Real-Time Genomic Surveillance of Bacterial Outbreaks
By Dr. Zubair Khalid, DVM, MS, PhD ·
Quick Answer
- Real-time genomic surveillance of bacterial outbreaks is achievable with Oxford Nanopore sequencing, which produces sequence data during the run instead of after completion.
- The practical next step for a laboratory is to validate a standard operating procedure that links rapid DNA extraction, library preparation, basecalling, and phylogenetic placement within a single shift.
- A key limitation is that raw accuracy of Nanopore reads remains lower than short-read platforms, so confirmatory analysis or hybrid assembly may be required for high-confidence public health conclusions.
At a Glance
The table below summarizes the core components of a real-time genomic surveillance workflow and the decisions a laboratory must make at each stage.
| Workflow Stage | Primary Decision | Typical Output | Key Limitation |
|---|---|---|---|
| Sample preparation | Select extraction method that yields high molecular weight DNA | Purified genomic DNA | Time to extraction can exceed sequencing run time |
| Library preparation | Choose rapid kit versus ligation kit | Sequencing-ready library | Rapid kits reduce input requirements but may lower read length |
| Sequencing | Set run duration and read depth target | Raw signal data in FAST5 or POD5 format | Throughput per flow cell varies by pore count and chemistry |
| Basecalling | Select high accuracy mode versus fast mode | FASTQ files with quality scores | High accuracy mode requires more computational time |
| Assembly | Use hybrid assembly or long-read only assembly | Draft genome contigs | Short reads may still be needed for plasmid resolution |
| Phylogenetic analysis | Choose reference-based or reference-free approach | Tree or SNP distance matrix | Reference choice can bias placement of novel strains |
The Problem That Real-Time Genomic Surveillance Solves
Bacterial outbreak investigations have historically depended on culture, biochemical identification, and antimicrobial susceptibility testing. These methods produce actionable data but they operate on a time scale that can allow a cluster to expand before the source is identified. Genomic surveillance changes the timeline because it examines the genetic material of the organism directly, allowing laboratories to compare isolates from different patients, food samples, or environmental sources at the level of single nucleotide differences.
The World Health Organization identifies antimicrobial resistance as a global health threat that requires coordinated surveillance across human, animal, and environmental sectors. Genomic methods are central to that coordination because they can reveal whether a resistant strain is spreading or whether resistance is emerging independently in different locations. The WHO context for antimicrobial resistance emphasizes that surveillance is also about counting cases but about understanding the mechanisms of spread. Genomic data provides that mechanism-level view.
For a public health laboratory, the transition to real-time surveillance is not a simple purchase of a sequencing instrument. It is a workflow change that affects sample intake, data storage, staff training, and the way results are reported to epidemiologists. The laboratory must decide which samples are sequenced, how quickly the sequence data is analyzed, and what evidence standard is used to declare that two isolates are part of the same outbreak.
Core Principles of Bacterial Genomic Surveillance
What Genomic Surveillance Measures
Bacterial genomic surveillance compares the complete or near-complete DNA sequence of bacterial isolates to determine their relatedness. The comparison is usually made by identifying single nucleotide polymorphisms, which are positions in the genome where one base differs between isolates. Two isolates that differ by a small number of SNPs are likely to share a recent common ancestor. Two isolates that differ by a large number of SNPs are likely to be unrelated.
The same sequence data can be used to detect genes that confer antimicrobial resistance, genes that encode virulence factors, and the sequence type of the isolate. This means that one sequencing run can answer multiple questions that previously required separate tests. A laboratory can determine the species, the strain, the resistance profile, and the outbreak cluster from a single data set.
The resolution of genomic surveillance depends on the amount of the genome that is compared. Whole genome sequencing compares the entire chromosome and any plasmids that are present. Targeted sequencing compares only a set of selected genes. Whole genome sequencing provides the highest resolution because it captures the entire genetic content of the organism.
How Real-Time Sequencing Differs From Traditional Methods
Traditional sequencing methods, such as Sanger sequencing or short-read sequencing, require the sequencing reaction to be completed before the data is available. The data is then processed in batches. Real-time sequencing, as performed by Oxford Nanopore instruments, produces data continuously as the DNA molecule passes through a nanopore. The instrument detects changes in electrical current as the DNA moves through the pore, and the basecaller converts those current changes into nucleotide sequences.
