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Next-generation sequencing (NGS) has caused a revolution, yet left a gap: long-range genetic information from native, non-amplified DNA fragments is unavailable. It might be obtained by optical mapping of megabase-sized DNA molecules. Frequently only a specific genomic region is of interest, so here we introduce a method for selection and enrichment of megabase-sized DNA molecules intended for single-molecule optical mapping: DNA from a human cell line is digested by the NotI rare-cutting enzyme and size-selected by pulsed-field gel electrophoresis. For demonstration, more than 600 sub-megabase- to megabase-sized DNA molecules were recovered from the gel and analysed by denaturation-renaturation optical mapping. Size-selected molecules from the same gel were sequenced by NGS. The optically mapped molecules and the NGS reads showed enrichment from regions defined by NotI restriction sites. We demonstrate that the unannotated genome can be characterized in a locus-specific manner via molecules partially overlapping with the annotated genome. The method is a promising tool for investigation of structural variants in enriched human genomic regions for both research and diagnostic purposes. Our enrichment method could potentially work with other genomes or target specified regions by applying other genomic editing tools, such as the CRISPR/Cas9 system.

Humidity is a key parameter in controlling drying processes and ambient conditions in many industrial manufacturing, storage and test applications. Air humidity is routinely measured at temperatures above 100 °C and at conditions that are often challenging due to temporal and local variations. Calibrations of humidity sensors do not provide appropriate representativeness of measurement conditions because they are limited to temperatures below 100 °C and static conditions. A European metrology research project HIT (“Metrology for Humidity at High Temperatures and Transient conditions”) is developing improved humidity measurement and calibration techniques to temperatures up to 180 °C and non-static conditions. This paper summaries developments of the project: calibration and test facilities for industrial hygrometers, studies on humidity control in specific microbial transient processes and a new measurement approach for water activity measurements.

Bien que les mesures de teneur en eau soient largement utilisees dans l’industrie, les considerations metrologiques quant a cette mesure ne sont pas completement abouties de sorte a fournir des mesures fiables et tracables au SI. Afin de remedier a ceci, le projet de recherche conjoint, Joint Research Project SIB64 “METefnet - Metrology for moisture in materials”, est actuellement en cours, et contribue au programme europeen de recherche en metrologie European Metrology Research Programme. Le projet METefnet a pour objectifs de developper et d’ameliorer l’approche metrologique de ce sujet. Ceci inclus notamment: le travail sur de nouvelles methodes de reference pour evaluer la fraction massique en eau, l’amelioration des mesures mettant en œuvre la methode primaire de type titration Karl Fischer, le developpement de nouveaux materiaux de reference certifies presentant une tres bonne stabilite et permettant une tracabilite au SI, le developpement de nouveaux etalons de transfert, la realisation d’etudes visant a quantifier et reduire les effets lies a la prise d’echantillon, son transport et sa manipulation, le developpement d’une nouvelle methode pour etalonner les instruments mesurant l’humidite de surface, et l’amelioration des methodes d’estimation d’incertitudes de ces mesures. Ce travail, realise dans le domaine de la metrologie de l’humidite au sein des materiaux, couvre a la fois le mesurande decrit comme etant specifiquement la teneur en eau, seule, dans les materiaux, mais egalement un mesurande plus large pouvant inclure l’eau ainsi que d’autres liquides ou composes organiques volatiles; ceci afin de bien mettre en exergue la difference qui peut etre observee entre ces deux mesurandes. Le projet global a pour objectif de soutenir une action de dissemination et de tracabilite au systeme SI des mesures de teneur en eau dans les materiaux avec une exactitude optimale et de developper une infrastructure metrologique coherente pour ce type de mesures. Le travail technique ainsi que les dernieres avancees vous sont ainsi presentees.

