Novel bioinformatics programs for taxonomical classification and functional analysis of the whole genome sequencing data of arbuscular mycorrhizal fungi

Abstract Arbuscular Mycorrhizal Fungi (AMF) are obligate plant-root symbionts belonging to the phylum Glomeromycota. They form coenocytic hyphae and reproduce through large multinucleated asexual spores. Numerous studies have shown that AMF interact closely or loosely with a myriad of microorganisms, particularly bacteria and fungi that live on the surface of or inside of their mycelia and spores. Whole genome sequencing (WGS) data of the AMF grown in-vivo (typically grown in root of a host plant in pot filled with soil) contain a large amount of sequences from microorganisms inhabiting in their spore along with their own genome sequences, resulting in a metagenome.  The goal of my study was to develop bioinformatics programs for taxonomical classification and for functional analysis of the WGS data of the AMF. In the area of metagenomics, there are mainly two approaches for taxonomical classification: similarity-based (i.e., homology search) and composition-based (i.e., k-mers) methods. Similarity-based method solely depends on bioinformatics sequence databases and homology search programs such as BLAST program. The similarity-based method may not be suitable for ancient fungi AMF, because bioinformatics databases represent only a small fraction of the diversity of existing microorganisms, and gene prediction programs are highly biased towards intensively studied microorganisms. Considering that AMF have high inter/ intra genome variations, in addition to coenocytic and multi-genomic characteristics, probably due to their adaptation via various kinds of symbioses, composition-based method alone is not an effective solution for AMF, because it relies on base composition biases and focuses on taxonomical classification for prokaryotic organisms. In the first project, I a developed novel bioinformatics program, called SeSaMe (Spore associated Symbiotic Microbes), for taxonomical classification of the WGS data of the AMF. I selected microorganisms that were dominant in soil environment and grouped them into 54 genera which were used as references. I created a reference sequence database with a variable called Three codon DNA 9-mer. They were created based on a large number of structure files from Protein Data Bank (PDB): approx. 224,000 Three codon DNA 9-mers encoding for subunits of protein secondary structures. Based on the reference sequence database, I created genus specific usage databases containing codon usage and amino acid usage per taxonomic rank- genus. The program distinguishes between coding sequence (CDS) and non-CDS, detects an open reading frame, and classifies a query sequence into a genus group out of 54 genera used as reference. The developed program enables us to estimate relative abundances of taxonomic groups and to assess symbiotic roles of taxonomic groups associated with AMF. The program can be applied to other microorganisms as well as soil metagenome data. The program has applications in applied environmental microbiology. The developed program is available for free of charge at www.fungalsesame.org. In the second project, I developed another bioinformatics program, called SeSaMe PS Function, for position specific functional analysis of the WGS data of the AMF. AMF may contain a large portion of genes with unknown functions for which we may not be able to find homologues in existing sequence databases. While existing motif annotation programs rely on sequence alignment and have limitations for inferring functionality of novel genes, the developed program identifies potentially important interaction sites that are structurally and functionally distinctive from other subsequences, within a query sequence with exploratory data analysis. The program identifies matching Three codon DNA 9-mers in a query sequence, and dynamically creates comparative dataset of 54 genera, based on codon usage bias information retrieved from the genus specific usage databases. The program applies correlation Principal Component Analysis in conjunction with K-means clustering method to the comparative dataset. The program identifies outliers; Three codon DNA 9-mers, assigned into a cluster with a single member or with only a few members, are often outliers with important structures that may play roles in molecular interaction. In the third project, I developed a novel bioinformatics program called Posts (POsition Specific genetic code Tables) that assigns a codon into an amino acid group according to the codon position. The standard genetic code table may be more readily applicable to the genes whose genetic codes comply with the standard biological coding rules obtained from model organisms grown under laboratory condition. However, it may be insufficient for studying evolutions of genetic codes that may provide important information about codon properties. The mainstream hypotheses of genetic code origin suggested that codon position played important roles in the evolution of genetic codes. As a case study, we investigated irregular codons in 187 mitochondrial genomes of plants, lichen-forming fungi, endophytic fungi, and AMF. Each column of the Post contains 16 codons and the amino acids encoded by these are called an amino acid characteristics group (A.A. Char Group). Based on A.A. Char Group, an irregular codon can be classified into within-column type or trans-column type. The majority of the identified irregular codons belonged to the within-column type. The Post may offer new perspectives on codon property and codon assignment. The developed program is freely available at www.codon.kr. Taken together, the developed programs, the SeSaMe, the SeSaMe PS Function, and the Post, provide important research tools for advancing our knowledge of AMF genomics and for studying their symbiotic relations with associated microorganisms.  Keywords: Sesame; Spore associated Symbiotic Microbes; Symbiosis; Sesame PS function; Arbuscular mycorrhizal fungi; Three codon DNA 9-mer; Amino acid characteristics; Secondary structure; Taxonomical classification; Position specific functional analysis; Position specific genetic code tables; Post; Comparative study; Mitochondrial genome

Keyphrases: Sesame, Spore associated Symbiotic Microbes, Symbiosis, Sesame PS function, Arbuscular mycorrhizal fungi, Three codon DNA 9-mer, Amino acid characteristics, Secondary structure, Taxonomical classification, Position specific functional analysis, Position specific genetic code tables, Post, Comparative study, Mitochondrial genome