Massively parallel sequencing technologies, commonly known as next-generation sequencing (NGS), have revolutionized molecular diagnostic testing. Not since the advent of the polymerase chain reaction (PCR) has there been such a dramatic transformation in the capabilities of molecular analysis of DNA and RNA for clinical diagnostic testing.
Next-generation sequencing technology allows for the parallel generation of multiple DNA sequence reactions using template libraries generated from either selected genomic regions, with the use of capture probes or PCR amplification, or unselected total genomic nucleic acid. This generates massive amounts of sequence data, which are then assembled, mapped to a reference genome, and interrogated for nucleotide sequence differences from the reference genome using sophisticated bioinformatics solutions. Next-generation sequencing technologies far exceed previous DNA sequencing technologies, such as Maxim and Gilbert or Sanger dideoxy sequencing, both in the breadth of genomic real estate that can be sequenced in a single experiment and in the depth of coverage for each genomic region sequenced (resulting from multiple individual sequence reads generated), which allows for a superior limit of detection for low-level sequence variants.
Since the advent of NGS, there has been rapid uptake of this technology in biologic research, which has resulted in significant advancements across multiple fields, including novel identification of disease-causing mutations associated with multiple genetic diseases, understanding of the human microbiome associated with healthy normal flora and disease states in multiple body sites, and a rich classification of the genomic mutations associated with tumor biology, including those that are associated with response or resistance to molecularly targeted cancer therapies. These advancements have significant medical implications, and there has been a similarly rapid uptake of NGS technology across multiple disciplines of clinical molecular diagnostic testing.
In this special section of Archives of Pathology & Laboratory Medicine, members of the scientific resource committees within the College of American Pathologists (CAP) Molecular Pathology and Genomics Cluster provide examples of laboratory assay validation of NGS assays for multiple diagnostic applications where this powerful technology is being used clinically.
It is important to note that, as with many molecular diagnostic tests currently in clinical use, there are very few FDA-approved NGS assays, and thus clinical assay validation of laboratory-developed tests is essential to the application of NGS technology for clinical diagnostic testing. The articles in this series provide examples of the development and validation of NGS assays for clinical testing by individual laboratories that may be useful for other clinical molecular laboratories interested in developing and validating NGS assays. The CAP has implemented checklist requirements for CAP-accredited laboratories using NGS assays, and the articles in this series address specific checklist requirements for NGS assay validation. In addition, these articles emphasize important key aspects of NGS assay development and recommendations for assay validation.
Although NGS technology has applicability to all subdisciplines of molecular pathology, there are important key attributes specific to each clinical application that have implications for assay design and validation and that are detailed in the articles in this series.
The article by Garcia et al describes validation of an NGS assay for the detection of somatic variants in cancer, highlighting the need for validation of the limit of detection for somatic variants with low variant allele fraction and the importance of preanalytic parameters assuring that sufficient tumor cells are present. In addition, they address validation of NGS for copy number variant analysis of gene deletions and amplifications, which commonly occur in cancer.
Thomas et al describe validation of an NGS panel for detection of somatic mutations in myeloid malignancies that are of diagnostic and prognostic significance. Of special note is a thorough validation of quantitation of variant allele fraction.
Schlaberg et al describe validation of metagenomic NGS assays for detection of pathogens in cerebrospinal fluid and respiratory specimens. This manuscript is a joint publication of the CAP Microbiology Resource Committee and the American Society for Microbiology. Of note are challenges to interpretation due to incomplete reference sequence databases for pathogen identification and differentiation of human (host) sequence from nonpathogenic or pathogenic microorganisms.
Two papers address use of NGS for detection of germline variants in inherited diseases. Santani et al describe development and validation of NGS panels for inherited genetic diseases associated with mutations in multiple genes. Hegde et al discuss specific aspects of development and validation of whole-exome sequencing or whole-genome sequencing for genetic disease testing. Germane to both of these applications are the selection of genes for analysis that are consistent with the patient's phenotype, the challenges to interpretation of variants and detection of variants of uncertain significance, and the need for access to detailed clinical phenotypic information in order for the laboratory to be able to make a clinical interpretation.
Finally, Gandhi et al present the use of targeted NGS analysis for human leukocyte antigen (HLA) typing and discuss challenges that are related to the high homology among HLA genes, certain genomic regions that are difficult to sequence, and bioinformatics software solutions for HLA haplotype identification.
