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

ABSTRACT: Despite the improved accuracy of next-generation sequencing (NGS), it is widely accepted that variants need to be validated using Sanger sequencing before reporting. Validation of all NGS variants considerably increases the turnaround time and costs of clinical diagnosis. We comprehensively assessed this need in 1109 variants from 825 clinical exomes, the largest sample set to date assessed using Illumina chemistry reported. With a concordance of 100%, we conclude that Sanger sequencing can be very useful as an internal quality control, but not so much as a verification method for high-quality single-nucleotide and small insertion/deletions variants. Laboratories might validate and establish their own thresholds before discontinuing Sanger confirmation studies. We also expand and validate 23 copy number variations detected by exome sequencing in 20 samples, observing a concordance of 95.65% (22/23).

SUBMITTER: Arteche-Lopez A

PROVIDER: S-EPMC7952542 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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

Arteche-López A A Ávila-Fernández A A Romero R R Riveiro-Álvarez R R López-Martínez M A MA Giménez-Pardo A A Vélez-Monsalve C C Gallego-Merlo J J García-Vara I I Almoguera Berta B Bustamante-Aragonés A A Blanco-Kelly F F Tahsin-Swafiri S S Rodríguez-Pinilla E E Minguez P P Lorda I I Trujillo-Tiebas M J MJ Ayuso C C

Scientific reports 20210311 1

Despite the improved accuracy of next-generation sequencing (NGS), it is widely accepted that variants need to be validated using Sanger sequencing before reporting. Validation of all NGS variants considerably increases the turnaround time and costs of clinical diagnosis. We comprehensively assessed this need in 1109 variants from 825 clinical exomes, the largest sample set to date assessed using Illumina chemistry reported. With a concordance of 100%, we conclude that Sanger sequencing can be v ...[more]

PMID: 33707547

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Project description:BACKGROUND: Usually, next generation sequencing (NGS) technology has the property of ultra-high throughput but the read length is remarkably short compared to conventional Sanger sequencing. Paired-end NGS could computationally extend the read length but with a lot of practical inconvenience because of the inherent gaps. Now that Illumina paired-end sequencing has the ability of read both ends from 600 bp or even 800 bp DNA fragments, how to fill in the gaps between paired ends to produce accurate long reads is intriguing but challenging. RESULTS: We have developed a new technology, referred to as pseudo-Sanger (PS) sequencing. It tries to fill in the gaps between paired ends and could generate near error-free sequences equivalent to the conventional Sanger reads in length but with the high throughput of the Next Generation Sequencing. The major novelty of PS method lies on that the gap filling is based on local assembly of paired-end reads which have overlaps with at either end. Thus, we are able to fill in the gaps in repetitive genomic region correctly. The PS sequencing starts with short reads from NGS platforms, using a series of paired-end libraries of stepwise decreasing insert sizes. A computational method is introduced to transform these special paired-end reads into long and near error-free PS sequences, which correspond in length to those with the largest insert sizes. The PS construction has 3 advantages over untransformed reads: gap filling, error correction and heterozygote tolerance. Among the many applications of the PS construction is de novo genome assembly, which we tested in this study. Assembly of PS reads from a non-isogenic strain of Drosophila melanogaster yields an N50 contig of 190 kb, a 5 fold improvement over the existing de novo assembly methods and a 3 fold advantage over the assembly of long reads from 454 sequencing. CONCLUSIONS: Our method generated near error-free long reads from NGS paired-end sequencing. We demonstrated that de novo assembly could benefit a lot from these Sanger-like reads. Besides, the characteristic of the long reads could be applied to such applications as structural variations detection and metagenomics.

Project description:ImportanceDiagnostic genetic testing can lead to changes in management in the pediatric intensive care unit. Genetic risk in children with critical illness but nondiagnostic exome sequencing (ES) has not been explored.ObjectiveTo assess the association between loss-of-function (LOF) variants and pediatric critical illness.Design, setting, and participantsThis genetic association study examined ES first screened for causative variants among 267 children at the Morgan Stanley Children's Hospital of NewYork-Presbyterian, of whom 22 were otherwise healthy with viral respiratory failure; 18 deceased children with bronchiolitis from the Office of the Chief Medical Examiner of New York City, of whom 14 were previously healthy; and 9990 controls from the Institute for Genomic Medicine at Columbia University Irving Medical Center. The ES data were generated between January 1, 2015, and December 31, 2020, and analyzed between January 1, 2017, and September 2, 2022.ExposureCritical illness.Main outcomes and measuresOdds ratios and P values for genes and gene-sets enriched for rare LOF variants and the loss-of-function observed/expected upper bound fraction (LOEUF) score at which cases have a significant enrichment.ResultsThis study included 285 children with critical illness (median [range] age, 4.1 [0-18.9] years; 148 [52%] male) and 9990 controls. A total of 228 children (80%) did not receive a genetic diagnosis. After quality control (QC), 231 children harbored excess rare LOF variants in genes with a LOEUF score of 0.680 or less (intolerant genes) (P = 1.0 × 10-5). After QC, 176 children without a diagnosis harbored excess ultrarare LOF variants in intolerant genes but only in those without a known disease association (odds ratio, 1.8; 95% CI, 1.3-2.5). After QC, 25 children with viral respiratory failure harbored excess ultrarare LOF variants in intolerant genes but only in those without a known disease association (odds ratio, 2.8; 95% CI, 1.1-6.6). A total of 114 undiagnosed children were enriched for de novo LOF variants in genes without a known disease association (observed, 14; expected, 6.8; enrichment, 2.05).Conclusions and relevanceIn this genetic association study, excess LOF variants were observed among critically ill children despite nondiagnostic ES. Variants lay in genes without a known disease association, suggesting future investigation may connect phenotypes to causative genes.

Dataset Information

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

Publications

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

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