The practical consequence is that a laboratory can begin analyzing the first reads from a run while the instrument is still sequencing the rest of the sample. This allows a preliminary identification of the species and a preliminary resistance profile within the first hour of the run. The full genome sequence is available when the run is complete.
The tradeoff is that the accuracy of individual Nanopore reads is lower than the accuracy of short-read sequencing. The error rate is not uniform across the genome, and some regions are more difficult to sequence than others. The laboratory must decide whether the speed of the result is worth the additional effort required to correct errors in the data.
The Real-Time Sequencing Workflow
The workflow for real-time genomic surveillance can be divided into five stages. Each stage has a set of decisions that affect the speed and the quality of the final result.
Sample Intake and Culture
The sequencing workflow begins with a pure culture of the bacterial isolate. The culture is necessary because the sequencing instrument requires a sufficient quantity of high quality DNA. The laboratory must grow the isolate on a solid medium or in a liquid culture to obtain enough cells for extraction.
The choice of culture medium and incubation time affects the quality of the DNA. Some bacteria produce polysaccharides or other compounds that inhibit the sequencing reaction. The laboratory must choose a method that removes these inhibitors during the DNA extraction step.
The culture step is the slowest part of the workflow. A fast growing organism such as Escherichia coli can produce sufficient growth in a few hours, but a slow growing organism such as Mycobacterium tuberculosis can require weeks. The laboratory must decide whether the speed of the sequencing step is worth the time required for the culture step.
DNA Extraction and Quality Assessment
The DNA extraction step must produce DNA that is pure, concentrated, and of high molecular weight. The extraction method must be compatible with the sequencing platform. Some extraction methods produce DNA that is fragmented, which reduces the length of the reads that can be obtained.
The quality of the DNA is assessed by measuring the concentration and the purity. The concentration is measured by a fluorometric method or by a spectrophotometric method. The purity is assessed by measuring the ratio of absorbance at 260 nanometers and 280 nanometers. A ratio of approximately 1.8 indicates pure DNA, while a lower ratio indicates protein contamination.
The size of the DNA is assessed by gel electrophoresis or by a microfluidic instrument. The DNA must be long enough to produce reads that span the entire genome. The length of the reads is a key factor in the quality of the assembly.
Library Preparation
The library preparation step converts the purified DNA into a form that can be sequenced by the instrument. The DNA is repaired, and adapters are attached to the ends. The adapters are the sequences that allow the DNA to interact with the nanopore.
The library preparation kit determines the length of the reads that can be obtained. A ligation kit produces longer reads but requires more input DNA. A rapid kit produces shorter reads but requires less input DNA and less time. The laboratory must decide which kit is appropriate for the sample and the question.
The library preparation step is also the point at which a barcode can be added to the DNA. A barcode is a unique sequence that allows the laboratory to sequence multiple samples in a single run. The barcode is read by the instrument, and the data is separated by the barcode during the analysis step.
Sequencing Run
The sequencing run is the step where the instrument produces the data. The run is started by loading the library into the flow cell. The flow cell contains the nanopores that read the DNA.
The run duration is set by the laboratory. A longer run produces more data, but the rate of data production decreases over time. The laboratory must decide how much data is needed to answer the question. A species identification can be made with a small amount of data, but a high quality assembly requires more data.
The instrument produces raw signal data during the run. The raw data is stored in a format that contains the electrical current measurements. The raw data is converted to nucleotide sequences by a process called basecalling.
Basecalling and Data Analysis
Basecalling is the process of converting the raw signal data into nucleotide sequences. The basecaller is a software program that uses a model to interpret the electrical current changes. The basecaller can be run on the instrument computer or on a separate computer with a graphics processing unit.
The basecaller produces a FASTQ file that contains the nucleotide sequence and a quality score for each base. The quality score is a measure of the probability that the base is correct. The quality score is used to filter the data during the analysis step.
The analysis step is where the sequence data is used to answer the biological question. The analysis can be performed by a variety of software programs. The analysis includes the assembly of the reads into a genome, the comparison of the genome to a reference, and the identification of resistance genes and virulence factors.
