Development and validation of an expanded targeted sequencing panel for non-invasive prenatal diagnosis of sporadic skeletal dysplasia

Abstract Background Skeletal dysplasia (SD) is one of the most common inherited neonatal disorders worldwide, where the recurrent pathogenic mutations in the FGFR2, FGFR3, COL1A1, COL1A2 and COL2A1 genes are frequently reported in both non-lethal and lethal SD. The traditional prenatal diagnosis of...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Ching-Yuan Wang, Yen-An Tang, I-Wen Lee, Fong-Ming Chang, Chun-Wei Chien, Hsien-An Pan, H. Sunny Sun
Formato:	article
Lenguaje:	EN
Publicado:	BMC 2021
Materias:	Skeletal dysplasia Amplicon-based targeted sequencing Noninvasive prenatal testing (NIPT) Precision medicine Internal medicine RC31-1245 Genetics QH426-470
Acceso en línea:	https://doaj.org/article/ad5d500e713c435cb2652abefd56b047
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:ad5d500e713c435cb2652abefd56b047
record_format	dspace
spelling	oai:doaj.org-article:ad5d500e713c435cb2652abefd56b0472021-11-21T12:04:18ZDevelopment and validation of an expanded targeted sequencing panel for non-invasive prenatal diagnosis of sporadic skeletal dysplasia10.1186/s12920-021-01063-11755-8794https://doaj.org/article/ad5d500e713c435cb2652abefd56b0472021-11-01T00:00:00Zhttps://doi.org/10.1186/s12920-021-01063-1https://doaj.org/toc/1755-8794Abstract Background Skeletal dysplasia (SD) is one of the most common inherited neonatal disorders worldwide, where the recurrent pathogenic mutations in the FGFR2, FGFR3, COL1A1, COL1A2 and COL2A1 genes are frequently reported in both non-lethal and lethal SD. The traditional prenatal diagnosis of SD using ultrasonography suffers from lower accuracy and performed at latter gestational stage. Therefore, it remains in desperate need of precise and accurate prenatal diagnosis of SD in early pregnancy. With the advancements of next-generation sequencing (NGS) technology and bioinformatics analysis, it is feasible to develop a NGS-based assay to detect genetic defects in association with SD in the early pregnancy. Methods An ampliseq-based targeted sequencing panel was designed to cover 87 recurrent hotspots reported in 11 common dominant SD and run on both Ion Proton and NextSeq550 instruments. Thirty-six cell-free and 23 genomic DNAs were used for assay developed. Spike-in DNA prepared from standard sample harboring known mutation and normal sample were also employed to validate the established SD workflow. Overall performances of coverage, uniformity, and on-target rate, and the detecting limitations on percentage of fetal fraction and read depth were evaluated. Results The established targeted-seq workflow enables a single-tube multiplex PCR for library construction and shows high amplification efficiency and robust reproducibility on both Ion Proton and NextSeq550 platforms. The workflow reaches 100% coverage and both uniformity and on-target rate are > 96%, indicating a high quality assay. Using spike-in DNA with different percentage of known FGFR3 mutation (c.1138 G > A), the targeted-seq workflow demonstrated the ability to detect low-frequency variant of 2.5% accurately. Finally, we obtained 100% sensitivity and 100% specificity in detecting target mutations using established SD panel. Conclusions An expanded panel for rapid and cost-effective genetic detection of SD has been developed. The established targeted-seq workflow shows high accuracy to detect both germline and low-frequency variants. In addition, the workflow is flexible to be conducted in the majority of the NGS instruments and ready for routine clinical application. Taken together, we believe the established panel provides a promising diagnostic or therapeutic strategy for prenatal genetic testing of SD in routine clinical practice.Ching-Yuan WangYen-An TangI-Wen LeeFong-Ming ChangChun-Wei ChienHsien-An PanH. Sunny SunBMCarticleSkeletal dysplasiaAmplicon-based targeted sequencingNoninvasive prenatal testing (NIPT)Precision medicineInternal medicineRC31-1245GeneticsQH426-470ENBMC Medical Genomics, Vol 14, Iss S3, Pp 1-13 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Skeletal dysplasia Amplicon-based targeted sequencing Noninvasive prenatal testing (NIPT) Precision medicine Internal medicine RC31-1245 Genetics QH426-470
spellingShingle	Skeletal dysplasia Amplicon-based targeted sequencing Noninvasive prenatal testing (NIPT) Precision medicine Internal medicine RC31-1245 Genetics QH426-470 Ching-Yuan Wang Yen-An Tang I-Wen Lee Fong-Ming Chang Chun-Wei Chien Hsien-An Pan H. Sunny Sun Development and validation of an expanded targeted sequencing panel for non-invasive prenatal diagnosis of sporadic skeletal dysplasia
