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

Abstract

We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 microm diameter microbeads. After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing microbeads in a flow cell at a density greater than 3x10(6) microbeads/cm2. Sequences of the free ends of the cloned templates on each microbead were then simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragment separation. Signature sequences of 16-20 bases were obtained by repeated cycles of enzymatic cleavage with a type IIs restriction endonuclease, adaptor ligation, and sequence interrogation by encoded hybridization probes. The approach was validated by sequencing over 269,000 signatures from two cDNA libraries constructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1. The approach provides an unprecedented depth of analysis permitting application of powerful statistical techniques for discovery of functional relationships among genes, whether known or unknown beforehand, or whether expressed at high or very low levels.

A plant transformation system designed for high throughput genomics inGossypium hirsutumto study root–organism interactions

“…The ability to generate large amounts of genomics data has reached an unprecedented state, in part, to advancements in nucleotide sequencing technology (Brenner et al 2000). This ability to generate the data has been met with the capability to analyze and organize the data into meaningful expression maps using a variety of computer applications (Scheideler et al 2002;Harris et al 2004;Trapnell et al 2009Trapnell et al , 2010Trapnell et al , 2012Matsye et al 2011;Risso et al 2014).…”

Digital quantitative measurements of gene expression

“…The techniques currently in vogue are based on direct sequence analysis (Adams, 1996), differential display (Fislage, 1998), or specific hybridization of complex cDNA or mRNA probes to microarrays of oligonucleotides or cDNAs (Brown et al, 1998;Brown and Botstein, 1999;Elek et al, 2000;Ramsay, 1998). These approaches are all limited in the dynamic range of the measurements and by their inability to produce digital measurements (Brenner et al, 2000;Velculescu et al, 1995). Two high-throughput approaches have been developed to make ''digital'' measurements or measurements involving simple counting of gene expression, thus providing more accurate and more precise measurements (Brenner et al, 2000;Velculescu et al, 1995).…”

Problems on RNA Secondary Structure Prediction and Design

“…In the first context, described by Brenner et al [7], the goal is to sequence millions of short DNA fragments (these fragments could be in a gene expression sample). DNA sequencing machines handle one sequence at a time, and it would be infeasible to separate out the millions of short fragments and sequence each separately.…”