Article

Quality of massively parallel sequencing/nextgeneration sequencing (MPS/NGS) methods - Qualità dei metodi di sequenziamento massivo parallelo/sequenziamento di nuova generazione (MPS/NGS)

Authors:
  • Società Italiana di Patologia Clinica e Medicina di Laboratorio (SIPMeL)
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Abstract

ISO's concern about the neglected quality of MPS/NGS is more than well-founded. Laboratory operations are considered analogous to a manufacturing process where the industrial process is replaced by the measurement process, the output of which is the results of examinations. Process control is therefore an essential element of the quality management system. The complexity of the MPS/NGS process is obviously daunting for some medical laboratories. The expression of the final result in nominal format is misleading. The ISO 20397-2 document provides many indicators and useful examples for doing laboratory estimation of result uncertainty for MPS/NGS.

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Next-generation sequencing (NGS) of tumor cell-derived DNA/RNA to screen for targetable genomic alterations is now widely available and has become part of routine practice in oncology. NGS testing strategies depend on cancer type, disease stage and the impact of results on treatment selection. The European Society for Medical Oncology (ESMO) has recently published recommendations for the use of NGS in patients with advanced cancer. We complement the ESMO recommendations with a practical review of how oncologists should read and interpret NGS reports. A concise and straightforward NGS report contains details of the tumor sample, the technology used and highlights not only the most important and potentially actionable results, but also other pathogenic alterations detected. Variants of unknown significance should also be listed. Interpretation of NGS reports should be a joint effort between molecular pathologists, tumor biologists and clinicians. Rather than relying and acting on the information provided by the NGS report, oncologists need to obtain a basic level of understanding to read and interpret NGS results. Comprehensive annotated databases are available for clinicians to review the information detailed in the NGS report. Molecular tumor boards do not only stimulate debate and exchange, but may also help to interpret challenging reports and to ensure continuing medical education.
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Background: The performance assessment of tests that express qualitative results in the medical laboratory is of primary importance in characterization, diagnosis, follow-up, and screening. An important contribution to this type of assessment may be the publication of the Eurachem AQA 2021 guide. The text intends to principally discuss the consistency of the subclauses of this guide with ISO 15189 and CLSI EP12-A2. Methods: The study involves a literature review within the scope of qualitative tests. Results: Tables are used for crossing AQA. with ISO 15189 and CLSI EP12-A2. Conclusions: Consistency with ISO 15189 and CLSI EP12-A2 is demonstrated in the study. Introducing “uncertainty of proportion” reflects the necessity of assessing uncertainties when dealing with qualitative results.
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Science is defined in part by an honest exposition of the uncertainties that arise in measurements and propagate through calculations and inferences, so that the reliabilities of its conclusions are made apparent. The recent rapid development of high-throughput DNA sequencing technologies has dramatically increased the number of measurements made at the biochemical and molecular level. These data come from many different DNA-sequencing technologies, each with their own platform-specific errors and biases, which vary widely. Several statistical studies have tried to measure error rates for basic determinations, but there are no general schemes to project these uncertainties so as to assess the surety of the conclusions drawn about genetic, epigenetic, and more general biological questions. We review here the state of uncertainty quantification in DNA sequencing applications, describe sources of error, and propose methods that can be used for accounting and propagating these errors and their uncertainties through subsequent calculations. Copyright © 2014 Elsevier Ltd. All rights reserved.
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To the Editor Most test results in medical laboratories are numbers, known as “quantitative results” if they have linearity (a ratio or interval scale) and are related to the international/national reference standard. Results consisting of nouns or adjectives (known as “qualitative results”) are less frequent but no less important to service quality. The Eurachem/Cooperation on International Traceability in Analytical Chemistry (CITAC) guide “Evaluating Performance and Uncertainty in Qualitative Chemical Analysis” has recently been published (1). However, the Eurachem/CITAC guide states that it does not address all available tools for evaluating the performance of qualitative methods and the uncertainty of qualitative results. It is limited to comparison of results to a reference and does not consider, for example, concordance between qualitative methods or the classification on ordinal scales. In fact, a number of criticisms of the guide have recently been reported, including the lack of the repeatability and reproducibility components for the accuracy of the methods (2).
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ISO published the draft for final approval of the revision of ISO 15189 standard. Following ISO directives, ISO 15189 must be aligned with ISO/IEC 17025:2017 and should be less prescriptive. Draft ISO/DIS 15189 deviates in some points from ISO 17025 and the ISO indications to limit prescriptiveness: equipment, uncertainty, quality control. This do not seem to be justified by medical specificities and could complicate the understanding of the new requirements in medical laboratories.
