Rapid Whole-genome Sequencing Research Articles

The Role of Rapid Molecular Tests, Whole-genome Sequencing, and Phenotypic Drug Susceptibility Testing in the Detection and Control of Drug-resistant Tuberculosis

The Role of Rapid Molecular Tests, Whole-genome Sequencing, and Phenotypic Drug Susceptibility Testing in the Detection and Control of Drug-resistant Tuberculosis

Rapid Whole-Genome Sequencing and Clinical Management in the PICU: A Multicenter Cohort, 2016-2023.

Analysis of the clinical utility of rapid whole-genome sequencing (rWGS) outside of the neonatal period is lacking. We describe the use of rWGS in PICU and cardiovascular ICU (CICU) patients across four institutions. Ambidirectional multisite cohort study. Four tertiary children's hospitals. Children 0-18 years old in the PICU or CICU who underwent rWGS analysis, from May 2016 to June 2023. None. A total of 133 patients underwent clinical, phenotype-driven rWGS analysis, 36 prospectively. A molecular diagnosis was identified in 79 patients (59%). Median (interquartile range [IQR]) age was 6 months (IQR 1.2 mo-4.6 yr). Median time for return of preliminary results was 3 days (IQR 2-4). In 79 patients with a molecular diagnosis, there was a change in ICU management in 19 patients (24%); and some change in clinical management in 63 patients (80%). Nondiagnosis changed management in 5 of 54 patients (9%). The clinical specialty ordering rWGS did not affect diagnostic rate. Factors associated with greater odds ratio (OR [95% CI]; OR [95% CI]) of diagnosis included dysmorphic features (OR 10.9 [95% CI, 1.8-105]) and congenital heart disease (OR 4.2 [95% CI, 1.3-16.8]). Variables associated with greater odds of changes in management included obtaining a genetic diagnosis (OR 16.6 [95% CI, 5.5-62]) and a shorter time to genetic result (OR 0.8 [95% CI, 0.76-0.9]). Surveys of pediatric intensivists indicated that rWGS-enhanced clinical prognostication (p < 0.0001) and contributed to a decision to consult palliative care (p < 0.02). In this 2016-2023 multiple-PICU/CICU cohort, we have shown that timely genetic diagnosis is feasible across institutions. Application of rWGS had a 59% (95% CI, 51-67%) rate of diagnostic yield and was associated with changes in critical care management and long-term patient management.

Assessing computational predictions of antimicrobial resistance phenotypes from microbial genomes.

The advent of rapid whole-genome sequencing has created new opportunities for computational prediction of antimicrobial resistance (AMR) phenotypes from genomic data. Both rule-based and machine learning (ML) approaches have been explored for this task, but systematic benchmarking is still needed. Here, we evaluated four state-of-the-art ML methods (Kover, PhenotypeSeeker, Seq2Geno2Pheno and Aytan-Aktug), an ML baseline and the rule-based ResFinder by training and testing each of them across 78 species-antibiotic datasets, using a rigorous benchmarking workflow that integrates three evaluation approaches, each paired with three distinct sample splitting methods. Our analysis revealed considerable variation in the performance across techniques and datasets. Whereas ML methods generally excelled for closely related strains, ResFinder excelled for handling divergent genomes. Overall, Kover most frequently ranked top among the ML approaches, followed by PhenotypeSeeker and Seq2Geno2Pheno. AMR phenotypes for antibiotic classes such as macrolides and sulfonamides were predicted with the highest accuracies. The quality of predictions varied substantially across species-antibiotic combinations, particularly for beta-lactams; across species, resistance phenotyping of the beta-lactams compound, aztreonam, amoxicillin/clavulanic acid, cefoxitin, ceftazidime and piperacillin/tazobactam, alongside tetracyclines demonstrated more variable performance than the other benchmarked antibiotics. By organism, Campylobacter jejuni and Enterococcus faecium phenotypes were more robustly predicted than those of Escherichia coli, Staphylococcus aureus, Salmonella enterica, Neisseria gonorrhoeae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Streptococcus pneumoniae and Mycobacterium tuberculosis. In addition, our study provides software recommendations for each species-antibiotic combination. It furthermore highlights the need for optimization for robust clinical applications, particularly for strains that diverge substantially from those used for training.

A Practical Guide to Whole Genome Sequencing in the NICU.