Other clinical applications of NGS technology that are not included in this series include pharmacogenomic testing and noninvasive prenatal screening of cell-free DNA of fetal origin in maternal plasma.
Next-generation sequencing is a powerful new technology that has rapidly resulted in multiple advancements in biologic research and has already demonstrated the potential to improve molecular diagnostic testing. As the cost, speed, and ease of use of NGS continue to improve, this technology has the potential to replace many of the current molecular testing modalities. However, because NGS represents very-high-complexity testing, assays must be designed and validated by experienced molecular laboratories, and there are still caveats and limitations to its use. In addition, as with any new technology, it is imperative that the analytical and clinical aspects of NGS be carefully validated prior to clinical implementation.
Although it is unlikely to replace many of the common molecular technologies in current use in the near future, the day may come when almost all molecular testing incorporates NGS technology.
Molecular pathology remains one of the most rapidly advancing diagnostic fields, with changes and improvements to molecular technologies continuing to drive improvements in practice. The manuscripts in this special section are excellent examples of how quickly expert molecular pathology laboratories have been able to adapt and validate new technologies for clinical use.
Karen E. Weck, MD
Karen E. Weck, MD, is clinical professor of pathology & laboratory medicine and genetics and director of the Clinical Molecular Genetics Laboratory at the University of North Carolina in Chapel Hill. Dr Weck is the past chair of the College of American Pathologists Molecular Pathology and Genomics Cluster,
Biochemical and Molecular Genetics Committee, and Pharmacogenetics Workgroup. Dr Weck received bachelor of science and doctor of medicine degrees from Duke University and residency training in laboratory medicine at Washington University in St Louis, and is boarded in clinical pathology and molecular genetic pathology. She was assistant professor of pathology and assistant director of molecular diagnostics at the University of Pittsburgh Medical Center from 1999 to 2004.
She has served as a consultant or advisor to numerous commercial and professional organizations, has held several offices for the Association for Molecular Pathology, and is a member of the US Food and Drug Administration Molecular and Clinical Genetics Devices Panel. She serves as an editor for the Journal of Molecular Diagnostics and the American Journal of Pathology and is the associate editor of molecular genetics and pharmacogenetics for Genetics in Medicine. Dr Weck is co–principal investigator of a National Institutes of Health (NIH) National Human Genome Research Institute Clinical Sequencing Exploratory Research award to implement whole-exome sequencing for diagnosis of genetic diseases and has had recent grant funding from the NIH Office of Rare Diseases, the North Carolina Biotechnology Center, and institutionally funded initiatives to study the clinical utility of incorporating pharmacogenomic information into clinical management and to develop genetic diagnostic testing for multigenic inherited diseases.
Karen E. Weck, MD
Karen E. Weck, MD, is clinical professor of pathology & laboratory medicine and genetics and director of the Clinical Molecular Genetics Laboratory at the University of North Carolina in Chapel Hill. Dr Weck is the past chair of the College of American Pathologists Molecular Pathology and Genomics Cluster,
Biochemical and Molecular Genetics Committee, and Pharmacogenetics Workgroup. Dr Weck received bachelor of science and doctor of medicine degrees from Duke University and residency training in laboratory medicine at Washington University in St Louis, and is boarded in clinical pathology and molecular genetic pathology. She was assistant professor of pathology and assistant director of molecular diagnostics at the University of Pittsburgh Medical Center from 1999 to 2004.
She has served as a consultant or advisor to numerous commercial and professional organizations, has held several offices for the Association for Molecular Pathology, and is a member of the US Food and Drug Administration Molecular and Clinical Genetics Devices Panel. She serves as an editor for the Journal of Molecular Diagnostics and the American Journal of Pathology and is the associate editor of molecular genetics and pharmacogenetics for Genetics in Medicine. Dr Weck is co–principal investigator of a National Institutes of Health (NIH) National Human Genome Research Institute Clinical Sequencing Exploratory Research award to implement whole-exome sequencing for diagnosis of genetic diseases and has had recent grant funding from the NIH Office of Rare Diseases, the North Carolina Biotechnology Center, and institutionally funded initiatives to study the clinical utility of incorporating pharmacogenomic information into clinical management and to develop genetic diagnostic testing for multigenic inherited diseases.