Phylogenetic Analysis in Near Real Time
Reference-Based Analysis
The most common approach to phylogenetic analysis is to compare the sequenced genome to a reference genome. The reference genome is a complete sequence of a representative strain of the species. The reads are aligned to the reference, and the positions where the reads differ from the reference are identified.
The result is a set of SNPs that describe the relationship between the isolate and the reference. The SNPs can be used to construct a phylogenetic tree that shows the relationship between the isolate and other isolates that have been sequenced.
The reference-based approach is fast and computationally efficient. The limitation is that the reference genome may not be representative of the isolate. If the isolate is distantly related to the reference, the reads may not align well, and the SNP calls may be inaccurate.
Reference-Free Analysis
The reference-free approach does not require a reference genome. The reads are assembled into a genome, and the genome is compared to other genomes. The comparison is performed by identifying the regions of the genome that are shared and the regions that are unique.
The reference-free approach is more computationally intensive than the reference-based approach. The assembly step requires more time and more memory. The advantage is that the approach can identify novel sequences that are not present in the reference genome.
The choice between the reference-based and the reference-free approach depends on the question. The reference-based approach is appropriate for a rapid comparison of a known species. The reference-free approach is appropriate for a novel species or for a species with a high degree of genetic diversity.
Phylogenetic Tree Interpretation
The phylogenetic tree is a visual representation of the relationship between the isolates. The tree is composed of branches and nodes. The nodes represent the common ancestors of the isolates. The length of the branches represents the number of SNPs that separate the isolates.
The interpretation of the tree requires an understanding of the limitations of the data. The tree is a hypothesis about the relationship between the isolates. The tree is based on the SNPs that are identified in the analysis. The SNPs that are not identified, because they are in regions that are not sequenced or because they are in regions that are not aligned, are not represented in the tree.
The laboratory must decide the threshold for the number of SNPs that defines an outbreak. A small number of SNPs indicates that the isolates are closely related. A large number of SNPs indicates that the isolates are not related. The threshold is not a fixed number. The threshold depends on the species and the time scale of the outbreak.
Quality Controls and Validation
Positive and Negative Controls
The sequencing workflow must include positive and negative controls. The positive control is a sample with a known sequence. The negative control is a sample that does not contain DNA. The controls are processed through the entire workflow, from extraction to analysis.
The positive control is used to verify that the workflow is producing the correct sequence. The sequence of the positive control is compared to the known sequence. A discrepancy indicates a problem in the workflow.
The negative control is used to verify that the workflow is not contaminated. The negative control should not produce a sequence. A sequence in the negative control indicates contamination in the reagents or the environment.
Reference Materials
The laboratory should use authenticated reference materials for the validation of the workflow. The American Type Culture Collection provides authenticated biological materials that can be used as reference strains. The reference strains have a known sequence that can be used to verify the accuracy of the workflow.
The reference materials are also used to validate the analysis software. The software is run on the reference data, and the output is compared to the known result. The validation is performed before the software is used for the clinical or public health samples.
Data Storage and Records
The sequencing data is a large amount of data. The raw signal data is the largest file. The basecalled data is smaller. The assembled genome is the smallest. The laboratory must have a plan for the storage of the data.
The data must be stored in a secure location. The data is a record of the laboratory work. The data must be retained for a period of time that is determined by the laboratory policy or the regulatory requirement.
The laboratory must also keep a record of the workflow. The record includes the sample identifier, the extraction method, the library preparation method, the sequencing run parameters, and the analysis software. The record is used to trace the data back to the sample.
Common Failure Patterns
Low DNA Yield
The most common failure in the sequencing workflow is a low DNA yield. The DNA yield is the amount of DNA that is obtained from the extraction. A low yield can be caused by a poor culture, an inefficient extraction, or a loss of DNA during the purification step.
The laboratory must assess the DNA yield before the library preparation. The yield is measured by the concentration and the volume. The yield must be sufficient for the library preparation. If the yield is low, the laboratory must repeat the extraction or the culture.
Short Read Length
The read length is the length of the DNA that is sequenced in a single read. The read length is determined by the quality of the DNA and the library preparation. The DNA that is fragmented produces short reads. The short reads are more difficult to assemble.
