Abstract

Stored neonatal dried blood spot (DBS) samples from neonatal screening programmes are a valuable diagnostic and research resource. Combined with information from national health registries they can be used in population-based studies of genetic diseases. DNA extracted from neonatal DBSs can be amplified to obtain micrograms of an otherwise limited resource, referred to as whole-genome amplified DNA (wgaDNA). Here we investigate the robustness of exome sequencing of wgaDNA of neonatal DBS samples. We conducted three pilot studies of seven, eight and seven subjects, respectively. For each subject we analysed a neonatal DBS sample and corresponding adult whole-blood (WB) reference sample. Different DNA sample types were prepared for each of the subjects. Pilot 1: wgaDNA of 2x3.2mm neonatal DBSs (DBS_2x3.2) and raw DNA extract of the WB reference sample (WB_ref). Pilot 2: DBS_2x3.2, WB_ref and a WB_ref replica sharing DNA extract with the WB_ref sample. Pilot 3: DBS_2x3.2, WB_ref, wgaDNA of 2x1.6 mm neonatal DBSs and wgaDNA of the WB reference sample. Following sequencing and data analysis, we compared pairwise variant calls to obtain a measure of similarity—the concordance rate. Concordance rates were slightly lower when comparing DBS vs WB sample types than for any two WB sample types of the same subject before filtering of the variant calls. The overall concordance rates were dependent on the variant type, with SNPs performing best. Post-filtering, the comparisons of DBS vs WB and WB vs WB sample types yielded similar concordance rates, with values close to 100%. WgaDNA of neonatal DBS samples performs with great accuracy and efficiency in exome sequencing. The wgaDNA performed similarly to matched high-quality reference—whole-blood DNA—based on concordance rates calculated from variant calls. No differences were observed substituting 2x3.2 with 2x1.6 mm discs, allowing for additional reduction of sample material in future projects.

Highlights

  • The accurate genotyping of DNA of neonatal dried blood spot (DBS) samples has proven plausible, and opened new avenues in neonatal screening and for identification of determinants in complex genetic diseases [1, 2]

  • Several studies have shown that the whole-genome amplified DNA (wgaDNA) performs well compared to high-quality DNA of other sources, using low- and high-throughput genotyping platforms [1, 14, 15]: the wgaDNA has been used with great success in research projects looking at infantile hypertrophic pyloric stenosis [16], birth weight [17], schizophrenia [18, 19], psychosis [20] and cerebral palsy [21]

  • We recently found that the wgaDNA works for next-generation sequencing (NGS) [22]: an adult whole-blood (WB) reference sample (WB_ref), a three-year old DBS reference sample of the same person and the corresponding archived neonatal DBS sample were compared

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Summary

Introduction

The accurate genotyping of DNA of neonatal dried blood spot (DBS) samples has proven plausible, and opened new avenues in neonatal screening and for identification of determinants in complex genetic diseases [1, 2]. Danish citizens are assigned a unique person-identifying number used across all public registration systems including the DNSB and in the wellestablished public health care system This makes it possible to study the entire population under a certain age as a single cohort, and to identify the determinants of common and complex genetic diseases in the Caucasian ethnicity [11]. Most projects are limited to a maximum of two discs of 3.2 mm size per sample with each spot yielding a maximum of 60 ng of retrievable DNA [12] This restricts the use of neonatal DBS samples to a limited set of technologies/platforms, most often with no possibility of repeating the experiments. As this study only included two subjects, the robustness’s of the methodology still needs to be confirmed

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