Efficient small fragment sequencing of human, cattle, and bison miRNA, small RNA, or csRNA-seq libraries using AVITI

Authors: MCDONALD, ANNA - Washington State University; BODDICKER, ANDREW - Element Biosciences; SAVENKOVA, MARINA - Washington State University; BRABB, IAN - Washington State University; QI, XIAODONG - Element Biosciences; MORÉ, DANIELA - Washington State University; Cunha, Cristina; ZHAO, JUNHUA - Element Biosciences; DUTTKE, SASCHA - Washington State University

Submitted to: BMC Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/8/2024
Publication Date: 11/29/2024
Citation: Mcdonald, A.L., Boddicker, A.M., Savenkova, M.I., Brabb, I.M., Qi, X., Moré, D.D., Cunha, C.W., Zhao, J., Duttke, S.H. 2024. Efficient small fragment sequencing of human, cattle, and bison miRNA, small RNA, or csRNA-seq libraries using AVITI. BMC Genomics. (25):1157. https://doi.org/10.1186/s12864-024-11013-7.
DOI: https://doi.org/10.1186/s12864-024-11013-7

Interpretive Summary: Small RNA (sRNA) molecules, such as micro RNAs, small interfering RNA, and piwi-interacting RNA, are crucial regulators of gene expression and are involved in various biological processes. Identification and quantification of RNA molecules in humans and animals has revolutionized biology and biomedicine and led to considerable advancements in medical research with human, agricultural, and environmental applications. Next-Generation Sequencing (NGS) is a largely accessible and cost-efficient sequencing method that has long offered researchers and clinicians increased speed, throughput, and precision in obtaining RNA data. The Illumina sequencing method is a cornerstone in NGS, but new technologies, such as the AVITI platform, are now emerging. In this study, we compared the Illumina and the AVITI sequencing methods using human, cattle, and bison RNA. Illumina and AVITI sequencing technologies equivalently captured RNA sequencing libraries in tree species, augmenting the confidence in both approaches. Our study not only provides a novel protocol to sequencing sRNA libraries on the AVITI platform, but also demonstrates its applicability for livestock animals. Broader use of sequencing platforms can potentially lead to the identification of new genetic variants and better understanding of population genetics, breeding, disease development and therapeutic approaches in humans and animals.

Technical Abstract: Background: Next-Generation Sequencing (NGS) catalyzed breakthroughs across various scientific domains. Illumina’s sequencing by synthesis method has long been central to NGS, but new sequencing methods like Element Biosciences’ AVITI technology are emerging. AVITI is reported to offer improved signal-to-noise ratios and cost reductions. However, its reliance on rolling circle amplification, which can be affected by polymer size, raises questions about its effectiveness in sequencing small RNAs (sRNAs) such as microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), and many others. These sRNAs are crucial regulators of gene expression and involved in various biological processes. Additionally, capturing capped small RNAs (csRNA-seq) is a powerful method for mapping active or “nascent” RNA polymerase II transcription initiation in tissues and clinical samples. Results: Here, we report a new protocol for seamlessly sequencing short fragments on the AVITI and demonstrate that AVITI and Illumina sequencing technologies equivalently capture human, cattle (Bos taurus), and bison (Bison bison) sRNA or csRNA sequencing libraries, increasing confidence in both sequencing approaches. Additionally, analysis of generated nascent transcription start site (TSS) data for cattle and bison revealed inaccuracies in their current genome annotations, underscoring the potential and necessity to translate small and nascent RNA sequencing methodologies to livestock. Conclusions: Our accelerated and optimized protocol bridges the advantages of AVITI sequencing with critical methods that rely on sequencing short fragments. This advance bolsters the utility of AVITI technology alongside traditional Illumina platforms, offering new opportunities for NGS applications