Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules
De novo sequencing of complex genomes is one of the main challenges for researchers seeking high-quality reference sequences. Many de novo assemblies are based on short reads, producing fragmented genome sequences. Third-generation sequencing, with read lengths >10 kb, will improve the assembly o...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2016-10-01
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| Series: | BioTechniques |
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| Online Access: | https://www.future-science.com/doi/10.2144/000114460 |
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| author | Baptiste Mayjonade Jérôme Gouzy Cécile Donnadieu Nicolas Pouilly William Marande Caroline Callot Nicolas Langlade Stéphane Muños |
| author_facet | Baptiste Mayjonade Jérôme Gouzy Cécile Donnadieu Nicolas Pouilly William Marande Caroline Callot Nicolas Langlade Stéphane Muños |
| author_sort | Baptiste Mayjonade |
| collection | DOAJ |
| description | De novo sequencing of complex genomes is one of the main challenges for researchers seeking high-quality reference sequences. Many de novo assemblies are based on short reads, producing fragmented genome sequences. Third-generation sequencing, with read lengths >10 kb, will improve the assembly of complex genomes, but these techniques require high-molecular-weight genomic DNA (gDNA), and gDNA extraction protocols used for obtaining smaller fragments for short-read sequencing are not suitable for this purpose. Methods of preparing gDNA for bacterial artificial chromosome (BAC) libraries could be adapted, but these approaches are time-consuming, and commercial kits for these methods are expensive. Here, we present a protocol for rapid, inexpensive extraction of high-molecular-weight gDNA from bacteria, plants, and animals. Our technique was validated using sunflower leaf samples, producing a mean read length of 12.6 kb and a maximum read length of 80 kb. |
| format | Article |
| id | doaj-art-8365f5a865b6495eb914b0d2e77893e8 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2016-10-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-8365f5a865b6495eb914b0d2e77893e82025-08-20T02:25:37ZengTaylor & Francis GroupBioTechniques0736-62051940-98182016-10-0161420320510.2144/000114460Extraction of high-molecular-weight genomic DNA for long-read sequencing of single moleculesBaptiste Mayjonade0Jérôme Gouzy1Cécile Donnadieu2Nicolas Pouilly3William Marande4Caroline Callot5Nicolas Langlade6Stéphane Muños71LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosa, France1LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosa, France2Get-PLAGE, Université de Toulouse, INRA, CNRS, Castanet Tolosa, France1LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosa, France3CNRGV, Université de Toulouse, INRA, CNRS, Castanet Tolosa, France4CRCT, INSERM, Université de Toulouse, CNRS, Toulous, France1LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosa, France1LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosa, FranceDe novo sequencing of complex genomes is one of the main challenges for researchers seeking high-quality reference sequences. Many de novo assemblies are based on short reads, producing fragmented genome sequences. Third-generation sequencing, with read lengths >10 kb, will improve the assembly of complex genomes, but these techniques require high-molecular-weight genomic DNA (gDNA), and gDNA extraction protocols used for obtaining smaller fragments for short-read sequencing are not suitable for this purpose. Methods of preparing gDNA for bacterial artificial chromosome (BAC) libraries could be adapted, but these approaches are time-consuming, and commercial kits for these methods are expensive. Here, we present a protocol for rapid, inexpensive extraction of high-molecular-weight gDNA from bacteria, plants, and animals. Our technique was validated using sunflower leaf samples, producing a mean read length of 12.6 kb and a maximum read length of 80 kb.https://www.future-science.com/doi/10.2144/000114460DNA extractionlong-read sequencingPacBio |
| spellingShingle | Baptiste Mayjonade Jérôme Gouzy Cécile Donnadieu Nicolas Pouilly William Marande Caroline Callot Nicolas Langlade Stéphane Muños Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules BioTechniques DNA extraction long-read sequencing PacBio |
| title | Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules |
| title_full | Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules |
| title_fullStr | Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules |
| title_full_unstemmed | Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules |
| title_short | Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules |
| title_sort | extraction of high molecular weight genomic dna for long read sequencing of single molecules |
| topic | DNA extraction long-read sequencing PacBio |
| url | https://www.future-science.com/doi/10.2144/000114460 |
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