Yu-Huei Cheng

Combinational Polymorphisms of Seven CXCL12-Related Genes Are Protective against Breast Cancer in Taiwan

Many single nucleotide polymorphisms (SNPs) have been found to be associated with breast cancer, but their SNP interactions are seldom addressed. In this study, we focused on the joint effect for SNP combinations of seven CXCL12-related genes involved in major cancer-related pathways. SNP genotyping was determined by PCR-restriction fragment length polymorphism (RFLP) in this study (case = 220, control = 334). Different numbers of combinational SNPs with genotypes called the SNP barcodes from different chromosomes were used to evaluate their joint effect on breast cancer risk. Except for vascular endothelial growth factor (VEGF) rs3025039-CT, none of these SNPs were found to individually contribute to breast cancer risk. However, for two combined SNPs, the proportion of subjects with breast cancer was significantly low in the SNP barcode with CC-GG genotypes in rs2228014-1801157 (CXCR4-CXCL12) compared to those with non-CC-GG genotypes. Similarly, the SNP barcode of rs12812942-rs2228014-rs3025039 (CD4-CXCR4-VEGF) and rs12812942-rs3136685-rs2228014-rs1801157 (CD4- CCR7-CXCR4-CXCL12) with specific genotype patterns (AT-CC-CC and AT-AG-CC-GG) among three and four combinational SNPs were significantly low in breast cancer occurrence. More SNP combinations larger than five SNPs were also addressed, and these showed similar effects. After controlling for age, and comparing their corresponding non-SNP barcodes, the estimated odds ratios for breast cancer ranged between 0.20 and 0.71 for specific SNP barcodes with two to seven SNPs. In conclusion, we have associated the potential combined CXCL12-related SNPs with genotypes that were protective against breast cancer, and that may contribute to identification of a low-risk population for the development of breast cancer.

SNP-RFLPing 2: an updated and integrated PCR-RFLP tool for SNP genotyping.

BackgroundPCR-restriction fragment length polymorphism (RFLP) assay is a cost-effective method for SNP genotyping and mutation detection, but the manual mining for restriction enzyme sites is challenging and cumbersome. Three years after we constructed SNP-RFLPing, a freely accessible database and analysis tool for restriction enzyme mining of SNPs, significant improvements over the 2006 version have been made and incorporated into the latest version, SNP-RFLPing 2.ResultsThe primary aim of SNP-RFLPing 2 is to provide comprehensive PCR-RFLP information with multiple functionality about SNPs, such as SNP retrieval to multiple species, different polymorphism types (bi-allelic, tri-allelic, tetra-allelic or indels), gene-centric searching, HapMap tagSNPs, gene ontology-based searching, miRNAs, and SNP500Cancer. The RFLP restriction enzymes and the corresponding PCR primers for the natural and mutagenic types of each SNP are simultaneously analyzed. All the RFLP restriction enzyme prices are also provided to aid selection. Furthermore, the previously encountered updating problems for most SNP related databases are resolved by an on-line retrieval system.ConclusionsThe user interfaces for functional SNP analyses have been substantially improved and integrated. SNP-RFLPing 2 offers a new and user-friendly interface for RFLP genotyping that can be used in association studies and is freely available at http://bio.kuas.edu.tw/snp-rflping2.

Novel generating protective single nucleotide polymorphism barcode for breast cancer using particle swarm optimization.

BACKGROUND High-throughput single nucleotide polymorphism (SNP) genotyping generates a huge amount of SNP data in genome-wide association studies. Simultaneous analyses for multiple SNP interactions associated with many diseases and cancers are essential; however, these analyses are still computationally challenging. METHODS In this study, we propose an odds ratio-based binary particle swarm optimization (OR-BPSO) method to evaluate the risk of breast cancer. RESULTS BPSO provides the combinational SNPs with their corresponding genotype, called SNP barcodes, with the maximal difference of occurrence between the control and breast cancer groups. A specific SNP barcode with an optimized fitness value was identified among seven SNP combinations within the space of one minute. The identified SNP barcodes with the best performance between control and breast cancer groups were found to be control-dominant, suggesting that these SNP barcodes may prove protective against breast cancer. After statistical analysis, these control-dominant SNP barcodes were processed for odds ratio analysis for quantitative measurement with regard to the risk of breast cancer. CONCLUSION This study proposes an effective high-speed method to analyze the SNP-SNP interactions for breast cancer association study.

