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 requ...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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BMC
2020-01-01
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| Series: | BMC Genomics |
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| Online Access: | https://doi.org/10.1186/s12864-020-6481-8 |
| _version_ | 1818460776150597632 |
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| author | Jennifer Chiniquy Megan E. Garber Aindrila Mukhopadhyay Nathan J. Hillson |
| author_facet | Jennifer Chiniquy Megan E. Garber Aindrila Mukhopadhyay Nathan J. Hillson |
| author_sort | Jennifer Chiniquy |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-14T23:35:37Z |
| format | Article |
| id | doaj.art-e2fa13f807164c9c94360e2a386dad2b |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-14T23:35:37Z |
| publishDate | 2020-01-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-e2fa13f807164c9c94360e2a386dad2b2022-12-21T22:43:37ZengBMCBMC Genomics1471-21642020-01-012111910.1186/s12864-020-6481-8Fluorescent amplification for next generation sequencing (FA-NGS) library preparationJennifer Chiniquy0Megan E. Garber1Aindrila Mukhopadhyay2Nathan J. Hillson3Biological Systems & Engineering Division, Lawrence Berkeley National LaboratoryBiological Systems & Engineering Division, Lawrence Berkeley National LaboratoryBiological Systems & Engineering Division, Lawrence Berkeley National LaboratoryBiological Systems & Engineering Division, Lawrence Berkeley National LaboratoryAbstract 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.https://doi.org/10.1186/s12864-020-6481-8Library preparationNext generation sequencingNGSSYBR greenEchoHigh-throughput |
| spellingShingle | Jennifer Chiniquy Megan E. Garber Aindrila Mukhopadhyay Nathan J. Hillson Fluorescent amplification for next generation sequencing (FA-NGS) library preparation BMC Genomics Library preparation Next generation sequencing NGS SYBR green Echo High-throughput |
| title | Fluorescent amplification for next generation sequencing (FA-NGS) library preparation |
| title_full | Fluorescent amplification for next generation sequencing (FA-NGS) library preparation |
| title_fullStr | Fluorescent amplification for next generation sequencing (FA-NGS) library preparation |
| title_full_unstemmed | Fluorescent amplification for next generation sequencing (FA-NGS) library preparation |
| title_short | Fluorescent amplification for next generation sequencing (FA-NGS) library preparation |
| title_sort | fluorescent amplification for next generation sequencing fa ngs library preparation |
| topic | Library preparation Next generation sequencing NGS SYBR green Echo High-throughput |
| url | https://doi.org/10.1186/s12864-020-6481-8 |
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