Viral Metagenomics in the Clinical Realm: Lessons Learned from a Swiss-Wide Ring Trial

Abstract

Shotgun metagenomics using next generation sequencing (NGS) is a promising technique to analyze both DNA and RNA microbial material from patient samples. Mostly used in a research setting, it is now increasingly being used in the clinical realm as well, notably to support diagnosis of viral infections, thereby calling for quality control and the implementation of ring trials (RT) to benchmark pipelines and ensure comparable results. The Swiss NGS clinical virology community therefore decided to conduct a RT in 2018, in order to benchmark current metagenomic workflows used at Swiss clinical virology laboratories, and thereby contribute to the definition of common best practices. The RT consisted of two parts (increments), in order to disentangle the variability arising from the experimental compared to the bioinformatics parts of the laboratory pipeline. In addition, the RT was also designed to assess the impact of databases compared to bioinformatics algorithms on the final results, by asking participants to perform the bioinformatics analysis with a common database, in addition to using their own in-house database. Five laboratories participated in the RT (seven pipelines were tested). We observed that the algorithms had a stronger impact on the overall performance than the choice of the reference database. Our results also suggest that differences in sample preparation can lead to significant differences in the performance, and that laboratories should aim for at least 5–10 Mio reads per sample and use depth of coverage in addition to other interpretation metrics such as the percent of coverage. Performance was generally lower when increasing the number of viruses per sample. The lessons learned from this pilot study will be useful for the development of larger-scale RTs to serve as regular quality control tests for laboratories performing NGS analyses of viruses in a clinical setting.

Proficiency Testing of Metagenomics-Based Detection of Food-Borne Pathogens Using a Complex Artificial Sequencing Dataset

“…This emphasizes the impact of the database used for taxonomic classification to obtain a comprehensive classification. This result is in contrast to the results of another proficiency test ( Junier et al, 2019 ), where the impact of the database was negligible compared with the influence of the applied algorithms.…”

“…Some ring trials and proficiency tests have previously been conducted to push forward the standardization of HTS approaches and their implementation into clinical diagnostic routine and diagnosis reporting systems. Metagenomics-based proficiency tests for pathogen detection have often been focused on virus detection ( Brinkmann et al, 2019 ; Junier et al, 2019 ; Zamperin et al, 2019 ). These studies highlighted that recognition of viruses that exhibit high mutation rates can cause some difficulties and might only be possible by inclusion of protein-based alignment approaches ( Brinkmann et al, 2019 ).…”

Implementation of next-generation sequencing for virus identification in veterinary diagnostic laboratories

“…The first Swiss-wide viral metagenomic ring trial pointed out several general issues, such as the impact of the database used and the permanent risk of contamination. 25 Regarding our own performance, we easily detected 3 increasing dilutions of 4 human viruses in human serum, even human herpesvirus 5, but missed all herpesviruses in a serum-based multiplex reagent containing 11 human viruses. Unfortunately, we do not know the exact copy numbers of viruses in the samples but know that the Cq values of the herpesviruses in the multiplex reagent were ~ 30 and similar to the highest dilution of the spiked-in samples.…”

Identification of Viral Signatures Using High-Throughput Sequencing on Blood of Patients With Kawasaki Disease

“…One important finding was that some contaminating viral sequences were not observed in NTC due to their stochastic appearance, leading to the authors' recommendation to use a large number of NTC despite cost of sequencing. HTS overall performance (e.g., sensitivity and specificity) depends on several parameters such as the sample preparation, the algorithms and the choice of the database used to analyse HTS raw data (27). Therefore, in order to obtain an optimal HTS performance, we used a robust sample preparation protocol and a bioinformatics pipeline both previously published (7,28,29) and benchmarked (30), with an overall sensibility very close to that of real-time (RT-)PCR assays for most of viruses recognized of clinical significance.…”