Sequencing-based quantitative mapping of the cellular small RNA landscape

AbstractCurrent next-generation RNA sequencing methods cannot provide accurate quantification of the population of small RNAs within a sample due to strong sequence-dependent biases in capture, ligation, and amplification during library preparation. We report the development of an RNA sequencing method – AQRNA-seq – that minimizes biases and enables absolute quantification of all small RNA species in a sample mixture. Validation of AQRNA-seq library preparation and data mining algorithms using a 963-member microRNA reference library, RNA oligonucleotide standards of varying lengths, and northern blots demonstrated a direct, linear correlation between sequencing read count and RNA abundance. Application of AQRNA-seq to bacterial tRNA pools, a traditionally hard-to-sequence class of RNAs, revealed 80-fold variation in tRNA isoacceptor copy numbers, patterns of site-specific tRNA fragmentation caused by stress, and quantitative maps of ribonucleoside modifications, all in a single AQRNA-seq experiment. AQRNA-seq thus provides a means to quantitatively map the small RNA landscape in cells and tissues.

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Massively differential bias between two widely used Illumina library preparation methods for small RNA sequencing

10.1101/001479 ◽

2013 ◽

Cited By ~ 3

Author(s):

Jeanette Baran-Gale ◽

Michael R Erdos ◽

Christina Sison ◽

Alice Young ◽

Emily E Fannin ◽

...

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

Small Rnas ◽

Small Rna Sequencing ◽

Library Preparation ◽

Sequencing Technology ◽

Preparation Methods ◽

Sequencing Platforms ◽

Small Rna Library ◽

Illumina Library

Recent advances in sequencing technology have helped unveil the unexpected complexity and diversity of small RNAs. A critical step in small RNA library preparation for sequencing is the ligation of adapter sequences to both the 5’ and 3’ ends of small RNAs. Two widely used protocols for small RNA library preparation, Illumina v1.5 and Illumina TruSeq, use different pairs of adapter sequences. In this study, we compare the results of small RNA-sequencing between v1.5 and TruSeq and observe a striking differential bias. Nearly 100 highly expressed microRNAs (miRNAs) are >5-fold differentially detected and 48 miRNAs are >10-fold differentially detected between the two methods of library preparation. In fact, some miRNAs, such as miR-24-3p, are over 30-fold differentially detected. The results are reproducible across different sequencing centers (NIH and UNC) and both major Illumina sequencing platforms, GAIIx and HiSeq. While some level of bias in library preparation is not surprising, the apparent massive differential bias between these two widely used adapter sets is not well appreciated. As increasingly more laboratories transition to the newer TruSeq-based library preparation for small RNAs, researchers should be aware of the extent to which the results may differ from previously published results using v1.5.

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Comparative Analysis of Biochemical Biases by Ligation- and Template-Switch-Based Small RNA Library Preparation Protocols

Clinical Chemistry ◽

10.1373/clinchem.2019.305045 ◽

2019 ◽

Vol 65 (12) ◽

pp. 1581-1591 ◽

Cited By ~ 2

Author(s):

Morgane Meistertzheim ◽

Tobias Fehlmann ◽

Franziska Drews ◽

Marcello Pirritano ◽

Gilles Gasparoni ◽

...

Keyword(s):

Small Rna ◽

Small Rnas ◽

Regulation Of Gene Expression ◽

Biochemical Properties ◽

Library Preparation ◽

Key Players ◽

Rna Fragments ◽

Template Switch ◽

Small Rna Species ◽

Small Rna Library

Abstract BACKGROUND Small RNAs are key players in the regulation of gene expression and differentiation. However, many different classes of small RNAs (sRNAs) have been described with distinct biogenesis pathways and, as a result, with different biochemical properties. To analyze sRNAs by deep sequencing, complementary DNA synthesis requires manipulation of the RNA molecule itself. Thus, enzymatic activities during library preparation bias the library content owing to biochemical criteria. METHODS We compared 4 different manipulations of RNA for library preparation: (a) a ligation-based procedure allowing only 5′-mono-phosphorylated RNA to enter the library, (b) a ligation-based procedure allowing additional 5′-triphosphates and Cap structures, (c) a ligation-independent, template-switch-based library preparation, and (d) a template-switch-based library preparation allowing 3′-phosphorylated RNAs to enter the library. RESULTS Our data show large differences between ligation-dependent and ligation-independent libraries in terms of their preference for individual sRNA classes such as microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and transfer RNA fragments. Moreover, the miRNA composition is different between both procedures, and more microRNA isoforms (isomiRs) can be identified after pyrophosphatase treatment. piRNAs are enriched in template-switch libraries, and this procedure apparently includes more different RNA species. CONCLUSIONS Our data indicate that miRNAomics from both methods will hardly be comparable. Ligation-based libraries enrich for canonical miRNAs, which thus may be suitable methods for miRNAomics. Template-switch libraries contain increased numbers and different compositions of fragments and long RNAs. Following different interests for other small RNA species, ligation-independent libraries appear to show a more realistic sRNA landscape with lower bias against biochemical modifications.