The laboratory must assess the read length during the run. The read length is reported by the sequencing instrument. If the read length is short, the laboratory must adjust the DNA extraction or the library preparation.
Low Basecalling Quality
The basecalling quality is the accuracy of the nucleotide sequence. The quality is reported as a quality score. The quality score is used to filter the data. The low quality reads are removed from the analysis.
The basecalling quality is affected by the quality of the DNA, the library, and the flow cell. The laboratory must monitor the quality during the run. If the quality is low, the laboratory must adjust the basecalling parameters or the run.
Contamination
Contamination is the presence of DNA from a source other than the sample. The contamination can be from the environment, the reagents, or the other samples. The contamination is detected by the negative control or by the analysis of the sequence.
The contamination is a serious problem because it can lead to a false result. The laboratory must identify the source of the contamination and correct the problem. The correction may require a change in the workflow or a change in the environment.
Biosafety and Regulatory Context
The sequencing workflow is performed in a laboratory that is designed for the work. The laboratory must follow the biosafety principles that are described in the Biosafety in Microbiological and Biomedical Laboratories. The BMBL describes the risk assessment, the containment, and the decontamination that are required for the work.
The risk assessment is the process of identifying the hazards of the work. The hazard is the organism that is being sequenced. The risk is the likelihood of the hazard causing harm. The risk assessment is used to determine the biosafety level of the laboratory.
The containment is the set of practices and equipment that are used to prevent the release of the organism. The containment includes the biological safety cabinet, the personal protective equipment, and the decontamination procedures. The containment is designed to protect the worker and the environment.
The decontamination is the process of removing the organism from the equipment and the waste. The decontamination is performed by the chemical or the physical method. The decontamination is required before the equipment is used for the other work or before the waste is disposed.
The laboratory must also follow the standards for the antimicrobial susceptibility testing. The Clinical and Laboratory Standards Institute provides the standards for the testing. The standards describe the methods and the interpretation of the results. The standards are used to ensure the accuracy of the testing.
The Role of the Laboratory Professional
The success of the real-time genomic surveillance depends on the skill of the laboratory professional. The professional must be trained in the sequencing workflow and the analysis. The professional must also be trained in the interpretation of the results.
The American Society for Microbiology provides the education and the training for the microbiology professional. The ASM provides the resources for the laboratory practice and the workforce. The ASM also provides the context for the professional development.
The professional must also be able to communicate the results to the epidemiologist and the public health official. The communication is the final step of the workflow. The professional must explain the result in a way that is clear and actionable.
The Limitations of Real-Time Genomic Surveillance
The real-time genomic surveillance is a powerful tool, but it has limitations. The limitations must be understood by the laboratory and the public health official.
The first limitation is the accuracy of the data. The Nanopore data has a higher error rate than the other sequencing methods. The error rate can affect the SNP calls and the resistance gene identification. The laboratory must use the quality controls and the confirmatory methods to the accuracy.
The second limitation is the cost of the workflow. The sequencing instrument, the flow cells, and the reagents are expensive. The cost is a barrier for the laboratory with a limited budget. The laboratory must decide whether the benefit of the real-time data is worth the cost.
The third limitation is the need for the bioinformatics expertise. The analysis of the data requires the knowledge of the software and the algorithms. The laboratory must have the staff with the expertise or the access to the expertise.
The fourth limitation is the need for the reference data. The phylogenetic analysis requires the reference genome and the reference data. The reference data may not be available for the novel species or the species with the high diversity.
The Future of Real-Time Genomic Surveillance
The real-time genomic surveillance is a rapidly evolving field. The technology is improving, and the cost is decreasing. The future of the field is the integration of the sequencing with the other data.
The integration of the sequencing with the epidemiological data will allow the public health official to see the full picture of the outbreak. The integration of the sequencing with the clinical data will allow the clinician to make the treatment decision.
The future of the field is also the automation of the workflow. The automation will reduce the time and the cost of the workflow. The automation will also reduce the error and the variability of the workflow.
The future of the field is the point of care. The point of care is the sequencing that is performed at the patient bedside or in the field. The point of care will allow the rapid diagnosis and the rapid response.
