2. O AK R IDGE N ATIONAL L ABORATORY U.S. D EPARTMENT OF E NERGY A Comparison of Methods for Aligning Genomic Sequences Ja’Nera Mitchom Fisk University Research Alliance for Minorities Oak Ridge National Laboratory Dr. Mike Leuze http://www.csm.ornl.gov

3. O AK R IDGE N ATIONAL L ABORATORY U.S. D EPARTMENT OF E NERGY Outline  Introduction  Purpose of Genomic Sequence Analysis  Background Information  Central Dogma of Molecular Biology  Gene Regulation  Transcription Factor Binding Sites  Computational Methods of Aligning Genomic Sequences  CLUSTALW  DIALIGN  ORNL Method  Results  Acknowledgements

4. O AK R IDGE N ATIONAL L ABORATORY U.S. D EPARTMENT OF E NERGY Introduction  Genomic Sequence Analysis is used to identify genomic regulatory regions  Gene’s are regulated by proteins called transcription factors  Transcription factors bind to DNA sites found close to the gene- regulatory regions  Regions can be found by sequence comparison and may contain binding sites for proteins The importance of this research is a better understanding of the way genes are regulated, which will aid in the development of treatments for genetic diseases

5. O AK R IDGE N ATIONAL L ABORATORY U.S. D EPARTMENT OF E NERGY Background Information “ DNA specifies RNA, which specifies proteins” - Francis Crick Central Dogma of Molecular Biology

6. O AK R IDGE N ATIONAL L ABORATORY U.S. D EPARTMENT OF E NERGY Gene Regulation  Each cell has copy of entire genome.  Every gene not expressed in every cell  Mechanism in each cell that causes the gene to be expressed and inhibits expression of other genes

7. O AK R IDGE N ATIONAL L ABORATORY U.S. D EPARTMENT OF E NERGY Transcription Factor Binding Sites A section of DNA that transcription factors, proteins that control gene’s expression, bind to in order to help transcription of DNA to RNA, usually found in close proximity to the gene. Gene Direct contact: Transcription factor

8. O AK R IDGE N ATIONAL L ABORATORY U.S. D EPARTMENT OF E NERGY Computational Methods of Aligning Genomic Sequences Computational methods have been developed to identify genomic regulatory regions by sequence comparison. CLUSTALW and DIALIGN are two computational methods that were compared to a method developed at ORNL to determine which method is the best for finding these regions.

9. O AK R IDGE N ATIONAL L ABORATORY U.S. D EPARTMENT OF E NERGY FAS(Fatty Acid Synthase)  Chicken  Goose  Important implications in obesity, diabetes, artherosclerosis, and cancer > Chicken_FAS GGATCCCGTGGGGAGGTTGAGCTGTGCACTAAATGGAGGAGGTGAATTCT CACAGCTTTCTATTATGACCAAAATATTAACAAATAGTTCATTGATCTAA AAAACAGATGGCAAAGAAGGGTGGCGAGGTGGCACCGCGGCTGCTACCGG CTTGTGCAAGGAAAGGAGATCCTATAAGCAATGACTCCAGCCGACCACAG AGGAAGGCAGAACTCGCCTCACAGCTCACCCCGCCCAACCCCCCGCACTG > Goose_FAS AGTCACCACACAATCGCTTATCGCCTAGCAACACCTACCGGGCACGCCAT TGGCAGGCCGCGCCCCCGCCCAACGCCCCCGCCTGATTGGGTGCCGGCCC AGGACTGCGCGTCCAGCCGCGCGCCCCTTCTCTTTGTGCGCTGCCGGGTG CGCGGCGATCCGTGGCCCGGCGCCCGCCCGGCTTCAGCGCGCCCTGCCGA GCCACGGTGCCGGCGCAGTAGTAGTCCCAACCACAGTGTGCACATCCGCG GGGCGGGGGGAGAGGGACACAGAAAGGGACGCGGCGCTCGCTGCGATGGG

10. O AK R IDGE N ATIONAL L ABORATORY U.S. D EPARTMENT OF E NERGY CLUSTALW Uses progressive method of multiple sequence analysis Basic Alignment Procedure:  Performs pair-wise alignment of sequences – Produces phylogenetic tree based on alignment scores – Sequences progressively aligned according to branching order in phylogenetic tree

