Gene expression analysis by massively parallel signature sequencing (MPSS)on microbead arrays

We describe a novel sequencing approach that combines non-gel-based signatu re sequencing with in vitro cloning of millions of templates on separate 5 mu m diameter microbeads. After constructing a microbead library of DNA tem plates by in vitro cloning, we assembled a planar array of a million templa te-containing microbeads in a flow cell at a density greater than 3 x 10(6) microbeads/cm(2). Sequences of the free ends of the cloned templates on ea ch microbead were then simultaneously analyzed using a fluorescence-based s ignature sequencing method that does not require DNA fragment separation. S ignature sequences of 19-20 bases were obtained by repeated cycles of enzym atic cleavage with a type Ils restriction endonuclease, adaptor ligation, a nd sequence interrogation by encoded hybridization probes. The approach was validated by sequencing over 269,000 signatures from two cDNA libraries co nstructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1. The approac h provides an unprecedented depth of analysis permitting application of pow erful statistical techniques for discovery of functional relationships amon g genes, whether known or unknown beforehand, or whether expressed at high or very low levels.