Lessons learned: An Ring Trial for Complex Variant Detection in Clinical Testing
Speaker: Dan Brudzewsky, SeraCare Life Sciences
Next-generation sequencing (NGS) has rapidly advanced the genetic testing for inherited disorders. However, these NGS assays present challenges for test development, validation, and quality control, given these interrogate large genomic regions for assessment of a broad range of variant types. Therefore, it is critical to include complex variants such as large Indels, single exon CNV, homopolymer and Structural Variants in NGS assay validation.
SeraCare has developed a reference material technology that can be used to assess the assay design and bioinformatics pipeline used to detect these variants, that are clinical relevant, but rare and challenging. A material for Inherited Cancer was used in an international inter-laboratory study. Here, we will present some of the lessons participants learnt when analyzing a highly multiplexed reference material on their validated and clinical routine, NGS wet-lab procedures and subsequent software analysis.
Targeted analysis of structural rearrangements using Genomic Vision's molecular combing technology
Speaker: Dr. Janine Altmüller, Cologne Center for Genomics (CCG)
Point and small mutations like SNVs and deletions of a couple of base pairs can easily be detected by Sanger and Next Generation Sequencing – FISH and array CGH diagnostics usually take over for large chromosomal aberrations. The grey zone in between comprises structural and Copy Number Variations of medium size that could also be of clinical significance. Some of these could be analyzed by expensive Genome Sequencing or other techniques based on long-range PCR but few still stay in the grey zone as repetitive elements, pseudogenes and different haplotypes interfere with alignment procedures and PCR limitations. Challenging FSHD diagnostic is a perfect example. We want to introduce a technique called “molecular combing” and share the experience of the first year in use at the CCG. In one of few applications, it basically allows comprehensive analysis and physical mapping of target regions on stretched DNA. This “combed” DNA is hybridized with a combination of fluorescent probes of different colors and sizes, designed to recognize a selected region of interest.