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

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.

Opportunities and challenges in long-read sequencing data analysis

“…While nanopore and SMRT are true long-read sequencing technologies and the focus of this review, there are also synthetic long-read sequencing approaches. These include linked reads, proximity ligation strategies, and optical mapping [19][20][21][22][23][24][25][26][27][28], which can be employed in synergy with true long reads.…”

Tay-Sachs Disease: Two Novel Rare HEXA Mutations from Pakistan and Morocco

“…As a positive result will always be confirmed or denied by further specific tests, in this regard DBS testing may further supplement a valid molecular diagnosis via WES. Researchers worldwide have combined newborn screening (NBS) programs with NGS and explored potential advantages of the long-term collection of DBS samples [10]. Hence it is recommended to the clinicians to use the DBS testing as a primary diagnostic test for timely diagnosis of TSD.…”

FarGen: Elucidating the distribution of coding variants in the isolated population of the Faroe Islands

“…The FarGen cohort comprise 1541 individuals. The recruitment procedures, socio-demographics, availability of the bioresources, DNA extraction, DNA barcoding using the 10x Genomics-Chromium controller, library construction, and sequencing has been described previously [12,15,16]. In short, inclusion criteria were: 1) participants had to be 18 years or older, 2) had to live in the Faroe Islands or be of Faroese descent, and 3) had to join the project voluntarily.…”

“…LinkSeq was developed to process the linked-read data in a manner that is suitable for population-based analyses and adapts the GATK Best Practices guidelines [17] to linked-reads [16], applying available opensource tools developed for linked-reads. One of the primary benefits of linked-reads is the ability to unambiguously align reads in homologous regions of the genome [18,19].…”