Abstract
Viral metagenomics is increasingly applied in clinical diagnostic settings for detection of pathogenic viruses. While several benchmarking studies have been published on the use of metagenomic classifiers for abundance and diversity profiling of bacterial populations, studies on the comparative performance of the classifiers for virus pathogen detection are scarce. In this study, metagenomic data sets (n = 88) from a clinical cohort of patients with respiratory complaints were used for comparison of the performance of five taxonomic classifiers: Centrifuge, Clark, Kaiju, Kraken2, and Genome Detective. A total of 1144 positive and negative PCR results for a total of 13 respiratory viruses were used as gold standard. Sensitivity and specificity of these classifiers ranged from 83 to 100% and 90 to 99%, respectively, and was dependent on the classification level and data pre-processing. Exclusion of human reads generally resulted in increased specificity. Normalization of read counts for genome length resulted in a minor effect on overall performance, however it negatively affected the detection of targets with read counts around detection level. Correlation of sequence read counts with PCR Ct-values varied per classifier, data pre-processing (R2 range 15.1–63.4%), and per virus, with outliers up to 3 log10 reads magnitude beyond the predicted read count for viruses with high sequence diversity. In this benchmarking study, sensitivity and specificity were within the ranges of use for diagnostic practice when the cut-off for defining a positive result was considered per classifier.
Highlights
In the era of next-generation sequencing (NGS), clinical metagenomics, the analysis of all microbial genetic material in clinical samples, is being introduced in diagnostic laboratories and revolutionizing the diagnostics of infectious diseases [1–4]
Classification at species and genus levels tended to result in lower sensitivity and higher Receiver Operating Characteristic (ROC) distances, but higher selectivity when compared with family level classification, for most of the classifiers evaluated
We compared the performance of five taxonomic classification tools for virus pathogen detection, using datasets from well-characterized clinical samples
Summary
In the era of next-generation sequencing (NGS), clinical metagenomics, the analysis of all microbial genetic material in clinical samples, is being introduced in diagnostic laboratories and revolutionizing the diagnostics of infectious diseases [1–4]. As opposed to running a series of pathogen targeted diagnostic PCR assays to identify suspected pathogens, one single metagenomic run enables the detection of all potential pathogens in a clinical sample [5,6]. The use of this method, known as shotgun high-throughput sequencing, has resulted in the detection of several pathogens missed by current routine diagnostic procedures [1,7]. Patients with clinical syndromes suspected from an infectious disease but with negative conventional test results are increasingly considered as candidates for metagenomic testing. With sequencing costs decreasing and the significance of detection of unexpected, novel viruses being underscored by the currently pandemic SARS-CoV-2 [13], metagenomics is increasingly moving towards implementation in diagnostic laboratories
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