Unbiased metagenomic exploration of transfusion-transmitted infections with nanopore sequencing

Despite advances in blood safety, emerging infectious agents continue to pose risks to the blood supply. Traditional nucleic acid testing assays primarily target known pathogens, limiting the detection of novel microbes. Nanopore metagenomic sequencing enables agnostic identification of diverse pathogens, potentially enhancing transfusion safety surveillance.

We assessed the analytical performance and limit of detection (LoD) of a nanopore MinION metagenomic sequencing workflow for pathogen detection in clinical plasma samples. DNA/RNA was extracted from 14 archived samples from individuals with confirmed infections (HIV, HBV, HCV, WNV), followed by cDNA synthesis, barcoded library preparation, and nanopore sequencing. Quantitative reference panels were tested, and data were analyzed using a comprehensive bioinformatics pipeline.

Metagenomic sequencing generated over 2 million reads, with 2.3% of reads mapping to microbial sequences. Key transfusion-transmissible viruses, including West Nile virus (WNV), HIV, and hepatitis C virus (HCV), were reliably detected, while protocol modifications enabled HBV identification. Strain-level characterization identified HIV-1 subtype B, HCV genotypes 1a, 2b, 4a, HBV genotype C, and WNV lineage 1A. High viral loads produced extensive genome coverage, while lower loads yielded limited recovery. Co-infections, including human pegivirus (HPgV-2) and torque teno virus (TTV), were identified. The workflow detected viral targets at 10³ genome copy equivalents (GCEs)/mL, with > 50% genome coverage achieved at 10⁴ GCE/mL.

Nanopore metagenomic sequencing enables comprehensive detection of blood-borne pathogens in plasma samples. This approach offers a promising complementary strategy for enhancing transfusion safety surveillance, and the demonstrated strain-level characterization supports its potential utility for blood safety applications.