Multisite Evaluation of Next-Generation Methods for Small RNA Quantification

Abstract

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 in vivo. 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.

Quantitative mapping of the cellular small RNA landscape with AQRNA-seq

“…A direct comparison of AQRNA-seq to six commercial small RNA-seq kits ( Fig. 2d ), 27 as well as additional reports using the miRXplore reference, 5 , 21 , 28 established AQRNA-seq as the most quantitatively accurate RNA-seq workflow.…”

Small RNA-sequencing for Analysis of Circulating miRNAs: Benchmark Study

“…In addition, EdgeSeq, a platform using hybridization probes and targeted sequencing readout, speci cally designed for ease-of-use in clinical setting, is available as an alternative to small RNA-Seq. Previous comparative studies performed on a subsets of available methods revealed vast di erences in their performance (7,(9)(10)(11)(12)(13)(14)(15)(16)(17). However, how current commercial small RNA-Seq methods perform, particularly in challenging setting such as liquid biopsy samples, is not yet established.…”

Digital Multiplexed Gene Expression Analysis of mRNA and miRNA from Routinely Processed and Stained Cytological Smears: A Proof-of-Principle Study

“…Moreover, high-throughput assays for DNA and RNA extraction have been adopted in the recently updated molecular testing guidelines for the selection of patients with lung cancer for treatment with targeted tyrosine kinase inhibitors, jointly supported by the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology [29]. However, considering the critical consequences that the analysis of cytological samples may have for patients in terms of diagnosis, prognosis, disease monitoring, and treatment outcomes, we strongly recommend that the implementation of molecular testing as a standard-of-care be performed only after in-house validation, considerations regarding the bias of expression level normalization [30, 31], and under very strict technical QCs, such as those developed for other high-throughput tools [31-36].…”