Diagenode

Multisite Evaluation of Next-Generation Methods for Small RNA Quantification.


Herbert ZT, Thimmapuram J, Xie S, Kershner JP, Kolling FW, Ringelberg CS, LeClerc A, Alekseyev YO, Fan J, Podnar JW, Stevenson HS, Sommerville G, Gupta S, Berkeley M, Koeman J, Perera A, Scott AR, Grenier JK, Malik J, Ashton JM, Pivarski KL, Wang X, Kuffe

Small RNAs (smRNAs) are important regulators of many biologic processes and are now most frequently characterized using Illumina sequencing. However, although standard RNA sequencing library preparation has become routine in most sequencing facilities, smRNA sequencing library preparation has historically been challenging because of high input requirements, laborious protocols involving gel purifications, inability to automate, and a lack of benchmarking standards. Additionally, studies have suggested that many of these methods are nonlinear and do not accurately reflect the amounts of smRNAs . Recently, a number of new kits have become available that permit lower input amounts and less laborious, gel-free protocol options. Several of these new kits claim to reduce RNA ligase-dependent sequence bias through novel adapter modifications and to lessen adapter-dimer contamination in the resulting libraries. With the increasing number of smRNA kits available, understanding the relative strengths of each method is crucial for appropriate experimental design. In this study, we systematically compared 9 commercially available smRNA library preparation kits as well as NanoString probe hybridization across multiple study sites. Although several of the new methodologies do reduce the amount of artificially over- and underrepresented microRNAs (miRNAs), we observed that none of the methods was able to remove all of the bias in the library preparation. Identical samples prepared with different methods show highly varied levels of different miRNAs. Even so, many methods excelled in ease of use, lower input requirement, fraction of usable reads, and reproducibility across sites. These differences may help users select the most appropriate methods for their specific question of interest.

Tags
CATS

Share this article

Published
January, 2020

Source

Products used in this publication

  • Library prep kit icon
    C05010040
    CATS Small RNA-seq Kit x24

イベント

  • 10th Canadian Conference on Epigenetics
    Ontario, Canada
    Oct 1-Oct 4, 2024
  • Long-Read Sequencing Meeting 2024
    Uppsala, Sweden
    Oct 21-Oct 23, 2024
  • NextGen Omics 2024
    London, UK
    Oct 23-Oct 25, 2024
  • FEBS 2024
    Budapest, Hungary
    Oct 28-Oct 31, 2024
  • 5th Danube Conference on Epigenetics
    Budapest, Hungary
    Oct 28-Oct 31, 2024
 すべてのイベントを見る

 


       Site map   |   Contact us   |   Conditions of sales   |   Conditions of purchase   |   Privacy policy