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A novel NGS library preparation method to characterize native termini of fragmented DNA.

ABSTRACT: Biological and chemical DNA fragmentation generates DNA molecules with a variety of termini, including blunt ends and single-stranded overhangs. We have developed a Next Generation Sequencing (NGS) assay, XACTLY, to interrogate the termini of fragmented DNA, information traditionally lost in standard NGS library preparation methods. Here we describe the XACTLY method, showcase its sensitivity and specificity, and demonstrate its utility in in vitro experiments. The XACTLY assay is able to report relative abundances of all lengths and types (5' and 3') of single-stranded overhangs, if present, on each DNA fragment with an overall accuracy between 80-90%. In addition, XACTLY retains the sequence of each native DNA molecule after fragmentation and can capture the genomic landscape of cleavage events at single nucleotide resolution. The XACTLY assay can be applied as a novel research and discovery tool for fragmentation analyses and in cell-free DNA.

SUBMITTER: Harkins KM

PROVIDER: S-EPMC7192605 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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A novel NGS library preparation method to characterize native termini of fragmented DNA.

Harkins Kelly M KM Schaefer Nathan K NK Troll Christopher J CJ Rao Varsha V Kapp Joshua J Naughton Colin C Shapiro Beth B Green Richard E RE

Nucleic acids research 20200501 8

Biological and chemical DNA fragmentation generates DNA molecules with a variety of termini, including blunt ends and single-stranded overhangs. We have developed a Next Generation Sequencing (NGS) assay, XACTLY, to interrogate the termini of fragmented DNA, information traditionally lost in standard NGS library preparation methods. Here we describe the XACTLY method, showcase its sensitivity and specificity, and demonstrate its utility in in vitro experiments. The XACTLY assay is able to report ...[more]

PMID: 32112100

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Project description: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.

Dataset Information

A novel NGS library preparation method to characterize native termini of fragmented DNA.

Publications

A novel NGS library preparation method to characterize native termini of fragmented DNA.

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