Small RNAs are becoming increasingly important as clinical and diagnostic tools
RealSeq® proprietary technology allows for bias-free small RNA library construction
RealSeq® -Biofluids expands upon this technology to allow for low-input samples
Small RNAs are short non-coding RNA molecules that regulate gene expression at the post-transcriptional level. Typically ranging from 18 to 30 nucleotides in length, small RNAs can be classified into several categories, including microRNAs (miRNAs), small nuclear RNAs (snRNAs), tRNA derived small RNAs (tsRNAs), and piwi-interacting RNAs (piRNAs). These RNAs are generating increased interest as researchers scrutinize the potential of small RNAs and more recently RNA fragments (RNA fragmentomics) [1] as biomarkers for infections (e.g. SARS-CoV2) and progressive disease, (e.g. cancer) while also investigating the therapeutic potential of manipulating small RNA expression for the treatment of diseases and the development of novel therapeutic interventions.
The importance of optimized small RNA expression analysis
Given the increasing clinical significance of these small RNAs, accurate and reliable measurement of their expression is crucial because they are being investigated for several purposes, including disease biomarkers, therapeutic interventions, drug discovery, and basic research.
RealSeq® sequencing library preparation kits use innovative, proprietary technology for bias-free small RNA library construction. The technology relies on a novel single adapter and circularization, reducing incorporation bias in sequencing and improving detection of many small RNAs (Figure 1). The RealSeq®- Biofluids sequencing library preparation kit has been developed to address the unique challenges of working with biofluids and low-input samples while maintaining transcript representation accuracy. Sample inputs for the RealSeq®-Biofluids kit include plasma, serum, urine, whole blood, saliva and cerebrospinal fluid, and the kit is compatible with RNA isolated by any standard method. In the study herein, we evaluated the RealSeq®-Biofluids kit for small RNA detection and representation, focusing on number of reads, consisitency and pool diversity using porcine plasma samples.
Figure 1 | RealSeq® technology shows significantly less bias than competitor kits. For each kit, sequencing libraries were prepared from the miRXplore™ pool and sequenced; the sequence data were then used to calculate fold-deviations from the equimolar input and plotted as log2 values. Densities of miRNAs within a two-fold deviation from the expected values (between vertical lines) are considered un- biased. Under-represented, over-represented, and accurately quantified percentages (within the two horizontal lines) of miRNAs are shown. (Adapted from Barberan- Soler S, et al. Genome Biology. 2018)
Figure 2 | Library preparation and analysis workflow for porcine plasma case study. In this study, we evaluated the RealSeq®- Biofluids kit for small RNA detection and performance in a side-by-side comparison to another commercially available kit that relies on the standard sequential ligation of adapters to the 5’ and 3’ ends of small RNAs. Briefly, sequencing libraries were prepared from RNA extracted from 5 replicates of porcine plasma using the RealSeq®-Biofluids kit or the competitor kit. Sequencing was performed on a NextSeq 550 system sequencer (Illumina). Reads were mapped per 25 million randomly collected reads from each sequencing library and analyzed to determine similarities and differences in output between RealSeq®-Biofluids and a competitor library preparation.
Figure 3| RealSeq®-Biofluids sequencing library preparation kit delivers higher and more consistent reads. Libraries were prepared from 5 replicates of porcine plasma using the RealSeq®-Biofluids kit and a kit from a leading supplier. Subsampling of 25 million randomly collected reads from each library showed that the RealSeq®- Biofluids kit produced 1.5 to 3.5 times more reads than the comparison kit. (left panel). The reads from the RealSeq®-Biofluids kit were also more consistent (two-tailed test p = 0.00122). Much of this can be attributed to increase incorporation of “hard to map” reads (below 10 nts in length) in competitor libraries (right panel).
Figure 4 |RealSeq®-Biofluids sequencing library preparation kit results in increased small RNAs diversity with less inter-library variability.
Sequencing libraries prepared from 5 replicates of porcine plasma using the RealSeq®-Biofluids kit and a kit from a leading supplier and the data were analyzed using an in-house bioinfomatics pipeline. The RealSeq®-Biofluids kit allowed for detection of a more diverse pool of small and non-coding RNAs, particularly rRNA, tsRNA, and piRNA, among others (left panel). Principle component analysis (right panel) between the RealSeq®-Biofluids libraries and competitor libraries showed less variability for the RealSeq® kit when analyzing the total pool of small RNA molecules mapped to the porcine genome.
Summary
Given the increasing clinical significance of small RNAs and RNA fragments in biomarker discovery [2-6], therapeutic development, and drug discovery [7], among others, accurate and reliable measurement of their expression is crucial. The RealSeq®-Biofluids sequencing library preparation kit was evaluated for its ability to enable reliable detection of small RNA molecules from biofluids and low-input samples, using porcine plasma as the sample material. A widely used, commercially available small RNA library preparation kit was used as a reference for performance evaluation. Libraries prepared with the RealSeq kit delivered more sequencing reads and more consistency than the competitor kit. Both kits performed well at generating libraries for reproducible sequencing of miRNAs. The RealSeq® kit outperformed the competitor kit for analyzing other small RNA molecules such as tsRNAs and piRNAs, detecting more of these molecules with less variability and expanding the types of small RNAs that can be explored for biomarker, diagnostic, and therapeutic use. These data support the use of the RealSeq®-Biofluids sequencing library preparation kit for small RNA analysis and pave the way for the use of RealSeq® technology in RNA fragmentomics–based biomarker discovery and diagnostics.
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