chromosomal Preimplantation Genetic Diagnosis: 25 Years and Counting. JOURNAL OF FETAL MEDICINE 2017. [DOI: 10.1007/s40556-017-0123-5] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]
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Lian M, Zhao M, Lee CG, Chong SS. Single-Tube Dodecaplex PCR Panel of Polymorphic Microsatellite Markers Closely Linked to the DMPK CTG Repeat for Preimplantation Genetic Diagnosis of Myotonic Dystrophy Type 1. Clin Chem 2017; 63:1127-1140. [PMID: 28428361 DOI: 10.1373/clinchem.2017.271528] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2017] [Accepted: 03/08/2017] [Indexed: 12/16/2022]
Abstract
BACKGROUND Preimplantation genetic diagnosis (PGD) of myotonic dystrophy type 1 (DM1) currently uses conventional PCR to detect nonexpanded dystrophia myotonica protein kinase (DMPK) alleles or triplet-primed PCR to detect the CTG-expanded alleles, coupled with analysis of linked microsatellite markers to increase diagnostic accuracy. We aimed to simplify the process of identification and selection of informative linked markers for application to DM1 PGD. METHODS An in silico search was performed to identify all markers within 1-1.5 Mb flanking the DMPK gene. Five previously known (D19S559, APOC2, D19S543, D19S112, and BV209569) and 7 novel (DM45050, DM45178, DM45209, DM45958, DM46513, DM46892, and DM47004.1) markers with potentially high heterozygosity values and polymorphism information content were selected and optimized in a single-tube multiplex PCR panel. RESULTS Analysis of 184 DNA samples of Chinese and Caucasian individuals (91 from unrelated, anonymized cord blood of Chinese babies born at the National University Hospital, Singapore, and 93 Caucasian DNA samples from the Human Variation Panel HD100CAU) confirmed the high polymorphism indices of all markers (polymorphism information content >0.5), with observed heterozygosity values ranging from 0.62-0.93. All individuals were heterozygous for at least 6 markers, with 99.5% of individuals heterozygous for at least 2 markers on either side of the DMPK CTG repeat. The dodecaplex marker assay was successfully validated on 42 single cells and 12 whole genome amplified single cells. CONCLUSIONS The DM1 multiplex PCR panel is suitable for use in DM1 PGD either as a standalone linkage-based assay or as a complement to DMPK CTG repeat expansion-mutation detection.
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Affiliation(s)
- Mulias Lian
- Khoo Teck Puat - National University Children's Medical Institute, National University Health System, Singapore
| | | | - Caroline G Lee
- Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.,Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore.,Division of Medical Sciences, National Cancer Center, Singapore
| | - Samuel S Chong
- Khoo Teck Puat - National University Children's Medical Institute, National University Health System, Singapore; .,Departments of Pediatrics and.,Department of Laboratory Medicine, National University Hospital, National University Health System, Singapore
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Zhang C, Cai H, Huang J, Song Y. nbCNV: a multi-constrained optimization model for discovering copy number variants in single-cell sequencing data. BMC Bioinformatics 2016; 17:384. [PMID: 27639558 PMCID: PMC5027123 DOI: 10.1186/s12859-016-1239-7] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2016] [Accepted: 09/04/2016] [Indexed: 02/02/2023] Open
Abstract
Background Variations in DNA copy number have an important contribution to the development of several diseases, including autism, schizophrenia and cancer. Single-cell sequencing technology allows the dissection of genomic heterogeneity at the single-cell level, thereby providing important evolutionary information about cancer cells. In contrast to traditional bulk sequencing, single-cell sequencing requires the amplification of the whole genome of a single cell to accumulate enough samples for sequencing. However, the amplification process inevitably introduces amplification bias, resulting in an over-dispersing portion of the sequencing data. Recent study has manifested that the over-dispersed portion of the single-cell sequencing data could be well modelled by negative binomial distributions. Results We developed a read-depth based method, nbCNV to detect the copy number variants (CNVs). The nbCNV method uses two constraints-sparsity and smoothness to fit the CNV patterns under the assumption that the read signals are negatively binomially distributed. The problem of CNV detection was formulated as a quadratic optimization problem, and was solved by an efficient numerical solution based on the classical alternating direction minimization method. Conclusions Extensive experiments to compare nbCNV with existing benchmark models were conducted on both simulated data and empirical single-cell sequencing data. The results of those experiments demonstrate that nbCNV achieves superior performance and high robustness for the detection of CNVs in single-cell sequencing data. Electronic supplementary material The online version of this article (doi:10.1186/s12859-016-1239-7) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Changsheng Zhang
- School of Computer Science & Engineering, South China University of Technology, Guangzhou, 510006, China
| | - Hongmin Cai
- School of Computer Science & Engineering, South China University of Technology, Guangzhou, 510006, China.
