Detection of Viral Pathogens With Multiplex Nanopore MinION Sequencing: Be Careful With Cross-Talk

Metagenomic sequencing with the Oxford Nanopore MinION sequencer offers potential for point-of-care testing of infectious diseases in clinical settings. To improve cost-effectiveness, multiplexing of several, barcoded samples upon a single flow cell will be required during sequencing. We generated a unique sequencing dataset to assess the extent and source of cross barcode contamination caused by multiplex MinION sequencing. Sequencing libraries for three different viruses, including influenza A, dengue, and chikungunya, were prepared separately and sequenced on individual flow cells. We also pooled the respective libraries and performed multiplex sequencing. We identified 0.056% of total reads in the multiplex sequencing data that were assigned to incorrect barcodes. Chimeric reads were the predominant source of this error. Our findings highlight the need for careful filtering of multiplex sequencing data before downstream analysis, and the trade-off between sensitivity and specificity that applies to the barcode demultiplexing methods