A Decision Framework for Selecting Real-Time Surveillance Depth
The decision to sequence an isolate in real time is not a single choice between sequencing and not sequencing. It is a series of choices about how much data is needed, how quickly the result must be available, and what level of certainty is acceptable for the public health action that will follow. A laboratory that sequences every isolate with the same depth and the same turnaround target will waste resources on samples that need only a species confirmation and will underpower the analysis of samples that require high confidence phylogenetic placement. A structured decision framework helps the laboratory match the sequencing effort to the question being asked.
The Three Surveillance Tiers
A practical framework divides real-time surveillance into three tiers based on the urgency and the consequence of the result. Each tier has a distinct sequencing depth, a distinct analysis pipeline, and a distinct reporting pathway.
Tier one is the rapid screen. The question is simple: what species is this isolate, and does it carry a known high consequence resistance gene or virulence marker? The sequencing run is short, often less than two hours. The analysis is limited to species identification and a targeted search for a small set of genes. The result is reported as a preliminary finding that requires confirmation before it is used for a regulatory action or a clinical decision. This tier is appropriate for a busy reference laboratory that needs to triage a large number of isolates and identify the ones that require immediate attention.
Tier two is the cluster investigation. The question is whether two or more isolates are part of the same outbreak. The sequencing run is longer, typically four to eight hours, and the analysis includes a reference-based SNP comparison or a core genome multilocus sequence typing approach. The result is a phylogenetic placement that allows the epidemiologist to decide whether to expand or narrow an outbreak investigation. This tier is the core of real-time genomic surveillance because it produces the data that changes the course of an investigation while the investigation is still active.
Tier three is the high confidence characterization. The question requires a complete or near complete genome assembly with accurate resistance gene detection and plasmid resolution. The sequencing run is extended, and the analysis may include hybrid assembly with short-read data or additional polishing steps. The result is used for a published report, a regulatory submission, or a legal proceeding. This tier is not real time in the strict sense because the analysis may take more than a day, but it is the tier that produces the detailed explanation.
The laboratory should document which tier applies to each sample at the time of intake. The tier assignment is made by the laboratory director or the designated supervisor in consultation with the epidemiologist who requested the testing. The assignment is recorded in the laboratory information management system and is reviewed if the preliminary results change the risk picture.
Criteria for Tier Assignment
The tier assignment is based on four criteria: the clinical severity of the case, the potential for spread, the availability of a reference genome, and the regulatory or legal consequence of the result.
Clinical severity is the first consideration. An isolate from a bloodstream infection or a case of meningitis warrants a higher tier than an isolate from a routine stool sample. The laboratory should have a written list of specimen types and clinical presentations that trigger tier two or tier three. The list is developed with the input of the hospital epidemiologist or the public health medical officer and is reviewed annually.
Potential for spread is the second consideration. An isolate from a long term care facility, a neonatal intensive care unit, or a food production facility has a higher potential for spread than an isolate from a single community case. The laboratory should have a written list of settings that trigger tier two. The list is based on the local epidemiology and is updated when new transmission settings are identified.
Reference genome availability is the third consideration. A species with a well curated reference genome and a large database of comparator isolates is well suited to tier two analysis. A species with no reference genome or a highly diverse species requires tier three analysis because the reference based approach will not produce a reliable result. The laboratory should maintain a list of species for which tier two analysis is validated and a list of species that require tier three.
Regulatory or legal consequence is the fourth consideration. An isolate that is part of a foodborne outbreak investigation with regulatory implications requires tier three analysis because the result may be used in a recall or a legal action. An isolate that is part of an epidemiologic investigation with no regulatory consequence can be analyzed at tier two. The laboratory should have a written policy that defines which results require tier three documentation.
The Decision Matrix
The table below presents the decision matrix that the laboratory uses to assign a tier to each sample. The matrix is applied at sample intake and is recorded in the laboratory information management system.
| Criterion | Tier One | Tier Two | Tier Three |
|---|---|---|---|
| Clinical severity | Routine specimen | Serious infection | Life threatening infection |
| Spread potential | Single community case | Healthcare or food setting | Outbreak with regulatory action |
| Reference genome | Not required | Available and validated | Not available or highly diverse |
| Regulatory consequence | None | Possible | Confirmed or likely |
| Sequencing run target | Less than 2 hours | 4 to 8 hours | Extended run with polishing |
| Analysis output | Species and marker screen | SNP comparison or cgMLST | Complete assembly with resistance genes |
| Reporting pathway | Preliminary verbal report | Written preliminary report | Final written report with documentation |
The matrix is a guide, not a rule. The laboratory director can override the tier assignment when the circumstances require it. The override is documented with the reason and the date.