SNP-Flankplus: SNP ID-centric retrieval for SNP flanking sequences.

The flanking sequences provided by dbSNP of NCBI are usually short and fixed length without further extension, thus making the design of appropriate PCR primers difficult. Here, we introduce a tool named “SNP-Flankplus” to provide a web environment for retrieval of SNP flanking sequences from both the dbSNP and the nucleotide databases of NCBI. Two SNP ID types, rs# and ss#, are acceptable for querying SNP flanking sequences with adjustable lengths for at least sixteen organisms. Availability This software is freely available at http://bio.kuas.edu.tw/snp-flankplus/

Confronting two-pair primer design for enzyme-free SNP genotyping based on a genetic algorithm

BackgroundPolymerase chain reaction with confronting two-pair primers (PCR-CTPP) method produces allele-specific DNA bands of different lengths by adding four designed primers and it achieves the single nucleotide polymorphism (SNP) genotyping by electrophoresis without further steps. It is a time- and cost-effective SNP genotyping method that has the advantage of simplicity. However, computation of feasible CTPP primers is still challenging.ResultsIn this study, we propose a GA (genetic algorithm)-based method to design a feasible CTPP primer set to perform a reliable PCR experiment. The SLC6A4 gene was tested with 288 SNPs for dry dock experiments which indicated that the proposed algorithm provides CTPP primers satisfied most primer constraints. One SNP rs12449783 in the SLC6A4 gene was taken as an example for the genotyping experiments using electrophoresis which validated the GA-based design method as providing reliable CTPP primer sets for SNP genotyping.ConclusionsThe GA-based CTPP primer design method provides all forms of estimation for the common primer constraints of PCR-CTPP. The GA-CTPP program is implemented in JAVA and a user-friendly input interface is freely available at http://bio.kuas.edu.tw/ga-ctpp/.

V-MitoSNP: visualization of human mitochondrial SNPs

BackgroundMitochondrial single nucleotide polymorphisms (mtSNPs) constitute important data when trying to shed some light on human diseases and cancers. Unfortunately, providing relevant mtSNP genotyping information in mtDNA databases in a neatly organized and transparent visual manner still remains a challenge. Amongst the many methods reported for SNP genotyping, determining the restriction fragment length polymorphisms (RFLPs) is still one of the most convenient and cost-saving methods. In this study, we prepared the visualization of the mtDNA genome in a way, which integrates the RFLP genotyping information with mitochondria related cancers and diseases in a user-friendly, intuitive and interactive manner. The inherent problem associated with mtDNA sequences in BLAST of the NCBI database was also solved.DescriptionV-MitoSNP provides complete mtSNP information for four different kinds of inputs: (1) color-coded visual input by selecting genes of interest on the genome graph, (2) keyword search by locus, disease and mtSNP rs# ID, (3) visualized input of nucleotide range by clicking the selected region of the mtDNA sequence, and (4) sequences mtBLAST. The V-MitoSNP output provides 500 bp (base pairs) flanking sequences for each SNP coupled with the RFLP enzyme and the corresponding natural or mismatched primer sets. The output format enables users to see the SNP genotype pattern of the RFLP by virtual electrophoresis of each mtSNP. The rate of successful design of enzymes and primers for RFLPs in all mtSNPs was 99.1%. The RFLP information was validated by actual agarose electrophoresis and showed successful results for all mtSNPs tested. The mtBLAST function in V-MitoSNP provides the gene information within the input sequence rather than providing the complete mitochondrial chromosome as in the NCBI BLAST database. All mtSNPs with rs number entries in NCBI are integrated in the corresponding SNP in V-MitoSNP.ConclusionV-MitoSNP is a web-based software platform that provides a user-friendly and interactive interface for mtSNP information, especially with regard to RFLP genotyping. Visual input and output coupled with integrated mtSNP information from MITOMAP and NCBI make V-MitoSNP an ideal and complete visualization interface for human mtSNPs association studies.

