Preimplantation Genetic Diagnosis in Genomic Regions with Duplications and Pseudogenes: Long-Range PCR in the Single-Cell Assay

2013 ◽  
Vol 34 (5) ◽  
pp. 792-799 ◽  
Author(s):  
David A. Zeevi ◽  
Paul Renbaum ◽  
Raphael Ron-El ◽  
Talia Eldar-Geva ◽  
Arieh Raziel ◽  
...  
Keyword(s):  
Single Cell ◽  
Long Range ◽  
Preimplantation Genetic Diagnosis ◽  
Genetic Diagnosis ◽  
Cell Assay ◽  
Long Range Pcr ◽  
Genomic Regions

scholarly journals Long-Range PCR-Based NGS Applications to Diagnose Mendelian Retinal Diseases

2021 ◽  
Vol 22 (4) ◽  
pp. 1508
Author(s):  
Jordi Maggi ◽  
Samuel Koller ◽  
Luzy Bähr ◽  
Silke Feil ◽  
Fatma Kivrak Pfiffner ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Long Range ◽  
Copy Number Variants ◽  
Retinal Disease ◽  
Promoter Regions ◽  
Long Range Pcr ◽  
Cost Efficient ◽  
Nucleotide Resolution ◽  
Genomic Regions ◽  
Next Generation Sequencing Ngs

The purpose of this study was to develop a flexible, cost-efficient, next-generation sequencing (NGS) protocol for genetic testing. Long-range polymerase chain reaction (PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel (n = 35) of inherited retinal disease (IRD)-associated loci. Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with IRD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, and (C) 25 undiagnosed after whole-exome sequencing (WES). The method was validated with 100% sensitivity on cohort A. Long-range PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 copy number variants (CNVs) could be characterized. Long-range PCR libraries spike-in extended coverage of WES. Read phasing confirmed compound heterozygosity in 5 probands. The proposed sequencing protocol provided deep coverage of the entire gene, including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, (ii) to elucidate missing heritability cases, (iii) to characterize breakpoints of CNVs at nucleotide resolution, (iv) to extend WES data to non-coding regions by spiking-in long-range PCR libraries, and (v) to help with phasing of candidate variants.

Towards single cell ad hoc protocols: First report of a healthy birth after preimplantation genetic diagnosis for methylmalonic acidemia

2015 ◽  
Vol 2 (2) ◽  
pp. 40-45
Author(s):  
Rosa Bautista-Llácer ◽  
Merche Pardo-Belenguer ◽  
Elena García-Mengual ◽  
Cristina Sánchez-Matamoros ◽  
Empar Raga ◽  
...  
Keyword(s):  
Single Cell ◽  
Preimplantation Genetic Diagnosis ◽  
Ad Hoc ◽  
Genetic Diagnosis ◽  
Methylmalonic Acidemia ◽  
First Report

Preimplantation Genetic Diagnosis for β-Thalassemia Using Single-cell DNA Analysis for Codons 17 and 26 of β-globin Gene

2009 ◽  
Vol 40 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Noor Wahidah Mohd Nasri ◽  
A. Rahman A. Jamal ◽  
Nurshaireen Chue Abdullah ◽  
Zainul Rashid Mohd Razi ◽  
Norfilza Mohd Mokhtar
Keyword(s):  
Single Cell ◽  
Preimplantation Genetic Diagnosis ◽  
Dna Analysis ◽  
Globin Gene ◽  
Genetic Diagnosis

Embryo Genome Profiling by Single-Cell Sequencing for Preimplantation Genetic Diagnosis in a β-Thalassemia Family

2015 ◽  
Vol 61 (4) ◽  
pp. 617-626 ◽  
Author(s):  
Yanwen Xu ◽  
Shengpei Chen ◽  
Xuyang Yin ◽  
Xiaoting Shen ◽  
Xiaoyu Pan ◽  
...  
Keyword(s):  
Single Cell ◽  
Preimplantation Genetic Diagnosis ◽  
Genetic Diagnosis ◽  
Human Leukocyte ◽  
Nucleotide Polymorphisms ◽  
Clinical Practices ◽  
Mendelian Disorder ◽  
Leukocyte Antigen ◽  
Single Cell Sequencing ◽  
Embryonic Genome

Abstract BACKGROUND The embryonic genome, including genotypes and haplotypes, contains all the information for preimplantation genetic diagnosis, representing great potential for mendelian disorder carriers to conceive healthy babies. METHODS We developed a strategy to obtain the full embryonic genome for a β-thalassemia–carrier couple to have a healthy second baby. We carried out sequencing for single blastomere cells and the family trio and further developed the analysis pipeline, including recovery of the missing alleles, removal of the majority of errors, and phasing of the embryonic genome. RESULTS The final accuracy for homozygous and heterozygous single-nucleotide polymorphisms reached 99.62% and 98.39%, respectively. The aneuploidies of embryos were detected as well. Based on the comprehensive embryonic genome, we effectively performed whole-genome mendelian disorder diagnosis and human leukocyte antigen matching tests. CONCLUSIONS This retrospective study in a β-thalassemia family demonstrates a method for embryo genome recovery through single-cell sequencing, which permits detection of genetic variations in preimplantation genetic diagnosis. It shows the potential of single-cell sequencing technology in preimplantation genetic diagnosis clinical practices.

scholarly journals Long-range PCR-based NGS applications to diagnose Mendelian retinal diseases

2020 ◽  
Author(s):  
Jordi Maggi ◽  
Samuel Koller ◽  
Luzy Bähr ◽  
Silke Feil ◽  
Fatma Kivrak Pfiffner ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Long Range ◽  
Retinal Diseases ◽  
Promoter Regions ◽  
Coding Regions ◽  
Polymerase Chain ◽  
Long Range Pcr ◽  
Cost Efficient ◽  
Genomic Regions ◽  
Next Generation Sequencing Ngs

AbstractBackgroundThe purpose of this study was to develop a flexible, cost-efficient next-generation sequencing (NGS) protocol for genetic testing.MethodsLong-range polymerase chain reaction (LR PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel (n=35) of loci associated with retinal diseases (RDs). Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with RD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, (C) 25 undiagnosed after exome sequencing (ES).ResultsThe method was validated with 100% sensitivity on cohort A. LR PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 CNVs could be characterized. LR PCR libraries spike-in extended coverage data of ES. Read phasing confirmed compound heterozygosity in 5 probands.ConclusionThe proposed sequencing protocol provided deep coverage of the entire gene including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, ii) to elucidate missing heritability cases, iii) to characterize breakpoints of CNVs at the nucleotide level, iv) to extend WES data to non-coding regions by spiking-in LR libraries, and v) to help with phasing of candidate variants.

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