Single molecule sequencer

A gene expression technique adapted for single molecule sequencing has enabled researchers at the Riken Omics Science Center (OSC) to accurately and quantitatively measure gene expression levels using only 100 ng of total RNA. The technique, which pairs Riken’s Cap Analysis of Gene Expression (CAGE) protocol with the Helicos Genetic Analysis System opens the door to the detailed analysis of gene expression networks and rare cell populations.

Figure 1: HeliScopeCAGE protocol workflow. (A) Reverse transcription. cDNA is synthesised using SuperScript III and random N15 primer. (B) Oxidation/biotinylation. The cap structure is oxidised with sodium peroxide and biotinylated with biotin (long arm) hydrazine. (C) RNase I digestion. Single-strand RNA is digested with RNase I. (D) Capture on magnetic streptavidin beads. Biotinylated RNA/cDNA hybrid molecules are captured using magnetic streptavidin beads. (E) Wash unbound molecules. Unbound RNA/DNA hybrid molecules are washed away. (F) Release ss-cDNA. Captured RNA/DNA hybrid molecules are treated with RNase H and RNase I, then heat treated. (G) Poly-A tailing/blocking. Released cDNA is poly-A tailed using terminal deoxynucleotidyl transferase and dATP, then blocked with biotin-ddATP. (H) Load on flow cell. Blocked poly-A tailed cDNA is loaded on the HeliScope flow cell channel and anneals with the dT 50 surface. (I) Fill with dTTP/locked with A/G/C virtual terminator. After annealing of cDNA, the single-strand poly-A tail part is filled with DNA polymerase, dTTP and an A/G/C virtual terminator which is used for the HeliScope sequencing to lock the poly-T termini. The library is then ready for sequencing.

In recent years, next-generation DNA sequencers have produced an increasingly detailed picture of how genes are expressed at the molecular level. The transcriptional output of these genes - the RNA copies produced from DNA - has revealed a richness of complexity in transcript structure and function, providing insights into the molecular-level properties of cancers and other diseases.

One of the most powerful methods for analysing RNA transcripts is the Cap Analysis of Gene Expression (CAGE) protocol developed at the Riken OSC. A unique approach, CAGE enables not only high-throughput gene expression profiling, but also simultaneous identification of transcriptional start sites (TSS) specific to each tissue, cell or condition.

With HeliScopeCAGE, the OSC research team has adapted the existing CAGE protocol for use with the HeliScope Single Molecule Sequencer. Unlike earlier sequencers, the HeliScope sequencer does not employ polymerase chain reaction (PCR) amplification to multiply a small number of DNA strands for analysis, a process which can introduce biases into data. Instead, the HeliScope Sequencer actually sequences the DNA strand itself, enabling direct, high-precision measurement.

In a paper published in Genome Research, Riken researchers confirm that this direct approach reduces biases and generates highly reproducible data from between 5 µg to as little as 100 ng of total RNA. A comparison using a leukaemia cell line (THP-1) and a human cervical cancer cell line (HeLa) further shows that results from the technique are closely correlated to those from traditional microarray analysis. By making possible high-precision gene expression analysis from tiny samples, HeliScopeCAGE greatly expands the scope of research at the OSC, strengthening the institute’s role in Japan as a hub for next-generation genome analysis.

LSA Technology Development Group

Riken Omics Science Center (OSC)

www.osc.riken.jp