Ordered catenation of sequence-tagged sites and multiplexed SNP genotyping by sequencing.
We describe a method for the efficient genotyping of SNPs, involving sequencing of ordered and catenated sequence-tagged sites (OCS). In OCS, short genomic segments, each containing an SNP, are amplified by PCR using primers that carry specially designed extra nucleotides at their 5'-ends. Amplification products are then combined and converted to a concatamer in a defined order by a second round of thermal cycling. The concatenation takes place because the 5'-ends of each amplicon are designed to be complementary to the ends of the presumptive neighboring amplicons. The primer sequences for OCS are chosen using newly developed dedicated software, OCS Optimizer. Using sets of SNPs, we show that at least 10 STSs can be concatenated in a predefined order and all SNPs in the STSs are accurately genotyped by one two-way sequencing reaction.
Higasa
Koichiro
K
Division of Genome Analysis, Research Center for Genetic Information, Medical Institute of Bioregulation, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
Hayashi
Kenshi
K
D044466:Asian People / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D023061:Gene Order / Q000235:genetics; D005838:Genotype; D006579:Heterozygote; D006801:Humans; D007564:Japan; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*; D016324:Sequence Tagged Sites; D012984:Software
D017931:DNA Primers; D004247:DNA