| | - Jingying Huang
- School of Computer Science & Engineering, South China University of Technology, Guangzhou, 510006, China
| | - Yan Song
- School of Computer Science & Engineering, South China University of Technology, Guangzhou, 510006, China
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Zhao M, Chen M, Lee CG, Chong SS. Identification of Novel Microsatellite Markers <1 Mb from the HTT CAG Repeat and Development of a Single-Tube Tridecaplex PCR Panel of Highly Polymorphic Markers for Preimplantation Genetic Diagnosis of Huntington Disease. Clin Chem 2016; 62:1096-105. [PMID: 27335079 DOI: 10.1373/clinchem.2016.255711] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2016] [Accepted: 05/11/2016] [Indexed: 12/11/2022]
Abstract
BACKGROUND Preimplantation genetic diagnosis (PGD) of Huntington disease (HD) generally employs linkage analysis of flanking microsatellite markers to complement direct mutation testing, as well as for exclusion testing. Thus far, only 10 linked markers have been developed for use in HD PGD, with a maximum of 3 markers coamplified successfully. We aimed to develop a single-tube multiplex PCR panel of highly polymorphic markers to simplify HD PGD. METHODS An in silico search was performed to identify all markers within 1 Mb flanking the huntingtin (HTT) gene. Selected markers were optimized in a single-tube PCR panel, and their polymorphism indices were determined in 2 populations. The panel was tested on 63 single cells to validate its utility in PGD. RESULTS We identified 102 markers in silico, of which 56 satisfied the selection criteria. After initial testing, 12 markers with potentially high heterozygosity were optimized into a single-tube PCR panel together with a 13th more distally located marker. Analysis of DNA from 183 Chinese and Caucasian individuals revealed high polymorphism indices for all markers (polymorphism information content >0.5), with observed heterozygosities ranging from 0.5-0.92. All individuals were heterozygous for at least 5 markers, with 99.5% of individuals heterozygous for at least 2 markers upstream and downstream of the HTT CAG repeat. CONCLUSIONS The tridecaplex marker assay amplified reliably from single cells either directly or after whole genome amplification, thus validating its standalone use in HD exclusion PGD or as a complement to HTT CAG repeat expansion-mutation detection.
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Affiliation(s)
| | | | - Caroline G Lee
- Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Division of Medical Sciences, National Cancer Center, Singapore; Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore
| | - Samuel S Chong
- Departments of Pediatrics and Preimplantation Genetic Diagnosis Center, Khoo Teck Puat-National University Children's Medical Institute, National University Health System, Singapore; Molecular Diagnosis Center and Clinical Cytogenetics Service, Department of Laboratory Medicine, National University Hospital, National University Health System, Singapore.
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Handyside AH. Live births following karyomapping - a "key" milestone in the development of preimplantation genetic diagnosis. Reprod Biomed Online 2016; 31:307-8. [PMID: 26353913 DOI: 10.1016/j.rbmo.2015.07.003] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
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Goldman KN, Nazem T, Berkeley A, Palter S, Grifo JA. Preimplantation Genetic Diagnosis (PGD) for Monogenic Disorders: the Value of Concurrent Aneuploidy Screening. J Genet Couns 2016; 25:1327-1337. [DOI: 10.1007/s10897-016-9975-4] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2015] [Accepted: 05/16/2016] [Indexed: 01/16/2023]
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Abstract
There have been tremendous advances in the ability to screen for the "odds" of having a genetic disorder (both mendelian and chromosomal). With microarray analyses on fetal tissue now showing a minimum risk for any pregnancy being at least 1 in 150 and ultimately greater than 1%, it is thought that all patients, regardless of age, should be offered chorionic villus sampling/amniocentesis and microarray analysis. As sequencing techniques replace other laboratory methods, the only question will be whether these tests are performed on villi, amniotic fluid cells, or maternal blood.
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Patel NH, Bhadarka HK, Patel KB, Vaniawala SN, Acharya A, Mukhopadhyaya PN, Sodagar NR. Embryo genome profiling by single-cell sequencing for successful preimplantation genetic diagnosis in a family harboring COL4A1 c.1537G>A; p.G513S mutation. J Hum Reprod Sci 2016; 9:200-206. [PMID: 27803589 PMCID: PMC5070403 DOI: 10.4103/0974-1208.192072] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022] Open
Abstract
CONTEXT Genetic profiling of embryos (also known as preimplantation genetic diagnosis) before implantation has dramatically enhanced the success quotient of in vitro fertilization (IVF) in recent times. The technology helps in avoiding selective pregnancy termination since the baby is likely to be free of the disease under consideration. AIM Screening of embryos free from c.1537G>A; p.G513S mutation within the COL4A1 gene for which the father was known in before be in heterozygous condition. SUBJECTS AND METHODS Processing of trophectoderm biopsies was done from twelve embryos for c.1537G>A; p.G513S mutation within the COL4A1 gene. DNA extracted from isolated cells were subjected to whole genome amplification using an isothermal amplification and strand displacement technology. Oligonucleotide primers bracketing the mutation were synthesized and used to amplify 162 base pairs (bp) polymerase chain reaction amplicons originating from each embryo which were subsequently sequenced to detect the presence or absence of the single base polymorphism. RESULTS Three out of 12 embryos interrogated in this study were found to be normal while 9 were found to harbor the mutation in heterozygous condition. Implantation of one of the normal embryos following by chorionic villus sampling at 11th week of pregnancy indicated that the baby was free from c.1537G>A; p.G513S mutation within the COL4A1 gene. CONCLUSIONS Single-cell sequencing is a helpful tool for preimplantation embryo profiling. This is the first report from India describing the birth of a normal child through IVF procedure where a potential pathogenic COL4A1 allele was avoided using this technology.
