The second wave of next generation sequencing technologies, referred to as single-molecule sequencing (SMS), carries the promise of profiling samples directly without employing polymerase chain reaction steps used by amplification-based sequencing (AS) methods. With the goal of uncovering the merits of both technologies, researchers at the University of Michigan examined mRNA sequencing results from single-molecule and amplification-based sequencing in a set of human cancer cell lines and tissues.
They observed a characteristic coverage bias towards high abundance transcripts in amplification-based sequencing. A larger fraction of AS reads cover highly expressed genes, such as those associated with translational processes and housekeeping genes, resulting in relatively lower coverage of genes at low and mid-level abundance. In contrast, the coverage of high abundance transcripts plateaus off using SMS. Consequently, SMS is able to sequence lower- abundance transcripts more thoroughly, including some that are undetected by AS methods; however, these include many more mapping artifacts.
A better understanding of the technical and analytical factors introducing platform specific biases in high throughput transcriptome sequencing applications will be critical in cross platform meta-analytic studies.
Sam LT, Lipson D, Raz T, Cao X, Thompson J, et al. (2011) A Comparison of Single Molecule and Amplification Based Sequencing of Cancer Transcriptomes. PLoS ONE 6(3), e17305. [article]