scholarly journals Modified TruSeq Small RNA Library Prep using Randomized 4N Adapters: In house 4N Protocol D

2018 ◽  
Author(s):  
Roger P. Alexander ◽  
MD Giraldez ◽  
RM Spengler ◽  
A Etheridge ◽  
PM Godoy ◽  
...  
Keyword(s):  
Small Rna ◽  
Small Rna Library

scholarly journals Deep sequencing and in silico analysis of small RNA library reveals novel miRNA from leaf Persicaria minor transcriptome

3 Biotech ◽  
2018 ◽  
Vol 8 (3) ◽  
Author(s):  
Abdul Fatah A. Samad ◽  
Nazaruddin Nazaruddin ◽  
Abdul Munir Abdul Murad ◽  
Jaeyres Jani ◽  
Zamri Zainal ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Small Rna ◽  
In Silico ◽  
In Silico Analysis ◽  
Novel Mirna ◽  
Persicaria Minor ◽  
Silico Analysis ◽  
Small Rna Library

scholarly journals An improved method of constructing degradome library suitable for sequencing using Illumina platform

Plant Methods ◽  
2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Yong-Fang Li ◽  
Miao Zhao ◽  
Menglei Wang ◽  
Junqiang Guo ◽  
Li Wang ◽  
...  
Keyword(s):  
Gene Family ◽  
Small Rna ◽  
Messenger Rna ◽  
High Throughput Sequencing ◽  
Mirna Family ◽  
Mirna Biogenesis ◽  
Improved Method ◽  
Illumina Platform ◽  
Transcriptional Gene Regulation ◽  
Small Rna Library

Abstract Background Post-transcriptional gene regulation is one of the critical layers of overall gene expression programs and microRNAs (miRNAs) play an indispensable role in this process by guiding cleavage on the messenger RNA targets. The transcriptome-wide cleavages on the target transcripts can be identified by analyzing the degradome or PARE or GMUCT libraries. However, high-throughput sequencing of PARE or degradome libraries using Illumina platform, a widely used platform, is not so straightforward. Moreover, the currently used degradome or PARE methods utilize MmeI restriction site in the 5′ RNA adapter and the resulting fragments are only 20-nt long, which often poses difficulty in distinguishing between the members of the same target gene family or distinguishing miRNA biogenesis intermediates from the primary miRNA transcripts belonging to the same miRNA family. Consequently, developing a method which can generate longer fragments from the PARE or degradome libraries which can also be sequenced easily using Illumina platform is ideal. Results In this protocol, 3′ end of the 5′RNA adaptor of TruSeq small RNA library is modified by introducing EcoP15I recognition site. Correspondingly, the double-strand DNA (dsDNA) adaptor sequence is also modified to suit with the ends generated by the restriction enzyme EcoP15I. These modifications allow amplification of the degradome library by primer pairs used for small RNA library preparation, thus amenable for sequencing using Illumina platform, like small RNA library. Conclusions Degradome library generated using this improved protocol can be sequenced easily using Illumina platform, and the resulting tag length is ~ 27-nt, which is longer than the MmeI generated fragment (20-nt) that can facilitate better accuracy in validating target transcripts belonging to the same gene family or distinguishing miRNA biogenesis intermediates of the same miRNA family. Furthermore, this improved method allows pooling and sequencing degradome libraries and small RNA libraries simultaneously using Illumina platform.

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.

scholarly journals Depletion of erythropoietic miR-486-5p and miR-451a improves detectability of rare microRNAs in peripheral blood-derived small RNA sequencing libraries

2019 ◽  
Author(s):  
Simonas Juzenas ◽  
Carl Mårten Lindqvist ◽  
Go Ito ◽  
Yewgenia Dolshanskaya ◽  
Jonas Halfvarson ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Research Question ◽  
Cost Effective ◽  
Small Rna Sequencing ◽  
Negative Effects ◽  
Mirna Species ◽  
Small Rna Library

AbstractErythroid-specific miR-451a and miR-486-5p are two of the most dominant microRNAs (miRNAs) in human peripheral blood. In small RNA sequencing libraries, their overabundance reduces diversity as well as complexity and consequently causes negative effects such as missing detectability and inaccurate quantification of low abundant miRNAs. Here we present a simple, cost-effective and easy to implement hybridization-based method to deplete these two erythropoietic miRNAs from blood-derived RNA samples. By utilization of blocking oligonucleotides, this method provides a highly efficient and specific depletion of miR-486-5p and miR-451a, which leads to a considerable increase of measured expression as well as detectability of low abundant miRNA species. The blocking oligos are compatible with common 5’ ligation-dependent small RNA library preparation protocols, including commercially available kits, such as Illumina TruSeq and Perkin Elmer NEXTflex. Furthermore, the here described method and oligo design principle can be easily adapted to target many other miRNA molecules, depending on context and research question.

scholarly journals Placental Exosomes as Early Biomarker of Preeclampsia: Potential Role of Exosomal MicroRNAs Across Gestation

2017 ◽  
Vol 102 (9) ◽  
pp. 3182-3194 ◽  
Author(s):  
Carlos Salomon ◽  
Dominic Guanzon ◽  
Katherin Scholz-Romero ◽  
Sherri Longo ◽  
Paula Correa ◽  
...  
Keyword(s):  
Small Rna ◽  
Operating Characteristic ◽  
Maternal Plasma ◽  
Maternal Circulation ◽  
Receiver Operating Characteristic Curves ◽  
Early Biomarker ◽  
Severity Of The Disease ◽  
Small Rna Library ◽  
Time Point

AbstractContextThere is a need to develop strategies for early prediction of patients who will develop preeclampsia (PE) to establish preventive strategies to reduce the prevalence and severity of the disease and their associated complications.ObjectiveThe objective of this study was to investigate whether exosomes and their microRNA cargo present in maternal circulation can be used as early biomarker for PE.Design, Setting, Patients, and InterventionsA retrospective stratified study design was used to quantify total exosomes and placenta-derived exosomes present in maternal plasma of normal (n = 32 per time point) and PE (n = 15 per time point) pregnancies. Exosomes present in maternal circulation were determined by nanoparticle tracking analysis. An Illumina TruSeq® Small RNA Library Prep Kit was used to construct a small RNA library from exosomal RNA obtained from plasma samples.ResultsIn presymptomatic women, who subsequently developed PE, the concentration of total exosomes and placenta-derived exosomes in maternal plasma was significantly greater than those observed in controls, throughout pregnancy. The area under the receiver operating characteristic curves for total exosome and placenta-derived exosome concentrations were 0.745 ± 0.094 and 0.829 ± 0.077, respectively. In total, over 300 microRNAs were identified in exosomes across gestation, where hsa-miR-486-1-5p and hsa-miR-486-2-5p were identified as the candidate microRNAs.ConclusionsAlthough the role of exosomes during PE remains to be fully elucidated, we suggest that the concentration and content of exosomes may be of diagnostic utility for women at risk for developing PE.

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.

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