Abstract
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.
Highlights
Molecular testing has been used with increasing frequency in newborn screening (NBS) for both primary screening (i.e., severe combined immunodeficiency (SCID) and recently spinal muscular atrophy) and second-tier testing to add clarity or specificity to the primary biochemical test [1,2]
Since there are >2000 variants that have been identified in the CFTR gene [8,9], a panel of pathogenic variants will not be inclusive of all variants that can result in cystic fibrosis (CF)
In quarter 1 of 2020, there were 77 participants including both domestic and international programs enrolled in the CF DNA PT program, eight of which identified that their programs use next-generation sequencing (NGS) technology either to sequence the coding regions of the CFTR gene or to identify a large number of pathogenic variants [17]
Summary
Molecular testing has been used with increasing frequency in newborn screening (NBS) for both primary screening (i.e., severe combined immunodeficiency (SCID) and recently spinal muscular atrophy) and second-tier testing to add clarity or specificity to the primary biochemical test (i.e., hemoglobinopathies, Pompe, mucopolysaccharidosis I, adrenoleukodystrophy and cystic fibrosis) [1,2]. CDC’s CF DNA DBS repository is made from anonymous donor-CF-patient and family-donor blood samples and contains a diverse number of CFTR pathogenic variants including the 23 recommended by the American College of Medical Genetics (ACMG), as well as 58 expanded pathogenic variants These samples are characterized extensively by CDC to ensure robust performance in NBS laboratories. In quarter 1 of 2020, there were 77 participants including both domestic and international programs enrolled in the CF DNA PT program, eight of which identified that their programs use NGS technology either to sequence the coding regions of the CFTR gene or to identify a large number of pathogenic variants [17] As this technology is incorporated into additional programs, it will be critical to understand how to best perform NGS in high-throughput laboratories responsible for screening entire populations of newborns while still meeting short turnaround requirements needed for NBS. The CDC’s NSMBB Molecular Quality Improvement Program laboratory designed a study to evaluate the utility of nine DBS DNA extraction methods that range from a highly purified column extraction to a crude detergent-based no-wash boil prep and examines their utility in NGS using two assays to sequence the CFTR gene, MiSeqDx cystic fibrosis clinical sequencing assay (Illumina, San Diego, CA, USA) and Accel-AmpliconTM CFTR panel for Illumina platforms (Swift Biosciences, Ann Arbor, MI, USA)
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