METHODS AND COMPOSITIONS FOR HIGH-THROUGHPUT BISULPHITE DNA-SEQUENCING AND UTILITIES

The invention relates to novel methods and compositions to produce DNA templates suitable for chemical modifications and high-throughput DNA-sequencing. A method of the invention relates to a DNA adaptor design where constituent deoxycytosines are substituted with 5-methyl-deoxycytosines rendering the resulting adaptor resistant to bisulphite mediated deamination. When said adaptor is ligated onto double stranded DNA template, subsequent DNA denaturation and bisulphite treatment deaminates template DNA deoxycytosine differentially to deoxyuraeil whilst the 5-methyl-deoxycytosines of the ligated adaptor resist chemical conversion resulting in the adaptor sequence remaining unaltered. Both strands of bisulphite treated DNA can thus be amplified with a single primer set that hybridizes to the unaltered adaptor sequence. The invention also relates to methods to produce control template of a defined methylation composition to optimize conditions for the bisulphite reaction. In a preferred embodiment, the present invention can be used to produce templates suitable for genome-wide bisulphite-DNA sequencing using conventional, Solexa(TM), SOLiD(TM) or 454(TM)-type DNA sequencing platforms to study DNA methylation.published_or_final_versio

Methods and compositions for high-throughput bisulphite DNA-sequencing and utilities

The novel methods and compositions to produce DNA templates suitable for chemical modifications and high-throughput DNA-sequencing are disclosed. A method of a DNA adaptor design where constituent cytosines are substituted with 5-methylcytosines rendering the resulting adaptor resistant to bisulphite mediated deamination is also disclosed. When said adaptor is ligated onto double stranded DNA template, subsequent DNA denaturation and bisulphite treatment deaminates template DNA cytosine differentially to uracil whilst the 5-methylcytosines of the ligated adaptor resist chemical conversion resulting in the adaptor sequence remaining unaltered. Both strands of bisulphite treated DNA can thus be amplified with a single primer set that hybridizes to the unaltered adaptor sequence. Also the methods to produce control template of a defined methylation composition to optimize conditions for the bisulphite reaction are disclosed. In a preferred embodiment, the present invent can be used to produce templates suitable for genome-wide bisulphite-DNA sequencing using conventional, SolexaTM, SOLiDTM or 454TM_ type DNA sequencing platforms to study DNA methylation.published_or_final_versio

METHODS AND COMPOSITIONS FOR HIGH-THROUGHPUT BISULPHITE DNA-SEQUENCING AND UTILITIES

The novel methods and compositions to produce DNA templates suitable for chemical modifications and high-throughput DNA-sequencing are disclosed. A method of a DNA adaptor design where constituent cytosines are substituted with 5-methylcytosines rendering the resulting adaptor resistant to bisulphite mediated deamination is also disclosed. When said adaptor is ligated onto double stranded DNA template, subsequent DNA denaturation and bisulphite treatment deaminates template DNA cytosine differentially to uracil whilst the 5-methylcytosines of the ligated adaptor resist chemical conversion resulting in the adaptor sequence remaining unaltered. Both strands of bisulphite treated DNA can thus be amplified with a single primer set that hybridizes to the unaltered adaptor sequence. Also the methods to produce control template of a defined methylation composition to optimize conditions for the bisulphite reaction are disclosed. In a preferred embodiment, the present invent can be used to produce templates suitable for genome-wide bisulphite-DNA sequencing using conventional, SolexaTM, SOLiDTM or 454TM_ type DNA sequencing platforms to study DNA methylation.published_or_final_versio

PlantID – DNA-based identification of multiple medicinal plants in complex mixtures

Background An efficient method for the identification of medicinal plant products is now a priority as the global demand increases. This study aims to develop a DNA-based method for the identification and authentication of plant species that can be implemented in the industry to aid compliance with regulations, based upon the economically important Hypericum perforatum L. (St John’s Wort or Guan ye Lian Qiao). Methods The ITS regions of several Hypericum species were analysed to identify the most divergent regions and PCR primers were designed to anneal specifically to these regions in the different Hypericum species. Candidate primers were selected such that the amplicon produced by each species-specific reaction differed in size. The use of fluorescently labelled primers enabled these products to be resolved by capillary electrophoresis. Results Four closely related Hypericum species were detected simultaneously and independently in one reaction. Each species could be identified individually and in any combination. The introduction of three more closely related species to the test had no effect on the results. Highly processed commercial plant material was identified, despite the potential complications of DNA degradation in such samples. Conclusion This technique can detect the presence of an expected plant material and adulterant materials in one reaction. The method could be simply applied to other medicinal plants and their problem adulterants

