Leveraging the fundamentals of heat transfer and fluid mechanics in microscale geometries for automated next-generation sequencing library preparation
Abstract Next-generation sequencing (NGS) is emerging as a powerful tool for molecular diagnostics but remains limited by cumbersome and inefficient sample preparation. We present an innovative automated NGS library preparation system with a simplified mechanical design that exploits both macro- and...
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| Format: | Article |
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Nature Portfolio
2024-05-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-024-63014-x |
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| author | Olivia Ott Sabrina Tolppi Jennifer Figueroa-Cruz Khaliun Myagmar Khulan Unurbuyan Anubhav Tripathi |
| author_facet | Olivia Ott Sabrina Tolppi Jennifer Figueroa-Cruz Khaliun Myagmar Khulan Unurbuyan Anubhav Tripathi |
| author_sort | Olivia Ott |
| collection | DOAJ |
| description | Abstract Next-generation sequencing (NGS) is emerging as a powerful tool for molecular diagnostics but remains limited by cumbersome and inefficient sample preparation. We present an innovative automated NGS library preparation system with a simplified mechanical design that exploits both macro- and microfluidic properties for optimizing heat transfer, reaction kinetics, mass transfer, fluid mechanics, adsorption–desorption rates, and molecular thermodynamics. Our approach introduces a unique two-cannula cylindrical capillary system connected to a programmable syringe pump and a Peltier heating element able to execute all steps with high efficiency. Automatic reagent movement, mixing, and magnetic bead-based washing with capillary-based thermal cycling (capillary-PCR) are completely integrated into a single platform. The manual 3-h library preparation process is reduced to less than 15 min of hands-on time via optimally pre-plated reagent plates, followed by less than 6 h of instrument run time during which no user interaction is required. We applied this method to two library preparation assays with different DNA fragmentation requirements (mechanical vs. enzymatic fragmentation), sufficiently limiting consumable use to one cartridge and one 384 well-plate per run. Our platform successfully prepared eight libraries in parallel, generating sequencing data for both human and Escherichia coli DNA libraries with negligible coverage bias compared to positive controls. All sequencing data from our libraries attained Phred (Q) scores > 30, mapping to reference genomes at 99% confidence. The method achieved final library concentrations and size distributions comparable with the conventional manual approach, demonstrating compatibility with downstream sequencing and subsequent data analysis. Our engineering design offers repeatability and consistency in the quality of sequence-able libraries, asserting the importance of mechanical design considerations that employ and optimize fundamental fluid mechanics and heat transfer properties. Furthermore in this work, we provide unique insights into the mechanisms of sample loss within NGS library preparation assays compared with automated adaptations and pinpoint areas in which the principles of thermodynamics, fluid mechanics, and heat transfer can improve future mechanical design iterations. |
| format | Article |
| id | doaj-art-8e49bc9948944de4a961cf1870d2b4c2 |
| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2024-05-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-8e49bc9948944de4a961cf1870d2b4c22025-01-12T12:25:20ZengNature PortfolioScientific Reports2045-23222024-05-0114112210.1038/s41598-024-63014-xLeveraging the fundamentals of heat transfer and fluid mechanics in microscale geometries for automated next-generation sequencing library preparationOlivia Ott0Sabrina Tolppi1Jennifer Figueroa-Cruz2Khaliun Myagmar3Khulan Unurbuyan4Anubhav Tripathi5Center for Biomedical Engineering, School of Engineering, Brown UniversityCenter for Biomedical Engineering, School of Engineering, Brown UniversityCenter for Biomedical Engineering, School of Engineering, Brown UniversityCenter for Biomedical Engineering, School of Engineering, Brown UniversityCenter for Biomedical Engineering, School of Engineering, Brown UniversityCenter for Biomedical Engineering, School of Engineering, Brown UniversityAbstract Next-generation sequencing (NGS) is emerging as a powerful tool for molecular diagnostics but remains limited by cumbersome and inefficient sample preparation. We present an innovative automated NGS library preparation system with a simplified mechanical design that exploits both macro- and microfluidic properties for optimizing heat transfer, reaction kinetics, mass transfer, fluid mechanics, adsorption–desorption rates, and molecular thermodynamics. Our approach introduces a unique two-cannula cylindrical capillary system connected to a programmable syringe pump and a Peltier heating element able to execute all steps with high efficiency. Automatic reagent movement, mixing, and magnetic bead-based washing with capillary-based thermal cycling (capillary-PCR) are completely integrated into a single platform. The manual 3-h library preparation process is reduced to less than 15 min of hands-on time via optimally pre-plated reagent plates, followed by less than 6 h of instrument run time during which no user interaction is required. We applied this method to two library preparation assays with different DNA fragmentation requirements (mechanical vs. enzymatic fragmentation), sufficiently limiting consumable use to one cartridge and one 384 well-plate per run. Our platform successfully prepared eight libraries in parallel, generating sequencing data for both human and Escherichia coli DNA libraries with negligible coverage bias compared to positive controls. All sequencing data from our libraries attained Phred (Q) scores > 30, mapping to reference genomes at 99% confidence. The method achieved final library concentrations and size distributions comparable with the conventional manual approach, demonstrating compatibility with downstream sequencing and subsequent data analysis. Our engineering design offers repeatability and consistency in the quality of sequence-able libraries, asserting the importance of mechanical design considerations that employ and optimize fundamental fluid mechanics and heat transfer properties. Furthermore in this work, we provide unique insights into the mechanisms of sample loss within NGS library preparation assays compared with automated adaptations and pinpoint areas in which the principles of thermodynamics, fluid mechanics, and heat transfer can improve future mechanical design iterations.https://doi.org/10.1038/s41598-024-63014-x |
| spellingShingle | Olivia Ott Sabrina Tolppi Jennifer Figueroa-Cruz Khaliun Myagmar Khulan Unurbuyan Anubhav Tripathi Leveraging the fundamentals of heat transfer and fluid mechanics in microscale geometries for automated next-generation sequencing library preparation Scientific Reports |
| title | Leveraging the fundamentals of heat transfer and fluid mechanics in microscale geometries for automated next-generation sequencing library preparation |
| title_full | Leveraging the fundamentals of heat transfer and fluid mechanics in microscale geometries for automated next-generation sequencing library preparation |
| title_fullStr | Leveraging the fundamentals of heat transfer and fluid mechanics in microscale geometries for automated next-generation sequencing library preparation |
| title_full_unstemmed | Leveraging the fundamentals of heat transfer and fluid mechanics in microscale geometries for automated next-generation sequencing library preparation |
| title_short | Leveraging the fundamentals of heat transfer and fluid mechanics in microscale geometries for automated next-generation sequencing library preparation |
| title_sort | leveraging the fundamentals of heat transfer and fluid mechanics in microscale geometries for automated next generation sequencing library preparation |
| url | https://doi.org/10.1038/s41598-024-63014-x |
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