Future diagnostic of sepsis : Evaluating nanopore sequencing and adaptive sampling aproach for earlier sepsis diagnostic

Sepsis is a serious life-threatening condition caused by excessive immune response to infections. In the context of sepsis treatment, early diagnosis is a crucial objective, as a delay in suitable antibiotic therapy is associated with higher mortality in patients. Today, standard diagnostics of sepsis in hospitals involve blood culturing, which is a time-consuming process. This project aimed to assess the potential of a metagenomics sequencing approach for earlier sepsis diagnostics utilising nanopore third-generation sequencing. In this project, the whole blood from a healthy donor was spiked with microbial community standard and a set of DNA isolates extracted from various bacteria. The Quick-DNA Miniprep Plus and UCP Pathogen Mini Kits were used to perform DNA extractions from spiked whole blood. Both extraction methods yielded genomic DNA of adequate purity and concentration to support downstream nanopore sequencing. A MinION device with a Rapid Barcoding kit was used to perform nanopore sequencing. EPI2ME and BV-BRC tools were used for downstream bioinformatics analysis. Taxonomic identification revealed considerable variation between the two classification software tools, likely due to differences in their reference databases. However, the usability of nanopore sequencing for taxonomic identification of pathogens was demonstrated, with bacterial and yeast species identified by sequencing using MinION and Flongle flowcell. Identifying AMR genes resulted in only a small percentage of the expected AMR genes for the species included in the metagenomic samples. Despite this, the identification of AMR genes revealed several multidrug resistance genes, which could be particularly relevant in clinical contexts. Furthermore, the promising results of adaptive sampling for depleting human DNA were demonstrated.