Small RNA Library Construction from Minute Biological Samples

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.

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

3 Biotech ◽

10.1007/s13205-018-1164-8 ◽

2018 ◽

Vol 8 (3) ◽

Cited By ~ 4

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

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Small RNA Library Preparation and Illumina Sequencing in Plants

Plant Epigenetics - Methods in Molecular Biology ◽

10.1007/978-1-4899-7708-3_15 ◽

2016 ◽

pp. 189-196 ◽

Cited By ~ 1

Author(s):

Andriy Bilichak ◽

Andrey Golubov ◽

Igor Kovalchuk

Keyword(s):

Illumina Sequencing ◽

Small Rna ◽

Library Preparation ◽

Small Rna Library

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

Plant Methods ◽

10.1186/s13007-019-0524-7 ◽

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

BMC Genomics ◽

10.1186/s12864-018-4491-6 ◽

2018 ◽

Vol 19 (1) ◽

Cited By ~ 40

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

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

10.1101/001479 ◽

2013 ◽

Cited By ~ 3

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.

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

10.1101/789891 ◽

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.

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