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Affiliation(s)
- Nayana H Patel
- Department of IVF, Akanksha Hospital and Research Institute, Near Shrushti English Medium School, Lambhvel-387310, Anand, Gujarat, India
| | - Harsha K Bhadarka
- Department of IVF, Akanksha Hospital and Research Institute, Near Shrushti English Medium School, Lambhvel-387310, Anand, Gujarat, India
| | - Kruti B Patel
- Department of IVF, Akanksha Hospital and Research Institute, Near Shrushti English Medium School, Lambhvel-387310, Anand, Gujarat, India
| | - Salil N Vaniawala
- SN Gene Lab, Floor, A, Above Kansaar Hotel, President Plaza, Ring Rd, Aveerbhav Society 1, Nanpura, Surat, Gujarat, India
| | - Arpan Acharya
- SN Gene Lab, Floor, A, Above Kansaar Hotel, President Plaza, Ring Rd, Aveerbhav Society 1, Nanpura, Surat, Gujarat, India
| | - Pratap N Mukhopadhyaya
- SN Gene Lab, Floor, A, Above Kansaar Hotel, President Plaza, Ring Rd, Aveerbhav Society 1, Nanpura, Surat, Gujarat, India
| | - Nilofar R Sodagar
- Department of IVF, Akanksha Hospital and Research Institute, Near Shrushti English Medium School, Lambhvel-387310, Anand, Gujarat, India
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Alberter B, Klein CA, Polzer B. Single-cell analysis of CTCs with diagnostic precision: opportunities and challenges for personalized medicine. Expert Rev Mol Diagn 2015; 16:25-38. [PMID: 26567956 DOI: 10.1586/14737159.2016.1121099] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The generation of variant cancer cells is the major cause of acquired resistance against systemic therapies and consequently, of our inability to cure advanced cancer patients. Circulating tumor cells are gaining increasing clinical attention because they may enable the monitoring cancer progression and adjustment of treatment. In recent years multiple technologies for enrichment, isolation as well as molecular and functional analysis of circulating tumor cells have been developed. Implementation of these technologies in standardized and automated workflows in clinical diagnostics could provide valuable information for real-time monitoring of cancer and eventually new therapeutic strategies for the benefit of patients.
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Affiliation(s)
- Barbara Alberter
- a Project Group "Personalized Tumor Therapy" , Fraunhofer Institute for Toxicology and Experimental Medicine , Regensburg , Germany
| | - Christoph A Klein
- a Project Group "Personalized Tumor Therapy" , Fraunhofer Institute for Toxicology and Experimental Medicine , Regensburg , Germany.,b Experimental Medicine and Therapy Research , University of Regensburg , Regensburg , Germany
| | - Bernhard Polzer
- a Project Group "Personalized Tumor Therapy" , Fraunhofer Institute for Toxicology and Experimental Medicine , Regensburg , Germany
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Contemporary molecular tools in microbial ecology and their application to advancing biotechnology. Biotechnol Adv 2015; 33:1755-73. [DOI: 10.1016/j.biotechadv.2015.09.005] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2015] [Revised: 09/19/2015] [Accepted: 09/20/2015] [Indexed: 12/30/2022]
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Guignot F, Reigner F, Perreau C, Tartarin P, Babilliot JM, Bed'hom B, Vidament M, Mermillod P, Duchamp G. Preimplantation genetic diagnosis in Welsh pony embryos after biopsy and cryopreservation1,2. J Anim Sci 2015; 93:5222-31. [DOI: 10.2527/jas.2015-9469] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
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Cápal P, Blavet N, Vrána J, Kubaláková M, Doležel J. Multiple displacement amplification of the DNA from single flow-sorted plant chromosome. THE PLANT JOURNAL : FOR CELL AND MOLECULAR BIOLOGY 2015; 84:838-844. [PMID: 26400218 DOI: 10.1111/tpj.13035] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/11/2015] [Revised: 09/13/2015] [Accepted: 09/17/2015] [Indexed: 06/05/2023]
Abstract
A protocol is described for production of micrograms of DNA from single copies of flow-sorted plant chromosomes. Of 183 single copies of wheat chromosome 3B, 118 (64%) were successfully amplified. Sequencing DNA amplification products using an Illumina HiSeq 2000 system to 10× coverage and merging sequences from three separate amplifications resulted in 60% coverage of the chromosome 3B reference, entirely covering 30% of its genes. The merged sequences permitted de novo assembly of 19% of chromosome 3B genes, with 10% of genes contained in a single contig, and 39% of genes covered for at least 80% of their length. The chromosome-derived sequences allowed identification of missing genic sequences in the chromosome 3B reference and short sequences similar to 3B in survey sequences of other wheat chromosomes. These observations indicate that single-chromosome sequencing is suitable to identify genic sequences on particular chromosomes, to develop chromosome-specific DNA markers, to verify assignment of DNA sequence contigs to individual pseudomolecules, and to validate whole-genome assemblies. The protocol expands the potential of chromosome genomics, which may now be applied to any plant species from which chromosome samples suitable for flow cytometry can be prepared, and opens new avenues for studies on chromosome structural heterozygosity and haplotype phasing in plants.
