Extended Data Fig. 5: Schematic overview of the workflow used to sequence the metagenomic DNA fragments.

The pipeline resembles a previously published workflow (Dual Barcoded Shotgun Expression Library Sequencing pipeline (Mutalik et al.³⁴)) with a modification that avoids PCR amplification of resistance-conferring metagenomic DNA fragments, and therefore, preserves the original composition of the samples (Methods). The workflow consists of the following steps. First, all the functional metagenomic plasmids obtained from the screens were pooled and then linearized using SrfI restriction endonuclease. SrfI has an eight base-pair-long recognition sequence to minimize the digestion of the metagenomic insert. The linearized plasmids are then subjected to Nanopore long-read sequencing (Methods). Long-read sequencing identifies the metagenomic DNA fragment (insert) and the two 10 nucleotide long random barcodes pre-cloned up- and down-stream (Uptag and Downtag, respectively) of each metagenomic DNA fragment (Methods). Parallel, prior pooling the metagenomic plasmids from each screen, a multiplexed short-read deep-sequencing was applied to read out the plasmid-encoded unique barcodes on each side of the metagenomic fragments in each functional metagenomic screen. Specifically, the Uptag and Downtag sequences were PCR amplified with barcoded Illumina sequencing compatible primers (BC). Following illumina sequencing and demultiplexing of the samples using the BC barcodes, the Nanopore and Illumina datasets are combined to assign each plasmid (identified by the Up- and Downtags) to a screening batch that is a unique host, antibiotic and library combination.