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

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.

scholarly journals Scalable Newborn Screening Solutions: Bioinformatics and Next-Generation Sequencing

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.

scholarly journals Next generation sequencing as second-tier test in high-throughput newborn screening for nephropathic cystinosis

2019 ◽  
Vol 28 (2) ◽  
pp. 193-201 ◽  
Author(s):  
Tobias Fleige ◽  
Siegfried Burggraf ◽  
Ludwig Czibere ◽  
Julia Häring ◽  
Birgit Glück ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Newborn Screening ◽  
High Throughput ◽  
Nephropathic Cystinosis ◽  
Next Generation ◽  
Second Tier ◽  
Generation Sequencing

Next-generation sequencing as a second-tier diagnostic test for newborn screening

2018 ◽  
Vol 31 (8) ◽  
pp. 927-931 ◽  
Author(s):  
Xiaomei Luo ◽  
Ruifang Wang ◽  
Yanjie Fan ◽  
Xuefan Gu ◽  
Yongguo Yu
Keyword(s):  
Next Generation Sequencing ◽  
Newborn Screening ◽  
Diagnostic Test ◽  
Genomic Dna ◽  
Sanger Sequencing ◽  
Dried Blood Spots ◽  
Next Generation ◽  
Blood Spots ◽  
Second Tier ◽  
Generation Sequencing

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.

scholarly journals Assessing the Performance of Dried-Blood-Spot DNA Extraction Methods in Next Generation Sequencing

2020 ◽  
Vol 6 (2) ◽  
pp. 36 ◽  
Author(s):  
Miyono M. Hendrix ◽  
Carla D. Cuthbert ◽  
Suzanne K. Cordovado
Keyword(s):  
Cystic Fibrosis ◽  
Next Generation Sequencing ◽  
Newborn Screening ◽  
Dna Extraction ◽  
Technical Assistance ◽  
The United States ◽  
Extraction Methods ◽  
Next Generation ◽  
Dna Extraction Methods ◽  
Generation Sequencing

An increasing number of newborn screening laboratories in the United States and abroad are moving towards incorporating next-generation sequencing technology, or NGS, into routine screening, particularly for cystic fibrosis. As more programs utilize this technology for both cystic fibrosis and beyond, it is critical to identify appropriate DNA extraction methods that can be used with dried blood spots that will result in consistent, high-quality sequencing results. To provide comprehensive quality assurance and technical assistance to newborn screening laboratories wishing to incorporate NGS assays, CDC’s Newborn Screening and Molecular Biology Branch designed a study to evaluate the performance of nine commercial or laboratory-developed DNA extraction methods that range from a highly purified column extraction to a crude detergent-based no-wash boil prep. The DNA from these nine methods was used in two NGS library preparations that interrogate the CFTR gene. All DNA extraction methods including the cruder preps performed reasonably well with both library preps. One lower-concentration, older sample was excluded from one of the assay evaluations due to poor performance across all DNA extraction methods. When 84 samples, versus eight, were run on a flow cell, the DNA quality and quantity were more significant variables.

Sign in / Sign up

Export Citation Format

Share Document