Improved precision, sensitivity, and adaptability of ordered two-template relay cDNA library preparation for RNA...

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Standard RNA sequencing methods for small RNAs (<100 nt) suffer from low yields, bias, and information loss due to multistep protocols involving ligation or polynucleotide tailing.
• Ordered Two-Template Relay (OTTR) was previously developed to capture end-to-end RNA sequences and append sequencing adapters in a single step, showing promise for microRNA, tRNA, and ribosome footprint libraries.

Purpose of the Study:

• To characterize, quantify, and reduce any remaining bias or imprecision in the end-to-end capture of RNA sequences using the OTTR method.
• To optimize reaction conditions, enzyme variants, and adapter oligonucleotides for improved RNA sequence capture fidelity.

Main Methods:

• Development of new metrics for evaluating RNA sequence capture efficiency and precision.
• Optimization of OTTR reaction buffers, reverse transcriptase sequence, and adapter oligonucleotides.
• Improvement of recombinant expression and purification of the truncated Bombyx mori R2 reverse transcriptase.
• Introduction of a rapid, automation-compatible, gel-free OTTR protocol for cDNA enrichment.

Main Results:

• Modified reverse transcriptase and adapter oligonucleotides significantly increased 3' and 5' end-precision and minimized library bias.
• Enhanced purification of R2 reverse transcriptase reduced bacterial nucleic acid contamination in low-input RNA samples to <10%.
• The new protocol enables gel-free, length-agnostic enrichment of cDNA duplexes, efficiently separating them from adapter-only products.

Conclusions:

• The optimized OTTR method provides highly precise, unbiased, end-to-end RNA sequence capture, suitable for diverse RNA types.
• Improvements in enzyme purification and workflow reduce technical artifacts and increase the reliability of small RNA sequencing.
• This refined OTTR protocol facilitates accurate RNA annotation regardless of sequence, structure, or modifications, advancing transcriptomic studies.