Comparing DNA Isolation and Preparation Protocols for Dried Blood Spots in the Context of Genomic Newborn Screening

Abstract

Due to rapid technical advancements and increasing cost-effectiveness, the potential application of next-generation sequencing (NGS) in newborn screening (NBS) has raised great interest worldwide. Genomic NBS offers the possibility to improve current NBS programs when applied as follow-up tier, and, as first-tier, allows for inclusion of conditions lacking a detectable biomarker for conventional NBS. Obtaining enough high-quality DNA from typically limited dried blood spot (DBS) material to meet NGS requirements can be challenging. Selecting a DNA isolation method for genomic NBS requires balancing technical performance and laboratory feasibility with optimal cost-effectiveness. Ten DNA isolation protocols, including two column-based, five lysis-based, and three semi-automated magnetic bead-based protocols, were evaluated on technical outcomes and performance in targeted amplicon sequencing. Additionally, estimated costs, hands-on time, turnaround time, scalability, and plastic footprint were assessed. Although technical outcomes, including yield, purity, and molecular weight, differed between methods, qualitative results in amplicon sequencing, as defined by read output, mapping, and coverage depth, were found sufficient and comparable for various protocols. In conclusion, both technical requirements and operational parameters are crucial when selecting a DNA isolation protocol and will depend on the NGS application as well as the NBS approach, as either first-tier or follow-up tier.

Keywords: DBS; DNA isolation; DNA preparation; NBS; NGS; dried blood spot; genomic newborn screening; newborn screening; next-generation sequencing.

Figure 4

Sequencing performance based on yield, mapping, and coverage. DBS = dried blood spot. Comparison of amplicon sequencing performance between seven different DNA isolation protocols applied on one and two DBS punches (3.2 mm) of starting material, including the generated sequence reads in millions (A), percentage unmapped reads (B), mean read depth over target region (C) and percentage of amplicons (N total = 3003) with >30× read depth (D). The numbers above the columns correspond to the DNA isolation protocols described in Table 1. The bar reaches the median from all samples per protocol, with colors indicating different techniques used, including column-based (blue), lysis-based (orange), and magnetic bead-based (green). The dotted line depicts outcomes for the GIAB control sample.

Figure 1

DNA yield and A260/A280 ratio. DBS = dried blood spot. Comparison of DNA yield (ng) (A) and A260/A280 (B) ratio between ten different DNA isolation protocols using one, two, and three DBS punches (3.2 mm). The dotted line depicts the optimal A260/A280 ratio (B) with a 10% interval around it in grey. The numbers above the columns correspond to the DNA isolation protocols as described in Table 1. The median and range for the measurements per protocol are depicted by the bars, with colors indicating different techniques used, including column-based (blue), lysis-based (orange), and magnetic bead-based (green).

Figure 2

Mean DNA yield with increasing number of DBS punches. DBS = dried blood spot. Comparison of the mean DNA yield (ng) and 95% CI observed with increasing numbers of DBS punches (3.2 mm) used as starting material. The dotted line represents a hypothetical function if the yield from two and three DBS punches increased in direct proportion to the yield from one DBS punch. The colors indicate different techniques used, including column-based (blue), lysis-based (orange), and magnetic bead-based (green).

Figure 3

Molecular weight of isolated DNA from one DBS punch. DBS = dried blood spot. The analysis was performed with the Femto Pulse System on DNA isolated from one DBS punch (3.2 mm). For each DNA isolation protocol, results from two different donor samples are presented.