Multiplex dispensation order generation for pyrosequencing

This paper introduces the multiplex dispensation order generation problem, a real-life combinatorial problem that arises in the context of analyzing large numbers of short to medium length DNA sequences. The problem is modeled as a constraint optimization problem (COP). We present the COP, its constraint programming formulation, and a custom search procedure. We give some experimental data supporting our design decisions. One of the lessons learnt from this study is that the ease with which the relevant constraints are expressed can be a crucial factor in making design decisions in the COP model

Screening for the optimal siRNA targeting a novel gene (HA117) and construction and evaluation of a derivative recombinant adenovirus

We found a novel gene named as HA117 in our previous research. At this study, we screened for an optimal siRNA targeting the novel gene HA117 using the pSOS-HUS method, verified the results of pSOS-HUS siRNA screening for optimal affinity for the target gene, and constructed and evaluated a recombinant adenovirus carrying the DNA template for transcription of the optimal HA117 siRNA. The pSOS-HUS vector method was successfully utilized as a rapid and effective screen for an optimal siRNA for a target gene. Among five pairs of DNA templates, siRNA transcribed from HAi5 gave the strongest interference with the novel gene HA117; a HAi5-carrying recombinant adenovirus (Ad-HAi5) was successfully constructed and evaluated, laying a foundation for the further study of HA117 gene function with RNAi technology

Design, modeling and synthesis of an in vitro transcription rate regulatory circuit

This paper describes the design, modeling and realization of a synthetic in vitro circuit that aims at regulating the rate of mRNA transcription. Two DNA templates are designed to interact through their transcripts, creating negative feedback loops that will equate their transcription rates at steady state. A mathematical model is developed for this circuit, consisting of a set of ODEs derived from the mass action laws and Michaelis-Menten kinetics involving all the present chemical species. The DNA strands were accordingly designed, following thermodynamics principles and minimizing unwanted interactions. Preliminary experimental results show that the circuit is performing the expected task, by matching at steady state the transcription rates of the two DNA templates

Reprogramming human T cell function and specificity with non-viral genome targeting.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells

Synthetic in vitro transcriptional oscillators

The construction of synthetic biochemical circuits from simple components illuminates how complex behaviors can arise in chemistry and builds a foundation for future biological technologies. A simplified analog of genetic regulatory networks, in vitro transcriptional circuits, provides a modular platform for the systematic construction of arbitrary circuits and requires only two essential enzymes, bacteriophage T7 RNA polymerase and Escherichia coli ribonuclease H, to produce and degrade RNA signals. In this study, we design and experimentally demonstrate three transcriptional oscillators in vitro. First, a negative feedback oscillator comprising two switches, regulated by excitatory and inhibitory RNA signals, showed up to five complete cycles. To demonstrate modularity and to explore the design space further, a positive-feedback loop was added that modulates and extends the oscillatory regime. Finally, a three-switch ring oscillator was constructed and analyzed. Mathematical modeling guided the design process, identified experimental conditions likely to yield oscillations, and explained the system's robust response to interference by short degradation products. Synthetic transcriptional oscillators could prove valuable for systematic exploration of biochemical circuit design principles and for controlling nanoscale devices and orchestrating processes within artificial cells

BATCH-GE : batch analysis of next-generation sequencing data for genome editing assessment

Targeted mutagenesis by the CRISPR/Cas9 system is currently revolutionizing genetics. The ease of this technique has enabled genome engineering in-vitro and in a range of model organisms and has pushed experimental dimensions to unprecedented proportions. Due to its tremendous progress in terms of speed, read length, throughput and cost, Next-Generation Sequencing (NGS) has been increasingly used for the analysis of CRISPR/Cas9 genome editing experiments. However, the current tools for genome editing assessment lack flexibility and fall short in the analysis of large amounts of NGS data. Therefore, we designed BATCH-GE, an easy-to-use bioinformatics tool for batch analysis of NGS-generated genome editing data, available from https://github.com/WouterSteyaert/BATCH-GE.git. BATCH-GE detects and reports indel mutations and other precise genome editing events and calculates the corresponding mutagenesis efficiencies for a large number of samples in parallel. Furthermore, this new tool provides flexibility by allowing the user to adapt a number of input variables. The performance of BATCH-GE was evaluated in two genome editing experiments, aiming to generate knock-out and knock-in zebrafish mutants. This tool will not only contribute to the evaluation of CRISPR/Cas9-based experiments, but will be of use in any genome editing experiment and has the ability to analyze data from every organism with a sequenced genome