Assembling genomes from metagenomic short-read data can be challenging due to sequencing errors, depth of sequencing coverage and the unknown and uneven complexity of microbial communities. Reconstructing high-quality genomes is a crucial part of the metagenomic workflow as subsequent ecological and metabolic inferences depend on the accuracy, quality, and completeness of genomes. Genomes are often reconstructed by assembling all samples together (co-assembly) or creating individual assemblies. Co-assembly is a computationally intensive approach that involves the pooling of multiple samples to permit higher sequence coverage, which allows for the identification of populations that are present at lower abundances. In contrast, single sample assembly is computationally less intensive; however, lower sequence coverage resulting from this approach makes genome reconstruction difficult, as coverage heuristics to accurately disentangle repetitive sequences and differentiate between strain variants cannot be properly applied. In this study, we assessed the performance of a combination of selected assembly and binning strategies for time-series drinking water metagenomic data that were collected over 6 months, to determine the combination of assembly/binning approaches that result in the highest quality and quantity metagenome-assembled genomes (MAGs). Our findings suggest that the metaSPAdes co-assembly strategies had the best performance as it produced larger assemblies that were less fragmented, encompassing a lower proportion short contigs (N50 = 5.85 ± 0.63 kbp) that retained at least 80% of the sequence data. Furthermore, the MAGs that were reconstructed from the metaSPAdes co-assembly strategies, using CONCOCT and MetaBAT2, produced the highest quantity good-quality MAGs with mapping rates of at least 60%. These assembly/binning approaches also permitted the identification of unique MAGs and the differentiation between strain variants. Overall, this study suggest that multiple assembly and binning approaches are required to assist in the recovery of a greater proportion high-quality MAGs.
