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

Content Provider	Springer Nature : BioMed Central
Author	Chiniquy, Jennifer Garber, Megan E. Mukhopadhyay, Aindrila Hillson, Nathan J.
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.
Related Links	https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/s12864-020-6481-8.pdf
Ending Page	9
Page Count	9
Starting Page	1
File Format	HTM / HTML
ISSN	14712164
DOI	10.1186/s12864-020-6481-8
Journal	BMC Genomics
Issue Number	1
Volume Number	21
Language	English
Publisher	BioMed Central
Publisher Date	2020-01-28
Access Restriction	Open
Subject Keyword	Life Sciences Microarrays Proteomics Animal Genetics and Genomics Microbial Genetics and Genomics Plant Genetics and Genomics Library preparation Next generation sequencing NGS SYBR green Echo High-throughput qPCR
Content Type	Text
Resource Type	Article
Subject	Biotechnology Genetics
Journal Impact Factor	3.5/2023
5-Year Journal Impact Factor	4.1/2023

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