scholarly journals Method for improved Illumina sequencing library preparation using NuGEN Ovation RNA-Seq System

BioTechniques ◽  
2011 ◽  
Vol 50 (3) ◽  
pp. 177-181 ◽  
Author(s):  
Steven R. Head ◽  
H.Kiyomi Komori ◽  
G.Traver Hart ◽  
John Shimashita ◽  
Lana Schaffer ◽  
...  
Keyword(s):  
Illumina Sequencing ◽  
Library Preparation ◽  
Rna Seq ◽  
Sequencing Library ◽  
Sequencing Library Preparation

scholarly journals Methyltransferase-directed orthogonal tagging and sequencing of miRNAs and bacterial small RNAs

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Milda Mickutė ◽  
Kotryna Kvederavičiūtė ◽  
Aleksandr Osipenko ◽  
Raminta Mineikaitė ◽  
Saulius Klimašauskas ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Regulatory Networks ◽  
Library Preparation ◽  
Rna Seq ◽  
Basic Principles ◽  
Cofactor Binding ◽  
Sequencing Library ◽  
Sequencing Library Preparation ◽  
Target Rna

Abstract Background Targeted installation of designer chemical moieties on biopolymers provides an orthogonal means for their visualisation, manipulation and sequence analysis. Although high-throughput RNA sequencing is a widely used method for transcriptome analysis, certain steps, such as 3′ adapter ligation in strand-specific RNA sequencing, remain challenging due to structure- and sequence-related biases introduced by RNA ligases, leading to misrepresentation of particular RNA species. Here, we remedy this limitation by adapting two RNA 2′-O-methyltransferases from the Hen1 family for orthogonal chemo-enzymatic click tethering of a 3′ sequencing adapter that supports cDNA production by reverse transcription of the tagged RNA. Results We showed that the ssRNA-specific DmHen1 and dsRNA-specific AtHEN1 can be used to efficiently append an oligonucleotide adapter to the 3′ end of target RNA for sequencing library preparation. Using this new chemo-enzymatic approach, we identified miRNAs and prokaryotic small non-coding sRNAs in probiotic Lactobacillus casei BL23. We found that compared to a reference conventional RNA library preparation, methyltransferase-Directed Orthogonal Tagging and RNA sequencing, mDOT-seq, avoids misdetection of unspecific highly-structured RNA species, thus providing better accuracy in identifying the groups of transcripts analysed. Our results suggest that mDOT-seq has the potential to advance analysis of eukaryotic and prokaryotic ssRNAs. Conclusions Our findings provide a valuable resource for studies of the RNA-centred regulatory networks in Lactobacilli and pave the way to developing novel transcriptome and epitranscriptome profiling approaches in vitro and inside living cells. As RNA methyltransferases share the structure of the AdoMet-binding domain and several specific cofactor binding features, the basic principles of our approach could be easily translated to other AdoMet-dependent enzymes for the development of modification-specific RNA-seq techniques.

scholarly journals Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes

2009 ◽  
Vol 6 (4) ◽  
pp. 291-295 ◽  
Author(s):  
Iwanka Kozarewa ◽  
Zemin Ning ◽  
Michael A Quail ◽  
Mandy J Sanders ◽  
Matthew Berriman ◽  
...  
Keyword(s):  
Illumina Sequencing ◽  
Library Preparation ◽  
Sequencing Library ◽  
Sequencing Library Preparation

Abstract 556: Highly complex DNA sequencing library preparation for cfDNA enrichment panels using a single-stranded approach

2021 ◽  
Author(s):  
Charles J. Vaske ◽  
Chris Troll ◽  
Camille Schwartz ◽  
Colin Naughton ◽  
Abdullah Mahmood Ali ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Library Preparation ◽  
Sequencing Library ◽  
Sequencing Library Preparation
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