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J Biomol Tech. 2020 Jan 10. pii: jbt.20-3102-001. doi: 10.7171/jbt.20-3102-001. [Epub ahead of print]

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

Author information

1
Molecular Biology Core Facilities at Dana-Farber Cancer Institute, Boston, Massachusetts, USA.
2
Bioinformatics Core, Purdue University, West Lafayette, Indiana, USA.
3
Inscripta, Boulder, Colorado, USA.
4
Genomics and Molecular Biology Shared Resource, Norris Cotton Cancer Center, Geisel School of Medicine, Lebanon, New Hampshire, USA.
5
Microarray and Sequencing Resource Core Facility, Boston University, Boston, Massachusetts, USA.
6
Department of Pathology and Laboratory Medicine, Boston University, Boston, Massachusetts, USA.
7
Genomic Core Facility, Department of Biomedical Sciences, Joan C. Edwards School of Medicine, Marshall University, Huntington, West Virginia, USA.
8
Genomic Sequencing and Analysis Facility, University of Texas, Austin, Texas, USA.
9
Genomics Core Facility, Van Andel Institute, Grand Rapids, Michigan, USA.
10
Stowers Institute for Medical Research, Kansas City, Missouri, USA.
11
RNA Sequencing Core, Department of Biomedical Sciences, Cornell University, Ithaca, New York, USA.
12
Genomics Research Center, University of Rochester, Rochester, New York, USA.
13
NUSeq Core Research Facility, Northwestern University, Chicago, Illinois, USA.
14
Loyola Genomics Facility, Loyola University Chicago, Maywood, Illinois, USA.
15
Molecular Genomics Core, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.
16
Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Science Park, Smithville, Texas, USA.
17
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
18
Interdisciplinary Center for Biotechnology Research Gene Expression and Genotyping, University of Florida, Gainsville, Florida, USA.
19
Indiana University School of Medicine, Indianapolis, Indiana, USA.
20
MIT BioMicro Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

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.

KEYWORDS:

Illumina Library Prep; RNA sequencing; miRNA sequencing; small RNA sequencing

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