Showing papers by "Wellcome Trust Sanger Institute published in 1994"

An expert system for processing sequence homology data.

Abstract: When confronted with the task of finding homology to large numbers of sequences, database searching tools such as Blast and Fasta generate prohibitively large amounts of information. An automatic way of making most of the decisions a trained sequence analyst would make was developed by means of a rule-based expert system combined with an algorithm to avoid non-informative biased residue composition matches. The results found relevant by the system are presented in a very concise and clear way, so that the homology can be assessed with minimum effort. The expert system, HSPcrunch, was implemented to process the output to the programs in the BLAST suite. HSPcrunch embodies rules on detecting distant similarities when pairs of weak matches are consistent with a larger gapped alignment, i.e. when Blast has broken a longer gapped alignment up into smaller ungapped ones. This way, more distant similarities can be detected with no or little side-effects of more spurious matches. The rules for how small the gaps must be to be considered significant have been derived empirically. Currently a set of rules are used that operate on two different scoring levels, one for very weak matches that have very small gaps and one for medium weak matches that have slightly larger gaps. This set of rules proved to be robust for most cases and gives high fidelity separation between real homologies and spurious matches. One of the most important rules for reducing the amount of output is to limit the number of overlapping matches to the same region of the query sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation, digestion, and cloning of intact parasite chromosomes embedded in agarose.

Abstract: The chromosomes of most protozoan parasites cannot be visualized using conventional microscopy because they are too small and do not condense sufficiently at metaphase. Therefore, the development of pulsed field gel electrophoresis allowed the resolution of many parasite karyotypes for the first time. The ability to prepare intact chromosomes in agarose plugs and to isolate individual homologs by electrophoresis has led to many new applications in parasite genomic analysis. This chapter describes the preparation of chromosome plugs from single-celled protozoan parasites, providing numerous tips on how to achieve the highest-quality preparations that will last for years. We also provide detailed protocols for the manipulation of individual excised chromosomes, including restriction mapping and preparation of chromosome shotgun libraries as used in many of the genomic sequencing projects. The protocols provided here underpin several of the advanced methods of genomic analysis and manipulation described in this volume of parasite genomics protocols.