Standards and Guidelines for Validating Next-Generation Sequencing Bioinformatics Pipelines

Abstract

Bioinformatics pipelines are an integral component of next-generation sequencing (NGS). Processing raw sequence data to detect genomic alterations has significant impact on disease management and patient care. Because of the lack of published guidance, there is currently a high degree of variability in how members of the global molecular genetics and pathology community establish and validate bioinformatics pipelines. Improperly developed, validated, and/or monitored pipelines may generate inaccurate results that may have negative consequences for patient care. To address this unmet need, the Association of Molecular Pathology, with organizational representation from the College of American Pathologists and the American Medical Informatics Association, has developed a set of 17 best practice consensus recommendations for the validation of clinical NGS bioinformatics pipelines. Recommendations include practical guidance for laboratories regarding NGS bioinformatics pipeline design, development, and operation, with additional emphasis on the role of a properly trained and qualified molecular professional to achieve optimal NGS testing quality.

Targeted NGS Platforms for Genetic Screening and Gene Discovery in Primary Immunodeficiencies

“…In our experience, the use of selected NGS panels is useful and easy to handle for rapid diagnosis in clinically and immunologically wellcharacterized phenotypes. As compared to WES, targeted small NGS panels provide an important alternative for clinicians for direct sequencing of relevant genes, guaranteeing a high coverage and sequencing depth (64). On the contrary, their application in patients with atypical phenotypes could result in an incomplete and delayed diagnosis.…”

“…Many previously undetected variants do not have a well-defined role in our genome (1.5 × 10 6 million variants in each genome and lesser in exome). In this scenario, ethical and legal issues related to the disclosure of genetic information generated by NGS need to be considered and guidelines should be developed to help the different specialists to translate the genetic results into the clinics (64).…”

An interlaboratory study of complex variant detection

“…Most laboratories used an Illumina (San Diego, CA) platform with hybridization-based targeting. One workflow (6) used whole-genome sequencing and another (8) used the Ion Torrent platform with AmpliSeq PCR-based targeting (ThermoFisher, Waltham, MA). Each workflow used a unique bioinformatics pipeline, including custom software, thirdparty software, and sequencing vendor-supplied software (Supplemental Methods).…”

“…A recent systematic review found a "high degree of variability" among published validation studies, some of which did not even stratify performance by variant type (i.e. SNV versus indel) [8]. Indeed, the 2017 AMP (Association for Molecular Pathology) and CAP (College of American Pathologists) guidelines for NGS bioinformatics recommend that at least 59 variants of each type be included in validation studies [8].…”

“…SNV versus indel) [8]. Indeed, the 2017 AMP (Association for Molecular Pathology) and CAP (College of American Pathologists) guidelines for NGS bioinformatics recommend that at least 59 variants of each type be included in validation studies [8]. One difficulty in accomplishing this is that few positive controls containing challenging variants in specific genes are readily available, despite the otherwise excellent results of the Genome in a Bottle (GIAB) and Genetic Testing Reference Materials (GeT-RM) programs [9][10][11].…”