scholarly journals Next-generation sequencing library preparation method for identification of RNA viruses on the Ion Torrent Sequencing Platform

Virus Genes ◽  
2018 ◽  
Vol 54 (4) ◽  
pp. 536-542
Author(s):  
Guiqian Chen ◽  
Yuan Qiu ◽  
Qingye Zhuang ◽  
Suchun Wang ◽  
Tong Wang ◽  
...  
Keyword(s):  
Preparation Method ◽  
Rna Viruses ◽  
Ion Torrent ◽  
Library Preparation ◽  
Sequencing Platform ◽  
Sequencing Library ◽  
Ion Torrent Sequencing ◽  
Sequencing Library Preparation ◽  
Generation Sequencing ◽  
Library Preparation Method

Transcriptome-wide m6A detection with DART-Seq

2019 ◽  
Author(s):  
Kate D. Meyer
Keyword(s):  
Gene Expression Regulation ◽  
Preparation Method ◽  
Current Knowledge ◽  
Cross Reactivity ◽  
High Variability ◽  
Library Preparation ◽  
Rna Seq ◽  
Generation Sequencing ◽  
Library Preparation Method

Abstract m6A is the most abundant internal mRNA modification and plays diverse roles in gene expression regulation. Much of our current knowledge about m6A has been driven by recent advances in the ability to detect this mark transcriptome-wide. Antibody-based approaches have been the method of choice for global m6A mapping studies. These methods rely on m6A antibodies to immunoprecipitate methylated RNAs, followed by next-generation sequencing to identify m6A-containing transcripts1,2. While these methods enabled the first identification of m6A sites transcriptome-wide and have dramatically improved our ability to study m6A, they suffer from several limitations. These include requirements for high amounts of input RNA, costly and time-consuming library preparation, high variability across studies, and m6A antibody cross-reactivity with other modifications. Here, we describe DART-Seq (deamination adjacent to RNA modification targets), an antibody-free method for global m6A detection. In DART-Seq, the C to U deaminating enzyme, APOBEC1, is fused to the m6A-binding YTH domain. This fusion protein is then introduced to cellular RNA either through overexpression in cells or with in vitro assays, and subsequent deamination of m6A-adjacent cytidines is then detected by RNA sequencing to identify m6A sites. DART-Seq can successfully map m6A sites throughout the transcriptome using as little as 10 nanograms of total cellular RNA, and it is compatible with any standard RNA-seq library preparation method.

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 Screening ancient tuberculosis with qPCR: challenges and opportunities

2015 ◽  
Vol 370 (1660) ◽  
pp. 20130622 ◽  
Author(s):  
Kelly M. Harkins ◽  
Jane E. Buikstra ◽  
Tessa Campbell ◽  
Kirsten I. Bos ◽  
Eric D. Johnson ◽  
...  
Keyword(s):  
Screening Tool ◽  
Probe Design ◽  
Library Preparation ◽  
New Methods ◽  
Challenges And Opportunities ◽  
Sequencing Library ◽  
Sequencing Library Preparation ◽  
Skeletal Samples ◽  
Generation Sequencing ◽  
Targeted Enrichment

The field of ancient DNA (aDNA) has rapidly accelerated in recent years as a result of new methods in next-generation sequencing, library preparation and targeted enrichment. Such research is restricted, however, by the highly variable DNA preservation within different tissues, especially when isolating ancient pathogens from human remains. Identifying positive candidate samples via quantitative PCR (qPCR) for downstream procedures can reduce reagent costs, increase capture efficiency and maximize the number of sequencing reads of the target. This study uses four qPCR assays designed to target regions within the Mycobacterium tuberculosis complex (MTBC) to examine 133 human skeletal samples from a wide geographical and temporal range, identified by the presence of skeletal lesions typical of chronic disseminated tuberculosis. Given the inherent challenges working with ancient mycobacteria, strict criteria must be used and primer/probe design continually re-evaluated as new data from bacteria become available. Seven samples tested positive for multiple MTBC loci, supporting them as strong candidates for downstream analyses. Using strict and conservative criteria, qPCR remains a fast and effective screening tool when compared with screening by more expensive sequencing and enrichment technologies.

scholarly journals qPCR-based characterization of DNA fragmentation efficiency of Tn5 transposomes

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Vera Rykalina ◽  
Alexey Shadrin ◽  
Hans Lehrach ◽  
Tatiana Borodina
Keyword(s):  
Next Generation Sequencing ◽  
Dna Fragmentation ◽  
Fragment Size ◽  
Library Preparation ◽  
Next Generation ◽  
Amplification Curve ◽  
Fragmentation Efficiency ◽  
Sequencing Library ◽  
Sequencing Library Preparation ◽  
Generation Sequencing

Abstract Here, we describe an electrophoresis free assay for characterizing Tn5 transposomes fragmentation efficiency in a tagmentation reaction, in which double-stranded DNA is fragmented and tagged with adapter sequences. The assay uses plasmid DNA as a reference tagmentation substrate. Fragmentation efficiency is analyzed by comparative qPCR which measures the difference (ΔCt) in amplification of a specific plasmid region before and after tagmentation: more efficient fragmentation is characterized by a larger number of cleavage events within the amplified region, a delayed increase in the amplification curve and as a result, a larger ΔCt. Tagmentation reactions characterized with the same ΔCt exhibit the same fragment size distribution on an agarose gel. The ΔCt values measured can be used to quantitatively determine the relative performance of Tn5 transposome assemblies in optimization experiments and to standardize between batch variations in transposomes for use in next-generation sequencing library preparation. Moreover, the use of a reference tagmentation template added during next-generation sequencing library preparation enabled monitoring of the input DNA fragmentation. The presented qPCR-based assay is quick, contamination-safe, high-throughput and cost-efficient.

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