Systematic assessment of next generation sequencing for quantitative small RNA profiling: a multiple protocol study across multiple laboratories

AbstractSmall RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. We report here the results of a study using common references (synthetic RNA pools of defined composition, as well as plasma-derived RNA) to evaluate the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. We observed protocol-specific and sequence-specific bias, which was ameliorated using adapters for ligation with randomized end-nucleotides, and computational correction factors. Despite this technical bias, relative quantification using small RNA-seq was remarkably accurate and reproducible, even across multiple laboratories using different methods. These results provide strong evidence for the feasibility of reproducible cross-laboratory small RNA-seq studies, even those involving analysis of data generated using different protocols.

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Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing

Methods ◽

10.1016/j.ymeth.2012.07.030 ◽

2012 ◽

Vol 58 (2) ◽

pp. 164-170 ◽

Cited By ~ 86

Author(s):

Markus Hafner ◽

Neil Renwick ◽

Thalia A. Farazi ◽

Aleksandra Mihailović ◽

John T.G. Pena ◽

...

Keyword(s):

Next Generation Sequencing ◽

Cdna Library ◽

Small Rna ◽

Library Preparation ◽

Next Generation ◽

Rna Profiling ◽

Generation Sequencing ◽

Cdna Library Preparation

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Small RNA Profiling by Next-Generation Sequencing Using High-Definition Adapters

Methods in Molecular Biology - MicroRNA Detection and Target Identification ◽

10.1007/978-1-4939-6866-4_4 ◽

2017 ◽

pp. 45-57 ◽

Cited By ~ 2

Author(s):

Martina Billmeier ◽

Ping Xu

Keyword(s):

Next Generation Sequencing ◽

Small Rna ◽

Next Generation ◽

High Definition ◽

Rna Profiling ◽

Generation Sequencing

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Transcriptome-wide m6A detection with DART-Seq

10.21203/rs.2.12189/v1 ◽

2019 ◽

Author(s):

Kate D. Meyer

Keyword(s):

Gene Expression Regulation ◽

Preparation Method ◽

Current Knowledge ◽

Cross Reactivity ◽

High Variability ◽

Library Preparation ◽

Rna Seq ◽

Generation Sequencing ◽

Library Preparation Method

Abstract m6A is the most abundant internal mRNA modification and plays diverse roles in gene expression regulation. Much of our current knowledge about m6A has been driven by recent advances in the ability to detect this mark transcriptome-wide. Antibody-based approaches have been the method of choice for global m6A mapping studies. These methods rely on m6A antibodies to immunoprecipitate methylated RNAs, followed by next-generation sequencing to identify m6A-containing transcripts1,2. While these methods enabled the first identification of m6A sites transcriptome-wide and have dramatically improved our ability to study m6A, they suffer from several limitations. These include requirements for high amounts of input RNA, costly and time-consuming library preparation, high variability across studies, and m6A antibody cross-reactivity with other modifications. Here, we describe DART-Seq (deamination adjacent to RNA modification targets), an antibody-free method for global m6A detection. In DART-Seq, the C to U deaminating enzyme, APOBEC1, is fused to the m6A-binding YTH domain. This fusion protein is then introduced to cellular RNA either through overexpression in cells or with in vitro assays, and subsequent deamination of m6A-adjacent cytidines is then detected by RNA sequencing to identify m6A sites. DART-Seq can successfully map m6A sites throughout the transcriptome using as little as 10 nanograms of total cellular RNA, and it is compatible with any standard RNA-seq library preparation method.

