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Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs). In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron–exon boundary, although the distance between these SNPs and the intron–exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function., Author Summary Genetic variation, through its effects on gene expression, influences many aspects of the human phenotype. Understanding the impact of genetic variation on human disease risk has become a major goal for biomedical research and has the potential of revealing both novel disease mechanisms and novel functional elements controlling gene expression. Recent large-scale studies have suggested that a relatively high proportion of human genes show allele-specific variation in expression. Effects of common DNA polymorphisms on mRNA splicing are less well studied. Variation in splicing patterns is known to be tissue specific, and for a small number of genes has been shown to vary among individuals. What is not known is whether allele-specific splicing events are an important mechanism by which common genetic variation affects gene expression. In this study we show that allele-specific alternative splicing was observed in six out of 70 exon-skipping events. Sequence analysis of the relevant splice sites and of the regions surrounding single nucleotide polymorphisms correlated with the splicing events failed to identify any predictive bioinformatic signals. A genome-wide study of allele-specific splicing, using an experimental rather than a bioinformatic approach, is now required.

Motivation: Multi Locus Sequence Typing (MLST) is a newly developed typing method for bacteria based on the sequence determination of internal fragments of seven house-keeping genes. It has proved useful in characterizing and monitoring disease-causing and antibiotic resistant lineages of bacteria. The strength of this approach is that unlike data obtained using most other typing methods, sequence data are unambiguous, can be held on a central database and be queried through a web server. Results: A database-driven software system (mlstdb) has been developed, which is used by public health laboratories and researchers globally to query their nucleotide sequence data against centrally held databases over the internet. The mlstdb system consists of a set of perl scripts for defining the database tables and generating the database management interface and dynamic web pages for querying the databases. Availability: http://www.mlst.net. Contact: mchan@molbiol.ox.ac.uk * To whom correspondence should be addressed. 2 Present address: Department of Paediatrics, The Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK. 3 Present address: Department of Infectious Disease Epidemiology, Imperial College School of Medicine, University of London, St Mary’s Campus, Norfolk Place, London W2 1PG, UK.

Summary: The 32-bit Windows application START is implemented using Visual Basic and C ++ and performs analyses to aid in the investigation of bacterial population structure using multilocus sequence data. These analyses include data summary, lineage assignment, and tests for recombination and selection. Availability: START is available at http://outbreak.ceid.ox. ac.uk/software.htm. Contact: keith.jolley@ceid.ox.ac.uk Multilocus Sequence Typing (MLST) is a nucleotide sequence-based typing method that indexes the variation present in bacterial housekeeping genes, where most of the variation is selectively neutral (Maiden et al., 1998). Internal fragments of seven housekeeping genes, approximately 450–500 bp in length, are sequenced and novel alleles are assigned with arbitrary numbers sequentially to provide an allelic profile of seven integers that defines the Sequence Type (ST) of each isolate. The technique is designed primarily for global or long-term epidemiology and surveillance, and has the advantage over other typing methods, such as genetic fingerprinting, of electronic portability and unambiguous characterization of isolates. MLST schemes have been developed for a range of bacterial pathogens and databases for these organisms can be interrogated at the MLST web-site (http://www.mlst.net/) thus facilitating rapid comparisons of isolates typed using the method. A further advantage of MLST is that it provides large quantities of data that may be analyzed by a number of evolutionary approaches to yield insights into the structure of bacterial populations and the selective pressures which act upon them. With the increasing availability of MLST data, the need for software to describe and analyze datasets has become apparent. Sequence Type Analysis and Recombinational Tests (START) was written to address this need through the inclusion of multiple analytical techniques in an easyto-use and intuitive interface for Windows 95/98/NT/2000 operating systems.

Multi-locus sequence typing (MLST) is a method of typing that facilitates the discrimination of microbial isolates by comparing the sequences of housekeeping gene fragments. The mlstdbNet software enables the implementation of distributed web-accessible MLST databases that can be linked widely over the Internet. The software enables multiple isolate databases to query a single profiles database that contains allelic profile and sequence definitions. This separation enables isolate databases to be established by individual laboratories, each customised to the needs of the particular project and with appropriate access restrictions, while maintaining the benefits of a single definitive source of profile and sequence information. Databases are described by an XML file that is parsed by a Perl CGI script. The software offers a large number of ways to query the databases and to further break down and export the results generated. Additional features can be enabled by installing third-party (freely available) tools. Development of a distributed structure for MLST databases offers scalability and flexibility, allowing participating centres to maintain ownership of their own data, without introducing duplication and data integrity issues.