Low-bias ncRNA libraries using ordered two-template relay: Serial template jumping by a modified retroelement reverse transcriptase

Upton, Heather; Ferguson, Lucas; Temoche-Diaz, Morayma M.; Liu, Xiao Man; Pimentel, Sydney C.; Ingolia, Nicholas T.; Schekman, Randy; Collins, Kathleen

doi:10.1073/pnas.2107900118

Cited by 39 publications

(52 citation statements)

References 58 publications

(77 reference statements)

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“…Not only does OTTR accurately capture tRNAs with fidelity similar to that of protocols specifically optimized for tRNA sequencing, OTTR was previously shown to exhibit the best performance in small RNA sequencing when benchmarked using miRXplore -a synthetic miRNA reference standard of 962 small RNA oligos [28]. Here, we additionally show that OTTR more accurately captures tRNA fragments than three typical small RNA-Seq protocols, again highlighting the range of applications for this cloning protocol.…”

Section: Discussionsupporting

confidence: 55%

“…Small RNA sequencing. Small RNA sequencing was performed using one of four protocols: TruSeq Small RNA Library Preparation Kit (Illumina), NEBNext® Small RNA Library Prep Set for Illumina® (NEB), a standard in-house ligation-based cloning protocol [36], and Collins Lab OTTR Library Preparation Kit [28]. TruSeq and NEBNext library preparation was performed according to manufacturer instructions.…”

Section: Yeast Strains Parentmentioning

confidence: 99%

“…Ordered two-template relay OTTR was performed as described in Upton et al 2021 [28]. Briefly, total RNA was size selected either by mirVana (<200nt) or gel purification (60-100nt).…”

Section: Fu Et Al 2018 [36] Ligation-based Library Preparationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Most recently, Collins and colleagues developed a novel protocol – Ordered Two-Template Relay, or OTTR [28] – based on an engineered version of the B. Mori R2 retroelement reverse transcriptase [29]. Benchmarking a large number of protocols against a defined mixture of small RNAs revealed significantly lower cloning bias for OTTR than for any commercial protocol [28]. Moreover, characterization of RNA in tissue culture cell lines by OTTR revealed substantial levels of intact tRNAs, suggesting that OTTR could be a promising protocol for tRNA sequencing applications.…”

Section: Introductionmentioning

confidence: 99%

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Deep sequencing of yeast and mouse tRNAs and tRNA fragments using OTTR

Gustafsson

Galan

et al. 2022

Preprint

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Among the major classes of RNAs in the cell, tRNAs remain the most difficult to characterize via deep sequencing approaches, as tRNA secondary structure and nucleotide modifications can both interfere with cDNA synthesis by commonly-used reverse transcriptases (RTs). Here, we benchmark a recently-developed RNA cloning protocol, termed Ordered Two-Template Relay (OTTR), to characterize intact tRNAs and tRNA fragments in budding yeast and in mouse tissues. We find that OTTR robustly captures full-length tRNAs in budding yeast and in mouse testis, with relatively low levels of premature termination at known barriers - 1-methylguanine, N2,N2-dimethylguanine, and 1-methyladenine - to typical reverse transcriptases. Moreover, these and several other nucleotide modifications leave misincorporation signatures in OTTR datasets which enables their detection without any additional protocol steps. Turning to analysis of small RNAs such as tRNA cleavage products, we compare OTTR with several standard small RNA-Seq protocols, finding that OTTR provides the most accurate picture of tRNA fragment levels by comparison to "ground truth" Northern blots. Applying this protocol to mature mouse spermatozoa, our data dramatically alter our understanding of the small RNA cargo of mature mammalian sperm, revealing a far more complex population of tRFs - including both 5' and 3' tRNA halves derived from the majority of tRNAs - than previously appreciated. Taken together, our data confirm the superior performance of OTTR to commercial protocols in analysis of tRNA fragments, and force a reappraisal of potential epigenetic functions of the sperm small RNA payload.

