Fig. 1

Schematic workflow representing the main steps of the CMg workflow. A. Sample processing of respiratory samples during which sample is treated with saponin to lyse human cells followed by nuclease treatment of human DNA and microbial cells are bead-beaten and automated microbial NA extraction is carried out. B. DNA is prepared for nanopore sequencing using the Rapid PCR Barcoding kit (SQK-RPB004), then the library is sequenced either with a Flonge (1 sample only) or with a GridION (6 samples). C. Real-time data acquisition is carried out by MinKNOW during which squiggle plots are converted into raw sequencing data and low-quality reads are removed (qscore = > 6). Real-time basecalling and demultiplexing (multiplex runs only) of raw data are done simultaneously by Guppy. Human reads (if any) are then firstly subtracted via read-based alignment offline; pathogen identification (ID) and AMR gene detection are then followed in real-time after 2 h of sequencing. K-mer-based classification is used for microbial ID (WIMP within EPI2ME), and offline read-based alignment for AMR gene detection based on pathogen/s (if any) identified by the previous step (above pre-defined thresholds) is followed by Scagaire. After 24 h of sequencing, downstream offline analysis can be carried out (if needed) for molecular typing to characterised identified pathogens for public health purposes