The neonatal period is a peak time for the presentation of genetic disorders that can be diagnosed using whole genome sequencing (WGS). While any one genetic disorder is individually rare, they collectively contribute to significant morbidity, mortality, and health-care costs. As the cost of WGS continues to decline and becomes increasingly available, the ordering of rapid WGS for NICU patients with signs or symptoms of an underlying genetic condition is now feasible. However, many neonatal clinicians are not comfortable with the testing, and unfortunately, there is a dearth of geneticists to facilitate testing for every patient that needs it. Here, we will review the science behind WGS, diagnostic capabilities, limitations of testing, time to consider testing, test initiation, interpretation of results, developing a plan of care that incorporates genomic information, and returning WGS results to families.

Live tracking of a plant pathogen outbreak reveals rapid and successive, multidecade plasmid reduction.

Quickly understanding the genomic changes that lead to pathogen emergence is necessary to launch mitigation efforts and reduce harm. In this study, we tracked in real time a 2022 bacterial plant disease outbreak in U.S. geraniums (Pelargonium × hortorum) caused by Xhp2022, a novel lineage of Xanthomonas hortorum. Genomes from 31 Xhp2022 isolates from seven states showed limited chromosomal variation and all contained a single plasmid (p93). Time tree and single nucleotide polymorphism whole-genome analysis estimated that Xhp2022 emerged within the last decade. The phylogenomic analysis determined that p93 resulted from the cointegration of three plasmids (p31, p45, and p66) found sporadically across isolates from previous outbreaks. Although p93 had a 49 kb nucleotide reduction, it retained putative fitness genes, which became predominant in the 2022 outbreak. Overall, we demonstrated, through rapid whole-genome sequencing and analysis, a recent, traceable event of genome reduction for niche adaptation typically observed over millennia in obligate and fastidious pathogens.IMPORTANCEThe geranium industry, valued at $4 million annually, faces an ongoing Xanthomonas hortorum pv. pelargonii (Xhp) pathogen outbreak. To track and describe the outbreak, we compared the genome structure across historical and globally distributed isolates. Our research revealed Xhp population has not had chromosome rearrangements since 1974 and has three distinct plasmids. In 2012, we found all three plasmids in individual Xhp isolates. However, in 2022, the three plasmids co-integrated into one plasmid named p93. p93 retained putative fitness genes but lost extraneous genomic material. Our findings show that the 2022 strain group of the bacterial plant pathogen Xanthomonas hortorum underwent a plasmid reduction. We also observed several Xanthomonas species from different years, hosts, and continents have similar plasmids to p93, possibly due to shared agricultural settings. We noticed parallels between genome efficiency and reduction that we see across millennia with obligate parasites with increased niche specificity.

Development of an amplicon-based sequencing approach in response to the global emergence of mpox.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

A Multiplexed, Tiled PCR Method for Rapid Whole-Genome Sequencing of Infectious Spleen and Kidney Necrosis Virus (ISKNV) in Tilapia.

Tilapia farming is one of the most important sectors in aquaculture worldwide and of major importance to global food security. Infectious spleen and kidney necrosis virus (ISKNV) has been identified as an agent of high morbidity and mortality, threatening tilapia aquaculture. ISKNV was detected in Lake Volta, Ghana, in September 2018 and spread rapidly, with mortality rates between 60 and 90% and losses of more than 10 tonnes of fish per day. Understanding the spread and evolution of viral pathogens is important for control strategies. Here, we developed a tiled-PCR sequencing approach for the whole-genome sequencing of ISKNV, using long read sequencing to enable field-based, real-time genomic surveillance. This work represents the first use of tiled-PCR for whole genome recovery of viruses in aquaculture, with the longest genome target (>110 kb dsDNA) to date. Our protocol was applied to field samples collected from the ISKNV outbreaks from four intensive tilapia cage culture systems across Lake Volta, between October 2018 and May 2022. Despite the low mutation rate of dsDNA viruses, 20 single nucleotide polymorphisms accumulated during the sampling period. Droplet digital PCR identified a minimum requirement of template in a sample to recover 50% of an ISKNV genome at 275 femtograms (2410 viral templates per 5 µL sequencing reaction). Overall, tiled-PCR sequencing of ISKNV provides an informative tool to assist in disease control in aquaculture.

