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

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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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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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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Small RNA Library Preparation and Illumina Sequencing in Plants

Plant Epigenetics - Methods in Molecular Biology ◽

10.1007/978-1-4899-7708-3_15 ◽

2016 ◽

pp. 189-196 ◽

Cited By ~ 1

Author(s):

Andriy Bilichak ◽

Andrey Golubov ◽

Igor Kovalchuk

Keyword(s):

Illumina Sequencing ◽

Small Rna ◽

Library Preparation ◽

Small Rna Library

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Comparative profiling of small RNAs of pig seminal plasma and ejaculated and epididymal sperm

Reproduction ◽

10.1530/rep-17-0014 ◽

2017 ◽

Vol 153 (6) ◽

pp. 785-796 ◽

Cited By ~ 13

Author(s):

Cai Chen ◽

Han Wu ◽

Dan Shen ◽

Saisai Wang ◽

Li Zhang ◽

...

Keyword(s):

Seminal Plasma ◽

Small Rna ◽

Small Rnas ◽

Target Genes ◽

Large Population ◽

Small Nuclear Rna ◽

Epididymal Sperm ◽

Zygote Development ◽

Similarities And Differences ◽

Small Rna Species

The similarities and differences of small RNAs in seminal plasma, epididymal sperm and ejaculated sperm remain largely undefined. We conducted a systematic comparative analysis of small RNA profiles in pig ejaculated sperm, epididymal sperm and seminal plasma and found that the diversity distribution of small RNA species was generally similar, whereas the abundance of small RNAs is dramatically different across the three libraries; miRNAs and small RNAs derived from rRNA, tRNA, small nuclear RNA, 7SK RNA, NRON RNA and cis-regulatory RNA were enriched in the three libraries, but piRNA was absent. A large population of small RNAs from ejaculated sperm are ejaculated sperm specific, and only 8–30% of small RNAs overlapped with those of epididymal sperm or seminal plasma and a small proportion (5–18%) of small RNAs were shared in the three libraries, suggesting that, in addition to the testes, sperm RNAs may also originate from seminal plasma, epididymis as well as other resources. Most miRNAs were co-distributed but differentially expressed across the three libraries, with epididymal sperm exhibiting the highest abundance, followed by ejaculated sperm and seminal plasma. The prediction of target genes of the top 10 highly expressed miRNAs across the three libraries revealed that these miRNAs may be involved in spermatogenesis, zygote development and the interaction between the environment and animals. Our study provides the first description of the similarities and differences of small RNA profiles in ejaculated sperm, epididymal sperm and seminal plasma and indicates that sperm RNA may have origins other than the testes.

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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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A stress-induced Tyrosine tRNA depletion response mediates codon-based translational repression and growth suppression

10.1101/416727 ◽

2018 ◽

Cited By ~ 3

Author(s):

Doowon Huh ◽

Maria C. Passarelli ◽

Jenny Gao ◽

Shahnoza N Dusmatova ◽

Clara Goin ◽

...

Keyword(s):

Oxidative Stress ◽

Small Rna ◽

Small Rnas ◽

Oxidative Stress Response ◽

Translational Repression ◽

Dependent Manner ◽

Transfer Rnas ◽

C Elegans ◽

Metabolic Genes ◽

Rna Fragments

SUMMARYEukaryotic transfer RNAs can become selectively fragmented upon various stresses, generating tRNA-derived small RNA fragments. Such fragmentation has been reported to impact a small fraction of the tRNA pool and thus presumed to not directly impact translation. We report that oxidative stress can rapidly generate tyrosine tRNAGUA fragments in human cells—causing significant depletion of the precursor tRNA. Tyrosine tRNAGUA depletion impaired translation of growth and metabolic genes enriched in cognate tyrosine codons. Depletion of tyrosine tRNAGUA or its translationally regulated targets USP3 and SCD repressed proliferation—revealing a dedicated tRNA-regulated growth suppressive pathway for oxidative stress response. Tyrosine fragments are generated in a DIS3L2 exoribonuclease-dependent manner and inhibit hnRNPA1-mediated transcript destabilization. Moreover, tyrosine fragmentation is conserved in C. elegans. Thus, tRNA fragmentation can coordinately generate trans-acting small-RNAs and functionally deplete a tRNA. Our findings reveal the existence of an underlying adaptive codon-based regulatory response inherent to the genetic code.

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Functional analysis of virus and host components that mediate potyvirus-induced diseases

10.32747/2008.7591732.bard ◽

2008 ◽

Author(s):

Steven A. Whitham ◽

Amit Gal-On ◽

Tzahi Arazi

Keyword(s):

