1.
Identification of ARGs and MGEs
2. Metagenomic binning and quality control of MAGs
MAGs of same species
Metagenomics Data
CompareM (to calculate
amino- acid Mapping Quality check: FastQC
identity (AAI) between
genomes ) Trimming: Trimmomatic
AAI ≥99.5%
Quality Quality reads
MAGs Mapping: Bowtie2
Assembly: MEGAHIT (–k-min 61 –
(to generate a
QUAST min- contig-len 10 0 0)
coverage score for
CheckM individual contigs )
RefineM Contigs
BlastX: SARG e-value ≤10 −10 , a
Binning: MetaBAT2, similarity ≥80% and a query
MAGs
MaxBin, CONCOCT coverage ≥70%)
ARG-carrying contigs
PlasFlow Abundance
=∑(𝑁𝑚𝑎𝑝𝑝𝑒𝑑
Prodigal (coverage, x/GB)
∗𝐿𝑟𝑒𝑎𝑑𝑠/(𝐿𝑎𝑟𝑔−
ORFs of ARG-carrying 𝑙𝑖𝑘𝑒 𝑜𝑟𝑓))/𝑆
contigs
plasmid and
chromosomal sequences
BlastP: e-value ≤10 −5 , Abundance
identity and query
coverage threshold of
80% and 70%
keywords: transposase,
code the four components transposon, conjugative,
• the origin of transfer ( oriT ) integrase, integron,
• relaxase recombinase, conjugal,
• the type IV coupling proteins mobilization, recombination,
(T4CP) plasmid, resolvase, relaxase and
• the type IV secretion system invertase
(T4SS)),
To identify potential MGEs located on ACCs
mobility of ARGs carried were identified according
by plasmids. to the results of NR
annotation
3. Taxonomic assignment, relative abundance calculation and
functional annotation of MAGs
MAGs of the species
GTDB-Tk Taxonomic assignments
MAGs of each species
(or bin) Mapped the quality-controlled reads from each
sample against each genome bin fasta file
recovered from the samples (Bowtie2)
Sam files
Sample 1 quality reads
Sample 2 quality reads
calculate_breadth.py
Sample n quality reads
Average coverage of
each genome bin
Relative abundance of each MAG was calculated as the
number of reads (based on average coverage) aligning to the
MAG normalized by the total number of reads in the sample
Relative abundance of each MAG =
BLASTX against SARG: e-value cut-
off of 10 −10 , 80% similarity and
70% query coverage
ARG-carrying MAGs
Estimate the
ICEfinder transferability of
ARGs
• Those elements carrying integrase,
ICEs & IMEs
relaxase and T4SS gene clusters were
considered as T4SS-type ICEs.
• Those without T4SS but with integrase
and relaxase were identified as IMEs.
