Massively parallel sequencing of micro-manipulated cells targeting a comprehensive panel of disease-causing genes: A comparative evaluation of upstream whole-genome amplification methods
| Autor: | Yannick Gansemans, Filip Van Nieuwerburgh, Lieselot Deleye, Dieter De Coninck, Dieter Deforce |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Male lcsh:Medicine Gene Sequencing Genome Polymerase Chain Reaction Database and Informatics Methods 0302 clinical medicine Genomic library DNA sequencing Genome Sequencing lcsh:Science Whole Genome Amplification 030219 obstetrics & reproductive medicine Multidisciplinary Massive parallel sequencing High-Throughput Nucleotide Sequencing Genomics Gene Pool Neoplastic Cells Circulating ALIGNMENT Single-Cell Analysis Nucleic Acid Amplification Techniques Sequence Analysis Research Article Genotyping DNA Copy Number Variations Genotype Bioinformatics Nucleotide Sequencing Computational biology Biology ABERRATIONS Research and Analysis Methods Polymorphism Single Nucleotide Cell Line Molecular Genetics EMBRYOS 03 medical and health sciences Genetics Humans Molecular Biology Techniques Sequencing Techniques Molecular Biology Gene Library Evolutionary Biology Population Biology ANEUPLOIDY Genome Human lcsh:R Biology and Life Sciences Computational Biology Nucleic acid amplification technique DNA Sequence Analysis DNA Genome Analysis Genomic Libraries 030104 developmental biology Human genome lcsh:Q Sequence Alignment Population Genetics GENERATION |
| Zdroj: | PLoS ONE PLoS ONE, Vol 13, Iss 4, p e0196334 (2018) PLOS ONE |
| ISSN: | 1932-6203 |
| Popis: | Single Gene Disorders (SGD) are still routinely diagnosed using PCR-based assays that need to be developed and validated for each individual disease-specific gene fragment. The TruSight One sequencing panel currently covers 12 Mb of genomic content, including 4813 genes associated with a clinical phenotype. When only a limited number of cells are available, whole genome amplification (WGA) is required prior to DNA target capture techniques such as the TruSight One panel. In this study, we compared 4 different WGA methods in combination with the TruSight One sequencing panel to perform single nucleotide polymorphism (SNP) genotyping starting from 3 micro-manipulated cells. This setting simulates clinical settings such as day-5 blastocyst biopsy for Preimplantation Genetic Testing (PGT), liquid biopsy of circulating tumor cells (CTCs) and cancer-cell profiling. Bulk cell samples were processed alongside these WGA samples to serve as a performance reference. Target coverage, coverage uniformity and SNP calling accuracy obtained using any of the WGA, is inferior to the results obtained on bulk cell samples. However, results after REPLI-g come close. Compared to the other WGA methods, the method using REPLI-g WGA results in a better coverage of the targeted genomic regions with a more uniform read depth. Consequently, this method also results in a more accurate SNP calling and could be considered for clinical genotyping of a limited number of cells. |
| Databáze: | OpenAIRE |
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