An extension to: Systematic assessment of commercially available low-input miRNA library preparation kits

RNA Biol. 2020 Sep;17(9):1284-1292. doi: 10.1080/15476286.2020.1761081. Epub 2020 May 21.


High-throughput sequencing has emerged as the favoured method to study microRNA (miRNA) expression, but biases introduced during library preparation have been reported. We recently compared the performance (sensitivity, reliability, titration response and differential expression) of six commercially-available kits on synthetic miRNAs and human RNA, where library preparation was performed by the vendors. We hereby supplement this study with data from two further commonly used kits (NEBNext, NEXTflex) whose manufacturers initially declined to participate. NEXTflex demonstrated the highest sensitivity, which may reflect its use of partially-randomized adapter sequences, but overall performance was lower than the QIAseq and TailorMix kits. NEBNext showed intermediate performance. We reaffirm that biases are kit specific, complicating the comparison of miRNA datasets generated using different kits.

Keywords: NEBNext; NEXTflex; NGS; Next Generation Sequencing; library preparation; low RNA input; miRNA; microRNA; sequencing bias; small RNA-seq.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Library*
  • Genetic Engineering* / methods
  • High-Throughput Nucleotide Sequencing / methods
  • Laboratory Chemicals / standards
  • MicroRNAs / genetics*
  • Reproducibility of Results
  • Sequence Analysis, RNA / methods


  • Laboratory Chemicals
  • MicroRNAs

Grants and funding

This work was supported by Helse Sør-Øst RHF grants [2015034 and 2016122]. Sequencing was performed by the Norwegian Sequencing Centre (, a national technology platform hosted by Oslo University Hospital and supported by the ‘Functional Genomics’ and ‘Infrastructure’ programs of Norsk Forskningsrådet and Helse Sør-Øst RHF.