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Nucleic Acids Research, 2002, Vol. 30, No. 3 e11
© 2002 Oxford University Press

Ordered catenation of sequence-tagged sites and multiplexed SNP genotyping by sequencing

Koichiro Higasa and Kenshi Hayashi*

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

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.

* To whom correspondence should be addressed. Tel: +81 92 642 6170; Fax: +81 92 632 2375; Email: khayashi{at}gen.kyushu-u.ac.jp


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