Second-Tier Next Generation Sequencing Integrated in Nationwide Newborn Screening Provides Rapid Molecular Diagnostics of Severe Combined Immunodeficiency

Abstract Background Tandem mass spectrometry (MS/MS) has been used for newborn screening (NBS) of inherited metabolic diseases (IMDs) for decades. However, the traditional approach can yield false-positive or false-negative results and is affected by biochemical substrate-level fluctuations. To overcome the current limitations, we explored the possibility of using next-generation sequencing (NGS) as a second-tier diagnostic test to detect gene mutations in samples with abnormal MS/MS results. Methods Genomic DNA was extracted from dried blood spots and we designed a multigene panel, comprising 77 genes related to over 40 IMDs, for NBS. The prepared libraries were sequenced on the Ion Personal Genome Machine (PGM) platform. Thirty-eight samples identified as abnormal by MS/MS were tested for the diagnostic accuracy of NGS compared with Sanger sequencing. Results The concentration of DNA extracted from the 38 dried blood spots was sufficient for library preparation. The coverage and depth of the sequencing data were sufficient for the analysis. For all samples, the NGS results were consistent with the Sanger sequencing results. Conclusions The genomic DNA extracted from dried blood spots could be used for NGS, generating reliable sequencing results, and NGS may function as a second-tier diagnostic test for NBS. Ion PGM could facilitate the molecular diagnosis of IMDs with appropriate primers designed for candidate genes.

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Scalable Newborn Screening Solutions: Bioinformatics and Next-Generation Sequencing

International Journal of Neonatal Screening ◽

10.3390/ijns7040063 ◽

2021 ◽

Vol 7 (4) ◽

pp. 63

Author(s):

Nicole Ruiz-Schultz ◽

Bryce Asay ◽

Andreas Rohrwasser

Keyword(s):

Next Generation Sequencing ◽

Newborn Screening ◽

Screening Methods ◽

Next Generation ◽

Primary Screening ◽

Testing Method ◽

Second Tier ◽

Next Generation Sequencing Ngs ◽

Genomic Variant ◽

Generation Sequencing

Expansion of the newborn disorder panel requires the incorporation of new testing modalities. This is especially true for disorders lacking robust biomarkers for detection in primary screening methods and for disorders requiring genotyping or sequencing as a second-tier and/or diagnostic test. In this commentary, we discuss how next-generation sequencing (NGS) methods can be used as a secondary testing method in NBS. Additionally, we elaborate on the importance of genomic variant repositories for the annotation and interpretation of variants. Barriers to the incorporation of NGS and bioinformatics within NBS are discussed, and ideas for a regional bioinformatics model and shared variant repository are presented as potential solutions.

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Validation of a Custom Next-Generation Sequencing Assay for Cystic Fibrosis Newborn Screening

International Journal of Neonatal Screening ◽

10.3390/ijns7040073 ◽

2021 ◽

Vol 7 (4) ◽

pp. 73

Author(s):

Robert J. Sicko ◽

Colleen F. Stevens ◽

Erin E. Hughes ◽

Melissa Leisner ◽

Helen Ling ◽

...

Keyword(s):

Cystic Fibrosis ◽

New York ◽

Next Generation Sequencing ◽

Newborn Screening ◽

Disease Risk ◽

New York State ◽

Next Generation ◽

Second Tier ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Newborn screening (NBS) for Cystic Fibrosis (CF) is associated with improved outcomes. All US states screen for CF; however, CF NBS algorithms have high false positive (FP) rates. In New York State (NYS), the positive predictive value of CF NBS improved from 3.7% to 25.2% following the implementation of a three-tier IRT-DNA-SEQ approach using commercially available tests. Here we describe a modification of the NYS CF NBS algorithm via transition to a new custom next-generation sequencing (NGS) platform for more comprehensive cystic fibrosis transmembrane conductance regulator (CFTR) gene analysis. After full gene sequencing, a tiered strategy is used to first analyze only a specific panel of 338 clinically relevant CFTR variants (second-tier), followed by unblinding of all sequence variants and bioinformatic assessment of deletions/duplications in a subset of samples requiring third-tier analysis. We demonstrate the analytical and clinical validity of the assay and the feasibility of use in the NBS setting. The custom assay has streamlined our molecular workflow, increased throughput, and allows for bioinformatic customization of second-tier variant panel content. NBS aims to identify those infants with the highest disease risk. Technological molecular improvements can be applied to NBS algorithms to reduce the burden of FP referrals without loss of sensitivity.