11. O AK R IDGE N ATIONAL L ABORATORY U.S. D EPARTMENT OF E NERGY DIALIGN (Diagonal Alignment) Uses iterative method of multiple sequence alignment Basic Alignment Procedure:  Assembles pair-wise and multiple alignments of sequences  Assigns each possible fragment with weight score based on probability of random occurrence  Incorporates sequences into multiple alignment based on weight score

12. O AK R IDGE N ATIONAL L ABORATORY U.S. D EPARTMENT OF E NERGY Two Phase ORNL Method Developed within the last year. Phase One: collection of small subsequences that match specified criteria Phase Two: assembly of pieces from phase-one collection into larger patterns

13. O AK R IDGE N ATIONAL L ABORATORY U.S. D EPARTMENT OF E NERGY Results  Showed an importance of similarity in the regions closest to the gene  ORNL method found similarity in several regions  CLUSTALW and DIALIGN could not find those similarities-possibly due to their method of alignment The discovery of these potential regulatory regions can make the ORNL method a better method of genomic sequence analysis compared to CLUSTALW and DIALIGN.

14. O AK R IDGE N ATIONAL L ABORATORY U.S. D EPARTMENT OF E NERGY Results  Similar regions found by all three methods  CLUSTALW  Chicken_FAS TGGCCGCCGGAGGTGGTGGCTGCTTAAATAGCGGTGGGAGCTAGAGGGAGA  Goose_FAS TGGCCGCCGGAGGTGGCGGCTGCTTAAATAGCGGTGGGAGCTAGAGGGAGA  DIALIGN  Chicken_FAS GTGGCCGCCGGAGGTGGtG GCTGCTTAAA TAGCGGTGGG AGCTAGAGGG AGA  Goose_FAS GTGGCCGCCGGAGGTGGcG GCTGCTTAAA TAGCGGTGGG AGCTAGAGGG AGA  ORNL Method  Length: 52 Substitutions: 1 Score: 67.033 2 C: AGAGGGAGATCGAGGGTGGCGATAAATTCGTCGGTGGTGGAGGCCGCCGGTG 0 G: AGAGGGAGATCGAGGGTGGCGATAAATTCGTCGGCGGTGGAGGCCGCCGGTG

15. O AK R IDGE N ATIONAL L ABORATORY U.S. D EPARTMENT OF E NERGY Results  Region found by ORNL  Length: 28 Substitutions: 2 Score: 30.660 404 C: CCTGCGCGTCACGGCCCGGCCGTGGGTT 483 G: CCTGCGCGTCAGGACCCGGCCGTGGGTT  CLUSTALW match  Chicken_FAS …TTGGGTGCCGGCCCGGCACTGCGCGTCC…  Goose_FAS …CCGGTCGCAGAGCGCGGCCTTCCACGGC…  DIALIGN match  Chicken_FAS …TTG GGTGCCG-GC CCGGCACTGC GCGTCC…  Goose_FAS …CCG GTCGCAGaGC GCGGCCTTCC ACGGCC…

16. O AK R IDGE N ATIONAL L ABORATORY U.S. D EPARTMENT OF E NERGY Accomplishments  Learned PERL  Compared DNA sequences of two species using three computational methods  Discussed potential applications of comparison results  Prepared research document

17. O AK R IDGE N ATIONAL L ABORATORY U.S. D EPARTMENT OF E NERGY Acknowledgements  This research was performed under the Research Alliance for Minorities Program administered through the Computer Science and Mathematics Division, Oak Ridge National Laboratory. This program is sponsored by the Mathematical, Information and Computational Sciences Division, Office of Advanced Scientific Computing Research, U.S. Department of Energy. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05- 00OR22725.  This work has been authored by a contractor of the U.S. Government under contract DE-AC05- 00OR22725. Accordingly the U.S. Government retains a nonexclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do so, for U.S. Government purposes. I would like to thank: Dr. Mike Leuze Dr. Andrey Gorin Dr. Glenn Allgood Jason McKay Cheryl Hamby Debbie McCoy