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Section: Discussionsupporting

confidence: 55%

Section: Yeast Strains Parentmentioning

confidence: 99%

“…Ordered two-template relay OTTR was performed as described in Upton et al 2021 [28]. Briefly, total RNA was size selected either by mirVana (<200nt) or gel purification (60-100nt).…”

Section: Fu Et Al 2018 [36] Ligation-based Library Preparationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Deep sequencing of yeast and mouse tRNAs and tRNA fragments using OTTR

Gustafsson

Galan

et al. 2022

Preprint

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Cloning and Sequencing Eukaryotic Small RNAs

2022

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Small RNAs are ubiquitous regulators of gene expression that participate in nearly all aspects of physiology in a wide range of organisms. There are many different classes of eukaryotic small RNAs that play regulatory roles at every level of gene expression, including transcription, RNA stability, and translation. While eukaryotic small RNAs display diverse functions across and within classes, they are generally grouped functionally based on the machinery required for their biogenesis, the effector proteins they associate with, and their molecular characteristics. The development of techniques to clone and sequence small RNAs has been critical for their identification, yet the ligation-dependent addition of RNA adapters and the use of reverse transcriptase to generate cDNA in traditional library preparation protocols can be unsuitable to detect certain small RNA subtypes. In particular, 3 or 5 chemical modifications that are characteristic of specific types of small RNAs can impede the ligation-dependent addition of RNA adapters, while internal RNA modifications can interfere with accurate reverse transcription. The inability to clone certain small RNA subtypes with traditional protocols results in an inaccurate assessment of small RNA abundance and diversity, where some RNAs appear over-represented and others are not detected. This overview aims to guide users on how to design small RNA cloning workflows in eukaryotes to more accurately capture specific small RNAs of interest. Hence, we discuss the molecular biology underlying the identification and quantitation of small RNAs, explore the limitations of commonly used protocols, and detail the alternative approaches that can be used to enrich specific small RNA classes.

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Untacking small RNA profiling and RNA fragment footprinting: Approaches and challenges in library construction

Shen,

Naveed,

Bao

2024

WIREs RNA

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Small RNAs (sRNAs) with sizes ranging from 15 to 50 nucleotides (nt) are critical regulators of gene expression control. Prior studies have shown that sRNAs are involved in a broad range of biological processes, such as organ development, tumorigenesis, and epigenomic regulation; however, emerging evidence unveils a hidden layer of diversity and complexity of endogenously encoded sRNAs profile in eukaryotic organisms, including novel types of sRNAs and the previously unknown post‐transcriptional RNA modifications. This underscores the importance for accurate, unbiased detection of sRNAs in various cellular contexts. A multitude of high‐throughput methods based on next‐generation sequencing (NGS) are developed to decipher the sRNA expression and their modifications. Nonetheless, distinct from mRNA sequencing, the data from sRNA sequencing suffer frequent inconsistencies and high variations emanating from the adapter contaminations and RNA modifications, which overall skew the sRNA libraries. Here, we summarize the sRNA‐sequencing approaches, and discuss the considerations and challenges for the strategies and methods of sRNA library construction. The pros and cons of sRNA sequencing have significant implications for implementing RNA fragment footprinting approaches, including CLIP‐seq and Ribo‐seq. We envision that this review can inspire novel improvements in small RNA sequencing and RNA fragment footprinting in future.This article is categorized under: RNA Evolution and Genomics > Computational Analyses of RNA RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs

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Low-bias ncRNA libraries using ordered two-template relay: Serial template jumping by a modified retroelement reverse transcriptase

Cited by 39 publications

References 58 publications

Deep sequencing of yeast and mouse tRNAs and tRNA fragments using OTTR

Deep sequencing of yeast and mouse tRNAs and tRNA fragments using OTTR

Cloning and Sequencing Eukaryotic Small RNAs

Untacking small RNA profiling and RNA fragment footprinting: Approaches and challenges in library construction

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