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Fluorescent amplification for next generation sequencing (FA-NGS) library preparation.

ABSTRACT: BACKGROUND:Next generation sequencing (NGS) has become a universal practice in modern molecular biology. As the throughput of sequencing experiments increases, the preparation of conventional multiplexed libraries becomes more labor intensive. Conventional library preparation typically requires quality control (QC) testing for individual libraries such as amplification success evaluation and quantification, none of which occur until the end of the library preparation process. RESULTS:In this study, we address the need for a more streamlined high-throughput NGS workflow by tethering real-time quantitative PCR (qPCR) to conventional workflows to save time and implement single tube and single reagent QC. We modified two distinct library preparation workflows by replacing PCR and quantification with qPCR using SYBR Green I. qPCR enabled individual library quantification for pooling in a single tube without the need for additional reagents. Additionally, a melting curve analysis was implemented as an intermediate QC test to confirm successful amplification. Sequencing analysis showed comparable percent reads for each indexed library, demonstrating that pooling calculations based on qPCR allow for an even representation of sequencing reads. To aid the modified workflow, a software toolkit was developed and used to generate pooling instructions and analyze qPCR and melting curve data. CONCLUSIONS:We successfully applied fluorescent amplification for next generation sequencing (FA-NGS) library preparation to both plasmids and bacterial genomes. As a result of using qPCR for quantification and proceeding directly to library pooling, the modified library preparation workflow has fewer overall steps. Therefore, we speculate that the FA-NGS workflow has less risk of user error. The melting curve analysis provides the necessary QC test to identify and troubleshoot library failures prior to sequencing. While this study demonstrates the value of FA-NGS for plasmid or gDNA libraries, we speculate that its versatility could lead to successful application across other library types.

SUBMITTER: Chiniquy J

PROVIDER: S-EPMC6988211 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Fluorescent amplification for next generation sequencing (FA-NGS) library preparation.

Chiniquy Jennifer J Garber Megan E ME Mukhopadhyay Aindrila A Hillson Nathan J NJ

BMC genomics 20200128 1

<h4>Background</h4>Next generation sequencing (NGS) has become a universal practice in modern molecular biology. As the throughput of sequencing experiments increases, the preparation of conventional multiplexed libraries becomes more labor intensive. Conventional library preparation typically requires quality control (QC) testing for individual libraries such as amplification success evaluation and quantification, none of which occur until the end of the library preparation process.<h4>Results< ...[more]

PMID: 31992180

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Project description:BackgroundMassively parallel sequencing technology is revolutionizing approaches to genomic and genetic research. Since its advent, the scale and efficiency of Next-Generation Sequencing (NGS) has rapidly improved. In spite of this success, sequencing genomes or genomic regions with extremely biased base composition is still a great challenge to the currently available NGS platforms. The genomes of some important pathogenic organisms like Plasmodium falciparum (high AT content) and Mycobacterium tuberculosis (high GC content) display extremes of base composition. The standard library preparation procedures that employ PCR amplification have been shown to cause uneven read coverage particularly across AT and GC rich regions, leading to problems in genome assembly and variation analyses. Alternative library-preparation approaches that omit PCR amplification require large quantities of starting material and hence are not suitable for small amounts of DNA/RNA such as those from clinical isolates. We have developed and optimized library-preparation procedures suitable for low quantity starting material and tolerant to extremely high AT content sequences.ResultsWe have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing ~53% host contamination). By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates.ConclusionWe have developed a robust and optimized Next-Generation Sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due to low mass of DNA starting material.

Project description:BACKGROUND:Next-Generation Sequencing (NGS) technology has exceptionally increased the ability to sequence DNA in a massively parallel and cost-effective manner. Nevertheless, NGS data analysis requires bioinformatics skills and computational resources well beyond the possibilities of many "wet biology" laboratories. Moreover, most of projects only require few sequencing cycles and standard tools or workflows to carry out suitable analyses for the identification and annotation of genes, transcripts and splice variants found in the biological samples under investigation. These projects can take benefits from the availability of easy to use systems to automatically analyse sequences and to mine data without the preventive need of strong bioinformatics background and hardware infrastructure. RESULTS:To address this issue we developed an automatic system targeted to the analysis of NGS data obtained from large-scale transcriptome studies. This system, we named NGS-Trex (NGS Transcriptome profile explorer) is available through a simple web interface http://www.ngs-trex.org and allows the user to upload raw sequences and easily obtain an accurate characterization of the transcriptome profile after the setting of few parameters required to tune the analysis procedure. The system is also able to assess differential expression at both gene and transcript level (i.e. splicing isoforms) by comparing the expression profile of different samples.By using simple query forms the user can obtain list of genes, transcripts, splice sites ranked and filtered according to several criteria. Data can be viewed as tables, text files or through a simple genome browser which helps the visual inspection of the data. CONCLUSIONS:NGS-Trex is a simple tool for RNA-Seq data analysis mainly targeted to "wet biology" researchers with limited bioinformatics skills. It offers simple data mining tools to explore transcriptome profiles of samples investigated taking advantage of NGS technologies.

Dataset Information

Fluorescent amplification for next generation sequencing (FA-NGS) library preparation.

Publications

Fluorescent amplification for next generation sequencing (FA-NGS) library preparation.

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