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Biotechniques. 2016 Oct 1;61(4):203-205.

Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules.

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LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosan, France.
Get-PLAGE, Université de Toulouse, INRA, CNRS, Castanet Tolosan, France.
CNRGV, Université de Toulouse, INRA, CNRS, Castanet Tolosan, France.
CRCT, INSERM, Université de Toulouse, CNRS, Toulouse, France.


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.


DNA extraction; PacBio; long-read sequencing

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