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Background  Microbial genomes at the National Center for Biotechnology Information (NCBI) represent a large collection of more than 35,000 assemblies. There are several complexities associated with the data: a great variation in sampling density since human pathogens are densely sampled while other bacteria are less represented; different protein families occur in annotations with different frequencies; and the quality of genome annotation varies greatly. In order to extract useful information from these sophisticated data, the analysis needs to be performed at multiple levels of phylogenomic resolution and protein similarity, with an adequate sampling strategy.    Results  Protein clustering is used to construct meaningful and stable groups of similar proteins to be used for analysis and functional annotation. Our approach is to create protein clusters at three levels. First, tight clusters in groups of closely-related genomes (species-level clades) are constructed using a combined approach that takes into account both sequence similarity and genome context. Second, clustroids of conservative in-clade clusters are organized into  seed  global clusters. Finally, global protein clusters are built around the the  seed  clusters. We propose filtering strategies that allow limiting the protein set included in global clustering.  The in-clade clustering procedure, subsequent selection of clustroids and organization into  seed  global clusters provides a robust representation and high rate of compression. Seed protein clusters are further extended by adding related proteins. Extended seed clusters include a significant part of the data and represent all major known cell machinery. The remaining part, coming from either non-conservative (unique) or rapidly evolving proteins, from rare genomes, or resulting from low-quality annotation, does not group together well. Processing these proteins requires significant computational resources and results in a large number of questionable clusters.    Conclusion  The developed filtering strategies allow to identify and exclude such peripheral proteins limiting the protein dataset in global clustering. Overall, the proposed methodology allows the relevant data at different levels of details to be obtained and data redundancy eliminated while keeping biologically interesting variations.    Electronic supplementary material  The online version of this article (doi:10.1186/s12859-016-1112-8) contains supplementary material, which is available to authorized users.

Clustering analysis of proteins from microbial genomes at multiple levels of resolution