Integrated single-tube operation for rapid and uniform single-cell whole-genome sequencing

Single-cell whole-genome sequencing (scWGS) plays a critical role in revealing intercellular genome variations. However, the existing scWGS technologies suffer from tedious single-cell isolation and nonuniform amplification. Here, we present an Integrated single-tube operation for rapid and uniform single-cell whole-genome sequencing (Iso-seq) to overcome these limitations. The specially designed dual-well structure in the cap is based on size exclusion and locally quasi-hydrodynamic principles, which ensure that one cell is trapped in one cap, while the capillary-based centrifugal-driven droplet generator facilitates uniform droplet generation in the tube. Using Iso-seq, single cells can be isolated by the dual-well structure in an eight-cap strip within 3 min and the corresponding whole-genome of single cells can be readily compartmentalized in parallel droplets. The prepared single-cell samples show a high mapping ratio, broad coverage of the genome, and low coefficient of variation, demonstrating the good performance of Iso-seq in scWGS. With the advantages of rapid single-cell isolation, convenient droplet generation, broad coverage of the genome, and excellent sequencing uniformity, Iso-seq is promising for a wide variety of biological applications.

An Integrated Pipeline for Trio-Rapid Genome Sequencing in Critically Ill Infants.

Trio-rapid genome sequencing (trio-rGS) can assist the genetic diagnosis of critically ill infants given its ability to detect a broad range of pathogenic variants, as well as microbes, simultaneously with high efficiency. To achieve more comprehensive clinical diagnoses, it is essential to propose a recommended protocol in clinical practice. Here, we introduced an integrated pipeline to detect germline variants and microorganisms simultaneously from trio-RGS in critically ill infants, which provides step-by-step criteria for the semi-automatic processing procedures. With this pipeline in clinical application, only 1 ml of peripheral blood is needed for clinicians to provide both genetic and infectious causal information to a patient. The establishment and clinical practice of the method is of great significance for further mining of high-throughput sequencing data and for assisting clinicians in promoting diagnosis efficiency and accuracy. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Experimental pipeline for rapid whole-genome sequencing for the simultaneous detection of germline variants and microorganisms Basic Protocol 2: Computational pipeline for rapid whole-genome sequencing for the simultaneous detection of germline variants and microorganisms.

Using random forest to predict antimicrobial minimum inhibitory concentrations of nontyphoidal Salmonella in Taiwan

Antimicrobial resistance (AMR) is a global health issue and surveillance of AMR can be useful for understanding AMR trends and planning intervention strategies. Salmonella, widely distributed in food-producing animals, has been considered the first priority for inclusion in the AMR surveillance program by the World Health Organization (WHO). Recent advances in rapid and affordable whole-genome sequencing (WGS) techniques lead to the emergence of WGS as a one-stop test to predict the antimicrobial susceptibility. Since the variation of sequencing and minimum inhibitory concentration (MIC) measurement methods could result in different results, this study aimed to develop WGS-based random forest models for predicting MIC values of 24 drugs using data generated from the same laboratories in Taiwan. The WGS data have been transformed as a feature vector of 10-mers for machine learning. Based on rigorous validation and independent tests, a good performance was obtained with an average mean absolute error (MAE) less than 1 for both validation and independent test. Feature selection was then applied to identify top-ranked 10-mers that can further improve the prediction performance. For surveillance purposes, the genome sequence-based machine learning methods could be utilized to monitor the difference between predicted and experimental MIC, where a large difference might be worthy of investigation on the emerging genomic determinants.

Successful COVID-19 Contact Tracing of Crew from Two Cargo Ships at the Morowali Seaport, Indonesia.

This study aims to report a successful systematic quarantine of coronavirus disease (COVID-19) to detect Omicron, a new variant of concern, among 2 cargo ships in the Morowali seaport, Indonesia. An international standard entry point protocol had been followed in this study for all crew. Rapid diagnostic tests and whole-genome sequencing (WGS) tests have been conducted to identify Omicron, the variant of concern. Individual characteristics, laboratory result, and vaccine status were collected in this study. On December 9 and 18, 2021, there was an increase of 21 and 22 new cases, respectively, from 2 ships in the seaport. Both ships came from abroad, 43 new cases with a positivity rate increase from 0 to 13.4% and 13.7% within 2 weeks. A polymerase chain reaction (PCR) examination was carried out on all crews and obtained results 21 of the 22 positive COVID-19 crew (95.5%). The monitoring results showed that there was no fever in the entire crew, no symptoms of cough, runny nose, shortness of breath, and other symptoms pointing to COVID-19. Systematic quarantine has successfully contained the large clusters of COVID-19 in the crews of 2 ships and prevented further outbreaks in the local community.

