Bioinformatics for Clinical Next Generation Sequencing

Oliver, Gavin R.; Hart, Steven N.; Klee, Eric W.

doi:10.1373/clinchem.2014.224360

Cited by 127 publications

(90 citation statements)

References 75 publications

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“…The accelerated obsolescence of sequencing platforms presents several obstacles in bridging the gap between research and routine diagnostics, including standardization efforts (23). The downstream bioinformatics pipelines are also unique challenges for microbiology laboratories regarding both infrastructure and skilled operators (24–27). Overall, WGS “wet-bench” and “dry-bench” workflows represent integrated processes, which are not easily amenable to the traditional quality metrics used by microbiology laboratories (27–29).…”

Section: Introductionmentioning

confidence: 99%

Validation and Implementation of Clinical Laboratory Improvements Act-Compliant Whole-Genome Sequencing in the Public Health Microbiology Laboratory

et al. 2017

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Public health microbiology laboratories (PHLs) are on the cusp of unprecedented improvements in pathogen identification, antibiotic resistance detection, and outbreak investigation by using whole-genome sequencing (WGS). However, considerable challenges remain due to the lack of common standards. Here, we describe the validation of WGS on the Illumina platform for routine use in PHLs according to Clinical Laboratory Improvements Act (CLIA) guidelines for laboratory-developed tests (LDTs). We developed a validation panel comprising 10 Enterobacteriaceae isolates, 5 Gram-positive cocci, 5 Gram-negative nonfermenting species, 9 Mycobacterium tuberculosis isolates, and 5 miscellaneous bacteria. The genome coverage range was 15.71× to 216.4× (average, 79.72×; median, 71.55×); the limit of detection (LOD) for single nucleotide polymorphisms (SNPs) was 60×. The accuracy, reproducibility, and repeatability of base calling were >99.9%. The accuracy of phylogenetic analysis was 100%. The specificity and sensitivity inferred from multilocus sequence typing (MLST) and genome-wide SNP-based phylogenetic assays were 100%. The following objectives were accomplished: (i) the establishment of the performance specifications for WGS applications in PHLs according to CLIA guidelines, (ii) the development of quality assurance and quality control measures, (iii) the development of a reporting format for end users with or without WGS expertise, (iv) the availability of a validation set of microorganisms, and (v) the creation of a modular template for the validation of WGS processes in PHLs. The validation panel, sequencing analytics, and raw sequences could facilitate multilaboratory comparisons of WGS data. Additionally, the WGS performance specifications and modular template are adaptable for the validation of other platforms and reagent kits.

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Section: Introductionmentioning

confidence: 99%

Validation and Implementation of Clinical Laboratory Improvements Act-Compliant Whole-Genome Sequencing in the Public Health Microbiology Laboratory

et al. 2017

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“…While all methods have caveats and advantages to be aware of, it is also important to be aware that the data analysis pipeline is critical in any NGS platform. Both customized commercially available and in‐house analysis pipelines are commonly used . Indeed the performance of an NGS assay depends not only on the physical assay and instrumentation itself, but the computational/analytical tools being used or which have been developed.…”

Section: Dna and Rna Based Molecular Assaysmentioning

confidence: 99%

“…Both customized commercially available and in-house analysis pipelines are commonly used. [58][59][60] Indeed the performance of an NGS assay depends not only on the physical assay and instrumentation itself, but the computational/analytical tools being used or which have been developed. As such, it is critical to know the weaknesses and issues with any NGS workflow.…”

Section: Illumina Sequencing Technology-sequencing By Synthesismentioning

confidence: 99%

Molecular genetic testing methodologies in hematopoietic diseases: current and future methods

Sridhar

Singh

Butzmann

et al. 2019

Int J Lab Hematology

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Introduction Rapid technological advancements in clinical molecular genetics have increased our diagnostic and prognostic capabilities in health care. Understanding these assays, as well as how they may change over time, is critical for pathologists, clinicians, and translational researchers alike. Methods This review provides a practical summary and basic reference for current molecular genetic technologies, as well as new testing methodologies that are in use, gaining momentum, or anticipated to contribute more broadly in the future. Results Here, we discuss DNA and RNA based methodologies including classic assays such as the polymerase chain reaction (PCR), Sanger sequencing, and microarrays, to more cutting‐edge next‐generation sequencing (NGS) based assays and emerging molecular technologies such as cell‐free DNA (cfDNA) or circulating tumor DNA (ctDNA), and NGS‐based detection of infectious disease organisms. Conclusion This review serves as a basic foundation for knowledge in current and emerging clinical molecular genetic technologies.

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“…Increasing amounts of information at each level raises a number of issues for service delivery including adequately skilled personnel and infrastructure issues with associated costs of storage and processing power. A hybrid facility, as suggested by the ‘Belfast model' (Salto-Tellez et al , 2014), combines the skill sets of an academic bioinformatics department within a clinical environment to produce a cost-effective bioinformatics pipeline with clinical utility and appropriate TATs for application in a UHC setting (Oliver et al , 2015). It is clear from the ISO15189 standard for accreditation that the same rigorous validation is required for the bioinformatics pipeline and any change results in re-validation, although the raw FASTQ data can be used to condense this process considerably.…”

Section: Information Flow With Ngsmentioning

confidence: 99%

Tissue-based next generation sequencing: application in a universal healthcare system

et al. 2017

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In the context of solid tumours, the evolution of cancer therapies to more targeted and nuanced approaches has led to the impetus for personalised medicine. The targets for these therapies are largely based on the driving genetic mutations of the tumours. To track these multiple driving mutations the use of next generation sequencing (NGS) coupled with a morphomolecular approach to tumours, has the potential to deliver on the promises of personalised medicine. A review of NGS and its application in a universal healthcare (UHC) setting is undertaken as the technology has a wide appeal and utility in diagnostic, clinical trial and research paradigms. Furthermore, we suggest that these can be accommodated with a unified integromic approach. Challenges remain in bringing NGS to routine clinical use and these include validation, handling of the large amounts of information flow and production of a clinically useful report. These challenges are particularly acute in the setting of UHC where tests are not reimbursed and there are finite resources available. It is our opinion that the challenges faced in applying NGS in a UHC setting are surmountable and we outline our approach for its routine application in diagnostic, clinical trial and research paradigms.

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Bioinformatics for Clinical Next Generation Sequencing

Cited by 127 publications

References 75 publications

Validation and Implementation of Clinical Laboratory Improvements Act-Compliant Whole-Genome Sequencing in the Public Health Microbiology Laboratory

Validation and Implementation of Clinical Laboratory Improvements Act-Compliant Whole-Genome Sequencing in the Public Health Microbiology Laboratory

Molecular genetic testing methodologies in hematopoietic diseases: current and future methods

Tissue-based next generation sequencing: application in a universal healthcare system

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