Specific PCR product primer design using memetic algorithm

To provide feasible primer sets for performing a polymerase chain reaction (PCR) experiment, many primer design methods have been proposed. However, the majority of these methods require a relatively long time to obtain an optimal solution since large quantities of template DNA need to be analyzed. Furthermore, the designed primer sets usually do not provide a specific PCR product size. In recent years, evolutionary computation has been applied to PCR primer design and yielded promising results. In this article, a memetic algorithm (MA) is proposed to solve primer design problems associated with providing a specific product size for PCR experiments. The MA is compared with a genetic algorithm (GA) using an accuracy formula to estimate the quality of the primer design and test the running time. Overall, 50 accession nucleotide sequences were sampled for the comparison of the accuracy of the GA and MA for primer design. Five hundred runs of the GA and MA primer design were performed with PCR product lengths of 150–300 bps and 500–800 bps, and two different methods of calculating Tm for each accession nucleotide sequence were tested. A comparison of the accuracy results for the GA and MA primer design showed that the MA primer design yielded better results than the GA primer design. The results further indicate that the proposed method finds optimal or near‐optimal primer sets and effective PCR products in a dry dock experiment. Related materials are available online at http://bio.kuas.edu.tw/ma‐pd/.

Drug-SNPing

Many drug or single nucleotide polymorphism (SNP)-related resources and tools have been developed, but connecting and integrating them is still a challenge. Here, we describe a user-friendly web-based software package, named Drug-SNPing, which provides a platform for the integration of drug information (DrugBank and PharmGKB), protein-protein interactions (STRING), tagSNP selection (HapMap) and genotyping information (dbSNP, REBASE and SNP500Cancer). DrugBank-based inputs include the following: (i) common name of the drug, (ii) synonym or drug brand name, (iii) gene name (HUGO) and (iv) keywords. PharmGKB-based inputs include the following: (i) gene name (HUGO), (ii) drug name and (iii) disease-related keywords. The output provides drug-related information, metabolizing enzymes and drug targets, as well as protein-protein interaction data. Importantly, tagSNPs of the selected genes are retrieved for genotyping analyses. All drug-based and protein-protein interaction-based SNP genotyping information are provided with PCR-RFLP (PCR-restriction enzyme length polymorphism) and TaqMan probes. Thus, users can enter any drug keywords/brand names to obtain immediate information that is highly relevant to genotyping for pharmacogenomics research.

Seq-SNPing: Multiple-Alignment Tool for SNP Discovery, SNP ID Identification, and RFLP Genotyping

Many sequence-alignment tools were developed to discover single nucleotide polymorphisms (SNPs) derived from resequencing in genomic regions. Whether an identified SNP is indeed a novel SNP or is already contained in dbSNP is often difficult to answer. Here, we describe a freely available software, Seq-SNPing, which is a Java-based software for SNP discovery, and ID identification and editing and visualizating of sequence alignments. It is easy to use, fast, and provides an accurate method for searching and organizing SNP IDs from multiple sequence inputs, thereby greatly facilitating genetic studies. Seq-SNPing provides SNP identification by selecting any range of unaligned or aligned sequences in sequences that are similar. SNP IDs in the National Center for Biotechnology Information (NCBI) or user-defined SNPs within a selected sequence can be identified by Seq-SNPing. Information needed for SNP-RFLP (restriction fragment length polymorphism) genotyping is provided, such as SNP-REs (restriction enzymes), the sequence trimmer, sequence finder, BLAST (Basic Local Alignment Search Tool), SNP-BLAST, UCSC BLAT (BLAST-like alignment tool), RE mining, antisequencer (Anti-seq), and T(m) (melting temperature)/GC% of selected sequence. The thresholds for SNP calling are adjustable by selecting the height of the peak for each nucleotide representative curve in the chromatogram. Therefore, Seq-SNPing can discover SNPs and identify SNP IDs in both sequence text and chromatogram files in a fast and reliable way. The software is fully compatible with Microsoft Windows. The program and user manual are available at http://bio.kuas.edu.tw/Seq-SNPing for download.

Methyl-Typing: An improved and visualized COBRA software for epigenomic studies

Combined bisulfite restriction analysis (COBRA) is one of the most commonly used methylation quantification methods. However, it focuses on relatively few restriction enzymes. Here, we present Methyl‐Typing, a web‐based software that provides restriction enzyme mining data for methyl‐cytosine‐containing sequences following bisulfite‐conversion. Gene names, accession numbers, sequences, PCR primers, and file upload are accessible for input. Promoter sequences and restriction enzymes for CpG‐ and GpC‐containing recognition sites are retrieved. Four representative enzymes were tested successfully by COBRA on the experimental work. Therefore, the Methyl‐Typing tool provides a comprehensive COBRA‐restriction enzyme mining. It is freely available at http://bio.kuas.edu.tw/methyl‐typing.