tinyRNA-seq: An Optimized Approach To Sequencing Tiny RNAs And Primitive RNA Genomes

[biorxiv.org] Very short RNAs play critical roles in modern biology, and are thought to have been crucial for genome replication during the origin of life.

Next-generation sequencing is an essential tool for characterizing pools of small RNAs, but current library preparation methods suffer from strong size and sequence biases.

Here we present tinyRNA-seq, an optimized library preparation method designed to minimize length- and sequence-dependent capture bias enabling the sequencing of RNA fragments as short as 2 nucleotides. We use degenerate adaptor regions to reduce ligation sequence bias and facilitate unique molecular identifier (UMI) installation.

We benchmarked tinyRNA-seq against commercial kits using a model primordial RNA genome consisting of hundreds of defined oligonucleotides ranging from 2 to 12 nucleotides. tinyRNA-seq reproduced the input RNA distribution without the size and sequence bias of the commercial kits.

tinyRNA-seq also enables the detection of de novo oligonucleotide generation, an important process for the origins of life. Applied to biologically derived small RNAs including miRNAs, piRNAs, and cityRNAs, tinyRNA-seq showed significantly lower capture bias and recovered a wider range of sequences than commercial kits.

tinyRNA-seq may thus provide a more complete and quantitatively accurate representation of small RNAs from both biological and chemical sources.

tinyRNA-seq: An optimized approach to sequencing tiny RNAs and primitive RNA genomes, biorxiv.org

Astrobiology