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In 2002, our research group observed a gene clustering pattern based on the base frequency of A versus T at the second codon position in the genome of  Vibrio cholera  and found that the functional category distribution of genes in the two clusters was different. With the availability of a large number of sequenced genomes, we performed a systematic investigation of A 2 –T 2  distribution and found that 2694 out of 2764 prokaryotic genomes have an optimal clustering number of two, indicating a consistent pattern. Analysis of the functional categories of the coding genes in each cluster in 1483 prokaryotic genomes indicated, that 99.33% of the genomes exhibited a significant difference ( p  &lt; 0.01) in function distribution between the two clusters .  Specifically, functional category P was overrepresented in the small cluster of 98.65% of genomes, whereas categories J, K, and L were overrepresented in the larger cluster of over 98.52% of genomes. Lineage analysis uncovered that these preferences appear consistently across all phyla. Overall, our work revealed an almost universal clustering pattern based on the relative frequency of A 2  versus T 2  and its role in functional category preference. These findings will promote the understanding of the rationality of theoretical prediction of functional classes of genes from their nucleotide sequences and how protein function is determined by DNA sequence. Graphical abstract

interdisciplinary sciences: computational life sciences

Consistent Clustering Pattern of Prokaryotic Genes Based on Base Frequency at the Second Codon Position and its Association with Functional Category Preference