scholarly journals Ligation bias is a major contributor to nonstoichiometric abundances of secondary siRNAs and impacts analyses of microRNAs

2020 ◽  
Author(s):  
Patricia Baldrich ◽  
Saleh Tamim ◽  
Sandra Mathioni ◽  
Blake Meyers
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
High Throughput Sequencing ◽  
Library Construction ◽  
Construction Methods ◽  
Wide Range ◽  
Secondary Sirnas ◽  
Two Stages ◽  
Material Library ◽  
Small Rna Library

ABSTRACTPlant small RNAs are a diverse and complex set of molecules, ranging in length from 21 to 24 nt, involved in a wide range of essential biological processes. High-throughput sequencing is used for the discovery and quantification of small RNAs. However, several biases can occur during the preparation of small RNA libraries, especially using low input RNA. We used two stages of maize anthers to evaluate the performance of seven commercially-available methods for small RNA library construction, using different RNA input amounts. We show that when working with plant material, library construction methods have differing capabilities to capture small RNAs, and that different library construction methods provide better results when applied to the detection of microRNAs, phasiRNAs, or tRNA-derived fragment. We also observed that ligation bias occurs at both ends of miRNAs and phasiRNAs, suggesting that the biased compositions observed in small RNA populations, including nonstoichiometric levels of phasiRNAs within a locus, may reflect a combination of biological and technical influences.

scholarly journals unitas: the universal tool for annotation of small RNAs

2017 ◽  
Author(s):  
Daniel Gebert ◽  
Charlotte Hewel ◽  
David Rosenkranz
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
High Throughput Sequencing ◽  
Ease Of Use ◽  
Sequence Information ◽  
High Quality ◽  
Non Coding Rna ◽  
Wide Range ◽  
Rna Biology ◽  
User Friendly

AbstractBackgroundNext generation sequencing is a key technique in small RNA biology research that has led to the discovery of functionally different classes of small non-coding RNAs in the past years. However, reliable annotation of the extensive amounts of small non-coding RNA data produced by high-throughput sequencing is time-consuming and requires robust bioinformatics expertise. Moreover, existing tools have a number of shortcomings including a lack of sensitivity under certain conditions, limited number of supported species or detectable sub-classes of small RNAs.ResultsHere we introduce unitas, an out-of-the-box ready software for complete annotation of small RNA sequence datasets, supporting the wide range of species for which non-coding RNA reference sequences are available in the Ensembl databases (currently more than 800). unitas combines high quality annotation and numerous analysis features in a user-friendly manner. A complete annotation can be started with one simple shell command, making unitas particularly useful for researchers not having access to a bioinformatics facility. Noteworthy, the algorithms implemented in unitas are on par or even outperform comparable existing tools for small RNA annotation that base on available ncRNA sequence information.Conclusionsunitas brings together annotation and analysis features that hitherto required the installation of numerous different bioinformatics tools which can pose a challenge for the non-expert user. With this, unitas overcomes the problem of read normalization. Moreover, the high quality of sequence annotation and analysis, paired with the ease of use, make unitas a valuable tool for researchers in all fields connected to small RNA biology.

scholarly journals Validation and standardization of DNA extraction and library construction methods for metagenomics-based human fecal microbiome measurements

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Dieter M. Tourlousse ◽  
Koji Narita ◽  
Takamasa Miura ◽  
Mitsuo Sakamoto ◽  
Akiko Ohashi ◽  
...  
Keyword(s):  
Dna Extraction ◽  
High Throughput Sequencing ◽  
Performance Metrics ◽  
Collaborative Study ◽  
Human Microbiome ◽  
Second Phase ◽  
Intermediate Precision ◽  
Fecal Microbiome ◽  
Library Construction ◽  
Wide Range

Abstract Background Validation and standardization of methodologies for microbial community measurements by high-throughput sequencing are needed to support human microbiome research and its industrialization. This study set out to establish standards-based solutions to improve the accuracy and reproducibility of metagenomics-based microbiome profiling of human fecal samples. Results In the first phase, we performed a head-to-head comparison of a wide range of protocols for DNA extraction and sequencing library construction using defined mock communities, to identify performant protocols and pinpoint sources of inaccuracy in quantification. In the second phase, we validated performant protocols with respect to their variability of measurement results within a single laboratory (that is, intermediate precision) as well as interlaboratory transferability and reproducibility through an industry-based collaborative study. We further ascertained the performance of our recommended protocols in the context of a community-wide interlaboratory study (that is, the MOSAIC Standards Challenge). Finally, we defined performance metrics to provide best practice guidance for improving measurement consistency across methods and laboratories. Conclusions The validated protocols and methodological guidance for DNA extraction and library construction provided in this study expand current best practices for metagenomic analyses of human fecal microbiota. Uptake of our protocols and guidelines will improve the accuracy and comparability of metagenomics-based studies of the human microbiome, thereby facilitating development and commercialization of human microbiome-based products.

