title: Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules.
abstract: 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.
authors.affiliation: LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosan, France.
authors.forename: Baptiste
authors.initials: B
authors.lastname: Mayjonade
authors.affiliation: LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosan, France.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Gouzy
authors.affiliation: Get-PLAGE, Université de Toulouse, INRA, CNRS, Castanet Tolosan, France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Donnadieu
authors.affiliation: LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosan, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Pouilly
authors.affiliation: CNRGV, Université de Toulouse, INRA, CNRS, Castanet Tolosan, France.
authors.forename: William
authors.initials: W
authors.lastname: Marande
authors.affiliation: CRCT, INSERM, Université de Toulouse, CNRS, Toulouse, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Callot
authors.affiliation: LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosan, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Langlade
authors.affiliation: LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosan, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Muños
mesh_terms: D000818:Animals; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification; D016678:Genome / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D008970:Molecular Weight; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004269:DNA, Bacterial; D018744:DNA, Plant; D004247:DNA
keywords: DNA extraction; PacBio; long-read sequencing