Approaching the Hard-to-Diagnose

Examine the limitations of next generation sequencing, including short-read dead zones, exome vs genome coverage, and how depth (20x) affects SNV/INDEL and CNV detection, UPD, somatic variants, and orthogonal testing.

Compare exome sequencing and genome sequencing, highlighting genome's higher intronic and intergenic coverage. Explain CNV detection differences and that genome sequencing aids undiagnosed cases after exome and CMA.

Explore advanced variant interpretation of deep intronic variants using in silico tools like SpliceAI, and learn to detect and interpret copy number and structural variants, including splicing disruptions.

Learn how copy number variants, deletions and duplications, are detected by CMA, MLPA, exome and genome sequencing. Interpret CNVs with haploinsufficiency, triplosensitivity, and ACMG guidelines for pathogenicity.

Explore strategies for detecting and interpreting complex structural variants, including inversions and translocations, using split and discordant reads, and note limitations of short-read sequencing and copy-number neutral SV detection.

Define variants of uncertain significance (VUS) and their clinical implications in genetic testing. Explain how Bayesian classification quantifies VUS uncertainty and informs possible reclassification to likely pathogenic or benign.

Demonstrate reclassifying variants of uncertain significance (VUS) to likely pathogenic or likely benign through real-world cases, using de novo ARID1A and inherited SMARCC2 findings, DNM1 splicing, and Fabry biomarkers.

Learn how reanalysis turns unsolved rare disease cases into diagnoses by applying updated literature, improved tools, and clinical insights, to existing raw sequencing data.

Explore real-world case studies highlighting how reanalysis redefines genetic diagnoses through updated variants, phenotype matches, and family testing, including cystic fibrosis and developmental disorders.

Explore RNA sequencing, or transcriptome sequencing, to detect splicing changes, aberrant and allele-specific expression, and gene expression shifts, increasing diagnostic yield by 10–15% when used with genome or exome sequencing.

Long-read sequencing using PacBio and Oxford Nanopore spans repeats, improves genome assembly and phasing, and detects complex variants, enabling clearer clinical diagnoses and potential first-tier testing.