NGS-based Clinical Testing (Part III)

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Getting the NGS wet bench process right is not a small undertaking. Targeted NGS assays such as multigene panels or exome sequencing allow for the targeted analysis of genomic regions that are of particular interest. For every sample type, e.g. blood, formalin-fixed paraffin-embedded specimens, saliva etc, there must be a detailed protocol in place outlining how each sample type is going to be handled. The laboratory has to define and document clear and crisp criteria for the wet bench process summarized in a quality management program.

An acceptable quality assurance (QA) program has the following characteristics:

1. The quality assurance program outlines the specific steps of each workflow performed in the lab.
2. If the lab is part of a larger institution such as a hospital or medical center, then the quality assurance program for the lab needs to be integrated with the QA program of the larger organization.
3. The program should outline how to handle any outliers and respectively any problems that typically arise executing the workflow. It should also outline possible corrective actions, as well as the dismissal of a sample.
4. The QA program needs to ensure that the testing is for clinical purposes and the results of the tests need to be in sync with current medical science.
5. The program needs to outline how employees can voice any concerns about the work conducted in the lab.

Part of being an evolving set of technologies means that NGS doesn’t provide perfect results. It’s widely accepted that NGS-based sequencing assays will yield false-positive, as well as false-negative results. It’s the proverbial nature of the beast.

The College of American Pathologists has given laboratories the freedom to determine when confirming tests should be conducted, as well as how these confirming tests need to be carried out. For example, some labs decide that there is no need for a confirmatory test due to their very high coverage of 1500x of a single gene NGS-based assay. Others might opt to use a different method, such as Sanger sequencing, to reach the required confidence in order to report on a variant. Laboratories need to specify their confirmatory testing policy and/or outline in detail why such testing is not necessary based on their assay validation. Here are some key record keeping obligations that describe the wet lab process:

  1. Lab Records: This essentially outlines the overall framework leveraging any and all reagents, primers, sequencing chemistries and platforms utilized in the wet lab process. It describes the type of test performed, including the targeted sequence (e.g. a particular set of genes, whole genome or exome etc), coverage depth and any other parameter that defines the quality of the process. It outlines specific steps taken in the lab and puts them in the context of the currently available body of knowledge by referencing publications, websites etc. While these details are not incorporated in a clinical report the document is typically referenced accordingly.
  2. Exception Log: This part outlines any anticipated deviations from the standard process. Exceptions might be related to the specimen quality or to the process itself. Examples are (1) the handling of a sub-par sample, (2) changes to library preparation and (3) sequencing of libraries with sub-par concentrations. In addition, a communication protocol needs to be in place. For example, the lab has to ensure that employees are reporting exceptions to the lab supervisor.
  3. Upgrade Log: This is a crucial part of the ongoing documentation. As the overall field is evolving, labs have to ensure that all upgrades to instruments, sequencing chemistries, reagents or kits used in the process are thoroughly documented. The policy needs to outline how such upgrades are monitored and how their validation occurs before being used in a clinical setting.

Part IV of this series will outline the quality control issues vis a vis the bioinformatics process in further detail. You can find Part I of the series here and Part II here.

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