Cargando…

Assessment of clinical analytical sensitivity and specificity of next-generation sequencing for detection of simple and complex mutations

BACKGROUND: Detecting mutations in disease genes by full gene sequence analysis is common in clinical diagnostic laboratories. Sanger dideoxy terminator sequencing allows for rapid development and implementation of sequencing assays in the clinical laboratory, but it has limited throughput, and due...

Descripción completa

Detalles Bibliográficos
Autores principales: Chin, Ephrem LH, da Silva, Cristina, Hegde, Madhuri
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3599218/
https://www.ncbi.nlm.nih.gov/pubmed/23418865
http://dx.doi.org/10.1186/1471-2156-14-6
_version_ 1782262913332936704
author Chin, Ephrem LH
da Silva, Cristina
Hegde, Madhuri
author_facet Chin, Ephrem LH
da Silva, Cristina
Hegde, Madhuri
author_sort Chin, Ephrem LH
collection PubMed
description BACKGROUND: Detecting mutations in disease genes by full gene sequence analysis is common in clinical diagnostic laboratories. Sanger dideoxy terminator sequencing allows for rapid development and implementation of sequencing assays in the clinical laboratory, but it has limited throughput, and due to cost constraints, only allows analysis of one or at most a few genes in a patient. Next-generation sequencing (NGS), on the other hand, has evolved rapidly, although to date it has mainly been used for large-scale genome sequencing projects and is beginning to be used in the clinical diagnostic testing. One advantage of NGS is that many genes can be analyzed easily at the same time, allowing for mutation detection when there are many possible causative genes for a specific phenotype. In addition, regions of a gene typically not tested for mutations, like deep intronic and promoter mutations, can also be detected. RESULTS: Here we use 20 previously characterized Sanger-sequenced positive controls in disease-causing genes to demonstrate the utility of NGS in a clinical setting using standard PCR based amplification to assess the analytical sensitivity and specificity of the technology for detecting all previously characterized changes (mutations and benign SNPs). The positive controls chosen for validation range from simple substitution mutations to complex deletion and insertion mutations occurring in autosomal dominant and recessive disorders. The NGS data was 100% concordant with the Sanger sequencing data identifying all 119 previously identified changes in the 20 samples. CONCLUSIONS: We have demonstrated that NGS technology is ready to be deployed in clinical laboratories. However, NGS and associated technologies are evolving, and clinical laboratories will need to invest significantly in staff and infrastructure to build the necessary foundation for success.
format Online
Article
Text
id pubmed-3599218
institution National Center for Biotechnology Information
language English
publishDate 2013
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-35992182013-03-17 Assessment of clinical analytical sensitivity and specificity of next-generation sequencing for detection of simple and complex mutations Chin, Ephrem LH da Silva, Cristina Hegde, Madhuri BMC Genet Research Article BACKGROUND: Detecting mutations in disease genes by full gene sequence analysis is common in clinical diagnostic laboratories. Sanger dideoxy terminator sequencing allows for rapid development and implementation of sequencing assays in the clinical laboratory, but it has limited throughput, and due to cost constraints, only allows analysis of one or at most a few genes in a patient. Next-generation sequencing (NGS), on the other hand, has evolved rapidly, although to date it has mainly been used for large-scale genome sequencing projects and is beginning to be used in the clinical diagnostic testing. One advantage of NGS is that many genes can be analyzed easily at the same time, allowing for mutation detection when there are many possible causative genes for a specific phenotype. In addition, regions of a gene typically not tested for mutations, like deep intronic and promoter mutations, can also be detected. RESULTS: Here we use 20 previously characterized Sanger-sequenced positive controls in disease-causing genes to demonstrate the utility of NGS in a clinical setting using standard PCR based amplification to assess the analytical sensitivity and specificity of the technology for detecting all previously characterized changes (mutations and benign SNPs). The positive controls chosen for validation range from simple substitution mutations to complex deletion and insertion mutations occurring in autosomal dominant and recessive disorders. The NGS data was 100% concordant with the Sanger sequencing data identifying all 119 previously identified changes in the 20 samples. CONCLUSIONS: We have demonstrated that NGS technology is ready to be deployed in clinical laboratories. However, NGS and associated technologies are evolving, and clinical laboratories will need to invest significantly in staff and infrastructure to build the necessary foundation for success. BioMed Central 2013-02-19 /pmc/articles/PMC3599218/ /pubmed/23418865 http://dx.doi.org/10.1186/1471-2156-14-6 Text en Copyright ©2013 Chin et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Chin, Ephrem LH
da Silva, Cristina
Hegde, Madhuri
Assessment of clinical analytical sensitivity and specificity of next-generation sequencing for detection of simple and complex mutations
title Assessment of clinical analytical sensitivity and specificity of next-generation sequencing for detection of simple and complex mutations
title_full Assessment of clinical analytical sensitivity and specificity of next-generation sequencing for detection of simple and complex mutations
title_fullStr Assessment of clinical analytical sensitivity and specificity of next-generation sequencing for detection of simple and complex mutations
title_full_unstemmed Assessment of clinical analytical sensitivity and specificity of next-generation sequencing for detection of simple and complex mutations
title_short Assessment of clinical analytical sensitivity and specificity of next-generation sequencing for detection of simple and complex mutations
title_sort assessment of clinical analytical sensitivity and specificity of next-generation sequencing for detection of simple and complex mutations
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3599218/
https://www.ncbi.nlm.nih.gov/pubmed/23418865
http://dx.doi.org/10.1186/1471-2156-14-6
work_keys_str_mv AT chinephremlh assessmentofclinicalanalyticalsensitivityandspecificityofnextgenerationsequencingfordetectionofsimpleandcomplexmutations
AT dasilvacristina assessmentofclinicalanalyticalsensitivityandspecificityofnextgenerationsequencingfordetectionofsimpleandcomplexmutations
AT hegdemadhuri assessmentofclinicalanalyticalsensitivityandspecificityofnextgenerationsequencingfordetectionofsimpleandcomplexmutations