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Affiliation(s)
- Petr Cápal
- Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, CZ-78371, Olomouc, Czech Republic
| | - Nicolas Blavet
- Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, CZ-78371, Olomouc, Czech Republic
- Palacký University Olomouc, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 27, CZ-78371, Olomouc, Czech Republic
| | - Jan Vrána
- Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, CZ-78371, Olomouc, Czech Republic
| | - Marie Kubaláková
- Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, CZ-78371, Olomouc, Czech Republic
| | - Jaroslav Doležel
- Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, CZ-78371, Olomouc, Czech Republic
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Palini S, De Stefani S, Primiterra M, Galluzzi L. Pre-implantation genetic diagnosis and screening: now and the future. Gynecol Endocrinol 2015; 31:755-9. [PMID: 26291813 DOI: 10.3109/09513590.2015.1068752] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Since 1989, the year of the first pre-implantation genetic diagnosis (PGD), many developments occurred both in assisted reproduction techniques and in molecular tools. While PGD is a well-established and documented application, pre-implantation genetic screening (PGS) for the detection of aneuploid embryos is still debated due to the presence of mosaicism in the embryo, but especially to the knowledge of the limits that label an embryo as healthy or as appropriate to the life. The aim of this review is to present the state-of-the-art in the field of PGD and PGS, illustrating its benefits and limitations, along with biopsy techniques and the use of new high-throughput technologies.
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Affiliation(s)
- Simone Palini
- a IVF Unit, "Cervesi" Hospital Cattolica , Cattolica , Province of Rimini , Italy and
| | - Silvia De Stefani
- a IVF Unit, "Cervesi" Hospital Cattolica , Cattolica , Province of Rimini , Italy and
| | - Mariangela Primiterra
- a IVF Unit, "Cervesi" Hospital Cattolica , Cattolica , Province of Rimini , Italy and
| | - Luca Galluzzi
- b Department of Biomolecular Sciences , University of Urbino , Urbino , Province of Pesaro e Urbino , Italy
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Yalcin D, Hakguder ZM, Otu HH. Bioinformatics approaches to single-cell analysis in developmental biology. Mol Hum Reprod 2015; 22:182-92. [PMID: 26358759 DOI: 10.1093/molehr/gav050] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2015] [Accepted: 09/04/2015] [Indexed: 12/17/2022] Open
Abstract
Individual cells within the same population show various degrees of heterogeneity, which may be better handled with single-cell analysis to address biological and clinical questions. Single-cell analysis is especially important in developmental biology as subtle spatial and temporal differences in cells have significant associations with cell fate decisions during differentiation and with the description of a particular state of a cell exhibiting an aberrant phenotype. Biotechnological advances, especially in the area of microfluidics, have led to a robust, massively parallel and multi-dimensional capturing, sorting, and lysis of single-cells and amplification of related macromolecules, which have enabled the use of imaging and omics techniques on single cells. There have been improvements in computational single-cell image analysis in developmental biology regarding feature extraction, segmentation, image enhancement and machine learning, handling limitations of optical resolution to gain new perspectives from the raw microscopy images. Omics approaches, such as transcriptomics, genomics and epigenomics, targeting gene and small RNA expression, single nucleotide and structural variations and methylation and histone modifications, rely heavily on high-throughput sequencing technologies. Although there are well-established bioinformatics methods for analysis of sequence data, there are limited bioinformatics approaches which address experimental design, sample size considerations, amplification bias, normalization, differential expression, coverage, clustering and classification issues, specifically applied at the single-cell level. In this review, we summarize biological and technological advancements, discuss challenges faced in the aforementioned data acquisition and analysis issues and present future prospects for application of single-cell analyses to developmental biology.
