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

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

Laboratory implementation of Next Generation Sequencing for medium and high throughput whole genome analysis of clinical M. tuberculosis complex strains

10.26226/morressier.5991c409d462b80292388d15 ◽

2017 ◽

Author(s):

Christian Utpatel

Keyword(s):

Next Generation Sequencing ◽

High Throughput ◽

Genome Analysis ◽

Whole Genome ◽

Next Generation ◽

Tuberculosis Complex ◽

Whole Genome Analysis ◽

Generation Sequencing

Download Full-text

Next-Generation Sequencing: An Emerging Tool for Drug Designing

Current Pharmaceutical Design ◽

10.2174/1381612825666190911155508 ◽

2019 ◽

Vol 25 (31) ◽

pp. 3350-3357 ◽

Cited By ~ 1

Author(s):

Pooja Tripathi ◽

Jyotsna Singh ◽

Jonathan A. Lal ◽

Vijay Tripathi

Keyword(s):

Next Generation Sequencing ◽

High Throughput ◽

Large Scale ◽

Massively Parallel Sequencing ◽

Genomic Research ◽

Biological Research ◽

Next Generation ◽

Human Welfare ◽

Drug Designing ◽

Generation Sequencing

Background: With the outbreak of high throughput next-generation sequencing (NGS), the biological research of drug discovery has been directed towards the oncology and infectious disease therapeutic areas, with extensive use in biopharmaceutical development and vaccine production. Method: In this review, an effort was made to address the basic background of NGS technologies, potential applications of NGS in drug designing. Our purpose is also to provide a brief introduction of various Nextgeneration sequencing techniques. Discussions: The high-throughput methods execute Large-scale Unbiased Sequencing (LUS) which comprises of Massively Parallel Sequencing (MPS) or NGS technologies. The Next geneinvolved necessarily executes Largescale Unbiased Sequencing (LUS) which comprises of MPS or NGS technologies. These are related terms that describe a DNA sequencing technology which has revolutionized genomic research. Using NGS, an entire human genome can be sequenced within a single day. Conclusion: Analysis of NGS data unravels important clues in the quest for the treatment of various lifethreatening diseases and other related scientific problems related to human welfare.

Download Full-text

Advances in genetics and molecular breeding of three legume crops of semi-arid tropics using next-generation sequencing and high-throughput genotyping technologies

Journal of Biosciences ◽

10.1007/s12038-012-9228-0 ◽

2012 ◽

Vol 37 (5) ◽

pp. 811-820 ◽

Cited By ~ 44

Author(s):

Rajeev K Varshney ◽

Himabindu Kudapa ◽

Manish Roorkiwal ◽

Mahendar Thudi ◽

Manish K Pandey ◽

...

Keyword(s):

Next Generation Sequencing ◽

High Throughput ◽

Molecular Breeding ◽

Next Generation ◽

Legume Crops ◽

Generation Sequencing ◽

Semi Arid

Download Full-text

Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing

BMC Genomics ◽

10.1186/s12864-018-4677-y ◽

2018 ◽

Vol 19 (1) ◽

Cited By ~ 7

Author(s):

Wells W. Wu ◽

Je-Nie Phue ◽

Chun-Ting Lee ◽

Changyi Lin ◽

Lai Xu ◽

...

Keyword(s):

Next Generation Sequencing ◽

High Throughput ◽

Library Preparation ◽

Next Generation ◽

Generation Sequencing

Download Full-text

Evaluation of TypeSeq, a Novel High-Throughput, Low-Cost, Next-Generation Sequencing-Based Assay for Detection of 51 Human Papillomavirus Genotypes

The Journal of Infectious Diseases ◽

10.1093/infdis/jiz324 ◽

2019 ◽

Vol 220 (10) ◽

pp. 1609-1619 ◽

Cited By ~ 4

Author(s):

Sarah Wagner ◽

David Roberson ◽

Joseph Boland ◽

Aimée R Kreimer ◽

Meredith Yeager ◽

...

Keyword(s):

Human Papillomavirus ◽

Next Generation Sequencing ◽

High Throughput ◽

Vaccine Efficacy ◽

Linear Array ◽

Human Papillomaviruses ◽

Next Generation ◽

Hpv Genotyping ◽

Positive Agreement ◽

Generation Sequencing

AbstractBackgroundHuman papillomaviruses (HPV) cause over 500 000 cervical cancers each year, most of which occur in low-resource settings. Human papillomavirus genotyping is important to study natural history and vaccine efficacy. We evaluated TypeSeq, a novel, next-generation, sequencing-based assay that detects 51 HPV genotypes, in 2 large international epidemiologic studies.MethodsTypeSeq was evaluated in 2804 cervical specimens from the Study to Understand Cervical Cancer Endpoints and Early Determinants (SUCCEED) and in 2357 specimens from the Costa Rica Vaccine Trial (CVT). Positive agreement and risks of precancer for individual genotypes were calculated for TypeSeq in comparison to Linear Array (SUCCEED). In CVT, positive agreement and vaccine efficacy were calculated for TypeSeq and SPF10-LiPA.ResultsWe observed high overall and positive agreement for most genotypes between TypeSeq and Linear Array in SUCCEED and SPF10-LiPA in CVT. There was no significant difference in risk of precancer between TypeSeq and Linear Array in SUCCEED or in estimates of vaccine efficacy between TypeSeq and SPF10-LiPA in CVT.ConclusionsThe agreement of TypeSeq with Linear Array and SPF10-LiPA, 2 well established standards for HPV genotyping, demonstrates its high accuracy. TypeSeq provides high-throughput, affordable HPV genotyping for world-wide studies of cervical precancer risk and of HPV vaccine efficacy.

Download Full-text