1. Differential insertion of transposable elements in Anopheles gambiae M & S genomes Jenica L. Abrudan, Ryan C. Kennedy, Maria F. Unger, Michael R. Olson, Scott J. Emrich, Frank H. Collins, Nora J. Besansky Eck Institute of Global Health, University of Notre Dame Abstract : Mosquitoes in the Anopheles gambiae species complex are the major vectors of malaria in Africa. The original A. gambiae genome sequenced was the PEST strain, which was later discovered to be a composite of the A. gambiae M and S forms. These 2 sympatric forms demonstrate reproductive isolation and are believed to be incipient or different species. They have been individually sequenced recently, so we are performing computational analysis of the three genomes to identify sequence differences. We hypothesize that transposable elements may be influencing the speciation of A. gambiae. Insertions of transposable elements have been associated with alterations in chromosome structure, recombination, replication, and gene regulation. Recent studies have indicated the existence of “speciation islands” and numerous genes differentially expressed across multiple developmental stages between the M and S forms though many of those genes lie outside of the “speciation islands” implying there are more causal factors to be discovered. We have identified sequences differently inserted between the M & S genomes relative to PEST. We then identified the subset of those sequences that contain transposable elements using a discovery pipeline we have developed. We are currently using this subset of data to identify those sequences that are in close proximity (~1kb) to gene elements, and will perform experiments designed to measure the expression levels of those genes. We hope to find a correlation between the differentially inserted transposons and the observed gene expression differences. Input data: M genome assembly S genome assembly PEST genome assembly DNA sequences from RepBase and TEFam databases for the known transposable elements in Anopheles gambiae Bioperl Generating the start up data Indels between M/S and PEST were determined by mapping the reads of M/S to the PEST assembly and comparing the distance between the mate pairs. Sequences that are inserted into the S genome assembly, but not in the M or PEST assembly - 6,767 sequences Sequences that are inserted into the M genome assembly, but not in the S or PEST assembly - 6,792 sequences Sequences that are inserted into the M and PEST genome assemblies but not in S 1,301 sequences Sequences that are inserted into the S and PEST genome assemblies but not in M 2,128 sequences Steps in analyzing the potential differential insertions between M ans S Putative insertion in M relative to S Putative insertion in S relative to M Transposable elements compiled database 1,075 transposable element related putative insertions into M relative to S 1,146 transposable element related putative insertions into S relative to M Future plans: Verify that the insertions are fixed between M & S Look at the insertion site relative to genes Look at the possible influence of transposable elements related sequences on gene expression The distribution of the families of transposable elements derived indels between the two genomes seems to be highly similar References: D. Lawson, et al., VectorBase: a data resource for invertebrate vector genomics. Nucleic Acids Research, 37:D58307, 2009. Repbase. http://www.girinst.org/repbase/index.html.http://www.girinst.org/repbase/index.html VectorBase. http://www.vectorbase.org TEfam. http://tefam.biochem.vt.edu.http://tefam.biochem.vt.edu Bioperl. http://bioperl.org The VectorBase project is funded by the US National Institute of Allergy and Infectious Diseases (NIAID), contract HHSN266200400039C. The differentially inserted sequences are computed from the two genome assemblies by mapping the M and S reads to the PEST genome and measuring the distance between the mate pairs and comparing it to where the mate pair would map to PEST. Suspicious sequences fell into two categories : Sequences present in only one assembly (either M or S) Sequences present in either M or S and PEST but not in all three assemblies Sequences that were computationally found to be different between the M and S assemblies were further analyzed for presence of transposable elements. For this purpose a database was computed out of the transposable element known to be present in the A. gambiae. The sequences were blasted (blastn) against this database and only those with an e-value of 10 -26 or less were further considered. While the numbers differ between the two genomes, Mariner seems to be the most abundant DNA transposon for both, with 212 sequence for S and 189 for M. The next most abundant element from the same class in M, Tc1 has a much lower number for S (151 vs 94) Class I Non-LTR retrotransposons and Class II DNA transposons seem to have the highest representation among the potentially different insertions between the two genomes both as diversity of sequence and as number of sequences present.