scholarly journals Accurate detection for a wide range of mutation and editing sites of microRNAs from small RNA high-throughput sequencing profiles

2016 ◽  
Vol 44 (14) ◽  
pp. e123-e123 ◽  
Author(s):  
Yun Zheng ◽  
Bo Ji ◽  
Renhua Song ◽  
Shengpeng Wang ◽  
Ting Li ◽  
...  
Keyword(s):  
High Throughput ◽  
Small Rna ◽  
High Throughput Sequencing ◽  
Accurate Detection ◽  
Wide Range

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 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 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 Different classes of small RNAs are essential for head regeneration in the planarian Dugesia japonica

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Zhonghong Cao ◽  
David Rosenkranz ◽  
Suge Wu ◽  
Hongjin Liu ◽  
Qiuxiang Pang ◽  
...  
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
High Throughput Sequencing ◽  
Expression Profiles ◽  
Evolutionary Conservation ◽  
Body Parts ◽  
Promising Candidate ◽  
Dugesia Japonica ◽  
Head Regeneration

Abstract Background Planarians reliably regenerate all body parts after injury, including a fully functional head and central nervous system. But until now, the expression dynamics and functional role of miRNAs and other small RNAs during the process of head regeneration are not well understood. Furthermore, little is known about the evolutionary conservation of the relevant small RNAs pathways, rendering it difficult to assess whether insights from planarians will apply to other taxa. Results In this study, we applied high throughput sequencing to identify miRNAs, tRNA fragments and piRNAs that are dynamically expressed during head regeneration in Dugesia japonica. We further show that knockdown of selected small RNAs, including three novel Dugesia-specific miRNAs, during head regeneration induces severe defects including abnormally small-sized eyes, cyclopia and complete absence of eyes. Conclusions Our findings suggest that a complex pool of small RNAs takes part in the process of head regeneration in Dugesia japonica and provide novel insights into global small RNA expression profiles and expression changes in response to head amputation. Our study reveals the evolutionary conserved role of miR-124 and brings further promising candidate small RNAs into play that might unveil new avenues for inducing restorative programs in non-regenerative organisms via small RNA mimics based therapies.

scholarly journals Small RNA Library Construction from Minute Biological Samples

2013 ◽  
pp. 123-136 ◽  
Author(s):  
Jessica A. Matts ◽  
Yuliya Sytnikova ◽  
Gung-wei Chirn ◽  
Gabor L. Igloi ◽  
Nelson C. Lau
Keyword(s):  
Small Rna ◽  
Biological Samples ◽  
Library Construction ◽  
Small Rna Library

High Throughput Sequencing of Acute Lymphoblastic Leukemia Small RNAs.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3650-3650
Author(s):  
Michael C Wei ◽  
Elizabeth O Osborn ◽  
Michael Cleary
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
High Throughput ◽  
Small Rna ◽  
Small Rnas ◽  
High Throughput Sequencing ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Rna Expression ◽  
Leukemia Cell Lines

Abstract Abstract 3650 MicroRNAs are small, non-coding RNAs that regulate gene expression and play key roles in cancer by modulating oncogene and tumor suppressor pathways. We are investigating the clinical and prognostic roles of miRNA expression in pediatric leukemias using high-throughput sequencing as a profiling tool. To establish the methodology, we have utilized high-throughput sequencing to quantify small RNA expression from eight acute lymphoblastic leukemia cell lines and one MLL-rearranged infant ALL patient sample. We generated sequencing libraries from these cells, conducted high-throughput sequencing using the Illumina platform, and established a custom bioinformatics pipeline for data analysis. Over 50 million individual sequence reads were analyzed. These sequences were mapped against a database of human miRNAs, and the frequency of miRNA expression among samples was enumerated. Expression of hematopoietic-specific miR-142 and miR-181 cluster miRs was found in these leukemia samples, while the liver-specific miR-122 was not expressed. miR-196b, previously reported to be over-expressed in MLL-rearranged leukemias, was expressed in 3/3 MLL-rearranged leukemia cell lines and 1/5 non-MLL cell lines. Expression of individual miRNAs was validated by quantitative PCR. Additional analysis of MLL-associated miRNAs and novel small RNAs will be presented. Our results demonstrate the feasibility and potential of high-throughput sequencing to profile the expression of small RNAs from leukemia cells, and we plan to apply these methods to additional primary patient samples to examine prognostic and clinical correlations with small RNA expression patterns. Disclosures: No relevant conflicts of interest to declare.

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