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Inside the Lab Bioinformatics, Genetics and epigenetics, Biochemistry and molecular biology, Profession, Regulation and standards, Training and education, Quality assurance and quality control, Omics

A Guiding Light for Sequencing

At a Glance

  • Next generation sequencing (NGS) is being increasingly adopted, but variability between clinical laboratories remains high
  • New guidelines from the Association for Molecular Pathology hope to improve and standardize the NGS bioinformatics pipeline
  • The recommendations emphasize the crucial role of a properly trained and qualified molecular laboratory professional
  • They also provide practical guidance for NGS design, development and operation

More than ever, pathologists and laboratory medicine professionals are turning to next generation sequencing (NGS) to provide patients with diagnostic and prognostic information. But not every laboratory performs sequencing and analysis the same way, and variability in clinical laboratory practice can lead to problems. To tackle this, the Association of Molecular Pathology (AMP) has released several sets of guidelines for sequencing and panel validation, culminating in a new set of consensus recommendations for NGS bioinformatics pipelines (1). With this latest release, the association hopes to provide guidance throughout the NGS workflow, so that patients in need of genetic analysis can receive the best possible care.

What prior guidelines were used for NGS?

Guidelines for the analytical validation, interpretation and reporting of NGS tests were published in the medical literature earlier this year (2)(3), and the molecular community is progressively adapting them into practice. However, prior guidelines did not specifically address requirements for validating NGS bioinformatics pipelines. The limited number of NGS pipeline validation studies, and the high degree of variability between studies, certainly reinforced the need for guidance in NGS bioinformatics pipeline validation.

Why was it so important to plug the guideline gap?

NGS technologies are being rapidly adopted in the clinic, but the constant evolution of technology and the absence of clear recommendations for analytical validation of NGS bioinformatics pipelines are contributing to variabilities in clinical laboratory practice. With the lack of clear guidelines, each laboratory must figure out its own best practices, effectively reinventing the wheel. Good guidelines work on a couple of levels. Firstly, they help laboratories reduce the time it takes to implement NGS bioinformatics by giving them a checklist of requirements; moreover, an established, trustworthy list allows them to plan adequately for time, labor and resources. Secondly, peer-reviewed and approved guidelines – if properly followed – can help laboratories improve quality, by setting minimum thresholds for good practice.

Of course, laboratories may certainly exceed minimum thresholds to their own comfort level – or as appropriate to the nuances of their own testing.

AMP believes it is the responsibility of professional organizations to establish guidelines for professional practice and, as such, we routinely engage with other professional associations to publish evidence-based practice guidelines. Our members are among the early adopters and users of NGS technology in a clinical setting and have accumulated substantial knowledge and expertise as it relates to this novel and powerful technology. The 17 consensus recommendations in our latest report (see Table 1) are designed to help clinical laboratory professionals achieve high-quality sequencing results, with the ultimate end result of delivering better patient care.

How were the new guidelines determined?

AMP convened and led a multidisciplinary subject matter expert working group with representation from the College of American Pathologists and the American Medical Informatics Association to summarize current knowledge, expose challenges, and provide guidance on how to develop, implement and validate high-quality bioinformatics pipelines to ensure better overall patient care. The guidelines are based on evidence from a review of published literature, empirical data, current laboratory practice surveys, and expert professional experiences. Because these recommendations represent current best practice in a rapidly developing field, AMP anticipates the need for ongoing updates.

Table 1. A summary of the 17 consensus recommendations for the development, implementation and validation of NGS bioinformatics pipelines (adapted from 1).
What are the key take-home messages from the new guidelines?

First and foremost, the new recommendations emphasize the critical role of the properly trained and qualified molecular laboratory professional to achieve optimal NGS test quality.

As noted, the new guidelines represent a set of minimum standards for NGS bioinformatics pipelines, which we anticipate will help not only clinical laboratories, but also NGS instrument manufacturers, software companies and researchers to advance medical technology in a safe and responsible way.

These recommendations were developed to optimize patient care and, as a result, many clinical laboratories may find that they are already compliant with many of them. Laboratories should focus their efforts on determining what, if any, changes are needed to their existing pipelines and processes, and then appropriately plan to implement those changes in a stepwise manner. Not every change will be a simple one; some of the guideline recommendations, for instance, may require certain laboratories to make fundamental changes to sequence file content to standardize sample identification. These processes must be implemented carefully and with an appropriate amount of validation prior to being put into clinical use.

Some of the guidelines address the inner workings of the pipeline itself to ensure the safety, accuracy and security of the data as it flows through the process; those recommendations aim to reduce the likelihood of errors reaching the laboratory medicine professional for interpretation.

The NGS bioinformatics pipeline validation guidelines include a number of recommendations intended to help pathologists and laboratory medicine professionals communicate with bioinformaticians and technology professionals to ensure that they understand the limitations of their pipelines, appropriately interpret NGS data, and adopt best practices when developing or updating pipelines.

The full reports provide a thorough explanation of each recommendation to assist in comprehension and implementation into clinical molecular diagnostic laboratory practice.

How can pathologists who use NGS promote the new guidelines?

All of the guideline documents are freely available for download, so pathologists can easily share this information with their colleagues. After all, understanding and communicating the importance of adhering to published guidelines – and following the guidelines to the best of your ability yourself – are great ways to help promote best practices in laboratories around the world.

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  1. S Roy et al., “Standards and guidelines for validating next-generation sequencing bioinformatics pipelines”, J Mol Diagn, [Epub ahead of print] (2017). PMID: 29154853.
  2. MM Li et al., “Standards and guidelines for the interpretation and reporting of sequence variants in cancer”, J Mol Diagn, 19, 4–23 (2017). PMID: 27993330.
  3. LJ Jennings et al., “Guidelines for validation of next-generation sequencing-based oncology panels”, J Mol Diagn, 19, 341–365 (2017).PMID: 28341590.
About the Author
Somak Roy and Alexis Carter

Somak Roy is Assistant Professor of Pathology at the University of Pittsburgh Medical Center, Working Group Chair and Member of the Association for Molecular Pathology, USA.
Alexis Carter is Physician Informaticist at Children’s Healthcare of Atlanta, Working Group Member, 2017 AMP Informatics Subdivision Chair, and Board Member of the Association for Molecular Pathology, USA.

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