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Affiliation(s)
- Dicle Yalcin
- Department of Electrical and Computer Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588-0511, USA
| | - Zeynep M Hakguder
- Department of Electrical and Computer Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588-0511, USA
| | - Hasan H Otu
- Department of Electrical and Computer Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588-0511, USA
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Ottolini CS, Rogers S, Sage K, Summers MC, Capalbo A, Griffin DK, Sarasa J, Wells D, Handyside AH. Karyomapping identifies second polar body DNA persisting to the blastocyst stage: implications for embryo biopsy. Reprod Biomed Online 2015; 31:776-82. [PMID: 26380865 DOI: 10.1016/j.rbmo.2015.07.005] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2015] [Revised: 07/04/2015] [Accepted: 07/07/2015] [Indexed: 10/23/2022]
Abstract
Blastocyst biopsy is now widely used for both preimplantation genetic screening (PGS) and preimplantation genetic diagnosis (PGD). Although this approach yields good results, variable embryo quality and rates of development remain a challenge. Here, a case is reported in which a blastocyst was biopsied for PGS by array comparative genomic hybridization on day 6 after insemination, having hatched completely. In addition to a small trophectoderm sample, excluded cell fragments from the subzonal space from this embryo were also sampled. Unexpectedly, the array comparative genomic hybridization results from the fragments and trophectoderm sample were non-concordant: 47,XX,+19 and 46,XY, respectively. DNA fingerprinting by short tandem repeat and amelogenin analysis confirmed the sex chromosome difference but seemed to show that the two samples were related but non-identical. Genome-wide single nucleotide polymorphism genotyping and karyomapping identified that the origin of the DNA amplified from the fragments was that of the second polar body corresponding to the oocyte from which the biopsied embryo developed. The fact that polar body DNA can persist to the blastocyst stage provides evidence that excluded cell fragments should not be used for diagnostic purposes and should be avoided when performing embryo biopsies as there is a risk of diagnostic errors.
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Affiliation(s)
- Christian S Ottolini
- The Bridge Centre, London SE1 9RY, UK; School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK.
| | | | | | - Michael C Summers
- The Bridge Centre, London SE1 9RY, UK; School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK
| | - Antonio Capalbo
- G.E.N.E.R.A., Centers for Reproductive Medicine, Marostica, Umbertide, Rome, Italy; GENETYX, Molecular Genetics Laboratory, Marostica, Italy
| | - Darren K Griffin
- School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK
| | - Jonas Sarasa
- Reprogenetics UK, Institute of Reproductive Sciences, Oxford Business Park North, Oxford OX4 2HW, UK
| | - Dagan Wells
- Reprogenetics UK, Institute of Reproductive Sciences, Oxford Business Park North, Oxford OX4 2HW, UK; Nuffied Department of Obstetrics and Gynaecology, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK
| | - Alan H Handyside
- The Bridge Centre, London SE1 9RY, UK; School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK; Illumina, Capital Park CPC4, Fulbourn, Cambridge CB21 5XE, UK; Institute of Integrative and Comparative Biology, University of Leeds, Leeds LS2 9JT, UK
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Kurahashi H, Kato T, Miyazaki J, Nishizawa H, Nishio E, Furukawa H, Miyamura H, Ito M, Endo T, Ouchi Y, Inagaki H, Fujii T. Preimplantation genetic diagnosis/screening by comprehensive molecular testing. Reprod Med Biol 2015; 15:13-19. [PMID: 29259418 DOI: 10.1007/s12522-015-0216-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2015] [Accepted: 07/01/2015] [Indexed: 11/28/2022] Open
Abstract
Although embryo screening by preimplantation genetic diagnosis (PGD) has become the standard technique for the treatment of recurrent pregnancy loss in couples with a balanced gross chromosomal rearrangement, the implantation and pregnancy rates of PGD using conventional fluorescence in situ hybridization (FISH) remain suboptimal. Comprehensive molecular testing, such as array comparative genomic hybridization and next-generation sequencing, can improve these rates, but amplification bias in the whole genome amplification method remains an obstacle to accurate diagnosis. Recent advances in amplification procedures combined with improvements in the microarray platform and analytical method have overcome the amplification bias, and the data accuracy of the comprehensive PGD method has reached the level of clinical laboratory testing. Currently, comprehensive PGD is also applied to recurrent pregnancy loss due to recurrent fetal aneuploidy or infertility with recurrent implantation failure, known as preimplantation genetic screening. However, there are still numerous problems to be solved, including misdiagnosis due to somatic mosaicism, cell cycle-related background noise, and difficulty in diagnosis of polyploidy. The technology for comprehensive PGD also requires further improvement.
