Systematic comparison of small RNA library preparation protocols for next-generation sequencing

Abstract

BackgroundNext-generation sequencing technologies have revolutionized the study of small RNAs (sRNAs) on a genome-wide scale. However, classical sRNA library preparation methods introduce serious bias, mainly during adapter ligation steps. Several types of sRNA including plant microRNAs (miRNA), piwi-interacting RNAs (piRNA) in insects, nematodes and mammals, and small interfering RNAs (siRNA) in insects and plants contain a 2’-O-methyl (2’-OMe) modification at their 3′ terminal nucleotide. This inhibits 3′ adapter ligation and makes library preparation particularly challenging. To reduce bias, the NEBNext kit (New England Biolabs) uses polyethylene glycol (PEG), the NEXTflex V2 kit (BIOO Scientific) uses both randomised adapters and PEG, and the novel SMARTer (Clontech) and CATS (Diagenode) kits avoid ligation altogether. Here we compared these methods with Illumina’s classical TruSeq protocol regarding the detection of normal and 2’ OMe RNAs. In addition, we modified the TruSeq and NEXTflex protocols to identify conditions that improve performance.ResultsAmong the five kits tested with their respective standard protocols, the SMARTer and CATS kits had the lowest levels of bias but also had a strong formation of side products, and as a result performed relatively poorly with biological samples; NEXTflex detected the largest numbers of different miRNAs. The use of a novel type of randomised adapters called MidRand-Like (MRL) adapters and PEG improved the detection of 2’ OMe RNAs both in the TruSeq as well as in the NEXTflex protocol.ConclusionsWhile it is commonly accepted that biases in sRNA library preparation protocols are mainly due to adapter ligation steps, the ligation-free protocols were not the best performing methods. Our modified versions of the TruSeq and NEXTflex protocols provide an improved tool for the study of 2’ OMe RNAs.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4491-6) contains supplementary material, which is available to authorized users.

AQ-seq: Accurate quantification of microRNAs and their variants

“…Thus, we tested various combinations of randomized adapters and PEG (Supplemental Fig. S1, lanes 2, 4-15), and found that either applying PEG only to the 3′ adaptor ligation step or adopting less than 20% of PEG -as being used in currently available protocols (Zhang et al 2013;Xu et al 2015;Dard-Dascot et al 2018) -is not sufficient to abolish the ligation bias. Substantial improvement was achieved when we included 20% PEG at both 3′ and 5′ adapter ligation reactions, in combination with randomized adapters (Fig.…”

“…However, accurate miRNA profiling has been difficult because certain miRNAs are favored over others in an enzyme-dependent ligation reaction due to the preference of RNA ligases for some sequences and structures, leading to a skewed representation of miRNAs. This can severely compromise quantitative analysis of end heterogeneity and strand preference of a given miRNA (Hafner et al 2011;Jayaprakash et al 2011;Sun et al 2011;Sorefan et al 2012;Zhuang et al 2012;Zhang et al 2013;Song et al 2014;Dard-Dascot et al 2018). Recent studies have sought to minimize the ligation bias by adopting randomized adapters, presuming that the increased diversity of adapter sequences raises chances of capturing miRNAs with various sequences (Jayaprakash et al 2011;Sun et al 2011;Sorefan et al 2012;Zhuang et al 2012;Zhang et al 2013;Fuchs et al 2015;Xu et al 2015;Dard-Dascot et al 2018).…”

“…This can severely compromise quantitative analysis of end heterogeneity and strand preference of a given miRNA (Hafner et al 2011;Jayaprakash et al 2011;Sun et al 2011;Sorefan et al 2012;Zhuang et al 2012;Zhang et al 2013;Song et al 2014;Dard-Dascot et al 2018). Recent studies have sought to minimize the ligation bias by adopting randomized adapters, presuming that the increased diversity of adapter sequences raises chances of capturing miRNAs with various sequences (Jayaprakash et al 2011;Sun et al 2011;Sorefan et al 2012;Zhuang et al 2012;Zhang et al 2013;Fuchs et al 2015;Xu et al 2015;Dard-Dascot et al 2018). Another approach was to apply polyethylene glycol (PEG), which facilitates ligation reaction via molecular crowding effect (Harrison and Zimmerman 1984;Zhang et al 2013;Song et al 2014;Xu et al 2015;Shore et al 2016;Dard-Dascot et al 2018).…”

CRISPR/Cas9-targeted removal of unwanted sequences from small-RNA sequencing libraries

“…Strategies using locked-nucleic acid (LNA) oligos have been employed to prevent adapter dimer formation with some limited success (22). Currently, the most effective means of adapter dimer prevention or removal during smRNA sequencing has been demonstrated with ligation free templateswitching protocols or chemically modified adapters that sterically inhibit ligation of the 3' and 5' adapters to each other and inhibit adapter dimer reverse transcription (23,24). Because adapter dimer formation is limited, these strategies allow for the use of SPRI-bead based size selection in place of gel separation, which greatly increases library preparation throughput.…”

Evaluation of miRNA Detection Methods for the Analytical Characteristic Necessary for Clinical Utilization

“…High volume of sample is consumed in this process and sophisticated software is required for evaluating results. The combination of expensive chemistry and instrumentation means a high cost per analysis [10]. …”