I am working on a metagenomic study of ARGs in river water samples. I have already performed read-level ARG profiling using ARGs-OAP, and the three biological replicates from each sampling location/time point were analyzed separately.
For the assembly-based part of the study, I now want to investigate resistome mobility, including ARG-carrying contigs, ARG–MGE associations, host assignment, and MAG-based analysis.
My experimental design is as follows:
- 4 seasons
- 6 sampling locations
- 3 biological replicates per location per season
- Total = 72 metagenomic samples
- The three replicates were collected at the same time and from the same location, but DNA was extracted and sequenced independently.
- Thus, the three replicates are parallel biological replicates rather than longitudinal samples.
My question is:
For assembly-based resistome mobility analysis, should I perform individual assemblies for all 72 samples, or should I combine the three biological replicates from each location/season and perform 24 co-assemblies?
More specifically, should MAG reconstruction and ARG–MGE–host linkage also be performed independently for each replicate, or would it be more appropriate to use co-assembly/co-binning of the three biological replicates?
What would be the most scientifically defensible strategy for a publication-level metagenomic study?
I would particularly appreciate advice from researchers experienced with metagenomic assembly, MAG reconstruction, ARG–MGE linkage, and resistome mobility analysis.
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