Optimization of the Illumina COVIDSeq™ protocol for decentralized, cost-effective genomic surveillance

Carpenter, Rob E.; Tamrakar, Vaibhav K; Almas, Sadia; Sharma, Archana; Rowan, Chase; Sharma, Rahul

doi:10.1016/j.plabm.2023.e00311

Cited by 2 publications

(1 citation statement)

References 7 publications

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“…Implementing PM in the clinical laboratory is expensive and time-consuming and requires high-level expertise. Furthermore, production workflows may benefit from multiplexity optimization to reduce the cost ( Carpenter et al., 2023 ). Moreover, the extensive differentiation power of PM makes distinguishing between pathogenic and commensal microflora challenging, particularly for less studied and emerging pathogens.…”

Section: Discussionmentioning

confidence: 99%

Advantage of precision metagenomics for urinary tract infection diagnostics

Almas¹,

Carpenter

Rowan³

et al. 2023

Front. Cell. Infect. Microbiol.

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BackgroundUrinary tract infections (UTIs) remain a diagnostic challenge and often promote antibiotic overuse. Despite urine culture being the gold standard for UTI diagnosis, some uropathogens may lead to false-negative or inconclusive results. Although PCR testing is fast and highly sensitive, its diagnostic yield is limited to targeted microorganisms. Metagenomic next-generation sequencing (mNGS) is a hypothesis-free approach with potential of deciphering the urobiome. However, clinically relevant information is often buried in the enormous amount of sequencing data.MethodsPrecision metagenomics (PM) is a hybridization capture-based method with potential of enhanced discovery power and better diagnostic yield without diluting clinically relevant information. We collected 47 urine samples of clinically suspected UTI and in parallel tested each sample by microbial culture, PCR, and PM; then, we comparatively analyzed the results. Next, we phenotypically classified the cumulative microbial population using the Explify® data analysis platform for potential pathogenicity.ResultsResults revealed 100% positive predictive agreement (PPA) with culture results, which identified only 13 different microorganisms, compared to 19 and 62 organisms identified by PCR and PM, respectively. All identified organisms were classified into phenotypic groups (0–3) with increasing pathogenic potential and clinical relevance. This PM can simultaneously quantify and phenotypically classify the organisms readily through bioinformatic platforms like Explify®, essentially providing dissected and quantitative results for timely and accurate empiric UTI treatment.ConclusionPM offers potential for building effective diagnostic models beyond usual care testing in complex UTI diseases. Future studies should assess the impact of PM-guided UTI management on clinical outcomes.

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Section: Discussionmentioning

confidence: 99%

Advantage of precision metagenomics for urinary tract infection diagnostics

Almas¹,

Carpenter

Rowan³

et al. 2023

Front. Cell. Infect. Microbiol.

Self Cite

View full text Add to dashboard Cite

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Deciphering Microbiota of Acute Upper Respiratory Infections: A Comparative Analysis of PCR and mNGS Methods for Lower Respiratory Trafficking Potential

Almas¹,

Carpenter

Singh

et al. 2023

Advances in Respiratory Medicine

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Although it is clinically important for acute respiratory tract (co)infections to have a rapid and accurate diagnosis, it is critical that respiratory medicine understands the advantages of current laboratory methods. In this study, we tested nasopharyngeal samples (n = 29) with a commercially available PCR assay and compared the results with those of a hybridization-capture-based mNGS workflow. Detection criteria for positive PCR samples was Ct < 35 and for mNGS samples it was >40% target coverage, median depth of 1X and RPKM > 10. A high degree of concordance (98.33% PPA and 100% NPA) was recorded. However, mNGS yielded positively 29 additional microorganisms (23 bacteria, 4 viruses, and 2 fungi) beyond PCR. We then characterized the microorganisms of each method into three phenotypic categories using the IDbyDNA Explify® Platform (Illumina® Inc, San Diego, CA, USA) for consideration of infectivity and trafficking potential to the lower respiratory region. The findings are significant for providing a comprehensive yet clinically relevant microbiology profile of acute upper respiratory infection, especially important in immunocompromised or immunocompetent with comorbidity respiratory cases or where traditional syndromic approaches fail to identify pathogenicity. Accordingly, this technology can be used to supplement current syndrome-based tests, and data can quickly and effectively be phenotypically characterized for trafficking potential, clinical (co)infection, and comorbid consideration—with promise to reduce morbidity and mortality.

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Optimization of the Illumina COVIDSeq™ protocol for decentralized, cost-effective genomic surveillance

Cited by 2 publications

References 7 publications

Advantage of precision metagenomics for urinary tract infection diagnostics

Advantage of precision metagenomics for urinary tract infection diagnostics

Deciphering Microbiota of Acute Upper Respiratory Infections: A Comparative Analysis of PCR and mNGS Methods for Lower Respiratory Trafficking Potential

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