Les hygromètres sont largement mis en oeuvre pour des conditions de température supérieures à 100°C et dans des conditions de régimes dynamiques. Cependant, la plupart des laboratoires européens ne peuvent fournir d'étalonnages qu'à des températures relativement basses et dans des conditions de régimes établi et stables. Il apparaît donc nécessaire de développer de nouvelles techniques et méthodes d'étalonnage de sorte à élargir la gamme des possibilités d'étalonnage et de garantir ainsi une traçabilité qui soit en adéquation avec les besoins industriels. Ces développements sont d'autant plus importants vis-à-vis des besoins de mesure à hautes températures, en régimes dynamiques et pour les mesures d'activité de l'eau. Les conditions de mesures d'humidité en régimes transitoires et dynamiques sont une composante à part entière des environnements industriels d'essai et de test et il n'existe actuellement aucune méthode permettant l'estimation d'incertitudes dans de telles situations. Cet article met en avant un nouveau projet européen de recherche en métrologie - HIT: "Metrology for Humidity at high Temperature and Transient conditions". Ce projet a été construit de sorte à améliorer significativement l'exactitude des mesures d'humidité jusqu'à des températures de 180°C et en régimes dynamiques en contexte industriel, au travers de l'amélioration des techniques de mesure et d'étalonnage. Les principaux bénfices que doit apporter ce projet, sont l'extension des gammes d'étalonnages industriels à haute température et en régimes dynamiques, l'établissement de nouvelles méthodes de mesure d'humidité en régime dynamique et en présence de variations spatiales ainsi que le développement de nouvelles méthodes de mesure d'activité de l'eau "en ligne" sur les procédés industriels. L'ensemble de ces développements seront validés et illustrés au travers d'applications industrielles.

We demonstrate all-polymer injection molded devices for optical mapping of denaturation–renaturation (DR) patterns on long, single DNA-molecules from the human genome. The devices have channels with ultra-low aspect ratio, only 110 nm deep while 20 μm wide, and are superior to the silica devices used previously in the field. With these polymer devices, we demonstrate on-chip recording of DR images of DNA-molecules stretched to more than 95% of their contour length. The stretching is done by opposing flows Marie et al (2013 Proc. Natl Acad. Sci. USA 110 4893–8). The performance is validated by mapping 20 out of 24 Mbp-long DNA fragments to the human reference genome. We optimized fabrication of the devices to a yield exceeding 95%. This permits a substantial economies-of-scale driven cost-reduction, leading to device costs as low as 3 USD per device, about a factor 70 lower than the cost of silica devices. This lowers the barrier to a wide use of DR mapping of native, megabase-size DNA molecules, which has a huge potential as a complementary method to next-generation sequencing.

Rapid prototyping is desirable when developing products. One example of such a product is all-polymer, passive flow controlled lab-on-a-chip systems that are preferential when developing low-cost disposable chips for point-of-care use. In this paper we investigate the following aspects of going from rapid prototyping to pilot (mass) production. (1) Fabrication of an all-polymer microfluidic system using a rapid prototyped master insert for injection moulding and ultrasonic welding, including a systematic experimental characterisation of chip featured geometric capillary microvalve test structures. (2) Numerical modelling of the microvalve burst pressures. Numerical modelling of burst pressures is challenging due to its non-equilibrium nature. We have implemented and tested the level-set method modified with a damped driving term and show that the introduction of the damping term leads to numerically robust results with limited computational demands and a low number of iterations. Numerical and simplified analytical results are validated against the experimental results. We find that injection moulding and ultrasonic welding are effective for chip production and that the experimental burst pressures could be estimated with an average accuracy of 5% using the presented numerical model.

Nanochannel technology, coupled with a suitable DNA labeling chemistry, is a powerful approach for performing high-throughput single-molecule mapping of genomes. Yet so far nanochannel technology has remained inaccessible to the broader research community due to high fabrication cost and/or requirement of specialized facilities/skill-sets. In this article we show that nanochannel-based mapping can be performed in all polymer chips fabricated via injection molding: a fabrication process so inexpensive that the devices can be considered disposable. Fluorescent intensity variations can be obtained from molecules extended in the polymer nanochannels via chemical counterstaining against YOYO-1. In particular, we demonstrate that the counterstaining induced fluorescent intensity variations to a large degree appear to be proportional to the theoretically computed sequence-maps of both local AT and GC variation along DNA sequences.

Microfabricated single-cell capture and DNA stretching devices have been produced by injection molding. The fabrication scheme employed deep reactive ion etching in a silicon substrate, electroplating in nickel and molding in cyclic olefin polymer. This work proposes technical solutions to fabrication challenges associated with chip sealing and demolding of polymer high-volume replication methods. UV-assisted thermal bonding was found to ensure a strong seal of the microstructures in the molded part without altering the geometry of the channels. In the DNA stretching device, a low aspect ratio nanoslit (1/200) connecting two larger micro-channels was used to stretch a 168.5 kbp DNA molecule, while in the other device single-HeLa cells were captured against a micro-aperture connecting two larger microfluidic channels. Different dry etching processes have been investigated for the master origination of the cell-capture device. The combination of a modified Bosch process and an isotropic polysilicon etch was found to ensure the ease of demolding by resulting in slightly positively tapered sidewalls with negligible undercut at the mask interface. (Some figures may appear in colour only in the online journal)