Sanger sequencing is no longer always necessary based on a single-center validation of 1109 NGS variants in 825 clinical exomes

Arteche‐López, Ana; Ávila-Fernández, Almudena; Romero, Raquel Escutia; Riveiro-Álvarez, Rosa; López-Martínez, Miguel Ángel; Giménez-Pardo, A; Vélez-Monsalve, Camilo; Gallego-Merlo, J.; García-Vara, I; Almoguera, Berta; Bustamante-Aragonés, Ana; Blanco‐Kelly, Fiona; Tahsin-Swafiri, Saoud; Rodríguez‐Pinilla, Elvira; Mínguez, Pablo; Lorda, Isabel; Trujillo-Tiebas, María José; Ayuso, Carmen

doi:10.1038/s41598-021-85182-w

Cited by 39 publications

(34 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…By convention, NGS results necessitate validation by Sanger sequencing as the reference standard. However, recently the necessity of Sanger validation has been called into question for human genome sequencing by several studies [38,39]. Arteche-Lopez et al, reported their validation study of 1109 NGS variants in 825 clinical exomes, the largest sample set to date using Illumina technology [38].…”

Section: Discussionmentioning

confidence: 99%

“…However, recently the necessity of Sanger validation has been called into question for human genome sequencing by several studies [38,39]. Arteche-Lopez et al, reported their validation study of 1109 NGS variants in 825 clinical exomes, the largest sample set to date using Illumina technology [38]. Only three discrepancies were found, and all false negative results arose from Sanger sequencing [38].…”

Section: Discussionmentioning

confidence: 99%

“…Arteche-Lopez et al, reported their validation study of 1109 NGS variants in 825 clinical exomes, the largest sample set to date using Illumina technology [38]. Only three discrepancies were found, and all false negative results arose from Sanger sequencing [38]. Taken together, high-quality NGS and analytical methods offer higher resolution and accuracy than Sanger/BLAST and only selective validation may be necessary.…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

HPV DeepSeq: An Ultra-Fast Method of NGS Data Analysis and Visualization Using Automated Workflows and a Customized Papillomavirus Database in CLC Genomics Workbench

et al. 2021

View full text Add to dashboard Cite

Next-generation sequencing (NGS) has actualized the human papillomavirus (HPV) virome profiling for in-depth investigation of viral evolution and pathogenesis. However, viral computational analysis remains a bottleneck due to semantic discrepancies between computational tools and curated reference genomes. To address this, we developed and tested automated workflows for HPV taxonomic profiling and visualization using a customized papillomavirus database in the CLC Microbial Genomics Module. HPV genomes from Papilloma Virus Episteme were customized and incorporated into CLC “ready-to-use” workflows for stepwise data processing to include: (1) Taxonomic Analysis, (2) Estimate Alpha/Beta Diversities, and (3) Map Reads to Reference. Low-grade (n = 95) and high-grade (n = 60) Pap smears were tested with ensuing collective runtimes: Taxonomic Analysis (36 min); Alpha/Beta Diversities (5 s); Map Reads (45 min). Tabular output conversion to visualizations entailed 1–2 keystrokes. Biodiversity analysis between low- (LSIL) and high-grade squamous intraepithelial lesions (HSIL) revealed loss of species richness and gain of dominance by HPV-16 in HSIL. Integrating clinically relevant, taxonomized HPV reference genomes within automated workflows proved to be an ultra-fast method of virome profiling. The entire process named “HPV DeepSeq” provides a simple, accurate and practical means of NGS data analysis for a broad range of applications in viral research.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

HPV DeepSeq: An Ultra-Fast Method of NGS Data Analysis and Visualization Using Automated Workflows and a Customized Papillomavirus Database in CLC Genomics Workbench

et al. 2021

View full text Add to dashboard Cite

show abstract

“…By convention, NGS results necessitate validation by Sanger sequencing as the reference standard. However, recently the necessity of Sanger validation has been called into question for human genome sequencing by several studies [25,26]. Arteche-Lopez et al, reported their validation study of 1,109 NGS variants in 825 clinical exomes, the largest sample set to date using Illumina technology [25].…”

Section: Discussionmentioning

confidence: 99%

“…However, recently the necessity of Sanger validation has been called into question for human genome sequencing by several studies [25,26]. Arteche-Lopez et al, reported their validation study of 1,109 NGS variants in 825 clinical exomes, the largest sample set to date using Illumina technology [25]. Only 3 discrepancies were found, and all false negative results arose from Sanger sequencing [25].…”

