2020 Short Course - Essential Fundamentals - Next Generation Sequencing From The Beginning-Approaches to NGS Assay Design and Validation

Video Transcription

Video Summary

In this video, Mark Ewaldt, a hematopathologist and molecular genetic pathologist, discusses the design and validation of next-generation sequencing (NGS) assays. He starts by discussing the life cycle of a clinical test, which includes test development, test design, test validation, and quality management. He emphasizes the importance of considering the clinical questions that the test is designed to answer and the sample types that will be tested.<br /><br />Ewaldt explains the key factors to consider during the design of an NGS assay, such as sample matrix, variant types to detect, sensitivity of the assay, sample throughput, and turnaround time. He also mentions the guidelines papers published by professional societies like ACMG, AMP, and CAP that provide frameworks and resources for assay design and validation.<br /><br />In terms of validation, Ewaldt recommends an optimization and familiarization phase before final validation to improve assay performance. He discusses the wet lab validation, including sample requirements, library preparation methods, and assessing the performance of the sequencing run. He also explains the importance of bioinformatics validation using real and synthetic data sets.<br /><br />Ewaldt addresses the challenges of working with formalin-fixed paraffin-embedded (FFPE) tissues, which are commonly used in oncology testing, due to their degradation and fragmentation. He suggests different approaches, such as sequencing large non-coding regions or using RNA-based testing, to overcome these challenges and detect larger indels or fusion events.<br /><br />He highlights the need for analytical and clinical sensitivity and specificity, as well as the importance of assessing the reference and reportable range of the assay. Ewaldt also mentions the use of population databases to understand normal genetic variation.<br /><br />Lastly, he briefly discusses the ongoing quality management of NGS assays, including routine quality control, proficiency testing, and the incorporation of in silico validation methods.<br /><br />Overall, this video provides a comprehensive overview of the design and validation of NGS assays, highlighting the key considerations and steps involved in the process.

Asset Subtitle

Mark Ewalt

Keywords

NGS assays

test development

test design

test validation

quality management

sample types

sensitivity

sample throughput

turnaround time