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.

Keywords: Echo; High-throughput; Library preparation; NGS; Next generation sequencing; SYBR green; qPCR.

Fig. 1

FA-NGS Workflow: Following library preparation method of choice, amplification is applied with SYBR green mastermix, including polymerase (pink clouds), intercalating dye (green rectangles), and index primers (yellow, blue, purple rectangles). The fluorescence is monitored during library amplification. Melting curve analysis is then applied to an aliquot of the library to determine amplification success. If libraries pass the melting curve analysis QC, end RFU measurements are used to pool the amplified libraries in equal quantities. Libraries that fail melting curve analysis QC are omitted from subsequent steps. The pooled library is then purified and ready for NGS

Fig. 2

Input titration of AL-DNA monitored with FA-NGS: DNA diluted 2-fold starting at 1000 pg per reaction was amplified with SYBR green in duplicate and monitored with continuous fluorescence (a), end-fluorescence (b), and melting curve analysis (c)

Fig. 3

End RFU and transfer volumes of Nextera and AL library preparations: Equation for calculating transfer volumes from End RFU values, where Transfer Volume_max is determined by the user, and End RFU_min is the minimum End RFU value in the data set (a). Heatmaps of end RFU values and the corresponding calculated transfer volume of each well, 4 unique plasmids per quadrant of Nextera library prepared plasmids (b), 4 two-fold dilutions starting at 500 pg per quadrant of AL library prepared gDNA (c)

Fig. 4

Library pooling yields near even distribution of percent of the total reads: Optimal pooling is the expectation (1.04) of the percent reads if all 96 libraries pooled evenly. 4 unique plasmids of Nextera library prepared plasmids (a), 4 two-fold dilutions starting at 500 pg of AL library prepared gDNA (b). The bars are colored by the end RFU values of the qPCR, blue if the RFU was greater than 1000, yellow if the RFU was between 700 and 1000, and red if the RFU was less than 700. For gDNA library (b), samples below RFU 700 were likely to be underpooled because they were out of the linear range of the qPCR. This trend was not observed with the plasmid library (a)