Addressing ethical and laboratory challenges for initiation of a rapid whole genome sequencing program.
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ABSTRACT:
SUBMITTER: Malone Jenkins S
PROVIDER: S-EPMC8596074 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Emerging genomic technologies promise more efficient infectious disease control. Whole genome sequencing (WGS) is increasingly being used in tuberculosis (TB) diagnosis, surveillance, and epidemiology. However, while the use of WGS by public health agencies may raise ethical, legal, and socio-political concerns, these challenges are poorly understood. METHOD:Between November 2017 and April 2018, we conducted semi-structured interviews with 22 key stakeholders across the fields of governance and policy, public health, and laboratory sciences representing the major jurisdictions currently using WGS in national TB programs. Thematic analysis of the interviews was conducted using NVivo 11. RESULTS:Respondents identified several ethical and practical challenges associated with WGS in TB care and surveillance, all related to issues of trust, including: 1) the power of public health; 2) data sharing and profits derived from surveillance efforts; and 3) concerns regarding who has access to, and can benefit from, the technology. Additional challenges included: the potential utility that WGS adds to a public health program, the risks associated with linking necessary epidemiological metadata to the genomic data, and challenges associated with jurisdictional capacity to implement the technology. CONCLUSIONS:Successful implementation of WGS is dependent on fostering relationships of trust between those working with genomics technology and those directly impacted by it, including clinicians. Building trust (a) between the public and the public health agencies and (b) within public health agencies themselves is critical due to the inherent complexity of WGS and its implementation for communicable disease control purposes.
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Project description:Experimental evolution of microbial populations provides a unique opportunity to study evolutionary adaptation in response to controlled selective pressures. However, until recently it has been difficult to identify the precise genetic changes underlying adaptation at a genome-wide scale. New DNA sequencing technologies now allow the genome of parental and evolved strains of microorganisms to be rapidly determined.We sequenced >93.5% of the genome of a laboratory-evolved strain of the yeast Saccharomyces cerevisiae and its ancestor at >28x depth. Both single nucleotide polymorphisms and copy number amplifications were found, with specific gains over array-based methodologies previously used to analyze these genomes. Applying a segmentation algorithm to quantify structural changes, we determined the approximate genomic boundaries of a 5x gene amplification. These boundaries guided the recovery of breakpoint sequences, which provide insights into the nature of a complex genomic rearrangement.This study suggests that whole-genome sequencing can provide a rapid approach to uncover the genetic basis of evolutionary adaptations, with further applications in the study of laboratory selections and mutagenesis screens. In addition, we show how single-end, short read sequencing data can provide detailed information about structural rearrangements, and generate predictions about the genomic features and processes that underlie genome plasticity.
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Project description:Traditionally, genetic testing has been too slow or perceived to be impractical to initial management of the critically ill neonate. Technological advances have led to the ability to sequence and interpret the entire genome of a neonate in as little as 26 h. As the cost and speed of testing decreases, the utility of whole genome sequencing (WGS) of neonates for acute and latent genetic illness increases. Analyzing the entire genome allows for concomitant evaluation of the currently identified 5588 single gene diseases. When applied to a select population of ill infants in a level IV neonatal intensive care unit, WGS yielded a diagnosis of a causative genetic disease in 57% of patients. These diagnoses may lead to clinical management changes ranging from transition to palliative care for uniformly lethal conditions for alteration or initiation of medical or surgical therapy to improve outcomes in others. Thus, institution of 2-day WGS at time of acute presentation opens the possibility of early implementation of precision medicine. This implementation may create opportunities for early interventional, frequently novel or off-label therapies that may alter disease trajectory in infants with what would otherwise be fatal disease. Widespread deployment of rapid WGS and precision medicine will raise ethical issues pertaining to interpretation of variants of unknown significance, discovery of incidental findings related to adult onset conditions and carrier status, and implementation of medical therapies for which little is known in terms of risks and benefits. Despite these challenges, precision neonatology has significant potential both to decrease infant mortality related to genetic diseases with onset in newborns and to facilitate parental decision making regarding transition to palliative care.
