Deliverables High-throughput SNP mapping by tag-array minisequencing A promising approach towards high-throughput genotyping of SNPs is to use arrays of immobilised oligonucleotides in miniaturised assays. Significant advantages of performing the assays in microarray formats are the reduced costs of genotyping due to the simultaneous analysis of many SNPs in each sample, and the small reaction volumes employed. To facilitate positional cloning in Drosophila, we established a microarray system for a genome-wide genotyping panel of 300 SNPs, discriminating between each of the major chromosomes from commonly used laboratory strains of Drosophila. The Information for SNP selection and assay design was taken from the high-density SNP map. After assay development, sets of 60 markers from each chromosomal arm were selected, which resulted in genotyping assays for SNPs at an average spacing of less than 400kb. The genotyping system is based on multiplex, four-color fluorescent minisequencing. The minisequencing reaction uses a DNA polymerase to extend detection primers that anneal immediately adjacent to the sites of the SNPs. The primers are extended with fluorescently labeled, terminating nucleotide analogues that are complementary to the nucleotide at the SNP site. The reactions are performed in solution using detection primers with 5 -Tag sequences. Each SNP has its own specific tag that is complementary to one of the cTags that are immobilised to the microarray. When the extended detection primers are applied to the microarray, the tags will hybridise to their corresponding cTags. From the known locations of the cTags on the microarray, the genotypes of the SNPs can be deduced. By the array of arrays format of our method separate reaction chambers are formed on an array by a silicon rubber grid. In this manner a single standard microscope slide can be used to analyse up to 200 SNP positions in 80 samples simultaneously. The tag-array minisequencing method is particularly suitable for large-scale gene mapping in Drosophila due to its robustness, simplicity in assay design and low cost. To facilitate data analysis, IMP/IMBA has developed software, which automatically translates raw data from the microarray analysis to the genotypes. In addition, it provides a user-friendly, graphical output, which simplifies localization of the region of interest. The tag-array minisequencing system is based on standard instruments, which makes the system easily implemented at other laboratories. MEDSCI has an extensive experience from using the technique, particularly in human genetic applications. MEDSCI also has experiences from organizing practical courses on the technique. In the context of the FLYSNP project MEDSCI organized the First FLYSNP workshop in November 2004. Currently, MEDSCI provides three annual courses for graduate students which include the tag-array minisequencing system. These courses, one of which is sponsored by EMBO (the European Molecular Biology Organization), are open to interested Drosophila researchers. Moreover, MEDSCI can disseminate the technology by accepting visitors for training in our lab in Uppsala. Genotyping services for the Drosophila community can be arranged on collaborative basis on a small scale. If the interest in gene mapping is large enough, a genotyping service can be established at the existing national SNP genotyping service unit, which operates in close connection with the Molecular Medicine research group at MEDSCI. For more information, please visit www.medsci.uu.se/molmed/ Genome-wide high-density SNP map of Drosophila melanogaster In Drosophila melanogaster, single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) can be found in high frequency, in average once every 200-300bp, when comparing two or more strains. Since the majority of these polymorphisms are also phenotypically neutral, and existing in two codominant variants, they are highly suitable as marker for positional cloning of mutations. We have created a genome-wide, high-density SNP map of Drosophila melanogaster consisting of 2300 SNP markers (1kb amplicons containing at least one SNP), evenly spaced across all major chromosome arms (X, 2L, 2R, 3L, and 3R), with an average distance of 49.7kb. For SNP detection, 1kb regions from three to seven standard laboratory strains per site were selectively amplified, sequenced and compared to the Release 4 genomic sequence from FlyBase. Amplification primers were designed so that they preferentially lie in non-repetitive and non-coding regions of the genome. After automatic SNP detection by the PolyBayes software package, quality criteria were applied to filter the most probable SNPs, followed by visual inspection of the alignments to increase reliablity of the data. With this procedure, 200.000 allele calls were reduced by ca. 50% to give a high-quality, reliable SNP map. When two strains are compared, one can find at least one polymorphism in 66%-86% of all 1kb segments. The