Humanbody is a breeding ground for antimicrobial resistancegenes, shows new study EarlhamInst NatureComms
. COG numbers were annotated to the ORFs according to the reference COG protein displaying the highest bit score after filtering the hits by maximum e-value 1e-7. ARG family names were annotated to the ORFs according to the reference CARD protein displaying the highest bit score after filtering the hits by maximum e-value 1e-20, minimum identity 80% and minimum reference coverage 80%.
In the read-based approach, we first aligned raw reads of the sample against the reference proteins in our curated version of CARD, using ‘diamond blastx -k 1 -f 6 -e 1.e-10 --id 80 --query-cover 70’ using Diamond version v0.9.9. Then the blastx alignments were grouped by the reference ARG family, each ARG family was subsequently inspected for the coverage breadth by the collection of blastx alignments.
using ‘–min-seq-id 0.9 -c 0.8’ , ‘–min-seq-id 0.95 -c 0.8’ , ‘–min-seq-id 0.99 -c 0.9’ , and ‘–min-seq-id 1.0 -c 0.9’ , under ‘–cov-mode 0’.We determined the lowest common ancestor taxon for each ARG cluster using the application galaxy-tool-lca when the cluster contained at least one ORF derived from a HQ-MAG or RefSeq genome.
We validated this approach by clustering a panel of 40 SCGs at a range of nucleotide identities. These SCGs which are often core house-keeping genes will be in general vertically transmitted albeit more slowly evolving than accessory genes. This is confirmed by the fact that the majority of SCGs coalesced into multi-species clusters at cut-offs around 97%-98% below our 99% threshold for horizontal transfer.
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