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The new European regulations, and in particular that on in vitro diagnostics, have made the principles already developed by ISO and CEN mandatory obligations. In particular, in post-market surveillance laboratories and manufacturers are now obliged to share important information throughout the life cycle of devices. Both serious accidents with the relative corrective actions, and in general the performance and safety, user reports, the frequency and severity of events, the drifts over time. Documents such as a surveillance plan and periodic safety reports are expected. The manufacturer collects from laboratories and proactively evaluates performance data and relevant scientific data related to the use of a device that bears the CE marking, with the aim of confirming safety, performance and scientific validity for life expected device. This means concretely starting from the results of external quality assessments (VEQ) and from those of internal quality control (CQI), arriving at the reports of clinicians and patients up to real complaints. ISO/TR 20416 for its part covers the post-market phase of medical devices. Close collaboration between laboratories and manufacturers is already present in ISO 15189 for accreditation, but it is clearly explicit at least in ISO 22367 for risk management and ISO 15198 for internal quality control, not to mention operator safety standards ISO 15190 and ISO 35001. Manufacturers and laboratories cannot fail to undertake to develop this collaboration in the narrow period of time that separates us from the full application of the European Regulation. -- https://www.minervamedica.it/en/journals/medicina-laboratorio/article.php?cod=R54Y2020N04A0309
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ISO, the International Organization for Standardization, is preparing document ISO/TS 17822-2 on the quality control of nucleic acid amplification techniques (NAAT). ISO 17822-2 takes into consideration many aspects that affect quality. ISO 17822 prescribes the use of control materials and describes different types of them. ISO 17822-2 states that the laboratory shall decide the appropriate type and frequency of use of internal and external controls, but says however that the results must remain within the pre-established limits, without saying how the limits should be fixed. Worldwide quality control in medical laboratories is often not performed correctly. Quality costs represent 22% of the laboratory’s direct costs. The SIPMeL Quality and Accreditation Commission has therefore produced its recommendations on quality control, with the transposition of ISO 15198: 2004, reconfirmed also in 2018. Diagnostic system manufacturers have a primary role in this activity. The guidelines for quality control are in the document Clinical Laboratory Standards Institute (CLSI) C24.
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Comprehensive next-generation sequencing (NGS) tests are increasingly used as first-line tests in the evaluation of patients with suspected heritable disease. Despite major technical simplifications, these assays still pose significant challenges for molecular testing laboratories. Existing professional guidelines and recommendations provide a framework for laboratories implementing such tests, but in-depth, concrete guidance is generally not provided. Consequently, there is variability in how laboratories interpret and subsequently implement these regulatory frameworks. To address the need for more detailed guidance, the College of American Pathologists with representation from the Association for Molecular Pathologists assembled a working group to create a practical resource for clinical laboratories. This initial work is focused on variant detection in the setting of inherited disease and provides structured worksheets that guide the user through the entire life cycle of an NGS test, including design, optimization, validation, and quality management with additional guidance for clinical bioinformatics. This resource is designed to be a living document that is publicly available and will be updated with user and expert feedback as the wet bench and bioinformatic landscapes continue to evolve. It is intended to facilitate the standardization of NGS testing across laboratories and therefore to improve patient care. © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology
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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 advisement 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.
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Diverse microbial communities of bacteria, archaea, viruses and single-celled eukaryotes have crucial roles in the environment and in human health. However, microbes are frequently difficult to culture in the laboratory, which can confound cataloging of members and understanding of how communities function. High-throughput sequencing technologies and a suite of computational pipelines have been combined into shotgun metagenomics methods that have transformed microbiology. Still, computational approaches to overcome the challenges that affect both assembly-based and mapping-based metagenomic profiling, particularly of high-complexity samples or environments containing organisms with limited similarity to sequenced genomes, are needed. Understanding the functions and characterizing specific strains of these communities offers biotechnological promise in therapeutic discovery and innovative ways to synthesize products using microbial factories and can pinpoint the contributions of microorganisms to planetary, animal and human health.
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The National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) trial is a national signal-finding precision medicine study that relies on genomic assays to screen and enroll patients with relapsed or refractory cancer after standard treatments. We report the analytical validation processes for the next-generation sequencing (NGS) assay that was tailored for regulatory compliant use in the trial. The Oncomine Cancer Panel assay and the Personal Genome Machine were used in four networked laboratories accredited for the Clinical Laboratory Improvement Amendments. Using formalin-fixed paraffin-embedded clinical specimens and cell lines, we found that the assay achieved overall sensitivity of 96.98% for 265 known mutations and 99.99% specificity. High reproducibility in detecting all reportable variants was observed, with a 99.99% mean interoperator pairwise concordance across the four laboratories. The limit of detection for each variant type was 2.8% for single-nucleotide variants, 10.5% for insertion/deletions, 6.8% for large insertion/deletions (gap ≥4 bp), and four copies for gene amplification. The assay system from biopsy collection through reporting was tested and found to be fully fit for purpose. Our results indicate that the NCI-MATCH NGS assay met the criteria for the intended clinical use and that high reproducibility of a complex NGS assay is achievable across multiple clinical laboratories. Our validation approaches can serve as a template for development and validation of other NGS assays for precision medicine.