polymorphisms are evenly distributed except for the regions lying close to the tip and the centromer of each chromosome arm. Construction of the SNP map proceeded in three phases: an initial low-density map, an intermediate medium-density SNP map and a final high-density SNP map. The low-density map consisted of ca. 120 SNP markers, the medium-density map of >600 SNP markers, and the high-density map of 2300 SNP markers. The average distances between any two adjacent markers was 1 Mb, 200 kb, and 50 kb, respectively. Three different wild type, one balancer, one recessive marker, one FRT, and one dEP stock per chromosome arm were genotyped for the SNP map. For the low resolution map all listed stocks were analysed, for the medium resolution map all wild type and one marker, one FRT, and one dEP stock were used. For the high-resolution map, only the two most divergent stocks and a third stock which is useful for mapping purposes were analysed. All lines needed for the SNP map were isogenized and tested for the lack of any obvious developmental phenotype. Dissemeniation of the SNP map occurs via the FLYSNP Database (http://flysnp.imp.univie.ac.at/snpdb.php) at the FLYSNP Website. SNPs from a specified region can be selected, and the corresponding data (allelic variants, coordinates, flanking regions, amplification primer sequences, genotyping assays etc.) downloaded. A direct link to FlyBase-GBrowse enables the user to open the graphical representation of the specified chromosomal segment in a separate window. Attempts to link the FLYSNP Database directly to FlyBase, and to enter the data to NCBI-dbSNP, are ongoing. The website also includes an introduction to the FLYSNP project, a user guide, protocols, contact addresses, workshop information, and links to important relevant sites. Also, links to the FLYSNP Website were added to several other webpages, and the project was mentioned in listings of resources (e.g. FlyBase website, section External Resources, see http://www.flybase.net/allied-data/resources.html; publication "Research Resources for Drosophila: The Expanding Universe", Matthews K.A., Kaufman T.C., Gelbart W.M., Nature Reviews Genetics, 2005, Vol. 6, pp179-193.). Mapping mutations from a mutagenesis screen used to be a very time-consuming task, which usually lasted for several months or even years. With the SNP mapping technique, which is now the state-of-the-art mapping method, one can reduce the process to a few months. Once genes involved in a certain process are identified, researchers can focus on further analysis of these genes, giving them much more time for downstream analyses and conclusions from their screens. Therefore, the genome-wide high-density SNP map created by FLYSNP provides a valuable and useful resource for researchers who need to locate mutations. SNP genotyping by MALDI-TOF mass-spectrometry FlySNP aimed to introduce modern molecular biology methods and SNP genotyping in Drosophila basic research. Creation of a set of 2EP lines The goal was to set up a series of chromosomes, each carrying two EP elements that are 1-2 Mbp apart, which can be used to generate a series of recombinants between the mutation and the EP insertions for a molecular-scale mapping of the mutation relative to the SNP sites between the EP inserts. These recombinants can be identified based on eye-colour differences in animals carrying neither, one, or two of these elements. Using such stocks, it is a simple matter to isolate a set of recombinant chromosomes with an average of one recombination event every 10-20kb. We have accomplished the following tasks: - We selected 20 EP insertions for each of the 5 major chromosome arms. These insertions are homozygous viable, the flanking 3000bp DNA has no repetitive sequences and the adults carrying one or two EP elements can be distinguished based on their eye colours. - All of the planned sets of 2EP stocks were generated except for chromosome arm 3R. 18 stocks for chromosome X, 18 stocks for 2L, 17 stocks for 2R, 15 stocks for 3L, and 11 stocks for 3R are now available. - Verification of the 2EP insertions by PCR has been successfully performed with all available 2EP stocks. With these 2EP lines, it is possible to fine-map mutations to a resolution of 10-20kb. In particular, if the location of the mutation could be narrowed down by the newly developed "tag"-array minisequencing technique to ca. 2Mbp, the 2EP lines provide a useful tool for the next, fine-mapping step. Thus, these stocks provide a further tool, which facilitates localization of genes. Consequently, one of the major rate-limiting steps in the study of Drosophila functional genomics assigning known functions to specific genes - is greatly accelerated. The 2EP lines are freely available for all Drosophila researchers. Both the Szeged Drosophila Transposon Stock Center and IMP/IMBA (Vienna) are keeping at least one copy of each of the verified 2EP stocks. Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
