Abstract
BackgroundEvolving interest in comprehensively profiling the full range of small RNAs present in small tissue biopsies and in circulating biofluids, and how the profile differs with disease, has launched small RNA sequencing (RNASeq) into more frequent use. However, known biases associated with small RNASeq, compounded by low RNA inputs, have been both a significant concern and a hurdle to widespread adoption. As RNASeq is becoming a viable choice for the discovery of small RNAs in low input samples and more labs are employing it, there should be benchmark datasets to test and evaluate the performance of new sequencing protocols and operators. In a recent publication from the National Institute of Standards and Technology, Pine et al., 2018, the investigators used a commercially available set of three tissues and tested performance across labs and platforms.ResultsIn this paper, we further tested the performance of low RNA input in three commonly used and commercially available RNASeq library preparation kits; NEB Next, NEXTFlex, and TruSeq small RNA library preparation. We evaluated the performance of the kits at two different sites, using three different tissues (brain, liver, and placenta) with high (1 μg) and low RNA (10 ng) input from tissue samples, or 5.0, 3.0, 2.0, 1.0, 0.5, and 0.2 ml starting volumes of plasma. As there has been a lack of robust validation platforms for differentially expressed miRNAs, we also compared low input RNASeq data with their expression profiles on three different platforms (Abcam Fireplex, HTG EdgeSeq, and Qiagen miRNome).ConclusionsThe concordance of RNASeq results on these three platforms was dependent on the RNA expression level; the higher the expression, the better the reproducibility. The results provide an extensive analysis of small RNASeq kit performance using low RNA input, and replication of these data on three downstream technologies.
Highlights
Evolving interest in comprehensively profiling the full range of small RNAs present in small tissue biopsies and in circulating biofluids, and how the profile differs with disease, has launched small RNA sequencing (RNASeq) into more frequent use
Our results demonstrate that while small RNA expression differences between the three tissues, brain, liver and placenta are captured by all three small RNASeq kits tested (Illumina TruSeq, BiooScientific NEXTFlex, and New England Biolabs NEB ), there are measurable differences between the kits in terms of RNA diversity
The results indicate 4 important findings: 1) We recommend using the BiooScientific NEXTFlex kit, as it detects the largest number of micro RNA (miRNA), owing to its 4 N random adaptor sequence that ameliorates ligation bias
Summary
Evolving interest in comprehensively profiling the full range of small RNAs present in small tissue biopsies and in circulating biofluids, and how the profile differs with disease, has launched small RNA sequencing (RNASeq) into more frequent use. In a recent publication from the National Institute of Standards and Technology, Pine et al, 2018, the investigators used a commercially available set of three tissues and tested performance across labs and platforms. Due to their stability, clinical relevance, and functional role in disease pathogenesis, small RNAs have the potential to be important reporters of dysregulated cellular processes across a range of diseases [1,2,3]. To increase diversity, we used RNA isolated from three different tissues for comparison of low input RNA effects in the sequencing kits: brain, liver, and placenta, and one biofluid sample: plasma. While the inter-site variation was minimal for the 1 μg input samples, as the input amount was decreased to 10 ng, the effect of operator/site on the percentage of input reads mapping to RNA became more pronounced
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