Section: Discussionmentioning

confidence: 99%

HPV DeepSeq: Ultra-Fast Method of NGS Data Analysis and Visualization Using Automated Workflows and a Customized Papillomavirus Database in CLC Genomics Workbench

Shen-Gunther¹,

Xia²,

Cai³

et al. 2021

Preprint

View full text Add to dashboard Cite

Next-generation sequencing (NGS) has actualized human papillomavirus (HPV) virome profiling for in-depth investigation of viral evolution and pathogenesis. However, viral computational analysis remains a bottleneck due to semantic discrepancies between computational tools and curated reference genomes. To address this, we developed and tested automated workflows for HPV taxonomic profiling and visualization using a customized Papillomavirus database in CLC Microbial Genomics Module. HPV genomes from Papilloma Virus Episteme were customized and incorporated into CLC “ready-to-use” workflows for stepwise data processing to include: 1) Taxonomic Analysis, 2) Estimate Alpha/Beta Diversities, and 3) Map Reads to Reference. Low-grade (n = 95) and high-grade (n = 60) Pap smears were tested with ensuing collective runtimes: Taxonomic Analysis (36 min); Alpha/Beta Diversities (5 sec); Map Reads (45 min). Tabular output conversion to visualizations entailed 1-2 keystrokes. Biodiversity analysis between low- (LSIL) and high-grade squamous intraepithelial lesions (HSIL) revealed loss of species richness and gain of dominance by HPV-16 in HSIL. Integrating clinically relevant, taxonomized HPV reference genomes within automated workflows proved to be an ultra-fast method of virome profiling. The entire process named “HPV DeepSeq” provides a simple, accurate and practical means of NGS data analysis for a broad range of applications in viral research.

show abstract

Inconsistencies between prenatal diagnostic and genetic testing laboratories on variant validation of rare monogenic diseases

Lin,

Zhang,

Pan

et al. 2024

Prenatal Diagnosis

View full text Add to dashboard Cite

BackgroundThe advent of next‐generation sequencing (NGS) has enhanced the diagnostic efficacy for monogenic diseases, while presenting challenges in achieving consistent diagnoses.MethodWe retrospectively analyzed the concordance rate and reasons for the inconsistency between the original diagnostic result from the genetic testing laboratory and the variant validation result from the prenatal diagnostic center. The validation procedure comprised three stages: validation of variant detection, reevaluation of variant classification, and assessment of recurrence risk, which involved verifying the mode of inheritance and parental carriage.ResultIn total, 17 (6%) of the 286 families affected by rare monogenic diseases showed different results during the variant validation procedure. These cases comprised four (23.5%) with variant detection errors, 12 (70.5%) with inconsistent interpretation, and one (6%) with non‐Mendelian inheritance patterns. False‐positive NGS results confirmed by Sanger sequencing were related to pseudogenes and GC‐rich regions. The classification of the 17 variants was altered in the 12 cases owing to various factors. The case with an atypical inheritance pattern was originally considered autosomal recessive inheritance, but was diagnosed as maternal uniparental disomy after additional genetic analysis.ConclusionWe underscored the significance of variant validation by prenatal diagnostic centers. Families affected by monogenic diseases with reproductive plans should be referred to prenatal genetic centers as early as possible to avoid different results that may postpone subsequent prenatal diagnosis.

show abstract

Sanger sequencing is no longer always necessary based on a single-center validation of 1109 NGS variants in 825 clinical exomes

Cited by 39 publications

References 18 publications

HPV DeepSeq: An Ultra-Fast Method of NGS Data Analysis and Visualization Using Automated Workflows and a Customized Papillomavirus Database in CLC Genomics Workbench

HPV DeepSeq: An Ultra-Fast Method of NGS Data Analysis and Visualization Using Automated Workflows and a Customized Papillomavirus Database in CLC Genomics Workbench

HPV DeepSeq: Ultra-Fast Method of NGS Data Analysis and Visualization Using Automated Workflows and a Customized Papillomavirus Database in CLC Genomics Workbench

Inconsistencies between prenatal diagnostic and genetic testing laboratories on variant validation of rare monogenic diseases

Contact Info

Product

Resources

About