Comparative evaluation of RNA-Seq library preparation methods for strand-specificity and low input

Sci Rep. 2019 Sep 17;9(1):13477. doi: 10.1038/s41598-019-49889-1.


Library preparation is a key step in sequencing. For RNA sequencing there are advantages to both strand specificity and working with minute starting material, yet until recently there was no kit available enabling both. The Illumina TruSeq stranded mRNA Sample Preparation kit (TruSeq) requires abundant starting material while the Takara Bio SMART-Seq v4 Ultra Low Input RNA kit (V4) sacrifices strand specificity. The SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Pico) by Takara Bio claims to overcome these limitations. Comparative evaluation of these kits is important for selecting the appropriate protocol. We compared the three kits in a realistic differential expression analysis. We prepared and sequenced samples from two experimental conditions of biological interest with each of the three kits. We report differences between the kits at the level of differential gene expression; for example, the Pico kit results in 55% fewer differentially expressed genes than TruSeq. Nevertheless, the agreement of the observed enriched pathways suggests that comparable functional results can be obtained. In summary we conclude that the Pico kit sufficiently reproduces the results of the other kits at the level of pathway analysis while providing a combination of options that is not available in the other kits.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Computational Biology / methods
  • Gene Expression Profiling
  • Gene Library*
  • High-Throughput Nucleotide Sequencing* / methods
  • Liver / metabolism
  • Male
  • Mice
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Sequence Analysis, RNA* / methods
  • Transcriptome