Genomics Education Partnership: Evolution Of The Dot Chromosome

1. Genomics Education Partnership: Evolution Of The Dot Chromosome Dr. Maria Santisteban – maria.santisteban@uncp.edu, Justin Branch – JB0043@bravemail.uncp.edu Department of Biology, UNC Pembroke; Genomics Education Partnership, Washington University In St. Louis Abstract The Dot chromosome of Drosophila melanogaster is unique in that it exhibits an amalgamation of heterochromatic and euchromatic properties. In an attempt to study this peculiar domain, students in participation with the Genomics Education Partnership have contributed vast amounts of data and have been responsible for the sequence and annotation of various species of Drosophila. Students using evidence based annotation compile gene models that are sent to Washington University where the final models are placed in GenBank, a National Institute of Health Genetic Database, for future studies. This summer I have annotated six genomic sequences from Drosophila biarmipes both from the Dot and left arm of the third chromosome. By analyzing the Dot chromosome of D.melanogaster and D.virilis the Genomics Education Partnership have concluded that the two Dot chromosomes are more similar to each other than that of the reference heterochromatic and euchromatic domains. Introduction Genomics has become a fundamental aspect of research in biology and has made an ever-growing impact on the study of human disease. As genomic sequencing becomes less expensive and the impact genomics has on human health continues to grow, the desire to include bioinformatics and genomics into undergraduate studies propagates among institutions. Established in a laboratory at Washington University in St. Louis, by the Howard Hughes Medical Institute, the Genomics Education Partnership (GEP) has offered students the opportunity of independent genomic based research. Specifically working with fruit fly DNA, the aspirations of the GEP is to analyze the Dot chromosome of Drosophila melanogaster by means of comparative genomics. The distal portion of the Dot is unique in that it exhibits an amalgamation of heterochromatic (e.g. dense packaging; late replication) and euchromatic properties (e.g. gene density; loose packaging). Using high quality annotated DNA sequences, the GEP has compared the Dot chromosome along with reference heterochromatic and euchromatic domains of both D. melanogaster and D. virilis to understand the similarity of these domains and evolution of the Dot chromosome. Regions of interest in genome shotgun assemblies of various Drosophila species are chosen and divided into 40-60 kilobases of sequence for students who continue to contribute to the research initiated by the GEP. Identifying genes homologous to D. melanogaster, students map exon boundaries accordingly and in the process of evidence based annotation become introduced to how pseudogenes, missense mutations, and intron creation can affect the process of creating a gene model. Materials • Flybase.org – A Database of Drosophila Genes and Genomes This site offers an alignment tool, BLAST (Basic local Alignment Search Tool), that searches against a species specified genomic database along with GBrowse which allows for a visual representation of genes and gene isoforms as found on the chromosome • NCBI (National Center for Biotechnology Information) (www.ncbi.nlm.nih.gov) Offers BLAST alignment tools that allow regions of similarity between biological sequences to be located or searched against an array of databases • Genomics Education Partnership (www.gep.wustl.edu) The official GEP website contains various annotation resources • Gene Record Finder – A database of Drosophila melanogaster genes accompanied by a CDS usage map for exon mapping • Small Exon Finder – Used to find CDSs that are too small to be recognized by BLAST • UCSC Genome Browser Mirror – Displays draft DNA assemblies for genome annotation including BLASTX alignments to Drosophila melanogaster proteins, Gene Predictors that use Hidden Markov Models to infer locations of protein coding genes, RNAseq data that displays where transcription is occurring, and Conservation tracks that depict evolutionary conservation among other species of Drosophila • Gene Model Checker – allows annotator to input exon boundaries for proof reading. The model checker confirms that correct splice sites are chosen and for the presence of in frame stop codons. Figure 4; BLASTX Results • Annotation Method • According to the gene predictor “GenScan” there are five possible genes within the contig as shown in the Genome Browser (Fig.1) • The protein sequence from each possible gene is used to find the ortholog in Drosophila melanogaster by using BLASTP found in flybase.org • The gene ortholog is determined by location of the gene, the expect value, and degree of homology (fig2.) • To determine the gene structure Gene Record Finder (Fig.3) is utilized and the amino acid sequence is obtained • Each CDS obtained from Gene Record Finder is located within the fosmid sequence by means of the alignment tool BLASTX from NCBI (fig.4) • Figure 1; Genome Browser • Figure 2; BLASTP results • Figure 3; Gene Record Finder Results Once evidence based annotation is complete, nucleotide coordinates are submitted into the Gene Model Checker which proofreads the curated gene model for correct splice sites and any discrepancies that result in an inconsistent gene model. The gene model checker also allows for an upload of files that can be submitted to GEP’s Annotation Files Merger which allows the students gene model to be visualized on the genome browser (fig5). Figure 5; Gene Model Conclusion Working with Dr. Maria Santisteban, a member of the Genomics Education Partnership, six genomic sequences were successfully annotated from both the dot and left arm of the third chromosome. The GEP has analyzed the Dot chromosomes of D.melanogaster and D. virilis and has concluded that compared to the reference euchromatic domain, the Dot displays higher repeat density, larger gene size, and a higher rate of gene rearrangement. A higher rate of gene rearrangement, as seen on the X chromosome, suggest that gene rearrangements play a certain role in the evolution of the Dot chromosome. I have valued my time spent as a genomic annotator and expanding my basic understanding of genetics to include genomics and bioinformatics. I anticipate on reading further studies coming forth from the Dot chromosome in association with chromatin packaging. Acknowledgments GEP Program Director: Sarah C.R. Elgin Technical Director: Chris Shaffer Chief Technical/Teaching Assistant: Wilson Leung Sponsored by Washington University at St. Louis and HHMI This work was supported by grant  #5R25GM077634-04 from the NIGMS (National Institute of General Medical Sciences) supporting the UNCP RISE Program.