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CleanTag Adapters Improve Small RNA Next-Generation Sequencing Library Preparation by Reducing Adapter Dimers

Methods in Molecular Biology - Next Generation Sequencing ◽

10.1007/978-1-4939-7514-3_10 ◽

2017 ◽

pp. 145-161

Author(s):

Sabrina Shore ◽

Jordana M. Henderson ◽

Anton P. McCaffrey

Keyword(s):

Next Generation Sequencing ◽

Small Rna ◽

Library Preparation ◽

Next Generation ◽

Sequencing Library ◽

Sequencing Library Preparation ◽

Generation Sequencing

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Systematic comparison of small RNA library preparation protocols for next-generation sequencing

BMC Genomics ◽

10.1186/s12864-018-4491-6 ◽

2018 ◽

Vol 19 (1) ◽

Cited By ~ 40

Author(s):

Cloelia Dard-Dascot ◽

Delphine Naquin ◽

Yves d’Aubenton-Carafa ◽

Karine Alix ◽

Claude Thermes ◽

...

Keyword(s):

Next Generation Sequencing ◽

Small Rna ◽

Library Preparation ◽

Next Generation ◽

Systematic Comparison ◽

Generation Sequencing ◽

Small Rna Library

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Proximity‐CLIP and Expedited Non‐Radioactive Library Preparation of Small RNA Footprints for Next‐Generation Sequencing

Current Protocols in Molecular Biology ◽

10.1002/cpmb.120 ◽

2020 ◽

Vol 131 (1) ◽

Author(s):

Dimitrios Anastasakis ◽

Daniel Benhalevy ◽

Markus Hafner

Keyword(s):

Next Generation Sequencing ◽

Small Rna ◽

Library Preparation ◽

Next Generation ◽

Generation Sequencing

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Next-Generation Sequencing of Small RNAs from HIV-Infected Cells Identifies Phased microRNA Expression Patterns and Candidate Novel microRNAs Differentially Expressed upon Infection

mBio ◽

10.1128/mbio.00549-12 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 37

Author(s):

Stewart T. Chang ◽

Matthew J. Thomas ◽

Pavel Sova ◽

Richard R. Green ◽

Robert E. Palermo ◽

...

Keyword(s):

Transcriptional Regulation ◽

Next Generation Sequencing ◽

Hiv Infection ◽

Small Rna ◽

Microrna Expression ◽

Rna Seq ◽

Next Generation ◽

Dynamic Changes ◽

Infected Cells ◽

Generation Sequencing

ABSTRACT HIV infection of CD4+ T cells induces a range of host transcriptional changes in mRNAs as well as microRNAs that may coordinate changes in mRNAs. To survey these dynamic changes, we applied next-generation sequencing, analyzing the small RNA fraction of HIV-infected cells at 5, 12, and 24 h postinfection (RNA-Seq). These time points afforded a view of the transcriptomic changes occurring both before and during viral replication. In the resulting small RNA-Seq data set, we detected a phased pattern of microRNA expression. Largely distinct sets of microRNAs were found to be suppressed at 5 and 12 h postinfection, and both sets of changes rebounded later in infection. A larger set of microRNA changes was observed at 24 h postinfection. When integrated with mRNA expression data, the small RNA-Seq data indicated a role for microRNAs in transcriptional regulation, T cell activation, and cell cycle during HIV infection. As a unique benefit of next-generation sequencing, we also detected candidate novel host microRNAs differentially expressed during infection, including one whose downregulation at 24 h postinfection may allow full replication of HIV to proceed. Collectively, our data provide a uniquely comprehensive view of the changes in host microRNAs induced by HIV during cellular infection. IMPORTANCE New sequencing technologies allow unprecedented views into changes occurring in virus-infected cells, including comprehensive and largely unbiased measurements of different types of RNA. In this study, we used next-generation sequencing to profile dynamic changes in cellular microRNAs occurring in HIV-infected cells. The sensitivity afforded by sequencing allowed us to detect changes in microRNA expression early in infection, before the onset of viral replication. A phased pattern of expression was evident among these microRNAs, and many that were initially suppressed were later overexpressed at the height of infection, providing unique signatures of infection. By integrating additional mRNA data with the microRNA data, we identified a role for microRNAs in transcriptional regulation during infection and specifically a network of microRNAs involved in the expression of a known HIV cofactor. Finally, as a distinct benefit of sequencing, we identified candidate nonannotated microRNAs, including one whose downregulation may allow HIV-1 replication to proceed fully.

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