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Next-generation sequencing and its applications in molecular diagnostics

Expert Review of Molecular Diagnostics ◽

10.1586/erm.11.3 ◽

2011 ◽

Vol 11 (3) ◽

pp. 333-343 ◽

Cited By ~ 103

Author(s):

Zhenqiang Su ◽

Baitang Ning ◽

Hong Fang ◽

Huixiao Hong ◽

Roger Perkins ◽

...

Keyword(s):

Next Generation Sequencing ◽

Molecular Diagnostics ◽

Next Generation ◽

Generation Sequencing

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ICT Aspects of Next-Generation-Sequencing Applied to Molecular Diagnostics

Handbook of Research on ICTs and Management Systems for Improving Efficiency in Healthcare and Social Care ◽

10.4018/978-1-4666-3990-4.ch047 ◽

2013 ◽

pp. 912-921

Author(s):

Saskia Biskup

Keyword(s):

Next Generation Sequencing ◽

Molecular Diagnostics ◽

Next Generation ◽

Detection Rates ◽

Genetic Causes ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Next-Generation-Sequencing (NGS) techniques are currently on the rise. This is seen as a revolution by (most) geneticists. The wealth of data stemming from Next-Generation-Sequencing will without a doubt lead to significant advances in the field of molecular diagnostics. On the clinical side, this will be higher detection rates of the genetic causes of particular diseases in patients. On the scientific side, NGS techniques will lead to the discovering of genes related to certain diseases (see, for example, Mardis, et al., 2009; Haack, et al., 2010; Lupski, et al., 2010). However, these advances come at a price: geneticists will be confronted with different and new ICT issues related to NGS. Because of the so far unknown amount of data stemming from NGS, these ICT issues need to be taken seriously. The purpose of this chapter is to give an overview on the different ICT aspects that come with the introduction of Next-Generation-Sequencing in molecular diagnostics.

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The long tail of molecular alterations in non-small cell lung cancer: a single-institution experience of next-generation sequencing in clinical molecular diagnostics

Journal of Clinical Pathology ◽

10.1136/jclinpath-2018-205032 ◽

2018 ◽

Vol 71 (9) ◽

pp. 767-773 ◽

Cited By ~ 6

Author(s):

Caterina Fumagalli ◽

Davide Vacirca ◽

Alessandra Rappa ◽

Antonio Passaro ◽

Juliana Guarize ◽

...

Keyword(s):

Next Generation Sequencing ◽

Molecular Diagnostics ◽

Small Cell ◽

Next Generation ◽

Small Cell Lung ◽

Single Institution ◽

Lung Cancers ◽

Targeted Next Generation Sequencing ◽

Molecular Alterations ◽

Generation Sequencing

BackgroundMolecular profiling of advanced non-small cell lung cancers (NSCLC) is essential to identify patients who may benefit from targeted treatments. In the last years, the number of potentially actionable molecular alterations has rapidly increased. Next-generation sequencing allows for the analysis of multiple genes simultaneously.AimsTo evaluate the feasibility and the throughput of next-generation sequencing in clinical molecular diagnostics of advanced NSCLC.MethodsA single-institution cohort of 535 non-squamous NSCLC was profiled using a next-generation sequencing panel targeting 22 actionable and cancer-related genes.Results441 non-squamous NSCLC (82.4%) harboured at least one gene alteration, including 340 cases (63.6%) with clinically relevant molecular aberrations. Mutations have been detected in all but one gene (FGFR1) of the panel. Recurrent alterations were observed in KRAS, TP53, EGFR, STK11 and MET genes, whereas the remaining genes were mutated in <5% of the cases. Concurrent mutations were detected in 183 tumours (34.2%), mostly impairing KRAS or EGFR in association with TP53 alterations.ConclusionsThe study highlights the feasibility of targeted next-generation sequencing in clinical setting. The majority of NSCLC harboured mutations in clinically relevant genes, thus identifying patients who might benefit from different targeted therapies.

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