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

2020 ◽  
Vol 48 (14) ◽  
pp. e80-e80 ◽  
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.

scholarly journals miRquant 2.0: an Expanded Tool for Accurate Annotation and Quantification of MicroRNAs and their isomiRs from Small RNA-Sequencing Data

2016 ◽  
Vol 13 (5) ◽  
Author(s):  
Matthew Kanke ◽  
Jeanette Baran-Gale ◽  
Jonathan Villanueva ◽  
Praveen Sethupathy
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Cell Types ◽  
Small Rna Sequencing ◽  
Sequencing Data ◽  
Sequencing Technology ◽  
Additional Information ◽  
Disease States ◽  
Non Coding Rnas ◽  
The Rich

SummarySmall non-coding RNAs, in particular microRNAs, are critical for normal physiology and are candidate biomarkers, regulators, and therapeutic targets for a wide variety of diseases. There is an ever-growing interest in the comprehensive and accurate annotation of microRNAs across diverse cell types, conditions, species, and disease states. Highthroughput sequencing technology has emerged as the method of choice for profiling microRNAs. Specialized bioinformatic strategies are required to mine as much meaningful information as possible from the sequencing data to provide a comprehensive view of the microRNA landscape. Here we present miRquant 2.0, an expanded bioinformatics tool for accurate annotation and quantification of microRNAs and their isoforms (termed isomiRs) from small RNA-sequencing data. We anticipate that miRquant 2.0 will be useful for researchers interested not only in quantifying known microRNAs but also mining the rich well of additional information embedded in small RNA-sequencing data.

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

2018 ◽  
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.

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

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

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

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Simonas Juzenas ◽  
Carl M 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

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

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

scholarly journals COMPASS: a COMprehensive Platform for smAll RNA-Seq data analySis

2019 ◽  
Author(s):  
Jiang Li ◽  
Alvin T. Kho ◽  
Robert P. Chase ◽  
Lorena Pantano-Rubino ◽  
Leanna Farnam ◽  
...  
Keyword(s):  
Data Analysis ◽  
Rna Sequencing ◽  
Small Rna ◽  
Small Rnas ◽  
Small Rna Sequencing ◽  
Sequencing Data ◽  
Healthy Human ◽  
Data Set ◽  
Sequence Processing ◽  
Rna Analysis

Abstract Background Circulating RNAs are potential disease biomarkers and their function is being actively investigated. Next generation sequencing (NGS) is a common means to interrogate the small RNA'ome or the full spectrum of small RNAs (<200 nucleotide length) of a biological system. A pivotal problem in NGS based small RNA analysis is identifying and quantifying the small RNA'ome constituent components. Most existing NGS data analysis tools focus on the microRNA component and a few other small RNA types like piRNA, snRNA and snoRNA. A comprehensive platform is needed to interrogate the full small RNA'ome, a prerequisite for down-stream data analysis. Results We present COMPASS, a comprehensive modular stand-alone platform for identifying and quantifying small RNAs from small RNA sequencing data. COMPASS contains prebuilt customizable standard RNA databases and sequence processing tools to enable turnkey basic small RNA analysis. We evaluated COMPASS against comparable existing tools on small RNA sequencing data set from serum samples of 12 healthy human controls, and COMPASS identified a greater diversity and abundance of small RNA molecules. Conclusion COMPASS is modular, stand-alone and integrates multiple customizable RNA databases and sequence processing tool and is distributed under the GNU General Public License free to non-commercial registered users at https://regepi.bwh.harvard.edu/circurna/ and the source code is available at https://github.com/cougarlj/COMPASS.

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