Journal Article

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

Duttke, Sascha H., Brabb, Ian M., Zhao, Junhua, Boddicker, Andrew M., Qi, Xiaodong, Moré, Daniela D., McDonald, Anna L., Cunha, Cristina W., Savenkova, Marina I.

2024

Overview

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.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Animal Genetics and Genomics / Animals / Annotations / AVITI / Background noise / Biological activity / Biological apparatus and supplies / Biomedical and Life Sciences / Bison / Bison - genetics / Bison bison / Buffalo / Capped small RNA sequencing (csRNA-seq) / catalytic activity / Cattle / Chemical synthesis / DNA-directed RNA polymerase / Fragments / Gene expression / Gene Library / Gene mapping / Gene sequencing / Genetic aspects / Genomes / Genomic analysis / genomics / High-Throughput Nucleotide Sequencing - methods / Human genetics / Humans / Illumina / Life Sciences / Livestock / Methods / Microarrays / Microbial Genetics and Genomics / microRNA / MicroRNAs / MicroRNAs - genetics / miRNA / Molecular biology / Next-generation sequencing / Nucleoli / Plant Genetics and Genomics / Polymers / Proteomics / Ribonucleic acid / RNA / RNA polymerase / RNA polymerase II / RNA sequencing / RNA, Small Nucleolar - genetics / Sequence Analysis, RNA - methods / Small RNA sequencing (sRNA-seq) / Transcription initiation / Transcription Initiation Site