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FunOrder 2.0 – a fully automated method for the identification of co-evolved genes

biorxiv(2022)

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Abstract
Coevolution is an important biological process that shapes interacting species or even proteins – may it be physically interacting proteins or consecutive enzymes in a metabolic pathway. The detection of co-evolved proteins will contribute to a better understanding of biological systems. Previously, we developed a semi-automated method, termed FunOrder, for the detection of co-evolved genes from an input gene or protein set. We demonstrated the usability and applicability of FunOrder by identifying essential genes in biosynthetic gene clusters from different ascomycetes. A major drawback of this original method was the need for a manual assessment, which may create a user bias and prevents a high-throughput application. Here we present a fully automated version of this method termed FunOrder 2.0. To fully automatize the method, we used several mathematical indices to determine the optimal number of clusters in the FunOrder output, and a subsequent k-means clustering based on the first three principal components of a principal component analysis of the FunOrder output. Further, we replaced the BLAST with the DIAMOND tool, which enhanced speed and allows the future integration of larger proteome databases. The introduced changes slightly decreased the sensitivity of this method, which is outweighed by enhanced overall speed and specificity. Additionally, the changes lay the foundation for future high-throughput applications of FunOrder 2.0 in different phyla to solve different biological problems. AUTHOR SUMMARY Coevolution is a process which arises between different species or even different proteins that interact with each other. Any change occurring in one partner must be met by a corresponding change in the other partner to maintain the interaction throughout evolution. These interactions may occur in symbiotic relationships or between rivaling species. Within an organism, consecutive enzymes of metabolic pathways are also subjected to coevolution. We developed a fully automated method, FunOrder 2.0, for the detection of co-evolved proteins, which will contribute to a better understanding of protein interactions within an organism. We demonstrate that this method can be used to identify essential genes of the secondary metabolism of fungi, but FunOrder 2.0 may also be used to detect pathogenicity factors or remains of horizontal gene transfer next to many other biological systems that were shaped by coevolution. ### Competing Interest Statement The authors have declared no competing interest.
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Key words
genes,co-evolved
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