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Project description:Laboratory strains, cell lines, and other genetic materials change hands frequently in the life sciences. Despite evidence that such materials are subject to mix-ups, contamination, and accumulation of secondary mutations, verification of strains and samples is not an established part of many experimental workflows. With the plummeting cost of next generation technologies, it is conceivable that whole genome sequencing (WGS) could be applied to routine strain and sample verification in the future. To demonstrate the need for strain validation by WGS, we sequenced haploid yeast segregants derived from a popular commercial mutant collection and identified several unexpected mutations. We determined that available bioinformatics tools may be ill-suited for verification and highlight the importance of finishing reference genomes for commonly used laboratory strains.
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Project description:ObjectivesThe emerging SARS-CoV-2 variants of concern (VoC), B.1.1.7, B.1.351 and P.1, with increased transmission and/or immune evasion, emphasise the need for broad and rapid variant monitoring. Our high-volume laboratory introduced a PCR variant assay (Variant PCR) in January 2021 based on the protocol by Vogels et al. STUDY DESIGN: To assess whether Variant PCR could be used for rapid B.1.1.7, B.1.351 and P.1 screening, all positive SARS-CoV-2 airway samples were prospectively tested in parallel using both the Variant PCR and whole genome sequencing (WGS).ResultsIn total 1,642 SARS-CoV-2 positive samples from individual patients were tested within a time span of 4 weeks. For all samples with valid results from both Variant PCR and WGS, no VoC was missed by Variant PCR (totalling 399 VoC detected). Conversely, all of the samples identified as "other lineages" (i.e., "non-VoC lineages") by the Variant PCR, were confirmed by WGS.ConclusionsThe Variant PCR based on the protocol by Vogels et al., is an effective method for rapid screening for VoC, applicable for most diagnostic laboratories within a pandemic setting. WGS is still required to confirm the identity of certain variants and for continuous surveillance of emerging VoC.
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Project description:Whole-genome sequencing is a powerful technique for obtaining the reference sequence information of multiple organisms. Its use can be dramatically expanded to rapidly identify genomic variations, which can be linked with phenotypes to obtain biological insights. We explored these potential applications using the emerging next-generation sequencing platform Solexa Genome Analyzer, and the well-characterized model bacterium Bacillus subtilis. Combining sequencing with experimental verification, we first improved the accuracy of the published sequence of the B. subtilis reference strain 168, then obtained sequences of multiple related laboratory strains and different isolates of each strain. This provides a framework for comparing the divergence between different laboratory strains and between their individual isolates. We also demonstrated the power of Solexa sequencing by using its results to predict a defect in the citrate signal transduction pathway of a common laboratory strain, which we verified experimentally. Finally, we examined the molecular nature of spontaneously generated mutations that suppress the growth defect caused by deletion of the stringent response mediator relA. Using whole-genome sequencing, we rapidly mapped these suppressor mutations to two small homologs of relA. Interestingly, stable suppressor strains had mutations in both genes, with each mutation alone partially relieving the relA growth defect. This supports an intriguing three-locus interaction module that is not easily identifiable through traditional suppressor mapping. We conclude that whole-genome sequencing can drastically accelerate the identification of suppressor mutations and complex genetic interactions, and it can be applied as a standard tool to investigate the genetic traits of model organisms.
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Project description:ObjectivesAs a result of the introduction of rapid benchtop sequencers, the time required to subculture a bacterial pathogen to extract sufficient DNA for library preparation can now exceed the time to sequence said DNA. We have eliminated this rate-limiting step by developing a protocol to generate DNA libraries for whole-genome sequencing directly from single bacterial colonies grown on primary culture plates.MethodsWe developed our protocol using single colonies of 17 bacterial pathogens responsible for severe human infection that were grown using standard diagnostic media and incubation conditions. We then applied this method to four clinical scenarios that currently require time-consuming reference laboratory tests: full identification and genotyping of salmonellae; identification of blaNDM-1, a highly transmissible carbapenemase resistance gene, in Klebsiella pneumoniae; detection of genes encoding staphylococcal toxins associated with specific disease syndromes; and monitoring of vaccine targets to detect vaccine escape in Neisseria meningitidis.ResultsWe validated our single-colony whole-genome sequencing protocol for all 40 combinations of pathogen and selective, non-selective or indicator media tested in this study. Moreover, we demonstrated the clinical value of this method compared with current reference laboratory tests.ConclusionsThis advance will facilitate the implementation of whole-genome sequencing into diagnostic and public health microbiology.
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