Dataset Information

Amplicon Sequencing Minimal Information (ASqMI): Quality and Reporting Guidelines for Actionable Calls in Biodefense Applications.

ABSTRACT:

Background

Accurate, high-confidence data is critical for assessing potential biothreat incidents. In a biothreat event, false-negative and -positive results have serious consequences. Worst case scenarios can result in unnecessary shutdowns or fatalities at an exorbitant monetary and psychological cost, respectively. Quantitative PCR assays for agents of interest have been successfully used for routine biosurveillance. Recently, there has been increased impetus for adoption of amplicon sequencing (AS) for biosurveillance because it enables discrimination of true positives from near-neighbor false positives, as well as broad, simultaneous detection of many targets in many pathogens in a high-throughput scheme. However, the high sensitivity of AS can lead to false positives. Appropriate controls and workflow reporting can help address these challenges.

Objectives

Data reporting standards are critical to data trustworthiness. The standards presented herein aim to provide a framework for method quality assessment in biodetection.

Methods

We present a set of standards, Amplicon Sequencing Minimal Information (ASqMI), developed under the auspices of the AOAC INTERNATIONAL Stakeholder Program on Agent Detection Assays for making actionable calls in biosurveillance applications. In addition to the first minimum information guidelines for AS, we provide a controls checklist and scoring scheme to assure AS run quality and assess potential sample contamination.

Results

Adoption of the ASqMI guidelines will improve data quality, help track workflow performance, and ultimately provide decision makers confidence to trust the results of this new and powerful technology.

Conclusion

AS workflows can provide robust, confident calls for biodetection; however, due diligence in reporting and controls are needed. The ASqMI guideline is the first AS minimum reporting guidance document that also provides the means for end users to evaluate their workflows to improve confidence.

Highlights

Standardized reporting guidance for actionable calls is critical to ensuring trustworthy data.

SUBMITTER: Keenum I

PROVIDER: S-EPMC10472743 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Amplicon Sequencing Minimal Information (ASqMI): Quality and Reporting Guidelines for Actionable Calls in Biodefense Applications.

Keenum Ishi I Player Robert R Kralj Jason J Servetas Stephanie S Sussman Michael D MD Russell Joseph A JA Stone Jennifer J Chandrapati Sailaja S Sozhamannan Shanmuga S

Journal of AOAC International 20230901 5

<h4>Background</h4>Accurate, high-confidence data is critical for assessing potential biothreat incidents. In a biothreat event, false-negative and -positive results have serious consequences. Worst case scenarios can result in unnecessary shutdowns or fatalities at an exorbitant monetary and psychological cost, respectively. Quantitative PCR assays for agents of interest have been successfully used for routine biosurveillance. Recently, there has been increased impetus for adoption of amplicon ...[more]

PMID: 37067472

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Project description:BACKGROUND:Creation and use of ontologies has become a mainstream activity in many disciplines, in particular, the biomedical domain. Ontology developers often disseminate information about these ontologies in peer-reviewed ontology description reports. There appears to be, however, a high degree of variability in the content of these reports. Often, important details are omitted such that it is difficult to gain a sufficient understanding of the ontology, its content and method of creation. RESULTS:We propose the Minimum Information for Reporting an Ontology (MIRO) guidelines as a means to facilitate a higher degree of completeness and consistency between ontology documentation, including published papers, and ultimately a higher standard of report quality. A draft of the MIRO guidelines was circulated for public comment in the form of a questionnaire, and we subsequently collected 110 responses from ontology authors, developers, users and reviewers. We report on the feedback of this consultation, including comments on each guideline, and present our analysis on the relative importance of each MIRO information item. These results were used to update the MIRO guidelines, mainly by providing more detailed operational definitions of the individual items and assigning degrees of importance. Based on our revised version of MIRO, we conducted a review of 15 recently published ontology description reports from three important journals in the Semantic Web and Biomedical domain and analysed them for compliance with the MIRO guidelines. We found that only 41.38% of the information items were covered by the majority of the papers (and deemed important by the survey respondents) and a large number of important items are not covered at all, like those related to testing and versioning policies. CONCLUSIONS:We believe that the community-reviewed MIRO guidelines can contribute to improving significantly the quality of ontology description reports and other documentation, in particular by increasing consistent reporting of important ontology features that are otherwise often neglected.

Project description:BACKGROUND:The state-of-the-art in nucleic acid based biodetection continues to be polymerase chain reaction (PCR), and many real-time PCR assays targeting biodefense pathogens for biosurveillance are in widespread use. These assays are predominantly singleplex; i.e. one assay tests for the presence of one target, found in a single organism, one sample at a time. Due to the intrinsic limitations of such tests, there exists a critical need for high-throughput multiplex assays to reduce the time and cost incurred when screening multiple targets, in multiple pathogens, and in multiple samples. Such assays allow users to make an actionable call while maximizing the utility of the small volumes of test samples. Unfortunately, current multiplex real-time PCR assays are limited in the number of targets that can be probed simultaneously due to the availability of fluorescence channels in real-time PCR instruments. RESULTS:To address this gap, we developed a pipeline in which the amplicons produced by a 14-plex end-point PCR assay using spiked samples were subsequently sequenced using Nanopore technology. We used bar codes to sequence multiple samples simultaneously, leading to the generation and subsequent analysis of sequence data resulting from a short sequencing run time (< 10 min). We compared the limits of detection (LoD) of real-time PCR assays to Oxford Nanopore Technologies (ONT)-based amplicon sequencing and estimated the sample-to-answer time needed for this approach. Overall, LoDs determined from the first 10 min of sequencing data were at least one to two orders of magnitude lower than real-time PCR. Given enough time, the amplicon sequencing approach is approximately 100 times more sensitive than real-time PCR, with detection of amplicon specific reads even at the lowest tested spiking concentration (around 2.5-50 Colony Forming Units (CFU)/ml). CONCLUSIONS:Based on these results, we propose amplicon sequencing assay as a viable alternative to replace the current real-time PCR based singleplex assays for higher throughput biodefense applications. We note, however, that targeted amplicon specific reads were not detectable even at the highest tested spike concentrations (2.5 X 104-5.0 X105 CFU/ml) without an initial amplification step, indicating that PCR is still necessary when utilizing this protocol.

Dataset Information

Amplicon Sequencing Minimal Information (ASqMI): Quality and Reporting Guidelines for Actionable Calls in Biodefense Applications.

Background

Objectives

Methods

Results

Conclusion

Highlights

Publications

Amplicon Sequencing Minimal Information (ASqMI): Quality and Reporting Guidelines for Actionable Calls in Biodefense Applications.

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