Improved Small RNA Library Preparation Workflow for Next Generation Sequencing

Sabrina Shore, Jordana Henderson, Anton McCaffrey, Gerald Zon, Richard Hogrefe
TriLink Biotechnologies, United States of America

Abstract
Next generation sequencing (NGS) can be used to analyze microRNAs (miRNAs), small non-coding RNAs that are important therapeutic targets and diagnostic markers. Commercially available small RNA sequencing library preparation kits require large inputs (100 ng) and a laborious gel purification step, which is not amenable to automation. Additionally commercial kits are hindered by adapter dimer formation, where 5΄ and 3΄ adapters ligate without an intervening RNA insert. Adapter dimers preferentially amplify relative to the library during PCR amplification. This is exacerbated at low RNA inputs where adapter dimers can greatly diminish usable sequencing reads. We describe an optimized small RNA library preparation workflow which suppresses adapter dimers, allows for RNA inputs as low as 1 ng and eliminates the need for gel purification. Chemically modified adapters and an optimized protocol were employed to suppress adapter dimers while still allowing for efficient library ligation. Library preparation with modified adapters was compared to the Illumina TruSeq® Small RNA Sample Prep Kit. Non-gel purified samples were purified with the Agencourt® AMPure® XP Kit. Samples were sequenced on an Illumina HiSeq™. Our modified adapter workflow was benchmarked against the TruSeq® Kit at 100 and 10 ng inputs. The modified adapter workflow allows RNA inputs as low as 1 ng and generates less than 1% adapter dimer reads when gel purified (Table 1). In contrast, the TruSeq® Kit yields 14% and 51% adapter dimer reads at 100 and 10 ng inputs, respectively. TriLink’s modified adapter workflow with magnetic bead-based size selection yields less than 10% dimer for all input levels, while the TruSeq® Kit results in a minimum of 14% dimer reads at the highest input level. TriLink’s modified adapter workflow improves small RNA library preparation by significantly reducing adapter dimer formation. In fact, our improvements allow for sequencing from 1 ng of total RNA without compromising valuable sequencing reads, which was previously not feasible. TriLink’s workflow with magnetic bead-based size selection, an automatable technique, results in lower amounts of dimer reads than current methods using gel purification. Replacement of gel purification with an automatable purification step results in less hands-on time, better reproducibility and higher throughput. The modified adapter workflow surpasses other currently available technologies and provides significant improvement to small RNA NGS.

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Development and Optimisation of PCR Assays to Analyse MicroRNAs and their Target Genes

David Arthur Simpson
Queen’s University Belfast, United Kingdom

Abstract
Introduction: Endothelial progenitor cells (EPCs) isolated from blood release microRNA-containing extracellular vesicles (EVs) which can potentially be harnessed to modulate angiogenesis as a treatment for vascular disease. These EVs contribute to the pool of circulating microRNAs, which provide biomarkers for many conditions. Quantification of microRNAs is required both to study their role in vascular repair and to exploit their potential as biomarkers. Multiple issues need to be considered in the design of a successful assay. RT-PCR reagents and template concentrations must be optimised. The final reaction volume must be minimised to reduce reagent use and amount of template required. Increasing the rate of thermal cycling brings time-savings and can be critical for certain clinical applications. The specificity of the assay with regard to microRNA isomiRs must be defined.
Methods: Probe-based assays and polyadenylation followed by oligo-dT primed reverse transcription and PCR with SYBR Green were employed. PCR reactions were set up using an Echo liquid handler (Labcyte), which uses acoustic energy to transfer 25nl droplets. qPCR was performed using a 384 well LightCycler 480 (Roche) or a rapid thermal cycler (xxpress, BJS Biotechnologies). RT-PCR products were sequenced using the Ion Torrent platform (Life Technologies).
Results: The quality of data was maintained as qPCR volumes were reduced to 2 µl. MicroRNAs can be amplified from plasma in less than 10 min using the xxpress cycler. Sequencing of RT-PCR products provides a profile of isomiRs for specific microRNAs comparable to sequencing of entire small RNA libraries.
Conclusion: The ability to accurately transfer nanolitre volumes and therefore adopt very low reaction volumes facilitates rapid optimisation of PCR reaction conditions and saves reagents and template. Characterisation of microRNA isomiRs and rapid detection of them from plasma broadens their potential as clinical biomarkers.

