The Impact of Protocol on Next-Gen Sequencing Based Gene Expression Analysis

RNA-Seq provides unparalleled levels of information about the transcriptome including precise expression levels over a wide dynamic range.

However, as new techniques and protocols for RNA-Seq measurement of gene expression are established, it becomes essential to understand how the technical variation from protocol to protocol impacts the quality and interpretability of results.

Researchers from Helicos BioSciences used multiple human RNA samples to assess the impact of RNA fragmentation, RNA fractionation, cDNA synthesis, and single versus multiple tag counting on the conclusions drawn from RNA-Seq experiments.

They found:

  • Though protocols employing polyA RNA selection generate the highest number of non-ribosomal reads and the most precise measurements for coding transcripts, such protocols were found to detect only a fraction of the non-ribosomal RNA in human cells.
  • PolyA RNA excludes thousands of annotated and even more unannotated transcripts, resulting in an incomplete view of the transcriptome.
  • Ribosomal-depleted RNA provides a more cost-effective method for generating complete transcriptome coverage.
  • Expression measurements using single tag counting provided advantages for assessing gene expression and for detecting short RNAs relative to multi-read protocols.
  • Detection of short RNAs was also hampered by RNA fragmentation.

This work will help researchers choose from among a range of options when analyzing gene expression, each with its own advantages and disadvantages.

Raz T, Kapranov P, Lipson D, Letovsky S, Milos PM, et al. (2011) Protocol Dependence of Sequencing-Based Gene Expression Measurements. PLoS ONE 6(5), e19287. [article]