Methods and feasibility study for exome sequencing as a universal second-tier test in newborn screening



Newborn screening disorders increasingly require genetic variant analysis as part of second-tier or confirmatory testing. Sanger sequencing and gene-specific next-generation sequencing (NGS)-based tests, the current methods of choice, are costly and lack scalability when expanding to new conditions. We describe a scalable, exome sequencing–based NGS pipeline with a priori analysis restriction that can be universally applied to any NBS disorder.


De-identified abnormal newborn screening specimens representing severe combined immune deficiency (SCID), cystic fibrosis (CF), VLCAD deficiency, metachromatic leukodystrophy (MLD), and in silico sequence read data sets were used to validate the pipeline. To support interpretation and clinical decision-making within the bioinformatics pipeline, variants from multiple databases were curated and validated.


CFTR variant panel analysis correctly identified all variants. Concordance compared with diagnostic testing results for targeted gene analysis was between 78.6% and 100%. Validation of the bioinformatics pipeline with in silico data sets revealed a 100% detection rate. Varying degrees of overlap were observed between ClinVar and other databases ranging from 3% to 65%. Data normalization revealed that 11% of variants across the databases required manual curation.


This pipeline allows for restriction of analysis to variants within a single gene or multiple genes, and can be readily expanded to full exome analysis if clinically indicated and parental consent is granted.

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Fig. 1: Overview of the Utah Newborn Screening (NBS) Program exome sequencing and analysis pipeline.
Fig. 2: Variant database curation pipeline.
Fig. 3: Genomic variation overlap between ClinVar and Leiden Open Variation Databases (LOVDs).

Data availability

Sequence analysis pipelines and read simulation data sets are available at

Code availability

Code developed for the variant database curation pipeline is available at


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This work was supported by the Association of Public Health Laboratories through the Health Resources and Services Administration (HRSA), US Department of Health and Human Services (HHS UG9MC30369). This information or content and conclusions are those of the author and should not be construed as the official position or policy of, nor should any endorsements be inferred by HRSA, HHS, or the US Government. Funding was also provided by the National Institutes of Health, National Human Genome Research Institute (5U41HG009650 to K.E.) and National Library of Medicine (T15LM007124 to D.S.).

Author information




Conceptualization: A.R., K.E. Curation: D.S., J.L., K.C., N.R.-S. Formal analysis: D.S., J.L., K.C., N.R.-S. Funding acquisition: A.R., K.E. Investigation: A.R., D.S., J.L., K.C., K.E., K.H., N.R.-S., S.N., W.D. Methodology: A.R., D.S., E.L.Y., K.E., K.F.O., N.R.-S. Software: B.A., D.S., J.L., K.C., N.R.-S. Visualization: D.S., N.R.-S. Writing—original draft: A.R., D.S., K.E., N.R.-S., S.N. Writing—review & editing: A.R., B.A., D.S., E.L.Y., K.E., K.F.O., K.H., N.R.-S., S.N., W.D.

Corresponding author

Correspondence to Andreas Rohrwasser.

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Ethics Declaration

Institutional review board (IRB) approval for the analysis of MLD screen-positive and screen-negative samples was granted by the Washington state IRB Project B-062702-H07.23. The Utah Department of Health IRB determined that IRB approval was not required for work utilizing de-identified CF, SCID, and VLCAD deficiency specimens, as these were analyzed as a part of a validation and process improvement project.

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The authors declare no competing interests.

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Ruiz-Schultz, N., Sant, D., Norcross, S. et al. Methods and feasibility study for exome sequencing as a universal second-tier test in newborn screening. Genet Med (2021).

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