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Comparison of RNA isolation and library preparation methods for small RNA sequencing of canine biofluids

Veterinary Clinical Pathology ◽

10.1111/vcp.12743 ◽

2019 ◽

Vol 48 (2) ◽

pp. 310-319 ◽

Cited By ~ 1

Author(s):

Candice P. Chu ◽

Mary B. Nabity

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

Rna Isolation ◽

Small Rna Sequencing ◽

Library Preparation ◽

Preparation Methods

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A low-bias and sensitive small RNA library preparation method using randomized splint ligation

Nucleic Acids Research ◽

10.1093/nar/gkaa480 ◽

2020 ◽

Vol 48 (14) ◽

pp. e80-e80 ◽

Cited By ~ 3

Author(s):

Sean Maguire ◽

Gregory J S Lohman ◽

Shengxi Guan

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

Small Rnas ◽

Small Rna Sequencing ◽

Preparation Process ◽

Library Preparation ◽

Normal Tissues ◽

Levels Of Detail ◽

Low Sensitivity ◽

Next Generation Sequencing Ngs

Abstract Small RNAs are important regulators of gene expression and are involved in human development and disease. Next generation sequencing (NGS) allows for scalable, genome-wide studies of small RNA; however, current methods are challenged by low sensitivity and high bias, limiting their ability to capture an accurate representation of the cellular small RNA population. Several studies have shown that this bias primarily arises during the ligation of single-strand adapters during library preparation, and that this ligation bias is magnified by 2′-O-methyl modifications (2′OMe) on the 3′ terminal nucleotide. In this study, we developed a novel library preparation process using randomized splint ligation with a cleavable adapter, a design which resolves previous challenges associated with this ligation strategy. We show that a randomized splint ligation based workflow can reduce bias and increase the sensitivity of small RNA sequencing for a wide variety of small RNAs, including microRNA (miRNA) and tRNA fragments as well as 2′OMe modified RNA, including Piwi-interacting RNA and plant miRNA. Finally, we demonstrate that this workflow detects more differentially expressed miRNA between tumorous and matched normal tissues. Overall, this library preparation process allows for highly accurate small RNA sequencing and will enable studies of 2′OMe modified RNA with new levels of detail.

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RppH can faithfully replace TAP to allow cloning of 5’-triphosphate carrying small RNAs

10.1101/283077 ◽

2018 ◽

Cited By ~ 1

Author(s):

Miguel Vasconcelos Almeida ◽

António Miguel de Jesus Domingues ◽

Hanna Lukas ◽

Maria Mendez-Lago ◽

René F. Ketting

Keyword(s):

Small Rna ◽

Small Rnas ◽

Small Rna Sequencing ◽

Cloning Efficiency ◽

Library Preparation ◽

Small Interfering Rnas ◽

Rdrp Activity ◽

C Elegans ◽

Rna Pathways ◽

Small Rna Species

AbstractRNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. Much like plants, but unlike Drosophila and mammals, worms have RNA-dependent RNA Polymerases (RdRPs) that directly synthesize small RNAs using other transcripts as a template. The very prominent secondary small interfering RNAs, also called 22G-RNAs, produced by the RdRPs RRF-1 and EGO-1 in C. elegans, maintain the 5’ triphosphate group, stemming from RdRP activity, also after loading into an Argonaute protein. This creates a technical issue, since 5’PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5’ pyrophosphohydrolase (RppH), a commercially available pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison.

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Library Preparation for small RNA sequencing using 4N adapters: In house 4N Protocol C

Protocol Exchange ◽

10.1038/protex.2018.081 ◽

2018 ◽

Author(s):

Roger P. Alexander ◽

MD Giraldez ◽

RM Spengler ◽

A Etheridge ◽

PM Godoy ◽

...

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

Small Rna Sequencing ◽

Library Preparation

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Library Preparation for small RNA sequencing using 4N adapters: In house 4N Protocol B

Protocol Exchange ◽

10.1038/protex.2018.050 ◽

2018 ◽

Author(s):

Roger P. Alexander ◽

MD Giraldez ◽

RM Spengler ◽

A Etheridge ◽

PM Godoy ◽

...

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

Small Rna Sequencing ◽

Library Preparation

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Comprehensive assessment of multiple biases in small RNA sequencing reveals significant differences in the performance of widely used methods

10.1101/445437 ◽

2018 ◽

Cited By ~ 2

Author(s):

Carrie Wright ◽

Anandita Rajpurohit ◽

Emily E. Burke ◽

Courtney Williams ◽

Leonardo Collado-Torres ◽

...

Keyword(s):

Rna Sequencing ◽

High Throughput ◽

Small Rna ◽

Reverse Transcription ◽

High Throughput Sequencing ◽

Small Rna Sequencing ◽

Library Preparation ◽

Adapter Ligation ◽

Improve Accuracy ◽

Reliable Quantification

ABSTRACTHigh-throughput sequencing offers advantages over other quantification methods for microRNA (miRNA), yet numerous biases make reliable quantification challenging. Previous evaluations of the biases associated with small RNA sequencing have focused on adapter ligation bias with limited evaluation of reverse transcription or amplification biases. Furthermore, evaluations of the accuracy of quantifications of isomiRs (miRNA isoforms) or the influence of starting amount on performance have been very limited and no study has yet evaluated differences in the quantification of isomiRs of altered length. In addition, no studies have yet compared the consistency of results derived from multiple moderate starting inputs. We therefore evaluated quantifications of miRNA and isomiRs using four library preparation kits, with various starting amounts, as well as quantifications following removal of duplicate reads using unique molecular identifiers (UMIs) to mitigate reverse transcription and amplification biases. All methods resulted in false isomiR detection; however, the adapter-free method tested was especially prone to false isomiR detection. We demonstrate that using UMIs improves accuracy and we provide a guide for input amounts to improve consistency. Our data show differences and limitations of current methods, thus raising concerns about the validity of quantification of miRNA and isomiRs across studies. We advocate for the use of UMIs to improve accuracy and reliability of miRNA quantifications.

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