Abstract
Long-read sequencing technologies are having a major impact on our approaches to studying non-model organisms and microbial communities. By significantly reducing the cost and facilitating the genome assembly pipelines, any laboratory can now develop its own genomics program regardless of the complexity of the genome studied. The most crucial current challenge is to develop efficient protocols for extracting genomic DNA (gDNA) with high quality and integrity adapted to the organism of interest. This can be particularly complex for obligate pathogens that must maintain intimate interactions inside infected host tissues. Here we propose a simple and cost-effective method for high molecular weight gDNA extraction from spores of Plasmopara halstedii, an obligate biotroph oomycete pathogen responsible for downy mildew in sunflower. We optimized the yield, the quality and the integrity of the extracted gDNA by fine-tuning three critical parameters, the grinding, the lysis temperature and the lysis duration. We obtained gDNA with a fragment size distribution reaching a peak ranging from 79 to 145 kb. More than half of the extracted gDNA consisted of DNA fragments larger than 42 kb, with 23% of fragments larger than 100 kb. We then demonstrated the relevance of this protocol for long-read sequencing using PacBio RSII technology. With this protocol, we were able to obtain a mean read length of 9.3 kb, a max read length of 71 kb and an N50 of 13.3 kb. The development of such DNA extraction protocols is an essential prerequisite for fully exploiting technologies requiring high molecular weight gDNA (e.g. long-read sequencing or optical mapping). These technological advances will help generate data to answer questions such as the role of newly duplicated gene clusters, repeated regions, genomic structural variations or to define number of chromosomes that still remains undefined in many species of pathogenic fungi and oomycetes.
Highlights
The biological sciences have undergone a revolution thanks firstly to Sanger sequencing and to high-throughput second-generation sequencing called Generation Sequencing (NGS) which has enabled to decipher genomic DNA from any organism to be deciphered and to broaden the use of genome-wide studies
Grinding was essential for an efficient genomic DNA (gDNA) extraction since 10 s of grinding led to an extract with insufficient amounts of gDNA with an average of 19.3 ng/mg, while the yield increased from 38.4 ng/mg after 30 s to 76.5 ng/mg after 90 s of grinding (Fig. 1A)
The A260/A230 ratio measurements indicated that grinding had a significant impact on gDNA purity since a sufficient quality was only reached from 30 s of grinding
Summary
The biological sciences have undergone a revolution thanks firstly to Sanger sequencing and to high-throughput second-generation sequencing called Generation Sequencing (NGS) which has enabled to decipher genomic DNA (gDNA) from any organism to be deciphered and to broaden the use of genome-wide studies. The most widespread NGS technologies were based on short read sequencing, typically of 50–300 nucleotides. These technologies required the development of complex bioinformatic pipelines to obtain the assembly of millions of short sequences into genomes. The limit of NGS had been reached since no assembler program can solve the problem of repeated elements. Short read sequencing can only give a fragmented genome view since whole genomic regions can be masked and structural variations, such as indels or complex chromosomal rearrangements, are highly underestimated.
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