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Affiliation(s)
- Hiroki Kurahashi
- Division of Molecular Genetics, Institute for Comprehensive Medical Science Fujita Health University 1-98 Dengakugakubo, Kutsukake-cho 470-1192 Toyoake Aichi Japan.,Genome and Transcriptome Analysis Center Fujita Health University 470-1192 Toyoake Aichi Japan
| | - Takema Kato
- Division of Molecular Genetics, Institute for Comprehensive Medical Science Fujita Health University 1-98 Dengakugakubo, Kutsukake-cho 470-1192 Toyoake Aichi Japan
| | - Jun Miyazaki
- Division of Molecular Genetics, Institute for Comprehensive Medical Science Fujita Health University 1-98 Dengakugakubo, Kutsukake-cho 470-1192 Toyoake Aichi Japan.,Department of Obstetrics and Gynecology Fujita Health University School of Medicine 470-1192 Toyoake Aichi Japan
| | - Haruki Nishizawa
- Department of Obstetrics and Gynecology Fujita Health University School of Medicine 470-1192 Toyoake Aichi Japan
| | - Eiji Nishio
- Department of Obstetrics and Gynecology Fujita Health University School of Medicine 470-1192 Toyoake Aichi Japan
| | - Hiroshi Furukawa
- Department of Laboratory Medicine Fujita Health University Hospital 470-1192 Toyoake Aichi Japan
| | - Hironori Miyamura
- Department of Obstetrics and Gynecology Fujita Health University School of Medicine 470-1192 Toyoake Aichi Japan
| | - Mayuko Ito
- Department of Obstetrics and Gynecology Fujita Health University School of Medicine 470-1192 Toyoake Aichi Japan
| | - Toshiaki Endo
- Department of Obstetrics and Gynecology Sapporo Medical University 060-8543 Sapporo Hokkaido Japan
| | - Yuya Ouchi
- Genome and Transcriptome Analysis Center Fujita Health University 470-1192 Toyoake Aichi Japan
| | - Hidehito Inagaki
- Division of Molecular Genetics, Institute for Comprehensive Medical Science Fujita Health University 1-98 Dengakugakubo, Kutsukake-cho 470-1192 Toyoake Aichi Japan.,Genome and Transcriptome Analysis Center Fujita Health University 470-1192 Toyoake Aichi Japan
| | - Takuma Fujii
- Department of Obstetrics and Gynecology Fujita Health University School of Medicine 470-1192 Toyoake Aichi Japan
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Choi YH, Penedo MCT, Daftari P, Velez IC, Hinrichs K. Accuracy of preimplantation genetic diagnosis in equine in vivo-recovered and in vitro-produced blastocysts. Reprod Fertil Dev 2015; 28:RD14419. [PMID: 25775205 DOI: 10.1071/rd14419] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2014] [Accepted: 01/19/2015] [Indexed: 02/28/2024] Open
Abstract
Preimplantation genetic diagnosis has great potential in the horse, but information on evaluation of equine embryo biopsy samples is limited. Blastocysts were biopsied using a Piezo drill and methods for whole-genome amplification (WGA) investigated. Results for 33 genetic loci were then compared between biopsy samples from in vitro-produced (IVP) and in vivo-recovered (VIV) blastocysts. Under the experimental conditions described, WGA using the Qiagen Repli-g Midi kit was more accurate than that using the Illustra Genomiphi V2 kit (98.2% vs 25.8%, respectively). Using WGA with the Qiagen kit, three biopsy samples were evaluated from each of eight IVP and 19 VIV blastocysts, some produced using semen from stallions carrying the genetic mutations associated with the diseases hereditary equine regional dermal asthenia (HERDA), hyperkalemic periodic paralysis (HYPP) or polysaccharide storage myopathy 1 (PSSM1). Three of 81 biopsy samples (3.7%) returned 95% overall accuracy in IVP and VIV embryos, and this technique is suitable for use in a clinical setting.
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Blair C, Campbell CR, Yoder AD. Assessing the utility of whole genome amplified DNA for next-generation molecular ecology. Mol Ecol Resour 2015; 15:1079-90. [PMID: 25619406 DOI: 10.1111/1755-0998.12376] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2014] [Revised: 01/16/2015] [Accepted: 01/19/2015] [Indexed: 01/23/2023]
Abstract
DNA quantity can be a hindrance in ecological and evolutionary research programmes due to a range of factors including endangered status of target organisms, available tissue type, and the impact of field conditions on preservation methods. A potential solution to low-quantity DNA lies in whole genome amplification (WGA) techniques that can substantially increase DNA yield. To date, few studies have rigorously examined sequence bias that might result from WGA and next-generation sequencing of nonmodel taxa. To address this knowledge deficit, we use multiple displacement amplification (MDA) and double-digest RAD sequencing on the grey mouse lemur (Microcebus murinus) to quantify bias in genome coverage and SNP calls when compared to raw genomic DNA (gDNA). We focus our efforts in providing baseline estimates of potential bias by following manufacturer's recommendations for starting DNA quantities (>100 ng). Our results are strongly suggestive that MDA enrichment does not introduce systematic bias to genome characterization. SNP calling between samples when genotyping both de-novo and with a reference genome are highly congruent (>98%) when specifying a minimum threshold of 20X stack depth to call genotypes. Relative genome coverage is also similar between MDA and gDNA, and allelic dropout is not observed. SNP concordance varies based on coverage threshold, with 95% concordance reached at ~12X coverage genotyping de-novo and ~7X coverage genotyping with the reference genome. These results suggest that MDA may be a suitable solution for next-generation molecular ecological studies when DNA quantity would otherwise be a limiting factor.