description	Abstract Background Skeletal dysplasia (SD) is one of the most common inherited neonatal disorders worldwide, where the recurrent pathogenic mutations in the FGFR2, FGFR3, COL1A1, COL1A2 and COL2A1 genes are frequently reported in both non-lethal and lethal SD. The traditional prenatal diagnosis of SD using ultrasonography suffers from lower accuracy and performed at latter gestational stage. Therefore, it remains in desperate need of precise and accurate prenatal diagnosis of SD in early pregnancy. With the advancements of next-generation sequencing (NGS) technology and bioinformatics analysis, it is feasible to develop a NGS-based assay to detect genetic defects in association with SD in the early pregnancy. Methods An ampliseq-based targeted sequencing panel was designed to cover 87 recurrent hotspots reported in 11 common dominant SD and run on both Ion Proton and NextSeq550 instruments. Thirty-six cell-free and 23 genomic DNAs were used for assay developed. Spike-in DNA prepared from standard sample harboring known mutation and normal sample were also employed to validate the established SD workflow. Overall performances of coverage, uniformity, and on-target rate, and the detecting limitations on percentage of fetal fraction and read depth were evaluated. Results The established targeted-seq workflow enables a single-tube multiplex PCR for library construction and shows high amplification efficiency and robust reproducibility on both Ion Proton and NextSeq550 platforms. The workflow reaches 100% coverage and both uniformity and on-target rate are > 96%, indicating a high quality assay. Using spike-in DNA with different percentage of known FGFR3 mutation (c.1138 G > A), the targeted-seq workflow demonstrated the ability to detect low-frequency variant of 2.5% accurately. Finally, we obtained 100% sensitivity and 100% specificity in detecting target mutations using established SD panel. Conclusions An expanded panel for rapid and cost-effective genetic detection of SD has been developed. The established targeted-seq workflow shows high accuracy to detect both germline and low-frequency variants. In addition, the workflow is flexible to be conducted in the majority of the NGS instruments and ready for routine clinical application. Taken together, we believe the established panel provides a promising diagnostic or therapeutic strategy for prenatal genetic testing of SD in routine clinical practice.
format	article
author	Ching-Yuan Wang Yen-An Tang I-Wen Lee Fong-Ming Chang Chun-Wei Chien Hsien-An Pan H. Sunny Sun
author_facet	Ching-Yuan Wang Yen-An Tang I-Wen Lee Fong-Ming Chang Chun-Wei Chien Hsien-An Pan H. Sunny Sun
author_sort	Ching-Yuan Wang
title	Development and validation of an expanded targeted sequencing panel for non-invasive prenatal diagnosis of sporadic skeletal dysplasia
title_short	Development and validation of an expanded targeted sequencing panel for non-invasive prenatal diagnosis of sporadic skeletal dysplasia
title_full	Development and validation of an expanded targeted sequencing panel for non-invasive prenatal diagnosis of sporadic skeletal dysplasia
title_fullStr	Development and validation of an expanded targeted sequencing panel for non-invasive prenatal diagnosis of sporadic skeletal dysplasia
title_full_unstemmed	Development and validation of an expanded targeted sequencing panel for non-invasive prenatal diagnosis of sporadic skeletal dysplasia
title_sort	development and validation of an expanded targeted sequencing panel for non-invasive prenatal diagnosis of sporadic skeletal dysplasia
publisher	BMC
publishDate	2021
url	https://doaj.org/article/ad5d500e713c435cb2652abefd56b047
work_keys_str_mv	AT chingyuanwang developmentandvalidationofanexpandedtargetedsequencingpanelfornoninvasiveprenataldiagnosisofsporadicskeletaldysplasia AT yenantang developmentandvalidationofanexpandedtargetedsequencingpanelfornoninvasiveprenataldiagnosisofsporadicskeletaldysplasia AT iwenlee developmentandvalidationofanexpandedtargetedsequencingpanelfornoninvasiveprenataldiagnosisofsporadicskeletaldysplasia AT fongmingchang developmentandvalidationofanexpandedtargetedsequencingpanelfornoninvasiveprenataldiagnosisofsporadicskeletaldysplasia AT chunweichien developmentandvalidationofanexpandedtargetedsequencingpanelfornoninvasiveprenataldiagnosisofsporadicskeletaldysplasia AT hsienanpan developmentandvalidationofanexpandedtargetedsequencingpanelfornoninvasiveprenataldiagnosisofsporadicskeletaldysplasia AT hsunnysun developmentandvalidationofanexpandedtargetedsequencingpanelfornoninvasiveprenataldiagnosisofsporadicskeletaldysplasia
_version_	1718419266543288320

Development and validation of an expanded targeted sequencing panel for non-invasive prenatal diagnosis of sporadic skeletal dysplasia

Ejemplares similares