Thompson John F, Steinmann Kathleen E
Helicos BioSciences, Cambridge, Massachusetts, USA.
Curr Protoc Mol Biol. 2010 Oct;Chapter 7:Unit7.10. doi: 10.1002/0471142727.mb0710s92.
Helicos™ Single Molecule Sequencing (SMS) provides a unique view of genome biology through direct sequencing of cellular nucleic acids in an unbiased manner, providing both accurate quantitation and sequence information. Sample preparation does not require ligation or PCR amplification, avoiding the GC-content and size biases observed in other technologies. DNA is simply sheared, tailed with poly(A), and hybridized to a flow cell surface containing oligo(dT) for sequencing-by-synthesis of billions of molecules in parallel. This process also requires far less material than other technologies. Gene expression measurements can be done using first-strand cDNA-based methods (RNA-Seq) or using a novel approach that allows direct hybridization and sequencing of cellular RNA for the most direct quantitation possible. In this unit, principles and methods for using the Helicos® Genetic Analysis System are discussed.
Helicos™ 单分子测序（SMS）通过对细胞核酸进行无偏差的直接测序，为基因组生物学提供了独特的视角，可同时提供准确的定量和序列信息。样品制备无需连接或PCR扩增，避免了其他技术中观察到的GC含量和大小偏差。DNA只需简单剪切、加上poly(A)尾，然后与含有寡聚(dT)的流动池表面杂交，即可并行合成测序数十亿个分子。该过程所需的材料也比其他技术少得多。基因表达测量可以使用基于第一链cDNA的方法（RNA测序），或者使用一种新颖的方法，该方法允许对细胞RNA进行直接杂交和测序，以实现最直接的定量。在本单元中，将讨论使用Helicos® 遗传分析系统的原理和方法。
