Single-step electrohydrodynamic separation of 1-150 kbp in less than 5 min using homogeneous glass/adhesive/glass microchips.

Electrohydrodynamic migration, which is based on hydrodynamic actuation with an opposing electrophoretic force, enables the separation of DNA molecules of 3-100 kbp in glass capillary within 1 h. Here, we wish to enhance these performances using microchip technologies. This study starts with the fabrication of microchips with uniform surfaces, as motivated by our observation that band splitting occurs in microchannels made out of heterogeneous materials such as glass and silicon. The resulting glass-adhesive-glass microchips feature the highest reported bonding strength of 11 MPa for such materials (115 kgf/cm2), a high lateral resolution of critical dimension 5 µm, and minimal auto-fluorescence. These devices enable us to report the separation of 13 DNA bands in the size range of 1-150 kbp in one experiment of 5 min, i.e. 13 times faster than with capillary. In turn, we observe that bands split during electrohydrodynamic migration in heterogeneous glass-silicon but not in homogeneous glass-adhesive-glass microchips. We suggest that this effect arises from differential Electro-Osmotic Flow (EOF) in between the upper and lower walls of heterogeneous channels, and provide evidence that this phenomenon of differential EOF causes band broadening in electrophoresis during microchip electrophoresis. We finally prove that our electrohydrodynamic separation compares very favorably to microchip technologies in terms of resolution length and features the broadest analytical range reported so far.

A tunable filter for high molecular weight DNA selection and linked-read sequencing.

In third generation sequencing, the production of quality data requires the selection of molecules longer than ∼20 kbp, but the size selection threshold of most purification technologies is smaller than this target. Here, we describe a technology operated in a capillary with a tunable selection threshold in the range of 3 to 40 kbp controlled by an electric field. We demonstrate that the selection cut-off is sharp, the purification yield is high, and the purification throughput is scalable. We also provide an analytical model that the actuation settings of the filter. The selection of high molecular weight genomic DNA from the melon Cucumis melo L., a diploid organism of ∼0.45 Gbp, is then reported. Linked-read sequencing data show that the N50 phase block size, which scores the correct representation of two chromosomes, is enhanced by a factor of 2 after size selection, establishing the relevance and versatility of our technology.

A new Multi Locus Variable Number of Tandem Repeat Analysis Scheme for epidemiological surveillance of Xanthomonas vasicola pv. musacearum, the plant pathogen causing bacterial wilt on banana and enset.

Xanthomonas vasicola pv. musacearum (Xvm) which causes Xanthomonas wilt (XW) on banana (Musa accuminata x balbisiana) and enset (Ensete ventricosum), is closely related to the species Xanthomonas vasicola that contains the pathovars vasculorum (Xvv) and holcicola (Xvh), respectively pathogenic to sugarcane and sorghum. Xvm is considered a monomorphic bacterium whose intra-pathovar diversity remains poorly understood. With the sudden emergence of Xvm within east and central Africa coupled with the unknown origin of one of the two sublineages suggested for Xvm, attention has shifted to adapting technologies that focus on identifying the origin and distribution of the genetic diversity within this pathogen. Although microbiological and conventional molecular diagnostics have been useful in pathogen identification. Recent advances have ushered in an era of genomic epidemiology that aids in characterizing monomorphic pathogens. To unravel the origin and pathways of the recent emergence of XW in Eastern and Central Africa, there was a need for a genotyping tool adapted for molecular epidemiology. Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA) is able to resolve the evolutionary patterns and invasion routes of a pathogen. In this study, we identified microsatellite loci from nine published Xvm genome sequences. Of the 36 detected microsatellite loci, 21 were selected for primer design and 19 determined to be highly typeable, specific, reproducible and polymorphic with two- to four- alleles per locus on a sub-collection. The 19 markers were multiplexed and applied to genotype 335 Xvm strains isolated from seven countries over several years. The microsatellite markers grouped the Xvm collection into three clusters; with two similar to the SNP-based sublineages 1 and 2 and a new cluster 3, revealing an unknown diversity in Ethiopia. Five of the 19 markers had alleles present in both Xvm and Xanthomonas vasicola pathovars holcicola and vasculorum, supporting the phylogenetic closeliness of these three pathovars. Thank to the public availability of the haplotypes on the MLVABank database, this highly reliable and polymorphic genotyping tool can be further used in a transnational surveillance network to monitor the spread and evolution of XW throughout Africa.. It will inform and guide management of Xvm both in banana-based and enset-based cropping systems. Due to the suitability of MLVA-19 markers for population genetic analyses, this genotyping tool will also be used in future microevolution studies.