Illumina mate-paired DNA sequencing-library preparation using Cre-Lox recombination

Standard Illumina mate-paired libraries are constructed from 3- to 5-kb DNA fragments by a blunt-end circularization. Sequencing reads that pass through the junction of the two joined ends of a 3-5-kb DNA fragment are not easy to identify and pose problems during mapping and de novo assembly. Longer read lengths increase the possibility that a read will cross the junction. To solve this problem, we developed a mate-paired protocol for use with Illumina sequencing technology that uses Cre-Lox recombination instead of blunt end circularization. In this method, a LoxP sequence is incorporated at the junction site. This sequence allows screening reads for junctions without using a reference genome. Junction reads can be trimmed or split at the junction. Moreover, the location of the LoxP sequence in the reads distinguishes mate-paired reads from spurious paired-end reads. We tested this new method by preparing and sequencing a mate-paired library with an insert size of 3 kb from Saccharomyces cerevisiae. We present an analysis of the library quality statistics and a new bio-informatics tool called DeLoxer that can be used to analyze an IlluminaCre-Lox mate-paired data set. We also demonstrate how the resulting data significantly improves a de novo assembly of the S. cerevisiae genome

Chemical synthesis, DNA incorporation and biological study of a new photocleavable 2′-deoxyadenosine mimic

The phototriggered cleavage of chemical bonds has found numerous applications in biology, particularly in the field of gene sequencing through photoinduced DNA strand scission. However, only a small number of modified nucleosides that are able to cleave DNA at selected positions have been reported in the literature. Herein, we show that a new photoactivable deoxyadenosine analogue, 3-nitro-3-deaza-2′-deoxyadenosine (d(3-NiA)), was able to induce DNA backbone breakage upon irradiation (λ > 320 nm). The d(3-NiA) nucleoside was chemically incorporated at desired positions into 40-mer oligonucleotides as a phosphoramidite monomer and subsequent hybridization studies confirmed that the resulting modified duplexes display a behaviour that is close to that of the related natural sequence. Enzymatic action of the Klenow fragment exonuclease free revealed the preferential incorporation of dAMP opposite the 3-NiA base. On the other hand, incorporation of the analogous 3-NiA triphosphate to a primer revealed high enzyme efficiency and selectivity for insertion opposite thymine. Furthermore, only the enzymatically synthesized base pair 3-NiA:T was a substrate for further extension by the enzyme. All the hybridization and enzymatic data indicate that this new photoactivable 3-NiA triphosphate can be considered as a photochemically cleavable dATP analogue

An MBSE Approach for Development of Resilient Automated Automotive Systems

Advanced driver assistance and automated driving systems must operate in complex environments and make safety-critical decisions. Resilient behavior of these systems in their targeted operation design domain is essential. In this paper, we describe developments in our Model-Based Systems Engineering (MBSE) approach to develop resilient safety-critical automated systems. An MBSE approach provides the ability to provide guarantees about system behavior and potentially reduces dependence on in-vehicle testing through the use of rigorous models and extensive simulation. We are applying MBSE methods to two key aspects of developing resilient systems: (1) ensuring resilient behavior through the use of Resilience Contracts for system decision making; and (2) applying simulation-based testing methods to verify the system handles all known scenarios and to validate the system against potential unknown scenarios. Resilience Contracts make use of contract-based design methods and Partially Observable Markov Decision Processes (POMDP), which allow the system to model potential uncertainty in the sensed environment and thus make more resilient decisions. The simulation-based testing methodology provides a structured approach to evaluate the operation of the target system in a wide variety of operating conditions and thus confirm that the expected resilient behavior has indeed been achieved. This paper provides details on the development of a utility function to support Resilience Contracts and outlines the specific test methods used to evaluate known and unknown operating scenarios. Document type: Articl

Quantitative Bias in Illumina TruSeq and a Novel Post Amplification Barcoding Strategy for Multiplexed DNA and Small RNA Deep Sequencing

Here we demonstrate a method for unbiased multiplexed deep sequencing of RNA and DNA libraries using a novel, efficient and adaptable barcoding strategy called Post Amplification Ligation-Mediated (PALM). PALM barcoding is performed as the very last step of library preparation, eliminating a potential barcode-induced bias and allowing the flexibility to synthesize as many barcodes as needed. We sequenced PALM barcoded micro RNA (miRNA) and DNA reference samples and evaluated the quantitative barcode-induced bias in comparison to the same reference samples prepared using the Illumina TruSeq barcoding strategy. The Illumina TruSeq small RNA strategy introduces the barcode during the PCR step using differentially barcoded primers, while the TruSeq DNA strategy introduces the barcode before the PCR step by ligation of differentially barcoded adaptors. Results show virtually no bias between the differentially barcoded miRNA and DNA samples, both for the PALM and the TruSeq sample preparation methods. We also multiplexed miRNA reference samples using a pre-PCR barcode ligation. This barcoding strategy results in significant bias

An Efficient Lanthanide-Dependent DNAzyme Cleaving 2 '-5 '-Linked RNA

This is the peer reviewed version of the following article: Zhou, W., Ding, J., & Liu, J. (2016). An Efficient Lanthanide-Dependent DNAzyme Cleaving 2 ’-5 ’-Linked RNA. Chembiochem, 17(10), 890–894, which has been published in final form at https://doi.org/10.1002/cbic.201500690. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.RNA can form two types of linkage. In addition to the predominant 3'-5' linkage, 2'-5'-linked RNA is also important in biology, medicine, and prebiotic studies. Here, in vitro selection was used to isolate a DNAzyme that specifically cleaves 2'-5' RNA by using Ce3+ as the metal cofactor, but leaves the 3'-5' counterpart intact. This Ce5 DNAzyme requires trivalent light lanthanide ions and shows a rate of 0.16 min(-1) in the presence of 10 mm Ce3+; the activity decreases with heavier lanthanide ions. This is the fastest DNAzyme reported for this reaction, and it might enable applications in chemical biology. As a proof-of-concept, using this DNAzyme, the reactions between phosphorothioate-modified RNA and strongly thiophilic metals (Hg2+ and Tl3+) were studied as a function of pH.University of Waterloo; Natural Sciences and Engineering Research Council of Canada (NSERC); Foundation for Shenghua Scholar of Central South University; National Natural Science Foundation of China [21301195]; China Scholarship Council (CSC) [201406370116

Model-Based Approach to Engineering Resilience in Multi-UAV Systems

Multi-UAV Operations are an area of great interest in government, industry, and research community. In multi-UAV operations, a group of unmanned aerial vehicles (UAVs) are deployed to carry out missions such as search and rescue or disaster relief. As multi-UAV systems operate in an open operational environment, many disrupting events can occur. To this end, resilience of these systems is of great importance. The research performed and reported in this paper utilizes simulation-based research methodology and demonstrates that resilience of multi-UAV systems can be achieved by real-time evaluation of resilience alternatives during system operation. This evaluation is done using a dynamic utility function where priorities change as a function of context. Simulation results show that resilience response can in fact change depending on the context