Small Rna ◽

Small Rnas ◽

Binding Activity ◽

Symptom Expression ◽

Wild Type ◽

Disease Symptoms ◽

Virus Accumulation ◽

Plant Genes ◽

Small Rna Species ◽

Host Genes

The mechanisms underlying the development of symptoms in response to virus infection remain to be discovered in plants. Insight into symptoms induced by potyviruses comes from evidence implicating the potyviral HC-Pro protein in symptom development. In particular, recent studies link the development of symptoms in infected plants to HC-Pro's ability to interfere with small RNA metabolism and function in plant hosts. Moreover, mutation of the highly conserved FRNK amino acid motif to FINK in the HC-Pro of Zucchini yellow mosaic virus (ZYMV) converts a severe strain into an asymptomatic strain, but does not affect virus accumulation in cucurbit hosts. The ability of this FINK mutation to uncouple symptoms from virus accumulation creates a unique opportunity to study symptom etiology, which is usually confounded by simultaneous attenuation of both symptoms and virus accumulation. Our goal was to determine how mutations in the conserved FRNK motif affect host responses to potyvirus infection in cucurbits and Arabidopsis thaliana. Our first objective was to define those amino acids in the FRNK motif that are required for symptoms by mutating the FRNK motif in ZYMV and Turnip mosaic virus (TuMV). Symptom expression and accumulation of resulting mutant viruses in cucurbits and Arabidopsis was determined. Our second objective was to identify plant genes associated with virus disease symptoms by profiling gene expression in cucurbits and Arabidopsis in response to mutant and wild type ZYMV and TuMV, respectively. Genes from the two host species that are differentially expressed led us to focus on a subset of genes that are expected to be involved in symptom expression. Our third objective was to determine the functions of small RNA species in response to mutant and wild type HC-Pro protein expression by monitoring the accumulation of small RNAs and their targets in Arabidopsis and cucurbit plants infected with wild type and mutant TuMV and ZYMV, respectively. We have found that the maintenance of the charge of the amino acids in the FRNK motif of HC-Pro is required for symptom expression. Reduced charge (FRNA, FRNL) lessen virus symptoms, and maintain the suppression of RNA silencing. The FRNK motif is involved in binding of small RNA species including microRNAs (miRNA) and short interfering RNAs (siRNA). This binding activity mediated by the FRNK motif has a role in protecting the viral genome from degradation by the host RNA silencing system. However, it also provides a mechanism by which the FRNK motif participates in inducing the symptoms of viral infection. Small RNA species, such as miRNA and siRNA, can regulate the functions of plant genes that affect plant growth and development. Thus, this binding activity suggests a mechanism by which ZYMVHC-Pro can interfere with plant development resulting in disease symptoms. Because the host genes regulated by small RNAs are known, we have identified candidate host genes that are expected to play a role in symptoms when their regulation is disrupted during viral infections. As a result of this work, we have a better understanding of the FRNK amino acid motif of HC-Pro and its contribution to the functions of HC-Pro, and we have identified plant genes that potentially contribute to symptoms of virus infected plants when their expression becomes misregulated during potyviral infections. The results set the stage to establish the roles of specific host genes in viral pathogenicity. The potential benefits include the development of novel strategies for controlling diseases caused by viruses, methods to ensure stable expression of transgenes in genetically improved crops, and improved potyvirus vectors for expression of proteins or peptides in plants.

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Salient Genetic Notes on Small-RNAs and their Applications in Agricultural Biotechnology

Journal of Advances in Biology & Biotechnology ◽

10.9734/jabb/2019/v22i430131 ◽

2020 ◽

pp. 1-17

Author(s):

Samuel Amiteye

Keyword(s):

Gene Regulation ◽

Small Rna ◽

Small Rnas ◽

Small Interfering Rna ◽

Crop Improvement ◽

Regulation Of Gene Expression ◽

Healthy Plant ◽

Biological Processes ◽

Protein Coding ◽

Interfering Rna

Small-RNAs are 20 to 27 nucleotides long non-protein-coding RNAs that act on either DNA or RNA to effect the regulation of gene expression. Small-RNAs are key in genetic and epigenetic regulation of diverse biological processes and pathways in response to biotic and abiotic environmental stresses. The gene regulatory functions of small-RNA molecules enhance healthy plant growth and normal development by controlling biological processes such as flowering programming, fruit development, disease and pests resistance. Small-RNAs comprise mainly microRNA and small interfering RNA species. MicroRNAs have been proven to primarily engage in posttranscriptional gene regulation while small interfering RNA have been implicated mainly in transcriptional gene regulation. This review covers the recent advancements in small-RNA research in plants, with emphasis on particularly microRNAs and small interfering RNA biogenesis, biological functions and their relevance in the regulation of traits of agronomic importance in plants. Also discussed extensively is the potential biotechnological applications of these small-RNAs for crop improvement.

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ILLUMINA®TRUSEQ® VS NEBNEXT® SMALL RNA LIBRARY PREPARATION KIT FOR MIRNA PROFILING IN PERSICARIA MINOR: WHICH BETTER?

Jurnal Teknologi ◽

10.11113/jt.v77.6720 ◽

2015 ◽

Vol 77 (24) ◽

Cited By ~ 1

Author(s):

Abdul Fatah A. Samad ◽

Nazaruddin Muhammad Ali ◽

Ismanizan Ismail

Keyword(s):

Small Rna ◽

Library Preparation ◽

Isolation Method ◽

Result Show ◽

Important Player ◽

Interfering Rna ◽

Persicaria Minor ◽

Mirna Library ◽

Small Rna Library ◽

Better Than

In plants, a group of non-coding small RNA (sRNA) has been provento be an important player in regulating gene expression that can govern network of genetic systems. The two major classes of sRNA which are very extensively studied through deep sequencing, microRNA (miRNA) and small-interfering RNA (siRNA) classes, are well documented. However, the isolation method of sRNA differs depending on the type of sample. Here, we demonstrate the miRNA library preparation using two different Small RNA Library preparation kit, Illumina®TruSeq® Small RNA Preparation and NEBNext® Multiplex Small RNA Library Preparation kit on a plant rich in secondary metabolite Persicaria minor using recommended protocol. The result show NEBNext® Multiplex Small RNA Library Preparation kit can recover small RNA better than Illumina®TruSeq® Small RNA Preparation kit. Thus, this study recommended NEBNext® Multiplex Small RNA Library Preparation kit for miRNA library preparation on Persicaria minor.

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