Extraction of High-Molecular-Weight Genomic DNA for Long-Read Sequencing of Single Molecules

Mayjonade, Baptiste; Gouzy, Jérôme; Donnadieu, Cécile; Pouilly, Nicolas; Marande, William; Callot, Caroline; Langlade, Nicolas; Muños, Stéphane

doi:10.2144/000114460

Cited by 178 publications

(139 citation statements)

References 8 publications

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“…Bacterial growth and genomic DNA extraction for the R. solanacearum GMI1000 strain was performed in the present work. The protocol used to extract DNA from the GMI1000 strain was derived from the protocol described in Mayjonade et al (2016). Briefly, bacteria were grown overnight in 50 ml MP minimal medium (FeSO 4 , 7H 2 O, 1.25 × 10 −4 g/l; (NH 4 ) 2 SO 4 , 0.5 g/l; MgSO 4 .…”

Section: Methodsmentioning

confidence: 99%

Comparative Analysis of Ralstonia solanacearum Methylomes

et al. 2017

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Ralstonia solanacearum is an important soil-borne plant pathogen with broad geographical distribution and the ability to cause wilt disease in many agriculturally important crops. Genome sequencing of multiple R. solanacearum strains has identified both unique and shared genetic traits influencing their evolution and ability to colonize plant hosts. Previous research has shown that DNA methylation can drive speciation and modulate virulence in bacteria, but the impact of epigenetic modifications on the diversification and pathogenesis of R. solanacearum is unknown. Sequencing of R. solanacearum strains GMI1000 and UY031 using Single Molecule Real-Time technology allowed us to perform a comparative analysis of R. solanacearum methylomes. Our analysis identified a novel methylation motif associated with a DNA methylase that is conserved in all complete Ralstonia spp. genomes and across the Burkholderiaceae, as well as a methylation motif associated to a phage-borne methylase unique to R. solanacearum UY031. Comparative analysis of the conserved methylation motif revealed that it is most prevalent in gene promoter regions, where it displays a high degree of conservation detectable through phylogenetic footprinting. Analysis of hyper- and hypo-methylated loci identified several genes involved in global and virulence regulatory functions whose expression may be modulated by DNA methylation. Analysis of genome-wide modification patterns identified a significant correlation between DNA modification and transposase genes in R. solanacearum UY031, driven by the presence of a high copy number of ISrso3 insertion sequences in this genome and pointing to a novel mechanism for regulation of transposition. These results set a firm foundation for experimental investigations into the role of DNA methylation in R. solanacearum evolution and its adaptation to different plants.

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Section: Methodsmentioning

confidence: 99%

Comparative Analysis of Ralstonia solanacearum Methylomes

et al. 2017

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“…However Vo and Jedlicka (2014) found SPRI to only have improved performance with less contaminated samples, such as avian oral and cloacal swab extractions. SPRI DNA purification is therefore best reserved for relatively clean environmental sample types, in particular clear lake and stream waters or tissue samples (see Mayjonade et al 2016).…”

Section: Adaptability Of Mu-dnamentioning

confidence: 99%

Mu-DNA: a modular universal DNA extraction method adaptable for a wide range of sample types

Sellers¹,

Muri²,

Gómez³

et al. 2018

MBMG

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Efficient DNA extraction is fundamental to molecular studies. However, commercial kits are expensive when a large number of samples need to be processed. Here we present a simple, modular and adaptable DNA extraction 'toolkit' for the isolation of high purity DNA from multiple sample types (modular universal DNA extraction method or Mu-DNA). We compare the performance of our method to that of widely used commercial kits across a range of soil, stool, tissue and water samples. Mu-DNA produced DNA extractions of similar or higher yield and purity to that of the commercial kits. As a proof of principle, we carried out replicate fish metabarcoding of aquatic eDNA extractions, which confirmed that the species detection efficiency of our method is similar to that of the most frequently used commercial kit. Our results demonstrate the reliability of Mu-DNA along with its modular adaptability to challenging sample types and sample collection methods. Mu-DNA can substantially reduce the costs and increase the scope of experiments in molecular studies.

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“…In genome sequencing, best practices for high molecular weight DNA extraction have often been discussed (e.g., [24]). This factor is fundamental to building longer contigs, whether employing short-read or long-read sequencing platforms.…”

Section: Starting Materials: Not Genomic Dna Extraction But In Situ Cmentioning

confidence: 99%

“…Statistics of Chinese softshell turtle draft genome assembly before Hi-C. Supplementary Table S2. HiC-Pro results of the human GM12878 HindIII Hi-C library with reduced reads 24 Supplementary Table S3. Quality control of Hi-C libraries.…”

Section: Continuity Assessment With Rna-seq Read Mappingmentioning

confidence: 99%

Multifaceted Hi-C benchmarking: what makes a difference in chromosome-scale genome scaffolding?

Kadota

Nishimura

Miura

et al. 2019

Preprint

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Background: Hi-C, a derivative of chromosome conformation capture (3C) targeting the whole genome, was originally developed as a means for characterizing chromatin conformation. More recently, this method has also been frequently employed in elongating nucleotide sequences obtained by de novo genome sequencing and assembly, in which the number of resultant sequences rarely converge into the chromosome number. Despite the prevailing and irreplaceable use, sample preparation methods for Hi-C have not been intensively discussed, especially from the standpoint of genome scaffolding. Results:To gain insights into the best practice of Hi-C scaffolding, we performed a multifaceted methodological comparison using vertebrate samples and optimized various factors during sample preparation, sequencing, and computation. As a result, we have identified some key factors that help improve Hi-C scaffolding including the choice and preparation of tissues, library preparation conditions, and restriction enzyme(s), as well as the choice of scaffolding program and its usage. Conclusions:This study provides the first comparison of multiple sample preparation kits/protocols and computational programs for Hi-C scaffolding, by an academic third party. We introduce a customized protocol designated the 'inexpensive and controllable Hi-C (iconHi-C) protocol', in which the optimal conditions revealed by this study have been incorporated, and release the resultant chromosome-scale genome assembly of the Chinese softshell turtle Pelodiscus sinensis.

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Extraction of High-Molecular-Weight Genomic DNA for Long-Read Sequencing of Single Molecules

Cited by 178 publications

References 8 publications

Comparative Analysis of Ralstonia solanacearum Methylomes

Comparative Analysis of Ralstonia solanacearum Methylomes

Mu-DNA: a modular universal DNA extraction method adaptable for a wide range of sample types

Multifaceted Hi-C benchmarking: what makes a difference in chromosome-scale genome scaffolding?

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