Small RNA Library Preparation and Illumina Sequencing in Plants

2016 ◽  
pp. 189-196 ◽  
Author(s):  
Andriy Bilichak ◽  
Andrey Golubov ◽  
Igor Kovalchuk
Keyword(s):  
Illumina Sequencing ◽  
Small Rna ◽  
Library Preparation ◽  
Small Rna Library

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

BMC Genomics ◽  
2018 ◽  
Vol 19 (1) ◽  
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

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

2013 ◽  
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.

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

2015 ◽  
Vol 77 (24) ◽  
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.

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

2019 ◽  
Vol 65 (12) ◽  
pp. 1581-1591 ◽  
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.

scholarly journals Optimization of small RNA library preparation protocol from human urinary exosomes

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Dolores Olivares ◽  
Javier Perez-Hernandez ◽  
Daniel Perez-Gil ◽  
Felipe J. Chaves ◽  
Josep Redon ◽  
...  
Keyword(s):  
Small Rna ◽  
Library Preparation ◽  
Urinary Exosomes ◽  
Small Rna Library ◽  
Library Preparation Protocol

scholarly journals Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation

PLoS ONE ◽  
2016 ◽  
Vol 11 (11) ◽  
pp. e0167009 ◽  
Author(s):  
Sabrina Shore ◽  
Jordana M. Henderson ◽  
Alexandre Lebedev ◽  
Michelle P. Salcedo ◽  
Gerald Zon ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Small Rna ◽  
Preparation Method ◽  
Library Preparation ◽  
Next Generation ◽  
Chemical Modifications ◽  
Dimer Formation ◽  
Generation Sequencing ◽  
Small Rna Library ◽  
Library Preparation Method

scholarly journals Addressing Bias in Small RNA Library Preparation for Sequencing: A New Protocol Recovers MicroRNAs that Evade Capture by Current Methods

2015 ◽  
Vol 6 ◽  
Author(s):  
Jeanette Baran-Gale ◽  
C. Lisa Kurtz ◽  
Michael R. Erdos ◽  
Christina Sison ◽  
Alice Young ◽  
...  
Keyword(s):  
Small Rna ◽  
Library Preparation ◽  
Small Rna Library
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