Article
Context: The higher throughput and lower per-base cost of next-generation sequencing (NGS) as compared to Sanger sequencing has led to its rapid adoption in clinical testing. The number of laboratories offering NGS-based tests has also grown considerably in the past few years, despite the fact that specific Clinical Laboratory Improvement Amendments of 1988/College of American Pathologists (CAP) laboratory standards had not yet been developed to regulate this technology. Objective: To develop a checklist for clinical testing using NGS technology that sets standards for the analytic wet bench process and for bioinformatics or "dry bench" analyses. As NGS-based clinical tests are new to diagnostic testing and are of much greater complexity than traditional Sanger sequencing-based tests, there is an urgent need to develop new regulatory standards for laboratories offering these tests. Design: To develop the necessary regulatory framework for NGS and to facilitate appropriate adoption of this technology for clinical testing, CAP formed a committee in 2011, the NGS Work Group, to deliberate upon the contents to be included in the checklist. Results . -A total of 18 laboratory accreditation checklist requirements for the analytic wet bench process and bioinformatics analysis processes have been included within CAP's molecular pathology checklist (MOL). Conclusions: This report describes the important issues considered by the CAP committee during the development of the new checklist requirements, which address documentation, validation, quality assurance, confirmatory testing, exception logs, monitoring of upgrades, variant interpretation and reporting, incidental findings, data storage, version traceability, and data transfer confidentiality.
Article
Next-generation sequencing (also known as massively parallel sequencing) technologies are revolutionising our ability to characterise cancers at the genomic, transcriptomic and epigenetic levels. Cataloguing all mutations, copy number aberrations and somatic rearrangements in an entire cancer genome at base pair resolution can now be performed in a matter of weeks. Furthermore, massively parallel sequencing can be used as a means for unbiased transcriptomic analysis of mRNAs, small RNAs and noncoding RNAs, genome-wide methylation assays and high-throughput chromatin immunoprecipitation assays. Here, I discuss the potential impact of this technology on breast cancer research and the challenges that come with this technological breakthrough.
Article
A method for DNA sequencing has been developed that utilises libraries of cloned randomly-fragmented DNA. The DNA to be sequenced is first subjected to limited attack by a non-specific endonuclease (DNase I in the presence of Mn++), fractionated by size and cloned in a single-stranded phage vector. Clones are then picked at random and used to provide a template for sequencing by the dideoxynucleotide chain termination method. This technique was used to sequence completely a 4257 bp EcoRI fragment of bovine mitochondrial DNA. The cloned fragments were evenly distributed with respect to the EcoRI fragment, and completion of the entire sequence required the construction of only a single library. In general, once a clone library has been prepared, the speed of this approach (>1000 nucleotides of randomly selected sequence per day) is limited mainly by the rate at which the data can be processed. Because the clones are selected randomly, however, the average amount of new sequence information per clone is substantially diminished as the sequence nears completion.
Next Generation Sequencing (NGS)
  • Istituto Zooprofilattico Sperimentale Delle Venezie
Istituto Zooprofilattico Sperimentale delle Venezie. Next Generation Sequencing (NGS). [Internet]. Disponibile alla pagina: https://www. izsvenezie.it/temi/tecnologia-innovazione/next-generation-sequencing/ [citato 3 aprile 2023].
al via il programma nazionale di test Ngs gratuiti
  • C Pinto
  • Maria Scatolini
  • M Tumori
Pinto C, Maria Scatolini M. Tumori, al via il programma nazionale di test Ngs gratuiti [Internet]. Disponibile alla pagina: https://www.sanita24. ilsole24ore.com/art/medicina-e-ricerca/2023-01-10/tumori-via-programma-nazionale-test-ngs-gratuiti-105645.php?uuid=AErT9cVC [citato 3 aprile 2023].
Guidelines for Validation of Next-Generation Sequencing-Based Oncology Panels: A Joint Consensus Recommendation of the Association for Molecular Pathology and College of American Pathologists
  • L J Jennings
  • M E Arcila
  • C Corless
  • S Kamel-Reid
  • I M Lubin
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