Turnaround Time Targets and Service Level Agreements
The tier framework is only useful if it is tied to measurable turnaround time targets. The laboratory should establish a service level agreement with the epidemiologists and the clinicians who request the testing. The agreement specifies the time from sample receipt to the preliminary report and the time from sample receipt to the final report for each tier.
A realistic target for tier one is a preliminary report within four hours of sample receipt. This target assumes that the culture is already available and that the DNA extraction and library preparation take approximately one hour. The sequencing run takes less than two hours, and the analysis takes less than one hour. The target is achievable with a dedicated instrument and a trained operator.
A realistic target for tier two is a preliminary report within eight hours of sample receipt. The additional time is required for the longer sequencing run and the more complex analysis. The target assumes that the reference genome is available and that the analysis pipeline is automated.
A realistic target for tier three is a final report within five working days. The extended time is required for the complete assembly, the polishing steps, and the additional quality controls. The target assumes that the laboratory has access to short-read sequencing for hybrid assembly or that the long-read assembly is sufficient for the question.
The service level agreement should include a provision for escalation. If the laboratory cannot meet the turnaround time target, the laboratory director must notify the requester within one hour of the anticipated delay. The notification includes the reason for the delay and the revised time of the report.
Records and Metrics for the Decision Framework
The laboratory must record the tier assignment and the outcome for every sample. The record includes the sample identifier, the date and time of receipt, the tier assignment, the reason for the tier assignment, the sequencing run start and end times, the analysis completion time, and the time of the preliminary and final reports.
The laboratory should review the tier assignments and the turnaround times on a quarterly basis. The review identifies the samples that were assigned to the wrong tier, the samples that missed the turnaround time target, and the samples that required an override. The review is documented and is used to update the decision matrix and the service level agreement.
A useful metric is the proportion of samples that are assigned to each tier. A laboratory that assigns most samples to tier three is overusing the high confidence workflow and is likely delaying results that could be delivered at tier two. A laboratory that assigns most samples to tier one is underusing the phylogenetic analysis and is likely missing cluster connections that would be identified at tier two. The target distribution depends on the laboratory mission, but a reference laboratory that serves outbreak investigations should expect a majority of samples at tier two.
A second useful metric is the proportion of tier one results that are changed after tier two or tier three analysis. A high rate of change indicates that the tier one analysis is not reliable for the species being tested. The laboratory should investigate the cause and adjust the tier one pipeline or the tier assignment criteria.
A third useful metric is the time from the preliminary report to the final report for tier two samples. A long gap indicates that the confirmation process is slow and that the epidemiologist is making decisions without the final result. The laboratory should identify the bottleneck and reduce the gap.
Common Failure Patterns in the Decision Framework
The most common failure is assigning every sample to the same tier. This pattern occurs when the laboratory does not have a written decision matrix or when the staff are not trained to apply it. The result is either a backlog of samples waiting for tier three analysis or a set of rapid results that do not answer the outbreak question. The correction is to implement the decision matrix and to train the staff on its use.
A second failure is assigning a sample to tier two when the reference genome is not available. The analysis fails or produces an unreliable result, and the sample must be reassigned to tier three. The delay is longer than if the sample had been assigned to tier three at intake. The correction is to verify the reference genome availability before the tier assignment is made.
A third failure is not documenting the override decisions. The laboratory director reassigns a sample to a higher tier, but the reason is not recorded. The result is that the review process cannot identify the patterns that drive the overrides. The correction is to require a written reason for every override.
A fourth failure is not reviewing the turnaround time metrics. The laboratory continues to miss the targets, but the misses are not identified because the data is not collected or reviewed. The correction is to automate the data collection and to schedule the quarterly review.
