Using dried blood spot samples from a trio for linked-read whole-exome sequencing

Area of Science:

• Genomics
• Molecular Biology
• Biotechnology

Background:

• Dried blood spot (DBS) samples are collected via newborn screening and offer long-term storage advantages.
• Short-read sequencing of DBS samples presents limitations, particularly for molecular phasing.
• Linked-read sequencing offers potential to overcome these limitations.

Purpose of the Study:

• To evaluate the efficacy of linked-read sequencing for molecular phasing using DNA from DBS samples.
• To compare the phasing capabilities of DBS and whole blood (WB) samples.
• To assess the potential of DBS samples for retrospective genetic analysis.

Main Methods:

• Whole-exome sequencing was performed on DNA extracted from DBS and WB reference samples from a trio (parents and proband with primary carnitine deficiency).
• Linked-read sequencing data was analyzed to determine phasing efficiency (genes, SNPs, phase block length).
• Genotype concordance for a known primary carnitine deficiency variant (rs72552725:A > G in SLC22A5) was assessed.

Main Results:

• Phasing of genes and SNPs was achieved for DBS samples, though with shorter phase blocks compared to WB samples.
• WB samples yielded higher percentages of phased genes (>85%) and SNPs (>75%) with significantly longer phase blocks (>915 kb).
• Full genotype concordance was observed between DBS and WB for the primary carnitine deficiency variant across all samples.

Conclusions:

• Linked-read sequencing demonstrates potential for molecular phasing with DBS samples, achieving significant phasing rates for genes and SNPs.
• While DBS samples show promise, current limitations in molecular length necessitate workflow optimization for comprehensive molecular phasing.
• The study confirms genotype concordance between DBS and WB, highlighting DBS utility for genetic variant detection, and suggests linked-read sequencing's potential for haplotype detection without trio data.