1. K-140 Email: zhili.he@ou.eduzhili.he@ou.edu Phone: 405-325-3958 Web: http://ieg.ou.edu A Software Package for the Design and Analysis of Microbial Functional Gene Arrays Zhili He 1,2, Ye Deng 1, Christopher W. Schadt 2, Xingyuan Li 3, Jizhong Zhou 1,2 A package has been developed to comprehensively analyze microbial functional genes for the design of functional gene arrays (FGAs). The package contains multiple modules to download sequences, remove low-homology sequences, design oligonucleotide probes, verify probe specificity, output selected oligonucleotide probes, and finally store data in local databases. The gene download module retrieves functional gene sequences from publicly available databases (e.g. GenBank) using BLAST algorithms and key words, or a combination of key words with AND, OR, and NOT operations as a query for a particular functional gene. Since some downloaded sequences may be mis-annotated or not related to the functional gene of interest, the homology search module removes those low-homology sequences, and also discards duplicate sequences. All processed sequences are used to design oligonucleotide probes using CommOligo, which designs single or multiple oligonucleotides for each sequence or each group of homologous sequences. Prior to oligonucleotide design, a multiple sequence alignment (MSA) may be conducted to select conserved regions to be used for probe selection. The probe check module confirms the specificity of all designed oligonucleotides using the same criteria as probe design against larger databases (e.g. GenBank). The plate production module outputs qualified probes in a 96-well plate format, which can be directly input into oligonucleotide synthesis systems of commercial companies. Finally, all information for sequences, probes and arrays are stored in databases, which can be easily retrieved by Search, Link and other functions. Since sequences are continuously deposited into public databases. This package is able to automatically update all information periodically so that it is kept consistent and up-to-date. Experimental details and analysis tools will be integrated into this package as well as development continues. This package will greatly facilitate the analysis of microbial functional gene sequences and arrays. ABSTRACT 1 University of Oklahoma, Norman, OK, 2 Oak Ridge National Laboratory, Oak Ridge, TN, 3 Perkin Elmer Life and Analytical Sciences, Boston, MA MODULES EXAMPLES Submit a search query to GenBank protein database Download the protein sequences Select the seed sequences to build an HMM model Download the CDS sequences for HMM-confirmed proteins Probe design by CommOligo Probes confirmation by using blastn with GenBank nr database Manage the verified probes MAJOR STEPS 1. Submit a query 2. List search results and select seed sequences 3. Do HMM search 4. Design oligonucleotide probes The functional gene nirK (nitrite reductase gene) was used as an example for probe design and verification procedures. 1. The summary of the whole process Query (nitrite reductase or nirK) AND nirK [gene] Total no. of sequences1208 No. of seed sequences87 No. of sequences confirmed by HMM global and local alignments 1137 No. of sequences confirmed only by HMM local alignments 59 No. of sequences excluded12 No. of genes for probe design1189 No. of sequences with unique probes219 No. of sequences with group probes419 No of sequences without probes551 Total no. of designed probes1561 No. of gene-specific probes1061 No. of group-specific probes500 No. of probes homology with other sequences in GenBank 178 No. of probes to the array703 No. of sequences covered587 The percentage of genes with probes48.6% 2. The summary of probe characteristics in functional gene array Unique ProbesGroup Probes Average GC content57% (44%~70%)58% (46%~70%) Average Tm92°C (87~97°C) Average maximal similarity80.0% (54%~90%)77.4% (56%~88%) Average minimal similarity among the same group \99.4%(96%~100%) Average minimal free energy-19.8 (-34.9~22.6)-79.4(-90.9~-66.6) Average maximal free energy among the same group \-22.9(-35.0~5.7) 5. Manage sequence and probe data ACKNOWLEDGEMENTS This research was funded by the U.S. Department of Energy (Office of Biological and Environmental Research, Office of Science) grants from the Genomes To Life Program and the ERSP Program. CONCLUSIONS 1.This package has been well integrated with multiple modules, sequence retrieval, homology confirmation, probe design, probe verification, data storage and data management. 2.This package greatly facilitate analysis of functional gene sequences, oligonucleotide probe design, and construction of functional gene arrays. 3.The package runs in Windows systems remotely or locally.