DP-Seq
DP-Seq
DP-Seq amplifies mRNA from limited starting material, as low as 50 pg. In this method, a specific set of heptamer primers is designed. The enriched poly(A)-selected mRNA undergoes first-strand cDNA synthesis. Next, the designed primers are hybridized to the first-strand cDNA, followed by second-strand synthesis and PCR. Deep sequencing of the amplified DNA allows for accurate detection of specific mRNA expression at the single-cell level.
Pros:
- As little as 50 pg of starting material can be used
- Little transcript-length bias
Cons:
- The sequences of the target areas must be known to design the heptamers
- Exponential amplification during PCR can lead to primer-dimers and spurious PCR products
- Some read-length bias
- Friedmann-Morvinski D., Bhargava V., Gupta S., Verma I. M. and Subramaniam S. Identification of therapeutic targets for glioblastoma by network analysis. Oncogene. 2015;
- Kolodziejczyk A. A., Kim J. K., Svensson V., Marioni J. C. and Teichmann S. A. The Technology and Biology of Single-Cell RNA Sequencing. Mol Cell. 2015;58:610-620
- Head S. R., Komori H. K., LaMere S. A., et al. Library construction for next-generation sequencing: overviews and challenges. Biotechniques. 2014;56:61-64, 66, 68, passim