Whole-genome amplified DNA from stored dried blood spots is reliable in high resolution melting curve and sequencing analysis

Abstract

BackgroundThe use of dried blood spots (DBS) samples in genomic workup has been limited by the relative low amounts of genomic DNA (gDNA) they contain. It remains to be proven that whole genome amplified DNA (wgaDNA) from stored DBS samples, constitutes a reliable alternative to gDNA.We wanted to compare melting curves and sequencing results from wgaDNA derived from DBS samples with gDNA derived from whole blood.MethodsgDNA was extracted from whole blood obtained from 10 patients with lone atrial fibrillation (mean age 22.3 years). From their newborn DBS samples, stored at -24°C, genomic DNA was extracted and whole-genome amplified in triplicates. Using high resolution melting curve analysis and direct sequencing in both wgaDNA and gDNA samples, all coding regions and adjacent intron regions of the genes SCN5A and KCNA5 were investigated.ResultsAltered melting curves was present in 85 of wgaDNA samples and 81 of gDNA samples. Sequence analysis identified a total of 31 variants in the 10 wgaDNA samples. The same 31 variants were found in the exact same pattern of samples in the gDNA group. There was no false positive or negative sequence variation in the wgaDNA group.ConclusionsThe use of DNA amplified in triplicates from DBS samples is reliable and can be used both for high resolution curve melting analysis as well as direct sequence analysis. DBS samples therefore can serve as an alternative to whole blood in sequence analysis.

The role of the sodium current complex in a nonreferred nationwide cohort of sudden infant death syndrome

“…The method has previously been described in detail and the use of wgaDNA for sequencing has previously been validated. 21,22 Individual exons with flanking intron sequences were amplified by PCR. Usually, at least 50 basepair of flanking intron sequences were included in the amplicons.…”

“…We have previously validated the use of whole-genome amplified DNA from DBS samples for genetic analysis. 21 The aim of this study was, for the first time ever to analyze the role of the sodium current in SIDS by investigating the presence of rare genetic variants in Nav1.5 and its interacting proteins in an unbiased (nonreferred) cohort of SIDS retrieved from a nationwide neonatal screening biobank.…”

High quality methylome-wide investigations through next-generation sequencing of DNA from a single archived dry blood spot

“…In contrast, the collected blood spots can be dried in room temperature and shipped without refrigeration. Furthermore, blood spots can be kept in a long-term storage facility (< -20°C) for years prior to DNA extraction without significant loss in quality 29 , 30 . For example, DNA extracted from blood spots, stored for up to 25 y, has successfully been used in whole genome amplification for various genetic investigations including direct sequencing and GWAS 29 - 31 .…”

Lessons that Newborn Screening in the USA Can Teach us about Biobanking and Large-Scale Genetic Studies

“…While the ability to extract and analyze DNA from dried blood spots has dramatically increased the potential utility of these dried blood spots for both NBS testing within the program and research outside the NBS program [15,16], the yield and quality of the DNA varies based on the storage conditions [13]. Medicine has a long tradition of using ‘leftover’ biomedical specimens (e.g., blood, DNA, organs and tissues) for research [17], and the ethical issues raised by the use of stored biomedical specimens – especially those that pertain to the use of specimens for genetic studies – are not new [18].…”