Small RNA Library Construction for High-Throughput Sequencing

2013 ◽  
pp. 195-208 ◽  
Author(s):  
Jon McGinn ◽  
Benjamin Czech
Keyword(s):  
High Throughput ◽  
Small Rna ◽  
High Throughput Sequencing ◽  
Library Construction ◽  
Small Rna Library

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 Millet Could Be both a Weed and Serve as a Virus Reservoir in Crop Fields

Plants ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 954
Author(s):  
György Pasztor ◽  
Zsuzsanna Galbacs N. ◽  
Tamas Kossuth ◽  
Emese Demian ◽  
Erzsebet Nadasy ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Seed Dormancy ◽  
High Throughput ◽  
Small Rna ◽  
Diagnostic Method ◽  
High Throughput Sequencing ◽  
Infection Risk ◽  
Viral Diseases ◽  
Crop Fields ◽  
Special Control

Millet is a dangerous weed in crop fields. A lack of seed dormancy helps it to spread easily and be present in maize, wheat, and other crop fields. Our previous report revealed the possibility that millet can also play a role as a virus reservoir. In that study, we focused on visual symptoms and detected the presence of several viruses in millet using serological methods, which can only detect the presence of the investigated pathogen. In this current work, we used small RNA high-throughput sequencing as an unbiased virus diagnostic method to uncover presenting viruses in randomly sampled millet grown as a volunteer weed in two maize fields, showing stunting, chlorosis, and striped leaves. Our results confirmed the widespread presence of wheat streak mosaic virus at both locations. Moreover, barley yellow striate mosaic virus and barley virus G, neither of which had been previously described in Hungary, were also identified. As these viruses can cause severe diseases in wheat and other cereals, their presence in a weed implies a potential infection risk. Our study indicates that the presence of millet in fields requires special control to prevent the emergence of new viral diseases in crop fields.

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 High-Throughput Sequencing of Small RNA Transcriptome Reveals Salt Stress Regulated MicroRNAs in Sugarcane

PLoS ONE ◽  
2013 ◽  
Vol 8 (3) ◽  
pp. e59423 ◽  
Author(s):  
Mariana Carnavale Bottino ◽  
Sabrina Rosario ◽  
Clicia Grativol ◽  
Flávia Thiebaut ◽  
Cristian Antonio Rojas ◽  
...  
Keyword(s):  
Salt Stress ◽  
High Throughput ◽  
Small Rna ◽  
High Throughput Sequencing

scholarly journals Differential Expression of Maize and Teosinte microRNAs under Submergence, Drought, and Alternated Stress

Plants ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 1367
Author(s):  
Edgar Baldemar Sepúlveda-García ◽  
José Francisco Pulido-Barajas ◽  
Ariana Arlene Huerta-Heredia ◽  
Julián Mario Peña-Castro ◽  
Renyi Liu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Zea Mays ◽  
High Throughput ◽  
Small Rna ◽  
Abiotic Stresses ◽  
Crop Production ◽  
High Throughput Sequencing ◽  
Expression Patterns ◽  
Evolutionary Divergence ◽  
Rna Profiling

Submergence and drought stresses are the main constraints to crop production worldwide. MicroRNAs (miRNAs) are known to play a major role in plant response to various stresses. In this study, we analyzed the expression of maize and teosinte miRNAs by high-throughput sequencing of small RNA libraries in maize and its ancestor teosinte (Zea mays ssp. parviglumis), under submergence, drought, and alternated stress. We found that the expression patterns of 67 miRNA sequences representing 23 miRNA families in maize and other plants were regulated by submergence or drought. miR159a, miR166b, miR167c, and miR169c were downregulated by submergence in both plants but more severely in maize. miR156k and miR164e were upregulated by drought in teosinte but downregulated in maize. Small RNA profiling of teosinte subject to alternate treatments with drought and submergence revealed that submergence as the first stress attenuated the response to drought, while drought being the first stress did not alter the response to submergence. The miRNAs identified herein, and their potential targets, indicate that control of development, growth, and response to oxidative stress could be crucial for adaptation and that there exists evolutionary divergence between these two subspecies in miRNA response to abiotic stresses.

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.

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