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Bacillus subtilis and Escherichia coli, as widely used microbial species, are of great significance in studying microbial community relationships, adaptive evolution in various niches, engineering cell factories that produce specific products, and designing genome reduction. The pan-genome analysis is an effective method for studying the characteristics and functions of genes among and within species. Many research directions and conclusions usually depend on accurate gene identification and reliable pan-genome results. However, ther

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