Biosafety and Professional Context for the Framework
The decision framework does not change the biosafety requirements of the laboratory. The risk assessment for the organism is performed before the work begins, and the containment and decontamination procedures are followed regardless of the tier assignment. The Biosafety in Microbiological and Biomedical Laboratories provides the principles for the risk assessment and the containment that apply to all tiers.
The decision framework also does not change the professional responsibilities of the laboratory staff. The American Society for Microbiology provides the education and the training context for the laboratory professional. The professional is responsible for applying the decision matrix correctly, for documenting the tier assignment, and for communicating the results within the time frame specified in the service level agreement.
The decision framework is a management tool that helps the laboratory use the real-time sequencing capability in a way that is consistent with the public health mission. The framework does not replace the judgment of the laboratory director or the epidemiologist. It provides a structure for that judgment and a record of the decisions that are made.
Frequently Asked Questions
What is the difference between real-time genomic surveillance and traditional surveillance?
Real-time genomic surveillance uses sequencing data that is produced during the run, allowing the laboratory to begin analysis before the run is complete. Traditional surveillance uses methods that produce data only after the entire process is finished, which can take days or weeks.
How long does it take to get a result from a Nanopore sequencing run?
The time to a result depends on the amount of data that is needed. A species identification can be made within the first hour of the run. A phylogenetic analysis requires more data and may take several hours. The time also depends on the basecalling and the analysis steps.
What is the difference between a reference-based and a reference-free analysis?
A reference-based analysis aligns the reads to a known reference genome and identifies the differences. A reference-free analysis assembles the reads into a genome and compares the genome to other genomes. The reference-based approach is faster, and the reference-free approach is more comprehensive.
What is the role of the quality score in the analysis?
The quality score is a measure of the probability that a base is incorrect. The quality score is used to filter the data. The low quality reads are removed from the analysis. The quality score is also used to assess the confidence of the SNP calls.
What is the difference between a barcode and a control?
A barcode is a unique sequence that is added to the DNA to identify the sample. A control is a sample that is used to verify the workflow. The positive control has a known sequence, and the negative control has no DNA.
What is the role of the reference material in the workflow?
The reference material is a sample with a known sequence. The reference material is used to verify the accuracy of the workflow. The reference material is also used to validate the analysis software.
What is the biosafety level for the sequencing workflow?
The biosafety level is determined by the risk of the organism that is being sequenced. The risk is assessed by the laboratory. The biosafety level is described in the Biosafety in Microbiological and Biomedical Laboratories.
What is the role of the laboratory professional in the workflow?
The laboratory professional performs the workflow and the analysis. The professional is also responsible for the interpretation of the results and the communication of the results to the public health official.
Related Bacteriology Guides
- Bacterial Pathogens in Poultry: Prevalence, Public Health Risks, and Control Strategies
- Bacterial Carriage in Ducks: Pathogens of Public Health Concern
- Bacterial Contamination of Chicken Meat: Food Safety and Public Health
- Salmonellosis in Reptilian Pets: Public Health and Diagnostic Challenges
- Bacterial Pathogens in Chicken Meat: Risk Assessment and Public Health Impact
References and Further Reading
- Biosafety in Microbiological and Biomedical Laboratories. CDC and NIH.
- Microbiology Resources. American Society for Microbiology.
- Antimicrobial Resistance. World Health Organization.
- Bacteriological Analytical Manual. U.S. Food and Drug Administration.
- Antimicrobial Susceptibility Testing. Clinical and Laboratory Standards Institute.
- Culture Collections. American Type Culture Collection.
- NCBI Pathogen Detection. National Center for Biotechnology Information.
- Real-Time Genomic Surveillance for Enhanced Healthcare Outbreak Detection and Control: Clinical and Economic Impact.. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 2026.
- Exploiting Real-Time Genomic Surveillance Data To Assess 4CMenB Meningococcal Vaccine Performance in Scotland, 2015 to 2022.. mBio, 2023.
- Real-Time Use of Monkeypox Virus Genomic Surveillance, King County, Washington, USA, 2022-2024.. Emerging infectious diseases, 2025.
- Evaluating metagenomics and targeted approaches for diagnosis and surveillance of viruses.. Genome medicine, 2024.
This article is educational and does not replace validated laboratory procedures, clinical diagnosis, institutional biosafety review, or public-health guidance.