Improved precision, sensitivity, and adaptability of Ordered Two-Template Relay cDNA library preparation for RNA sequencing

Sequencing of RNAs that are biologically processed and/or degraded to less than ~100 nucleotides typically involves the use of multi-step, low-yield protocols with bias or information loss inherent to ligation and/or polynucleotide tailing. We recently introduced Ordered Two-Template Relay (OTTR), an RNA sequencing method that not only captures the end-to-end sequences of input molecules but also appends 5' and 3' sequencing adapters by reverse transcription. OTTR has been benchmarked for microRNA, tRNA and tRNA fragment, and ribosome-protected mRNA footprint libraries. Here we sought to characterize, quantify, and ameliorate remaining sources of bias and imprecision of OTTR in the end-to-end capture of RNA sequences. We introduce new metrics for the evaluation of sequence capture and use them to optimize reaction buffers, reverse transcriptase sequence, adapter oligonucleotides, and overall workflow. Modifications of the reverse transcriptase and adapter oligonucleotides increased the 3' and 5' end-precision of sequence capture and minimized overall library bias. Improvements in recombinant expression and purification of the truncated Bombyx mori R2 reverse transcriptase used in OTTR reduced non-productive sequencing reads by minimizing bacterial nucleic acids that compete with low-input RNA molecules for cDNA synthesis, such that with miRNA input of 3 picograms (less than 1 fmol), fewer than 10% of sequencing reads are bacterial nucleic acid contaminants. We also introduce a rapid, automation-compatible OTTR protocol that enables gel-free, length-agnostic enrichment of cDNA duplexes from adaptor-only nucleic acids. Overall, this work informs considerations for unbiased end-to-end capture and annotation of RNAs independent of their sequence, structure, or post-transcriptional modifications.