Abstract
Long-term collection of dried blood spot (DBS) samples through newborn screening may have retrospective and prospective advantages, especially in combination with advanced analytical techniques. This work concerns whether linked-reads may overcome some of the limitations of short-read sequencing of DBS samples, such as performing molecular phasing. We performed whole-exome sequencing of DNA extracted from DBS and corresponding whole blood (WB) reference samples, belonging to a trio with unaffected parents and a proband affected by primary carnitine deficiency (PCD). For the DBS samples we were able to phase >21% of the genes under 100 kb, >40% of the SNPs, and the longest phase block was >72 kb. Corresponding results for the WB reference samples was >85%, >75%, and >915 kb, respectively. Concerning the PCD causing variant (rs72552725:A > G) in the SLC22A5 gene we observe full genotype concordance between DBS and WB for all three samples. Furthermore, we were able to phase all variants within the SLC22A5 gene in the proband’s WB data, which shows that linked-read sequencing may replace the trio information for haplotype detection. However, due to smaller molecular lengths in the DBS data only small phase blocks were observed in the proband’s DBS sample. Therefore, further optimisation of the DBS workflow is needed in order to explore the full potential of DBS samples as a test bed for molecular phasing.
Highlights
Whole-exome sequencing (WES) is increasingly being used in the clinic for diagnostic evaluation and variant detection, for genetically heterogeneous diseases [1, 2]
The benefit of using linked-reads is that we gain long-range information from short read sequencing, which could give a more accurate assembly and the ability to correctly assign the haplotypes of the variants; advantages that may especially apply to dried blood spot (DBS) samples as these samples may be more degraded than whole blood (WB) samples
One of the challenges working with DBS samples is to obtain sufficient amount of DNA required for next-generation sequencing (NGS) analyses, as well as obtaining high molecular weight DNA as DBS samples may be degraded
Summary
Whole-exome sequencing (WES) is increasingly being used in the clinic for diagnostic evaluation and variant detection, for genetically heterogeneous diseases [1, 2]. The use of trio information in this context has further improved diagnostic yield as haplotype information is obtained and novel candidate genes can be detected on the. These authors contributed : Ólavur Mortensen, Leivur Nattestad Lydersen
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