Establishing a canine genome sequencing protocol using Oxford Nanopore
The development of sequencing technologies has led to monumental ad-vances in the field of genomics, creating new areas of investigation and profoundly impacting our understanding of life itself. Presently, the “third generation” of these technologies is focused on improving the sequencing of long r...
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| Formato: | H2 |
| Lenguaje: | Inglés francés |
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SLU/Dept. of Animal Breeding and Genetics (until 231231)
2019
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| Materias: |
| _version_ | 1855572546329509888 |
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| author | Van Der Heiden, Anna Darlene |
| author_browse | Van Der Heiden, Anna Darlene |
| author_facet | Van Der Heiden, Anna Darlene |
| author_sort | Van Der Heiden, Anna Darlene |
| collection | Epsilon Archive for Student Projects |
| description | The development of sequencing technologies has led to monumental ad-vances in the field of genomics, creating new areas of investigation and profoundly impacting our understanding of life itself. Presently, the “third generation” of these technologies is focused on improving the sequencing of long reads, which allows for studying complex areas in the genome. A promising platform offering long-read sequencing at a comparatively low cost is the Oxford Nanopore Technologies “MinION,” a USB-connected device the size of an ordinary dongle, which can be used in as good as any laboratory setting with a consumer-grade computer. Given that the techno-logy is both recent and still under development, however, there is a need to formulate and verify adequate methodologies for a great variety of target species. In this thesis, a protocol for long-read sequencing of canine DNA using the MinION is presented. Four different HMW-gDNA extraction methods and five library preparation variants were evaluated in order to determine which approach would generate the best sequencing results. Additionally, a method for reusing flow cells in order to maximize data ge-nerated per cell and reducing costs was tested and deemed successful. Major challenges encountered throughout the project include DNA quality, fragment length, as well as high rates of pore loss and low pore occu-pancy. The best-performing DNA extraction protocol was an altered version of Qiagen's Genomic-tip 100/G. For library preparation, a modified version of Nanopore's Sequencing by Ligation kit (SQK-LSK109) had the most favourable results. The best sequencing run generated 14 Gbp of raw data in the span of 48 hours. The results presented herein constitute a first step towards the establishment of a method that leverages the MinION's advan-tages in canine genome sequencing projects. |
| format | H2 |
| id | RepoSLU15244 |
| institution | Swedish University of Agricultural Sciences |
| language | Inglés fre |
| publishDate | 2019 |
| publishDateSort | 2019 |
| publisher | SLU/Dept. of Animal Breeding and Genetics (until 231231) |
| publisherStr | SLU/Dept. of Animal Breeding and Genetics (until 231231) |
| record_format | eprints |
| spelling | RepoSLU152442023-07-20T06:47:19Z Establishing a canine genome sequencing protocol using Oxford Nanopore Van Der Heiden, Anna Darlene Oxford Nanopore long-read sequencing nanopore sequencing canine genome The development of sequencing technologies has led to monumental ad-vances in the field of genomics, creating new areas of investigation and profoundly impacting our understanding of life itself. Presently, the “third generation” of these technologies is focused on improving the sequencing of long reads, which allows for studying complex areas in the genome. A promising platform offering long-read sequencing at a comparatively low cost is the Oxford Nanopore Technologies “MinION,” a USB-connected device the size of an ordinary dongle, which can be used in as good as any laboratory setting with a consumer-grade computer. Given that the techno-logy is both recent and still under development, however, there is a need to formulate and verify adequate methodologies for a great variety of target species. In this thesis, a protocol for long-read sequencing of canine DNA using the MinION is presented. Four different HMW-gDNA extraction methods and five library preparation variants were evaluated in order to determine which approach would generate the best sequencing results. Additionally, a method for reusing flow cells in order to maximize data ge-nerated per cell and reducing costs was tested and deemed successful. Major challenges encountered throughout the project include DNA quality, fragment length, as well as high rates of pore loss and low pore occu-pancy. The best-performing DNA extraction protocol was an altered version of Qiagen's Genomic-tip 100/G. For library preparation, a modified version of Nanopore's Sequencing by Ligation kit (SQK-LSK109) had the most favourable results. The best sequencing run generated 14 Gbp of raw data in the span of 48 hours. The results presented herein constitute a first step towards the establishment of a method that leverages the MinION's advan-tages in canine genome sequencing projects. El desarrollo de tecnologías de secuenciación ha conducido a avances monumentales en el campo de la genómica, creando nuevas áreas de in-vestigación e impactando profundamente nuestro entendimiento de la vida misma. Actualmente, la "tercera generación" de estas tecnologías se con-centra en mejorar la secuenciación de lecturas largas, lo que permite estu-diar áreas complejas del genoma. Una nueva y prometedora plataforma que ofrece secuenciación de lecturas largas a un costo comparativamente bajo es el “MinION”, de la compañía Oxford Nanopore Technologies, cuyo tamaño, similar al de un adaptador USB, permite que pueda utilizarse en cualquier tipo de laboratorio. Sin embargo, dado que esta tecnología es relativamente reciente y aún se encuentra en desarrollo, es necesario formu-lar nuevas metodologías que sean adecuadas para diferentes tipos de especies. Esta tesis presenta un protocolo para la secuenciación de lectu-ras largas de ADN canino utilizando el dispositivo MinION. Se evaluaron cuatro métodos de extracción de ADN de alto peso molecular y cinco mé-todos de preparación de bibliotecas con el fin de determinar qué protocolo produce los mejores resultados. Asimismo, con el fin de maximizar los datos generados por celda de flujo y reducir costos, se analizó un método para reutilizar celdas de flujo, el cual fue considerado exitoso. Los principa-les desafíos que se encontraron a lo largo de este proyecto incluyen la obtención de ADN de calidad y de alto peso molecular, así como la alta tasa de pérdida de nanoporos. El protocolo de extracción de ADN que produjo los mejores resultados fue una versión alterada del kit de Qiagen Genomic-tip 100/G. Para la preparación de la biblioteca, una versión modi-ficada del kit de Secuenciación por Ligadura de Nanopore (SQK-LSK109) tuvo los resultados más favorables. El mejor experimento de secuencia-ción generó 14 Gbp en el lapso de 48 horas. Los resultados aquí presenta-dos constituyen un primer paso para el establecimiento de un método que aprovecha las ventajas del MinION para proyectos de secuenciación del genoma canino. SLU/Dept. of Animal Breeding and Genetics (until 231231) 2019 H2 eng fre https://stud.epsilon.slu.se/15244/ |
| spellingShingle | Oxford Nanopore long-read sequencing nanopore sequencing canine genome Van Der Heiden, Anna Darlene Establishing a canine genome sequencing protocol using Oxford Nanopore |
| title | Establishing a canine genome sequencing protocol using
Oxford Nanopore |
| title_full | Establishing a canine genome sequencing protocol using
Oxford Nanopore |
| title_fullStr | Establishing a canine genome sequencing protocol using
Oxford Nanopore |
| title_full_unstemmed | Establishing a canine genome sequencing protocol using
Oxford Nanopore |
| title_short | Establishing a canine genome sequencing protocol using
Oxford Nanopore |
| title_sort | establishing a canine genome sequencing protocol using
oxford nanopore |
| topic | Oxford Nanopore long-read sequencing nanopore sequencing canine genome |