SNP-RFLPing: restriction enzyme mining for SNPs in genomes

Abstract

Background: The restriction fragment length polymorphism (RFLP) is a common laboratory method for the genotyping of single nucleotide polymorphisms (SNPs). Here, we describe a web-based software, named SNP-RFLPing, which provides the restriction enzyme for RFLP assays on a batch of SNPs and genes from the human, rat, and mouse genomes.

Results: Three user-friendly inputs are included: 1) NCBI dbSNP "rs" or "ss" IDs; 2) NCBI Entrez gene ID and HUGO gene name; 3) any formats of SNP-in-sequence, are allowed to perform the SNP-RFLPing assay. These inputs are auto-programmed to SNP-containing sequences and their complementary sequences for the selection of restriction enzymes. All SNPs with available RFLP restriction enzymes of each input genes are provided even if many SNPs exist. The SNP-RFLPing analysis provides the SNP contig position, heterozygosity, function, protein residue, and amino acid position for cSNPs, as well as commercial and non-commercial restriction enzymes.

Conclusion: This web-based software solves the input format problems in similar softwares and greatly simplifies the procedure for providing the RFLP enzyme. Mixed free forms of input data are friendly to users who perform the SNP-RFLPing assay. SNP-RFLPing offers a time-saving application for association studies in personalized medicine and is freely available at http://bio.kuas.edu.tw/snp-rflp/.

Figure 1

SNP-RFLPing web-based flowchart. Three kinds of functions are incorporated in the SNP-RFLPing system, namely a user input interface, an analysis platform, and an output module. The user input interface contains 1.) Key search, 2.) Free format input, and 3.) Upload data for analysis. Only key search is mining from NCBI dbSNP [3]. The other interfaces of the input interface are programmed to input data preprocessing, and then transfer the data to the analysis platform. In the analysis platform, SNP-containing sequences are transferred to a local database downloaded from REBASE [2] and then the RFLP availability for the sense and antisense sequences are analyzed. Finally, the result is transferred to the output module. Under output, SNP-RFLPing availability is provided, as well as other SNP information and primer design.

Figure 2

Input items for RFLP availability and SNP related information. (A) SNP ID in rs# and ss# formats and gene in HUGO and ID formats are acceptable for SNP-RFLPing assay. Human, mouse and rat genomes are included. (B) Freely mixed forms of multiple inputs, including sequences and SNP ID with rs# are acceptable for SNP-RFLPing assay. Different events of input sequences and/or SNP ID are separated by the comma symbol "," or by different lines using the "Enter" key on the computer keyboard. IUPAC format or [dNTP1/dNTP2] format are acceptable in this software. Empty spaces while inputting data doesn't interfere with the screening. The IUPAC code is provided online. (C) All the results can be displayed online, by email or both.

Figure 3

Output items for RFLP availability and SNP related information. (A) Results of input for the gene name of HUGO. H19 (gene input in Figure 2A) is used as example. Here, only part of the SNP information is shown. The SNP ID in rs#, organism, contig-position, heterozygosity, function, protein residue, codon position, and amino acid position are shown. Each SNP in the SNP list for the input gene is shown in the order of its contig position. The system provides the partial or entire selections for SNPs. (B) Detailed information results are shown by inputting information from Figure 2B. SNP information from different species is acceptable. Each SNP-containing sequence was automatically transformed into sense and antisense strands marked with "+" and "-", respectively. After selection by the first sequence with SNP (= rs8144801), the results are shown in Figure 3C. (C) Standard results of SNP-RFLPing demonstrate detailed information of restriction enzymes and their target site in each strand if available. The system shows only the "+" (sense) strand by default. The hind "-" (antisense) strand can be shown by selection a checkbox. The alternative SNP-containing sequence is separated into two sequences marked with "0" and "1". When the sequences are suitable for restriction enzymes, the RFLP result shows "V". In contrast, if no RFLP is available in the restriction enzyme "X" is shown. Both commercial and non-commercial restriction enzymes are divided into two parts marked with blue and red colors to represent the recognition site for endonucleases with and without degenerated nucleotides, respectively.