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workflow

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12 views2 pages

workflow

Uploaded by

sammybackup2000
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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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.

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