As the study of the genome has progressed, the definition of a gene and the seemingly clear distinction between gene and “junk” has begun to blur. From large scale studies, such as the ENCODE project, it is evident that more of the genome is actively transcribed than ever imagined. Discovering these novel transcripts and elucidating their regulation and function in various tissue types and biological states is crucial for understanding genome biology and disease.

Helicos has developed an application for cDNA Sequencing of whole transcriptomes, utilizing its true direct DNA measurement technology.  Moving beyond digital gene expression, RNA-Seq offers full coverage of transcripts, and will allow you to:

• Discover new transcripts, study noncoding RNAs and retain strandedness
• Find transcribed mutations
• Identify new splice junctions and variants
• Quantify rare alleles and allele-specific expression

Only with the Helicos® Genetic Analysis Platform, can you perform RNA-Seq without the biases inherent to ligation or amplification. 

The Advantages
Helicos RNA-Seq, an amplification-free assay with millions of reads per sample, provides you with:

• The quantitative accuracy only possible with an amplification- and ligation-free approach
• The ability to perform RNA-Seq on hundreds of samples in a cost-effective manner
• The resolution to find changes not detectable on array-based platforms or amplification-based next-generation sequencers
• The precision to ensure that your results stay the same from run-to-run and from prep-to-prep
• The ability to utilize attomole-level (~500 picograms) of starting nucleic acid material

Recent publications utilizing Helicos RNA-Seq technology include:

Digital transcriptome profiling from attomole-level RNA samples. Genome Res 2010; 20, 519-525.

A Comparison of Single Molecule and Amplification Based Sequencing of Cancer Transcriptomes. Plos One, 2011;6: e17305.

The majority of total nuclear-encoded non-ribosomal RNA in a human cell is “dark matter” unannotated RNA. BMC Biology, 2010; 8:149.

Protocol dependence of sequencing-based gene expression measurements. PLoS One. 2011 May 6;6(5):e19287.

The Evidence: A high-resolution view of the transcriptome
Helicos has demonstrated the ability to provide a high resolution view of the transcriptome, by applying the tSMS approach to mRNA-Seq. Scientists at Helicos detected known and undiscovered transcripts throughout the genome of a chronic myelogenous leukemia (CML) cell line (see Figure 1).

Figure 1. RNA samples obtained from a CML cell line were converted to cDNA, which was processed using the Helicos DNA Sample Preparation methodology described in Figure 2. The modified cDNA sample was loaded into the Helicos Flow Cell, and analyzed using the HeliScope Single Molecule Sequencer. Single molecule reads were mapped to the human genome. Results from a representative gene are shown; Black lines: tSMS reads; Blue bars: Known exons; Solid black/grey bar: Human ESTs.

The Approach
Fragmented or unfragmented RNA from a sample is converted to first-strand cDNA, which is modified with a polyA tail and loaded onto the instrument. The tailed fragments hybridize to complementary poly-T strands anchored to the flow cell surface. Inside the HeliScope™ Single Molecule Sequencer, a series of nucleotide addition and detection cycles determine the sequence of each fragment. Open source data analysis software aligns the hundreds of millions of reads to a reference sequence.

Figure 2. Helicos mRNA-Seq Sample Preparation Methodology

Helicos mRNA-Seq Analysis – A virtually bias-free view of the transcriptome.