Systematic benchmarking of nanopore Q20+ kit in SARS-CoV-2 whole genome sequencing.

Whole genome sequencing provides rapid insight into key information about the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), such as virus typing and key mutation site, and this information is important for precise prevention, control and tracing of coronavirus disease 2019 (COVID-19) outbreak in conjunction with the epidemiological information of the case. Nanopore sequencing is widely used around the world for its short sample-to-result time, simple experimental operation and long sequencing reads. However, because nanopore sequencing is a relatively new sequencing technology, many researchers still have doubts about its accuracy. The combination of the newly launched nanopore sequencing Q20+ kit (LSK112) and flow cell R10.4 is a qualitative improvement over the accuracy of the previous kits. In this study, we firstly used LSK112 kit with flow cell R10.4 to sequence the SARS-CoV-2 whole genome, and summarized the sequencing results of the combination of LSK112 kit and flow cell R10.4 for the 1200bp amplicons of SARS-CoV-2. We found that the proportion of sequences with an accuracy of more than 99% reached 30.1%, and the average sequence accuracy reached 98.34%, while the results of the original combination of LSK109 kit and flow cell R9.4.1 were 0.61% and 96.52%, respectively. The mutation site analysis showed that it was completely consistent with the final consensus sequence of next generation sequencing (NGS). The results showed that the combination of LSK112 kit and flow cell R10.4 allowed rapid whole-genome sequencing of SARS-CoV-2 without the need for verification of NGS.

Whole-genome sequencing and genetic diversity of severe fever with thrombocytopenia syndrome virus using multiplex PCR-based nanopore sequencing, Republic of Korea.

BackgroundWhole-genome sequencing plays a critical role in the genomic epidemiology intended to improve understanding the spread of emerging viruses. Dabie bandavirus, causing severe fever with thrombocytopenia syndrome (SFTS), is a zoonotic tick-borne virus that poses a significant public health threat. We aimed to evaluate a novel amplicon-based nanopore sequencing tool to obtain whole-genome sequences of Dabie bandavirus, also known as SFTS virus (SFTSV), and investigate the molecular prevalence in wild ticks, Republic of Korea (ROK).Principal findingsA total of 6,593 ticks were collected from Gyeonggi and Gangwon Provinces, ROK in 2019 and 2020. Quantitative polymerase chain reaction revealed the presence of SFSTV RNA in three Haemaphysalis longicornis ticks. Two SFTSV strains were isolated from H. longicornis captured from Pocheon and Cheorwon. Multiplex polymerase chain reaction-based nanopore sequencing provided nearly full-length tripartite genome sequences of SFTSV within one hour running. Phylogenetic and reassortment analyses were performed to infer evolutionary relationships among SFTSVs. Phylogenetic analysis grouped SFTSV Hl19-31-4 and Hl19-31-13 from Pocheon with sub-genotype B-1 in all segments. SFTSV Hl20-8 was found to be a genomic organization compatible with B-1 (for L segment) and B-2 (for M and S segments) sub-genotypes, indicating a natural reassortment between sub-genotypes.Conclusion/SignificanceAmplicon-based next-generation sequencing is a robust tool for whole-genome sequencing of SFTSV using the nanopore platform. The molecular prevalence and geographical distribution of SFTSV enhanced the phylogeographic map at high resolution for sophisticated prevention of emerging SFTS in endemic areas. Our findings provide important insights into the rapid whole-genome sequencing and genetic diversity for the genome-based diagnosis of SFTSV in the endemic outbreak.