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Affiliation(s)
- Christopher Blair
- Department of Biology, Duke University, Box 90338, BioSci 130 Science Drive, Durham, NC, 27708, USA
| | - C Ryan Campbell
- Department of Biology, Duke University, Box 90338, BioSci 130 Science Drive, Durham, NC, 27708, USA
| | - Anne D Yoder
- Department of Biology, Duke University, Box 90338, BioSci 130 Science Drive, Durham, NC, 27708, USA
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Thornhill AR, Handyside AH, Ottolini C, Natesan SA, Taylor J, Sage K, Harton G, Cliffe K, Affara N, Konstantinidis M, Wells D, Griffin DK. Karyomapping-a comprehensive means of simultaneous monogenic and cytogenetic PGD: comparison with standard approaches in real time for Marfan syndrome. J Assist Reprod Genet 2015; 32:347-56. [PMID: 25561157 PMCID: PMC4363232 DOI: 10.1007/s10815-014-0405-y] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2014] [Accepted: 12/11/2014] [Indexed: 11/25/2022] Open
Affiliation(s)
- Alan R Thornhill
- School of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK
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Abstract
Whole genome amplification (WGA) is a widely used molecular technique that is becoming increasingly necessary in genetic research on a range of sample types including individual cells, fossilized remains and entire ecosystems. Multiple methods of WGA have been developed, each with specific strengths and weaknesses, but with a common defect in that each method distorts the initial template DNA during the course of amplification. The type, extent, and circumstance of the bias vary with the WGA method and particulars of the template DNA. In this review, we endeavor to discuss the types of bias introduced, the susceptibility of common WGA techniques to these bias types, and the interdependence between bias and characteristics of the template DNA. Finally, we attempt to illustrate some of the criteria specific to the analytical platform and research application that should be considered to enable combination of the appropriate WGA method, template DNA, sequencing platform, and intended use for optimal results.
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Affiliation(s)
| | - John H Leamon
- CyVek Inc., 2 Barnes Industrial Road South, Wallingford, CT, 06492, USA.
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Kim JY, Lee HS, Kang IS. Preimplantation genetic diagnosis. JOURNAL OF THE KOREAN MEDICAL ASSOCIATION 2015. [DOI: 10.5124/jkma.2015.58.11.979] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Affiliation(s)
- Jin Young Kim
- Department of Obstetrics and Gynecology of Fertility Center, CHA University, Seoul, Korea
| | - Hyoung-Song Lee
- Genetics Laboratory of Fertility Center, CHA Gangnam Medical Center, CHA University, Seoul, Korea
| | - Inn Soo Kang
- Department of Obstetrics and Gynecology of Fertility Center, CHA University, Seoul, Korea
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Parallel single cancer cell whole genome amplification using button-valve assisted mixing in nanoliter chambers. PLoS One 2014; 9:e107958. [PMID: 25233459 PMCID: PMC4169497 DOI: 10.1371/journal.pone.0107958] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2014] [Accepted: 08/16/2014] [Indexed: 12/20/2022] Open
Abstract
The heterogeneity of tumor cells and their alteration during the course of the disease urges the need for real time characterization of individual tumor cells to improve the assessment of treatment options. New generations of therapies are frequently associated with specific genetic alterations driving the need to determine the genetic makeup of tumor cells. Here, we present a microfluidic device for parallel single cell whole genome amplification (pscWGA) to obtain enough copies of a single cell genome to probe for the presence of treatment targets and the frequency of its occurrence among the tumor cells. Individual cells were first captured and loaded into eight parallel amplification units. Next, cells were lysed on a chip and their DNA amplified through successive introduction of dedicated reagents while mixing actively with the help of integrated button-valves. The reaction chamber volume for scWGA 23.85 nl, and starting from 6–7 pg DNA contained in a single cell, around 8 ng of DNA was obtained after WGA, representing over 1000-fold amplification. The amplified products from individual breast cancer cells were collected from the device to either directly investigate the amplification of specific genes by qPCR or for re-amplification of the DNA to obtain sufficient material for whole genome sequencing. Our pscWGA device provides sufficient DNA from individual cells for their genetic characterization, and will undoubtedly allow for automated sample preparation for single cancer cell genomic characterization.
