In the course of patient care, formalin-fixation and paraffin-embedding (FFPE) of biopsy tissue samples are routinely performed, where these samples can be analyzed by histology and archived to link the sample with clinical long-term follow-up. With the development of advanced NGS-based oncology gene panels, it is becoming increasingly important to consider pre-analytic variables when extracting nucleic acids from FFPE-treated samples. This post covers frequently asked questions (FAQs) around the extraction of nucleic acids from FFPE samples for downstream NGS analysis.
Answering frequently asked questions (FAQs)
Multiplexed Reference Materials as Controls for Cardiomyopathy Diagnostic Next-Generation Sequencing
A Journal for Molecular Diagnostics article describing ‘an attractive addition to the repertoire of materials for the development, validation, and quality monitoring of clinical NGS assays’
Hypertrophic Cardiomyopathy (HCM) is a disease where the heart muscle is enlarged and a significant cause of sudden cardiac death, and is frequently asymptomatic. HCM is commonly caused by a mutation in one of nine heart muscle genes that comprise the sarcomere, and occurs at a prevalence of about 1 in 500 in the general population. HCM is the leading cause of cardiac death in young athletes in the United States.
Clinical genetic testing for mutations in the HCM-related genes has been ongoing for over a decade; the GeneTest.org database reveals 105 laboratories offering some version of genetic testing. While knowledge of prevalent pathogenic variants are available, the majority of variants remain private (that is, unpublished and not widely available). The move to NGS-based gene panels for HCM testing has lead to new challenges for test development, validation and routine quality control due to the inherent scarcity of samples, the cost of including numerous single mutations from these individual samples, and the lack of these materials for laboratories without a long history of testing.
"The most effective ways to use QC data to monitor the health of your NGS assay."
Previously, we wrote about some of the Quality Control challenges that clinical laboratories performing Next Generation Sequencing (NGS) face towards ensuring their assays are safe and effective for guiding medical management decisions. Reliable access to high quality reference materials is necessary to help overcome these challenges; however, it is not sufficient. Insights that reference materials provide into the health of an NGS assay are only as good as laboratories’ ability to use their QC data effectively.
With limited time and resources to collect, organize, access, and analyze QC metrics, laboratories may frequently rely on reference materials as binary indicators of Pass/Fail: As long as the expected endpoint results are obtained, an assay is considered to be performing well. The drawback of this approach is that it is reactive, rather than proactive: A sufficient number of failures must occur within a given timeframe before a troubleshooting investigation is performed. By the time a problem is recognized, resources have been wasted and turnaround times (TAT) delayed; in some cases, fidelity of patient results may even have been put at risk. Additional time and costs are then incurred as the investigation proceeds.
Specimen analysis by NGS yields a wealth of information in addition to endpoint variant calls that is indicative of assay performance. Data such as nucleic acid quantity and quality at different steps throughout the workflow (PDF) and sequencing library characteristics are generated every time a reference material is tested. However, these data must be carefully tracked and trended to allow use as highly informative QC parameters. For clinical laboratories whose primary focus is on patient testing and reporting, granular QC metrics may not be captured and reviewed as part of routine test monitoring.
“Ensure NGS-based tests for Personalized Medicine are safe and effective for guiding medical management decisions”
Since the introduction of the GS20 in 2005 by 454 Life Sciences, Next Generation Sequencing (NGS) has found many applications in clinical diagnostics. As a result of this transition from the long-held gold standard, Sanger sequencing, the primary challenge for clinical laboratories has shifted from data acquisition to ensuring these tests are safe and effective for guiding medical management decisions.
Many laboratories struggle to gain a thorough understanding of the analytic performance characteristics of their NGS tests. The difficulty arises from the fact that these assays are comprised of highly complex, fragmented workflows, and have many different intended uses. However, across the various practices currently used for NGS assay development, validation, and performance monitoring, there is a common goal: results must be as accurate, precise, and consistent as possible.