A genome sequencing system for universal newborn screening, diagnosis, and precision medicine for severe genetic diseases

SummaryNewborn screening (NBS) dramatically improves outcomes in severe childhood disorders by treatment before symptom onset. In many genetic diseases, however, outcomes remain poor because NBS has lagged behind drug development. Rapid whole-genome sequencing (rWGS) is attractive for comprehensive NBS because it concomitantly examines almost all genetic diseases and is gaining acceptance for genetic disease diagnosis in ill newborns. We describe prototypic methods for scalable, parentally consented, feedback-informed NBS and diagnosis of genetic diseases by rWGS and virtual, acute management guidance (NBS-rWGS). Using established criteria and the Delphi method, we reviewed 457 genetic diseases for NBS-rWGS, retaining 388 (85%) with effective treatments. Simulated NBS-rWGS in 454,707 UK Biobank subjects with 29,865 pathogenic or likely pathogenic variants associated with 388 disorders had a true negative rate (specificity) of 99.7% following root cause analysis. In 2,208 critically ill children with suspected genetic disorders and 2,168 of their parents, simulated NBS-rWGS for 388 disorders identified 104 (87%) of 119 diagnoses previously made by rWGS and 15 findings not previously reported (NBS-rWGS negative predictive value 99.6%, true positive rate [sensitivity] 88.8%). Retrospective NBS-rWGS diagnosed 15 children with disorders that had been undetected by conventional NBS. In 43 of the 104 children, had NBS-rWGS-based interventions been started on day of life 5, the Delphi consensus was that symptoms could have been avoided completely in seven critically ill children, mostly in 21, and partially in 13. We invite groups worldwide to refine these NBS-rWGS conditions and join us to prospectively examine clinical utility and cost effectiveness.

EE144 Real-World Economic Evaluation of Prospective Rapid Whole-Genome Sequencing Compared to a Matched Retrospective Cohort of Critically Ill Pediatric Patients in the United States

In the United States, there is an increasing demand for evidence to support the adoption and use of rapid whole genome sequencing(rWGS) by rigorously demonstrating its real-world value. We aimed to assess the cost-effectiveness of rWGS in critically ill pediatric patients with diseases of unknown cause.

The Role of Genome Sequencing in Neonatal Intensive Care Units.

Genetic diseases disrupt the functionality of an infant's genome during fetal-neonatal adaptation and represent a leading cause of neonatal and infant mortality in the United States. Due to disease acuity, gene locus and allelic heterogeneity, and overlapping and diverse clinical phenotypes, diagnostic genome sequencing in neonatal intensive care units has required the development of methods to shorten turnaround times and improve genomic interpretation. From 2012 to 2021, 31 clinical studies documented the diagnostic and clinical utility of first-tier rapid or ultrarapid whole-genome sequencing through cost-effective identification of pathogenic genomic variants that change medical management, suggest new therapeutic strategies, and refine prognoses. Genomic diagnosis also permits prediction of reproductive recurrence risk for parents and surviving probands. Using implementation science and quality improvement, deployment of a genomic learning healthcare system will contribute to a reduction of neonatal and infant mortality through the integration of genome sequencing into best-practice neonatal intensive care.

Refinements and considerations for trio whole-genome sequence analysis when investigating Mendelian diseases presenting in early childhood

SummaryTo facilitate early deployment of whole-genome sequencing (WGS) for severely ill children, a standardized pipeline for WGS analysis with timely turnaround and primary care pediatric uptake is needed. We developed a bioinformatics pipeline for comprehensive gene-agnostic trio WGS analysis of children suspected of having an undiagnosed monogenic disease that included detection and interpretation of primary genetic mechanisms of disease, including SNVs/indels, CNVs/SVs, uniparental disomy (UPD), imprinted genes, short tandem repeat expansions, mobile element insertions, SMN1/2 copy number calling, and mitochondrial genome variants. We assessed primary care practitioner experience and competence in a large cohort of 521 families (comprising 90% WGS trios). Children were identified by primary practitioners for recruitment, and we used the UK index of multiple deprivation to confirm lack of patient socio-economic status ascertainment bias. Of the 521 children sequenced, 176 (34%) received molecular diagnoses, with rates as high as 45% for neurology clinics. Twenty-three of the diagnosed cases (13%) required bespoke methods beyond routine SNV/CNV analysis. In our multidisciplinary clinician user experience assessment, both pediatricians and clinical geneticists expressed strong support for rapid WGS early in the care pathway, but requested further training in determining patient selection, consenting, and variant interpretation. Rapid trio WGS provides an efficacious single-pass screening test for children when deployed by primary practitioners in clinical settings that carry high a priori risk for rare pediatric disease presentations.

Real-world economic evaluation of prospective rapid whole-genome sequencing compared to a matched retrospective cohort of critically ill pediatric patients in the United States.

