Abstract
BackgroundNext 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.ResultsIn 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.ConclusionsWe 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.
Highlights
Generation sequencing (NGS) has become a universal practice in modern molecular biology
Using an input titration assay, we determined that the concentration of input adapter-ligated (AL) genomic DNA (gDNA) or plasmid could be tracked by Fluorescent amplification for next generation sequencing (FA-Next generation sequencing (NGS)) with NGS primers (Fig. 2, Additional file 2: Fig. S2 and Additional file 3: Fig. S3)
We found that the detection limit of FA-NGS above a 1000 end relative fluorescence unit (RFU)
Summary
Generation sequencing (NGS) has become a universal practice in modern molecular biology. NGS libraries after amplification and purification include Quant-iT dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA), real-time qPCR-based library quantification methods like the KAPA Library Quantification Kit (Roche, Pleasanton, CA) [3], and digital PCR [4,5,6]. Our goal was to establish a modified library preparation workflow that eases multiplexing by limiting the number of steps required per individual library. We accomplished this by bridging amplification and QC steps to bypass individual DNA purification using real-time quantitative PCR (qPCR). We call this library preparation workflow modification fluorescent amplification for NGS (FA-NGS)
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