Helicos single-molecule sequencing technology provides the researcher with a comprehensive portfolio of robust sequencing applications for accurate quantitation of nucleic acids. The Helicos digital gene expression assay demonstrates the principles of this single-molecule sequencing technology and provides the researcher with a method for tag-based RNA quantitation. Utilizing total RNA or mRNA, a poly(U) primer is hybridized to the poly(A) region of mRNA and extended with reverse transcriptase, resulting in first-strand cDNA. After digestion of the RNA, the 3' end of the cDNA is poly(A)-tailed with terminal transferase, which is then hybridized for capture on the Helicos Sequencer Flow Cell oligo(dT) surface. cDNA molecules are then sequenced using the Helicos Genetic Analysis System, generating sequence reads from the 5' end of the original RNA transcript. One tag is generated from each RNA transcript and each of 50 flow cell channels yields 8-12 million alignable sequence tags or some 400-600 million sequence tags per run. Thus, the researcher is offered the flexibility of sequencing 50 independent samples or using multiple channels of individual samples to allow a deeper view for transcript quantitation. This method provides unique advantages relative to RNA-seq methods for the generation of accurate and reliable gene expression data from biological specimens.
- Helicos single-molecule sequencing
- Tag-based RNA