Optimizing Small RNA Sequencing for Salivary Biomarker Identification: A Comparative Study of Library Preparation Protocols

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and hold significant potential as biomarkers. Saliva, a non-invasive and easily accessible biofluid, offers a promising alternative to blood for miRNA-based diagnostics. However, miRNA profiling by next-generation sequencing (NGS) is highly influenced by library preparation protocol, which can introduce detection and quantification biases. This study compared four commercial small RNA library preparation kits-QIASeq miRNA library kit (Qiagen), RealSeq-Biofluids Plasma/Serum miRNA library kit (Somagenics), Small RNA-seq library prep kit (Lexogen) and NEBNext multiplex small RNA library prep set for illumina (set 1) (New England BioLabs)-to evaluate their performance in profiling miRNAs from cell-free saliva, plasma and their extracellular vesicles (EVs). Using both synthetic reference and biological samples, we assessed the kits' efficiency in handling low RNA input, minimizing bias and detecting diverse miRNAs. QIAseq outperformed the others, showing the highest miRNA mapping rates, minimal adapter dimers and the broadest miRNA detection, particularly in saliva. Moreover, substantial overlap between saliva- and plasma-derived miRNAs supports saliva's diagnostic potential. Overall, this study underscores the critical impact of library preparation on miRNA sequencing outcomes and offers guidance for selecting optimal protocols for biomarker discovery from non-invasive sample matrices.