Abstract
BackgroundWith the continuing decrease in cost of whole genome sequencing (WGS), we have already approached the point of inflection where WGS testing has become economically feasible, facilitating broader access to the benefits that are helping to define WGS as the new diagnostic standard. WGS provides unique opportunities for detection of structural variants; however, such analyses, despite being recognized by the research community, have not previously made their way into routine clinical practice.ResultsWe have developed a clinically validated pipeline for highly specific and sensitive detection of structural variants basing on 30X PCR-free WGS. Using a combination of breakpoint analysis of split and discordant reads, and read depth analysis, the pipeline identifies structural variants down to single base pair resolution. False positives are minimized using calculations for loss of heterozygosity and bi-modal heterozygous variant allele frequencies to enhance heterozygous deletion and duplication detection respectively. Compound and potential compound combinations of structural variants and small sequence changes are automatically detected. To facilitate clinical interpretation, identified variants are annotated with phenotype information derived from HGMD Professional and population allele frequencies derived from public and Variantyx allele frequency databases. Single base pair resolution enables easy visual inspection of potentially causal variants using the IGV genome browser as well as easy biochemical validation via PCR. Analytical and clinical sensitivity and specificity of the pipeline has been validated using analysis of Genome in a Bottle reference genomes and known positive samples confirmed by orthogonal sequencing technologies.ConclusionConsistent read depth of PCR-free WGS enables reliable detection of structural variants of any size. Annotation both on gene and variant level allows clinicians to match reported patient phenotype with detected variants and confidently report causative finding in all clinical cases used for validation.
Highlights
With the continuing decrease in cost of whole genome sequencing (WGS), we have already approached the point of inflection where WGS testing has become economically feasible, facilitating broader access to the benefits that are helping to define WGS as the new diagnostic standard
The Structural Variant (SV) component of Variantyx Genomic Intelligence Whole Genome Sequencing analysis workflow is comprised of three major parts: variant detection, annotation and filtering (Fig. 1)
Structural variants can be divided into two categories: those resulting in unbalanced changes in number of copies of human DNA, and those resulting in balanced changes so the total number of copies remains the same
Summary
With the continuing decrease in cost of whole genome sequencing (WGS), we have already approached the point of inflection where WGS testing has become economically feasible, facilitating broader access to the benefits that are helping to define WGS as the new diagnostic standard. Due to its uniformity and lack of pull down or amplification artifacts, WGS typically provides better than WES coverage of coding and adjacent regulatory regions. This approach, ignores many of the advantages of WGS which provides unique opportunities for detection of structural variants (SVs), pathologic short tandem repeats and mitochondrial variants, which otherwise require separate assays. Disease causing SVs in medical genetics are detected by karyotyping [1] and chromosomal microarrays (CMAs) [2]. These methods are limited in resolution and cannot identify all types of SVs
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