With the ever-increasing volume of single nucleotide polymorphisms (SNPs) deposited in publicly available databases such as National Center for Biotechnology Information (NCBI) dbSNP, laboratory geneticists are faced with the routine need of selecting an appropriate set of SNPs in both gene mapping and molecular evolution studies. The major bottleneck in the workflow for SNP-based studies has shifted away from SNP discovery toward SNP selection. Although it is beyond dispute that several web-based applications and stand-alone software packages are available for handling SNP data, including viewGene [1], Genotools SNP manager [2], SNPbox [3], SNPicker [4], and SNPper [5], these applications go off on a tangent when it comes to selecting the best SNP set because their applications focus on primer design (e.g. SNPbox and SNPicker), SNP visualization (e.g. viewGene), specific platform applications such as MassARRAY technology (e.g. Genotools SNP manager), and SNP search (e.g. SNPper). In light of the surging interest in haplotype inference [6, 7] and haplotype-based association studies, the power of a linkage disequlibrium (LD) study is determined not only by the number of SNPs used, but also by the quality. Contemporary geneticists aim to maximize the statistical power in detecting a disease-susceptible locus by selecting a "best set" of closely linked SNPs given a limited (and often fixed) genotyping budget [8].

In the case when a large number of SNPs are available for a susceptibility gene of interest, genotyping all SNPs on all samples is an inefficient utilization of resources. Recently, a cost-effective two-stage method has been proposed to identify disease-susceptibility markers [9]. In stage I, a set of SNPs (S₁), spaced in a predefined interval, is selected (e.g., evenly spaced every 3 to 5 Kb in and surrounding a candidate gene [10]). The genotypes of markers in S₁ are then used to define LD blocks and to reconstruct haplotypes within blocks across the candidate gene locus in a representative random sample, C₁, of the original source population (e.g., a multiethnic cohort of men and women [11]). In stage II, a representative set (S₂; S₂ ⊆ S₁) of htSNPs is selected on the basis of the LD characterization in the random sample of C₁, and S₂ is then genotyped in a much larger case-control set C₂ (C₁ ⊂ C₂), and haplotype-based association tests are performed in C₂, nested in the original source population.

Both stages are critical to the success of an association study. However, it is not a trivial task to select S₁ (i.e. a set of evenly spaced SNPs in and surrounding a candidate gene) because the number of available SNPs for each human gene varies dramatically (from <10 to >200) because of varying gene sizes and SNP densities. Furthermore, we certainly do not simply keep common SNPs in S₁ [i.e., minor allele frequency (MAF) ≥ 5%]; missense and regulatory SNPs should still be considered to be included in S₁ even if their MAFs fall below 5% [11]. Hand-picking S₁ by "eyeballing" is extremely labor-intensive, time-consuming, and error-prone for candidate genomic regions with hundreds and thousands of SNPs. Furthermore, obtaining a SNP flanking sequence long enough (~200 bp), and with annotation of nearby potential SNPs, is essential for the successful design of a SNP genotyping assay. Unfortunately, the flanking sequences of many SNPs recorded in dbSNP are short (<100 bp) and without any annotation of nearby SNPs.

NCBI's dbSNP offers a comprehensive SNP searching tool [12]. However, tools are still needed to easily and efficiently locate the desired SNPs, to evaluate their annotations, and to export them in suitable formats for downstream analyses. To meet such needs, we have developed the program SNPHunter, a tool with a friendly graphical user interface (GUI) that works as a portal between the user and NCBI dbSNP [13]. The program can extract and export SNP data retrieved from dbSNP, import saved SNP data, and offers a very flexible SNP selection function with graphic illustration of SNP position, function and heterozygosity. Furthermore, it retrieves any arbitrarily-defined, user-specified length of SNP flanking sequence with annotation of all nearby SNPs.

A detailed description of the implementation of the three modules has been presented in the User's Manual [15]. In brief, since retrieval of the flanking sequences of a desired SNP relies on knowledge of its genomic coordinate, in an ad hoc mode, SNPHunter first pinpoints the SNP's genomic coordinate from dbSNP's reference SNP (refSNP) record, strand orientation, and the SNP's corresponding contig number. Moreover, SNPHunter communicates with the NCBI MapViewer database and retrieves the corresponding sequence centering at the desired SNP, with the sequence lengths specified by users. SNPHunter will detect all neighboring SNPs located within a user-defined radius around the SNP of interest. Once the SNP's genomic coordinate and contig data are retreived, SNPHunter also obtains nearby SNP data on all neighboring SNPs by querying dbSNP for all available SNPs that lie within the user-defined radius. Once the starting and ending coordinates of a particular gene are determined by SNPHunter through NCBI's AceView, the 5' upstream and 3' downstream regions of the gene can be retrieved according to user-defined lengths. In a batch-mode, SNPHunter communicates with NCBI's LocusLink to fetch the SNPs that reside within each LocusLink gene. Since LocusLink has a curated SNP list for each gene included in the LocusLink database, this batch-mode search offers a reliable, efficient way to conduct a systematic SNP search for a large set of candidate genes (e.g. belonging to the same biological pathway/network). Furthermore, SNP data can be stored in the user's local directories, and SNP filtering can be performed automatically according to user-defined criteria.

Using these criteria and selection procedures, we selected a total of 670 SNPs for the 10 genes listed in Table 1.

Besides SNP selection, SNPHunter allows the retrieval of genomic coordinates and flanking sequences for specific SNPs and gives graphic illustration of all the SNPs within the gene of interest as well. Figure 2 gives an illustration of the 28 SNPs found in a 2.7 Kb region spanning the tumor necrosis factor (TNF) gene from NCBI dbSNP.

It is worth noting that SNPHunter relies on dbSNP for data retrieval, and thus is deprived of the independence whereas other application with local database support usually has. What's more, SNP selection should not be limited to NCBI dbSNP, although dbSNP represents the largest publicly available SNP database that can be accessed via the Internet worldwide. Some SNPs reported in the earlier literature have not yet been incorporated into dbSNP. Furthermore, there are several on-going gene re-sequencing projects for selected human genes, such as SeattleSNPs or SNP500Cancer [18]. Therefore, SNPs from these other sources, if not yet included in dbSNP, should also be considered in SNP selection. Nevertheless, NCBI dbSNP has been steadily updated and has gradually emerged as one of the most comprehensive SNP depositories.

In summary, SNPHunter allows for customized SNP searches (both ad hoc-mode and batch-mode) by directly retrieving and managing SNP information from the NCBI dbSNP database, eliminating tedious and costly local database maintenance on the user's side. To date, SNPHunter has received more than 1000 downloads worldwide. We hope this simple program can serve as an efficient and reliable tool for researchers everywhere to facilitate their genetic studies.

Project name: SNPHunter

Project home page: http://www.hsph.harvard.edu/ppg/software.htm

Operating system(s): Microsoft Windows

Programming language: Visual Basic .NET

Other requirements: Microsoft .NET Framework 1.0 or above.

License: None

Any restrictions to use by non-academics: Contact authors