International Mammalian Genome Society


The 13th International Mouse Genome Conference
October 31-November 3, 1999

Table of Contents * Structure * Bioinformatics * Sequence * Mapping * New Tools * Gene Discovery * Developmental * Mutagenesis * Functional Genomics

B14 EMPATH - EST Mapping Project at Harwell

S. Greenaway, C. Davison, C. Heuston, P. J. Trickett, A. Southwell, A. Haynes, M. A. Strivens, S. D. M. Brown and P. Denny. MRC UK Mouse Genome Centre and Mammalian Genetics Unit, Harwell, OX11 ORD, UK

We present as part of the EST Radiation Hybrid Mapping project (see abstract by Haynes et al.) a laboratory management system developed to manage, monitor and analyse mapping of ESTs. The system followed on from the earlier HOSEPIPE (Strivens et al. (1997) Mamm.Genome) system and is based around an industrial strength relational database accessible via the network using standard Web Browsers.

The system allows EST sequence entry either in the standard FASTA format, or as a Genbank accession number (with the sequence being fetched from Genbank as required) which is then masked for repeats and vector clipped before mouse and human homologies identified by BLAST analysis of the appropriate EST databases. Results from this section can then be viewed interactively allowing the user to view the original sequence, repeats and vector clipping. The top human, mouse and rat homologies can also be viewed as an aid to determining whether to proceed with primer design and synthesis.

After primer synthesis, the individual sets of primers are amplified in duplicate by PCR against the RH panel DNAs and visualised on agarose gels (each having an 8 x 12 well comb array). The gels are imaged (using a CCD camera), filtered to remove noise, rotated to line up the lanes vertically and scanned for the presence of bands (using a filter shaped for the 'optimum' band size). Any bands found are then compared to the control bands and a score is then calculated - no band, band, or error (inconclusive, a band was only found on one of the gels).

The scored gel is then presented to the user for final checking. The scores are then recorded on the database for future inclusion into the map. If any problems occur with adding an EST onto the map, the gel can be retrieved from the archive of gel images up to allow further checking.

Several mapping groups may use EMPATH at once, each group seeing only their data making it seem as if the system was dedicated to them. Each groups pipeline can be modified to their particular requirements. Two groups at Harwell currently use EMPATH.

 


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