Breakthroughs in DNA-based technologies, especially in metagenomic sequencing, have drastically enhanced researchers' ability to explore environmental microbiome and the associated interplays within. However, as new methodologies are being actively developed for improvements in different aspects, metagenomic workflows become diversified and heterogeneous. Through a single-variable control approach, we quantified the microbial profiling variations arising from 6 common technical variables associated with metagenomic workflows for both simple and complex samples. The incurred variations were constantly the lowest in replicates of DNA isolation and DNA sequencing library construction. Different DNA extraction kits often caused the highest variation among all the tested variables. Additionally, sequencing run batch was an important source of variability for targeted platforms. As such, the development of an environmental reference material for complex environmental samples could be beneficial in benchmarking accrued non-biological variability within and between protocols and insuring reliable and reproducible sequencing outputs immediately upstream of bioinformatic analysis. To develop an environment reference material, sequencing of a well-homogenized environmental sample composed of activated sludge was performed using different pre-analytical assays in replications. In parallel, a certified mock community was processed and sequenced. Assays were ranked based on the reconstruction of the theoretical mock community profile. The reproducibility of the best-performing assay and the microbial profile of the reference material were further ascertained. We propose the adoption of our complex environmental reference material, which could reflect the degree of diversity in environmental microbiome studies, to facilitate accurate, reproducible, and comparable environmental metagenomics-based studies.

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