Sequencing Library Preparation from Degraded Samples for Non-illumina Sequencing Platforms

2019 ◽  
pp. 85-92
Author(s):  
Renata F. Martins ◽  
Marie-Louise Kampmann ◽  
Daniel W. Förster
Keyword(s):  
Illumina Sequencing ◽  
Library Preparation ◽  
Degraded Samples ◽  
Sequencing Library ◽  
Sequencing Platforms ◽  
Sequencing Library Preparation

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 Miniaturization and optimization of 384-well compatible metagenomic sequencing library preparation

2018 ◽  
Author(s):  
Madeline Y Mayday ◽  
Lillian M Khan ◽  
Eric D Chow ◽  
Matt S Zinter ◽  
Joseph L DeRisi
Keyword(s):  
Metagenomic Sequencing ◽  
Library Preparation ◽  
Sequencing Library ◽  
Reaction Volumes ◽  
Sequencing Platforms ◽  
Time Required ◽  
The Cost ◽  
Sequencing Library Preparation ◽  
Full Volume ◽  
Generation Sequencing

AbstractPreparation of high-quality sequencing libraries is a costly and time-consuming component of metagenomic next generation sequencing (mNGS). While the overall cost of sequencing has dropped significantly over recent years, the reagents needed to prepare sequencing samples are likely to become the dominant expense in the process. Furthermore, libraries prepared by hand are subject to human variability and needless waste due to limitations of manual pipetting volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of reagents without consumable pipette tips, has the potential to provide significant advantages. Here, we describe the integration of several instruments, including the Labcyte Echo 525 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize and automate mNGS library preparation, significantly reducing the cost and the time required to prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full volume reactions. Furthermore, detection of viral and microbial species from cell culture and patient samples was also maintained in the miniaturized libraries. For 384-well mNGS library preparations, we achieved a savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library.

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

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.

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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