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MiRNA Profiling In Tumor Tissue, Body Fluids And Exosomes – A Combinational Techniques Approach Of NGS And QPCR.

Robert P. Loewe
GeneWake GmbH, Germany

Abstract
miRNA has gained a pivotal role in molecular diagnostics and disease analysis. Due its stable nature and an abundant presence either in the tissue of origin, in microparticles or free circulating, it is a viable and interesting analyte. To understand the dynamics and spectrum of this biological dilution – from the production site down to circulation – we analysed glioblastoma tissue in conjunction with cerebrospinal fluid (CSF) and serum from the same patients. As the generation of microvesicles, general gene expression and epigenetic changes (methylation and hydroxyl-methylation of DNA) might mechanistically contribute to the phenomena, these characteristics were also measured. miRNA was primarily analysed via miRNA-Seq on a HiSeq instrument to allow a certain depth of the data. A data convergence of the different biological levels and source materials in addition to further information via qPCR was gathered. The profiling procedure and results underlining the inherent dynamics will be presented.

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New sensitive and specific method for microRNA analysis

Robert Sjöback1, Lukáš Valihrach2, Mikael Kubista1,2
1TATAA Biocenter AB, Sweden; 
2Institute of Biotechnology, CAS, Czech Republic

Abstract
MicroRNAs (miRNAs) are small non-coding RNAs that function as important biological regulators in viruses, plants, and animals (1). The single-stranded miRNAs regulate gene expression at the post-transcription level and the dysregulation of miRNAs is associated with various human diseases (2-4). Recent reports show microRNAs are abundant not only in tissues but also in body fluids and they show great potential as minimum-invasive biomarkers in the diagnosis and prognosis of cancers and other diseases (5, 6). However, there are two main challenges when analyzing miRNAs by molecular methods: 1) miRNAs are short, usually not more than 22 nucleotides, which is the length of conventional PCR primers; 2) closely related miRNAs may differ in only a single nucleotide position. Current methods approach these challenges by extending the length of the miRNA using either miRNA specific RT primers or by non-specific RT primers, which compromises specificity and sensitivity (7). Here we present a novel method for the detection and quantification of short nucleic acids that has higher sensitivity than current approaches with concomitant enhanced specificity.
1) He et al., Nat Rev Genetics 2004, 5: 522-31. 2) Esquela-Kerscher A & Slack FJ, Nat. Rev. Cancer 2006, 6: 259-69. 3) Michael et al., Mol. Cancer Res. 2003, 1: 882-91. 4) Dimmeler S & Nicotera P, EMBO Mol. Med. 2013, 5: 180-90. 5) Brase JC et al., Mol. Cancer 2010, 26: 306. 6) Chen et al., Cell Res. 2008, 18: 997-1006. 7) Mestdagh P et al., Nature Methods 2014, 11:809-815.

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Decoding lncRNA functions using high-throughput pathway perturbation.

Pieter Mestdagh, Jan Hellemans, Ariane De Ganck, Jo Vandesompele
Biogazelle, Belgium

Abstract
Genome-wide studies have shown that our genome is pervasively transcribed, producing a complex pool of coding and non-coding transcripts that shape a cell’s transcriptome. Long non-coding RNAs or lncRNAs dominate the non-coding transcriptome and are emerging as key regulatory factors in human disease and development. Still, only a fraction of lncRNAs has been studied experimentally. In order to gain insights in lncRNA functions on a genome-wide scale, we performed high-throughput pathway perturbations followed by total RNA sequencing. Cells were treated with 90 targeted compounds and 90 transcription factor siRNAs, yielding a total of 180 individual perturbations. For each perturbation, differentially expressed lncRNAs were identified and mapped to pathways using matching protein-coding gene expression data. We define a functional context for thousands of lncRNAs that can serve as a starting point to guide more focused experimental studies and accelerate lncRNA research.

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Updated evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study

Pieter Mestdagh, Jo Vandesompele
Ghent University / Biogazelle, Belgium (on behalf of the microRNA quality control study consortium)

Abstract
MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription–quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 14 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.

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