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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.

Author information

1
LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosan, France.
2
Get-PLAGE, Université de Toulouse, INRA, CNRS, Castanet Tolosan, France.
3
CNRGV, Université de Toulouse, INRA, CNRS, Castanet Tolosan, France.
4
CRCT, INSERM, Université de Toulouse, CNRS, Toulouse, France.

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.

KEYWORDS:

DNA extraction; PacBio; long-read sequencing

PMID:
27712583
DOI:
10.2144/000114460
[Indexed for MEDLINE]
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