Next Generation Sequencing (NGS) Target Approach for Undiagnosed Dysglycaemia
Next-generation sequencing (NGS) has revolutionized the field of genomics and created new opportunities for basic research. We described the strategy for the NGS validation of the “dysglycaemia panel” composed by 44 genes related to glucose metabolism disorders (MODY, Wolfram syndrome) and familial...
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MDPI AG
2023-04-01
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| Online Access: | https://www.mdpi.com/2075-1729/13/5/1080 |
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| author | Concetta Aloi Alessandro Salina Francesco Caroli Renata Bocciardi Barbara Tappino Marta Bassi Nicola Minuto Giuseppe d’Annunzio Mohamad Maghnie |
| author_facet | Concetta Aloi Alessandro Salina Francesco Caroli Renata Bocciardi Barbara Tappino Marta Bassi Nicola Minuto Giuseppe d’Annunzio Mohamad Maghnie |
| author_sort | Concetta Aloi |
| collection | DOAJ |
| description | Next-generation sequencing (NGS) has revolutionized the field of genomics and created new opportunities for basic research. We described the strategy for the NGS validation of the “dysglycaemia panel” composed by 44 genes related to glucose metabolism disorders (MODY, Wolfram syndrome) and familial renal glycosuria using Ion AmpliSeq technology combined with Ion-PGM. Anonymized DNA of 32 previously genotyped cases with 33 different variants were used to optimize the methodology. Standard protocol was used to generate the primer design, library, template preparation, and sequencing. Ion Reporter tool was used for data analysis. In all the runs, the mean coverage was over 200×. Twenty-nine out of thirty three variants (96.5%) were detected; four frameshift variants were missed. All point mutations were detected with high sensitivity. We identified three further variants of unknown significance in addition to pathogenic mutations previously identified by Sanger sequencing. The NGS panel allowed us to identify pathogenic variants in multiple genes in a short time. This could help to identify several defects in children and young adults that have to receive the genetic diagnosis necessary for optimal treatment. In order not to lose any pathogenic variants, Sanger sequencing is included in our analytical protocol to avoid missing frameshift variants. |
| first_indexed | 2024-03-11T03:34:12Z |
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| id | doaj.art-2939ff361dbe4b1499f6ef1a039dd25d |
| institution | Directory Open Access Journal |
| issn | 2075-1729 |
| language | English |
| last_indexed | 2024-03-11T03:34:12Z |
| publishDate | 2023-04-01 |
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| record_format | Article |
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| spelling | doaj.art-2939ff361dbe4b1499f6ef1a039dd25d2023-11-18T02:08:23ZengMDPI AGLife2075-17292023-04-01135108010.3390/life13051080Next Generation Sequencing (NGS) Target Approach for Undiagnosed DysglycaemiaConcetta Aloi0Alessandro Salina1Francesco Caroli2Renata Bocciardi3Barbara Tappino4Marta Bassi5Nicola Minuto6Giuseppe d’Annunzio7Mohamad Maghnie8LABSIEM (Laboratory for the Study of Inborn Errors of Metabolism), IRCCS Istituto Giannina Gaslini, 16147 Genoa, ItalyLABSIEM (Laboratory for the Study of Inborn Errors of Metabolism), IRCCS Istituto Giannina Gaslini, 16147 Genoa, ItalyUOC Genetica Medica, IRCCS Istituto Giannina Gaslini, 16147 Genoa, ItalyUOC Genetica Medica, IRCCS Istituto Giannina Gaslini, 16147 Genoa, ItalyLABSIEM (Laboratory for the Study of Inborn Errors of Metabolism), IRCCS Istituto Giannina Gaslini, 16147 Genoa, ItalyDepartment of Neuroscience, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health (DINOGMI), University of Genoa, 16100 Genoa, ItalyDepartment of Pediatrics, IRCCS Istituto Giannina Gaslini, 16147 Genoa, ItalyDepartment of Pediatrics, IRCCS Istituto Giannina Gaslini, 16147 Genoa, ItalyDepartment of Neuroscience, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health (DINOGMI), University of Genoa, 16100 Genoa, ItalyNext-generation sequencing (NGS) has revolutionized the field of genomics and created new opportunities for basic research. We described the strategy for the NGS validation of the “dysglycaemia panel” composed by 44 genes related to glucose metabolism disorders (MODY, Wolfram syndrome) and familial renal glycosuria using Ion AmpliSeq technology combined with Ion-PGM. Anonymized DNA of 32 previously genotyped cases with 33 different variants were used to optimize the methodology. Standard protocol was used to generate the primer design, library, template preparation, and sequencing. Ion Reporter tool was used for data analysis. In all the runs, the mean coverage was over 200×. Twenty-nine out of thirty three variants (96.5%) were detected; four frameshift variants were missed. All point mutations were detected with high sensitivity. We identified three further variants of unknown significance in addition to pathogenic mutations previously identified by Sanger sequencing. The NGS panel allowed us to identify pathogenic variants in multiple genes in a short time. This could help to identify several defects in children and young adults that have to receive the genetic diagnosis necessary for optimal treatment. In order not to lose any pathogenic variants, Sanger sequencing is included in our analytical protocol to avoid missing frameshift variants.https://www.mdpi.com/2075-1729/13/5/1080performance validationNGSmonogenic diabetesMODYWolfram syndromecongenital hyperinsulinism |
| spellingShingle | Concetta Aloi Alessandro Salina Francesco Caroli Renata Bocciardi Barbara Tappino Marta Bassi Nicola Minuto Giuseppe d’Annunzio Mohamad Maghnie Next Generation Sequencing (NGS) Target Approach for Undiagnosed Dysglycaemia Life performance validation NGS monogenic diabetes MODY Wolfram syndrome congenital hyperinsulinism |
| title | Next Generation Sequencing (NGS) Target Approach for Undiagnosed Dysglycaemia |
| title_full | Next Generation Sequencing (NGS) Target Approach for Undiagnosed Dysglycaemia |
| title_fullStr | Next Generation Sequencing (NGS) Target Approach for Undiagnosed Dysglycaemia |
| title_full_unstemmed | Next Generation Sequencing (NGS) Target Approach for Undiagnosed Dysglycaemia |
| title_short | Next Generation Sequencing (NGS) Target Approach for Undiagnosed Dysglycaemia |
| title_sort | next generation sequencing ngs target approach for undiagnosed dysglycaemia |
| topic | performance validation NGS monogenic diabetes MODY Wolfram syndrome congenital hyperinsulinism |
| url | https://www.mdpi.com/2075-1729/13/5/1080 |
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