There is an increasing demand for supporting the adoption of rapid whole-genome sequencing (rWGS) by demonstrating its real-world value. We aimed to assess the cost-effectiveness of rWGS in critically ill pediatric patients with diseases of unknown cause. Data were collected prospectively of patients admitted to the Nicklaus Children's Hospital's intensive care units from March 2018 to September 2020, with rWGS (N = 65). Comparative data were collected in a matched retrospective cohort with standard diagnostic genetic testing. We determined total costs, diagnostic yield (DY), and incremental cost-effectiveness ratio (ICER) adjusted for selection bias and right censoring. Sensitivity analyses explored the robustness of ICER through bootstrapping. rWGS resulted in a diagnosis in 39.8% while standard testing in 13.5% (p = 0.026). rWGS resulted in a mean saving per person of $100,440 (SE = 26,497, p < 0.001) and a total of $6.53 M for 65 patients. rWGS in critically ill pediatric patients is cost-effective, cost-saving, shortens diagnostic odyssey, and triples the DY of traditional approaches.

Protocol for the COG-UK hospital-onset COVID-19 infection (HOCI) multicentre interventional clinical study: evaluating the efficacy of rapid genome sequencing of SARS-CoV-2 in limiting the spread of COVID-19 in UK NHS hospitals

ObjectivesNosocomial transmission of SARS-CoV-2 has been a significant cause of mortality in National Health Service (NHS) hospitals during the COVID-19 pandemic. The COG-UK Consortium Hospital-Onset COVID-19 Infections (COG-UK HOCI) study...

806. Uncluttering Case Clusters: Use of Rapid Sequencing to Exclude Transmission Events

Abstract Background Newly identified multi-drug resistant organisms (MDRO) isolated from hospitalized patients with shared epidemiological characteristics can either represent transmission events or independent, unrelated acquisitions. Whole genome sequencing (WGS) can improve the efficiency of investigations triggered by MDRO cases with apparent epidemiological linkages by early exclusion of clonality. We report an implementation of WGS to investigate a cluster of methicillin-resistant Staphylococcus aureus (MRSA) and a cluster of carbapenem-resistant Enterobacterales (CRE) inpatient nosocomial infections. Methods Study participants included five Neonatal ICU (NICU) patients with nosocomial MRSA isolates recovered between June and August 2020, and two Respiratory Acute Care Unit (RACU) patients with nosocomial CRE infections in October 2020. Routine unit surveillance activities and characterization using standard epidemiologic criteria identified the isolates as nosocomial to their respective unit. The isolates then underwent WGS and single nucleotide polymorphism (SNP)-based relatedness analysis. Results The MRSA cluster included five neonates with either clinical or surveillance isolates. WGS identified one of the five isolates as methicillin susceptible S. aureus due to the absence of the mecA or mecC resistance gene, despite growth on chromogenic MRSA screening agar. WGS revealed each of the five isolates as belonging to a distinct multi locus sequence type (MLST) group with thousands of SNP differences between samples. The CRE cluster included two patients with Klebsiella pneumoniae isolated from clinical cultures within five days of each other with identical antimicrobial susceptibility profiles. WGS of the two isolates revealed that they belonged to different MLSTs and had tens of thousands of differing SNPs. WGS results suggest that a nosocomial transmission linking these infections was highly unlikely. For both investigations, WGS returned results within thirty-six hours of sample receipt. Figure 1. Corynebacterium striatum Respiratory Cultures January 2020-February 2021 Panel showing single nucleotide polymorphism (SNP) differences between isolates for respective clusters. Conclusion Apparent transmission events can be resource intensive to investigate and manage. The application of rapid WGS allowed for early discontinuation of cluster investigations and conservation of resources. Disclosures Virginia M. Pierce, MD, UpToDate, Inc. (Other Financial or Material Support, Author) Mohamad Sater, PhD, Day Zero Diagnostics (Employee, Shareholder) Miriam Huntley, PhD, Day Zero Diagnostics (Employee, Shareholder) Ian Herriott, BS, Day Zero Diagnostics (Employee, Shareholder) Tim Farrell, MS, Day Zero Diagnostics, Inc. (Employee, Shareholder) David C. Rosenberg, MD, Cepheid Diagnostics (Consultant)Day Zero Diagnostics (Consultant)SeLux Diagnostics (Consultant) Erica S. Shenoy, MD, PhD, Vertex Pharmaceuticals (Individual(s) Involved: Self): I gave a single lecture in 3/2020 for which I received financial compensation, Other Financial or Material Support