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48
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Watanabe M, Kusano J, Ohtaki S, Ishikura T, Katayama J, Koguchi A, Paumen M, Hayashi Y. Simultaneous genomic identification and profiling of a single cell using semiconductor-based next generation sequencing. Appl Transl Genom 2014; 3:70-7. [PMID: 27294018 PMCID: PMC4887956 DOI: 10.1016/j.atg.2014.05.004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2014] [Revised: 05/19/2014] [Accepted: 05/29/2014] [Indexed: 12/02/2022]
Abstract
Combining single-cell methods and next-generation sequencing should provide a powerful means to understand single-cell biology and obviate the effects of sample heterogeneity. Here we report a single-cell identification method and seamless cancer gene profiling using semiconductor-based massively parallel sequencing. A549 cells (adenocarcinomic human alveolar basal epithelial cell line) were used as a model. Single-cell capture was performed using laser capture microdissection (LCM) with an Arcturus® XT system, and a captured single cell and a bulk population of A549 cells (≈ 106 cells) were subjected to whole genome amplification (WGA). For cell identification, a multiplex PCR method (AmpliSeq™ SNP HID panel) was used to enrich 136 highly discriminatory SNPs with a genotype concordance probability of 1031–35. For cancer gene profiling, we used mutation profiling that was performed in parallel using a hotspot panel for 50 cancer-related genes. Sequencing was performed using a semiconductor-based bench top sequencer. The distribution of sequence reads for both HID and Cancer panel amplicons was consistent across these samples. For the bulk population of cells, the percentages of sequence covered at coverage of more than 100 × were 99.04% for the HID panel and 98.83% for the Cancer panel, while for the single cell percentages of sequence covered at coverage of more than 100 × were 55.93% for the HID panel and 65.96% for the Cancer panel. Partial amplification failure or randomly distributed non-amplified regions across samples from single cells during the WGA procedures or random allele drop out probably caused these differences. However, comparative analyses showed that this method successfully discriminated a single A549 cancer cell from a bulk population of A549 cells. Thus, our approach provides a powerful means to overcome tumor sample heterogeneity when searching for somatic mutations.
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Affiliation(s)
- Manabu Watanabe
- Life Technologies Japan Ltd., a part of Thermo Fisher Scientific. 4-2-8 Shibaura, Minato-ku Tokyo 108-0023, Japan
| | - Junko Kusano
- Life Technologies Japan Ltd., a part of Thermo Fisher Scientific. 4-2-8 Shibaura, Minato-ku Tokyo 108-0023, Japan
| | - Shinsaku Ohtaki
- Life Technologies Japan Ltd., a part of Thermo Fisher Scientific. 4-2-8 Shibaura, Minato-ku Tokyo 108-0023, Japan
| | - Takashi Ishikura
- Life Technologies Japan Ltd., a part of Thermo Fisher Scientific. 4-2-8 Shibaura, Minato-ku Tokyo 108-0023, Japan
| | - Jin Katayama
- Life Technologies Japan Ltd., a part of Thermo Fisher Scientific. 4-2-8 Shibaura, Minato-ku Tokyo 108-0023, Japan
| | - Akira Koguchi
- Life Technologies Japan Ltd., a part of Thermo Fisher Scientific. 4-2-8 Shibaura, Minato-ku Tokyo 108-0023, Japan
| | - Michael Paumen
- Life Technologies Japan Ltd., a part of Thermo Fisher Scientific. 4-2-8 Shibaura, Minato-ku Tokyo 108-0023, Japan
| | - Yoshiharu Hayashi
- Life Technologies Japan Ltd., a part of Thermo Fisher Scientific. 4-2-8 Shibaura, Minato-ku Tokyo 108-0023, Japan
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A novel whole genome amplification method using type IIS restriction enzymes to create overhangs with random sequences. J Biotechnol 2014; 184:1-6. [PMID: 24833422 DOI: 10.1016/j.jbiotec.2014.04.020] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2014] [Revised: 04/25/2014] [Accepted: 04/28/2014] [Indexed: 11/22/2022]
Abstract
Ligation-mediated polymerase chain reaction (LM-PCR) is a whole genome amplification (WGA) method, for which genomic DNA is cleaved into numerous fragments and then all of the fragments are amplified by PCR after attaching a universal end sequence. However, the self-ligation of these fragments could happen and may cause biased amplification and restriction of its application. To decrease the self-ligation probability, here we use type IIS restriction enzymes to digest genomic DNA into fragments with 4-5nt long overhangs with random sequences. After ligation to an adapter with random end sequences to above fragments, PCR is carried out and almost all present DNA sequences are amplified. In this study, whole genome of Vibrio parahaemolyticus was amplified and the amplification efficiency was evaluated by quantitative PCR. The results suggested that our approach could provide sufficient genomic DNA with good quality to meet requirements of various genetic analyses.
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50
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Embryonic aneuploidy: overcoming molecular genetics challenges improves outcomes and changes practice patterns. Trends Mol Med 2014; 20:499-508. [PMID: 25113799 DOI: 10.1016/j.molmed.2014.06.006] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2014] [Revised: 06/18/2014] [Accepted: 06/26/2014] [Indexed: 11/20/2022]
Abstract
Since its inception, in vitro fertilization (IVF) has pursued molecular technology to improve patient outcomes, leading to enhanced methods of embryo selection. Comprehensive chromosomal screening (CCS) is a powerful tool that decreases maternal and neonatal morbidity due to multiple gestations by allowing the transfer of fewer embryos while maintaining success rates. To optimize this genetic test, physiological principles limiting the timing and type of cells to be removed had to be realized. Molecular barriers involved in genome amplification and ensuring the accuracy and validity of the CCS platform required a multistep approach to ensure that this technology was not used prematurely. Only after ensuring that the potential for harm was minimized and benefit maximized could clinicians use this technology to improve patient care.
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