Web-based primer design software for genome-scale genotyping by pyrosequencing.

Design of locus-specific primers for use during genetic analysis requires combining information from multiple sources and can be a time-consuming process when validating large numbers of assays. Data warehousing of genomic DNA sequences and genetic variations when coupled with software applications for optimizing the generation of locus-specific primers can increase the efficiency of assay development. Selection of oligonucleotide primers for PCR and Pyrosequencing (SOP3) software allows user-directed queries of warehoused data collected from the human and mouse genome sequencing projects. The software automates collection of DNA sequence flanking single-nucleotide polymorphisms (SNPs) as well as the incorporation of locus-associated functional information, such as whether the SNP occurs in an exon, intron, or untranslated region. SOP3 software accepts three types of user-directed input consisting of gene locus symbols, SNP reference sequence numbers, or chromosomal physical location. For human polymorphisms, SOP3 incorporates haplotype, ethnicity, and SNP validation attributes. The output is a list of oligonucleotide primers recommended for Pyrosequencing-based typing of genetic variations. SOP3 is available at the Division of Immunogenetics computational server found at http://imgen.ccbb.pitt.edu.

SOP3v2: web-based selection of oligonucleotide primer trios for genotyping of human and mouse polymorphisms.

SOP3v2 is a database-driven graphical web-based application for facilitating genotyping assay design. SOP3v2 accepts data input in numerous forms, including gene names, reference sequence numbers and physical location. For each entry, the application presents a set of recommended forward and reverse PCR primers, along with a sequencing primer, which is optimized for sequence-based genotyping assays. SOP3v2-generated oligonucleotide primer trios enable analysis of single nucleotide polymorphisms (SNPs) as well as insertion/deletion polymorphisms found in genomic DNA. The application's database was generated by warehousing information from the National Center for Biotechnology Information (NCBI) dbSNP database, genomic DNA sequences from human and mouse, and LocusLink gene attribute information. Query results can be sorted by their biological relevance, such as nonsynonymous coding changes or physical location. Human polymorphism queries may specify ethnicity, haplotype and validation status. Primers are developed using SOP3v2's core algorithm for evaluating primer candidates through stability tests and are suitable for use with sequence-based genotyping methods requiring locus-specific amplification. The method has undergone laboratory validation. Of the SOP3v2-designed primer trios that were tested, a majority (>80%) have successfully produced genotyping data. The application may be accessed via the web at http://imgen.ccbb.pitt.edu/sop3v2.

SOP3: a web-based tool for selection of oligonucleotide primers for single nucleotide polymorphism analysis by Pyrosequencing.

SOP3 is a web-based software tool for designing oligonucleotide primers for use in the analysis of single nucleotide polymorphisms (SNPs). Accessible via the Internet, the application is optimized for developing the PCR and sequencing primers that are necessary for Pyrosequencing. The application accepts as input gene name, SNP reference sequence number, or chromosomal nucleotide location. Output can be parsed by gene name, SNP reference number, heterozygosity value, location, chromosome, or function. The location of an individual polymorphism, such as an intron, exon, or 5' or 3' untranslated region is indicated, as are whether nucleotide changes in an exon are associated with a change in an amino acid sequence. SOP3 presents for each entry a set of forward and biotinylated reverse PCR primers as well as a sequencing primer for use during the analysis of SNPs by Pyrosequencing. Theoretical pyrograms for each allele are calculated and presented graphically. The method has been tested in the development of Pyrosequencing assays for determining SNPs and for deletion/insertion polymorphisms in the human genome. Of the SOP3-designed primer sets that were tested, a large majority of the primer sets have successfully produced PCR products and Pyrosequencing data.

HLA class II DRB high resolution genotyping by pyrosequencing: comparison of group specific PCR and pyrosequencing primers.

Sequencing of alleles of the highly polymorphic, multiple loci HLA-DRB gene family was performed by pyrosequencing using purified DNA from the 11(th) International Histocompatibility Workshop human lymphoblastiod cell lines as well as genomic DNA isolated from blood samples obtained from healthy adult volunteers. Genomic DNA was prepared from donors whose blood had been stored either frozen or as dried blood spots. Pyrosequence-based typing was optimized for identifying alleles of the HLA-DRB1, -3, -4, and -5 genes. The procedure should be applicable to other HLA loci including the class I genes HLA-A and -B that, along with HLA-DRB, are crucial for histocompatibility matching of tissue antigens during transplantation. Computer simulation of pyrosequencing data suggest that alleles of HLA-DRB1, -3, -4, and -5 were readily identifiable by pyrosequencing as were their heterozygous allelic combinations. Pyrosequencing primers were designed to specifically sequence HLA loci of interest even in a background of other amplified, closely related sequences such as alleles of the pseudogene HLA-DRB6, -7, -8, and -9. Polymorphic residues of HLA-DRB genes were identified within each pyrosequencing reaction, obtained by 50 to 70 nucleotide read lengths. Heterozygous allelic combinations of HLA genes were analyzed and compared successfully to genotyping of alleles by sequence-specific oligonucleotide probe hybridization as well as allele specific polymerase chain reaction protocols. Pyrosequence-based typing is compatible with genotyping of allelic combinations expected from heterozygous individuals, resulting in nucleotide resolution of the highly polymorphic HLA system. Using a single pyrosequence instrument, complete typing of HLA-DRB genes can be performed daily on hundreds of individuals for high resolution histocompatibility genotyping studies.