A toolkit for enhanced reproducibility of RNASeq analysis for synthetic biologists.

Sequencing technologies, in particular RNASeq, have become critical tools in the design, build, test and learn cycle of synthetic biology. They provide a better understanding of synthetic designs, and they help identify ways to improve and select designs. While these data are beneficial to design, their collection and analysis is a complex, multistep process that has implications on both discovery and reproducibility of experiments. Additionally, tool parameters, experimental metadata, normalization of data and standardization of file formats present challenges that are computationally intensive. This calls for high-throughput pipelines expressly designed to handle the combinatorial and longitudinal nature of synthetic biology. In this paper, we present a pipeline to maximize the analytical reproducibility of RNASeq for synthetic biologists. We also explore the impact of reproducibility on the validation of machine learning models. We present the design of a pipeline that combines traditional RNASeq data processing tools with structured metadata tracking to allow for the exploration of the combinatorial design in a high-throughput and reproducible manner. We then demonstrate utility via two different experiments: a control comparison experiment and a machine learning model experiment. The first experiment compares datasets collected from identical biological controls across multiple days for two different organisms. It shows that a reproducible experimental protocol for one organism does not guarantee reproducibility in another. The second experiment quantifies the differences in experimental runs from multiple perspectives. It shows that the lack of reproducibility from these different perspectives can place an upper bound on the validation of machine learning models trained on RNASeq data. Graphical Abstract.

Prediction of whole-cell transcriptional response with machine learning.

MOTIVATION: Applications in synthetic and systems biology can benefit from measuring whole-cell response to biochemical perturbations. Execution of experiments to cover all possible combinations of perturbations is infeasible. In this paper, we present the host response model (HRM), a machine learning approach that maps response of single perturbations to transcriptional response of the combination of perturbations. RESULTS: The HRM combines high-throughput sequencing with machine learning to infer links between experimental context, prior knowledge of cell regulatory networks, and RNASeq data to predict a gene's dysregulation. We find that the HRM can predict the directionality of dysregulation to a combination of inducers with an accuracy of >90% using data from single inducers. We further find that the use of prior, known cell regulatory networks doubles the predictive performance of the HRM (an R2 from 0.3 to 0.65). The model was validated in two organisms, Escherichia coli and Bacillus subtilis, using new experiments conducted after training. Finally, while the HRM is trained with gene expression data, the direct prediction of differential expression makes it possible to also conduct enrichment analyses using its predictions. We show that the HRM can accurately classify >95% of the pathway regulations. The HRM reduces the number of RNASeq experiments needed as responses can be tested in silico prior to the experiment. AVAILABILITY AND IMPLEMENTATION: The HRM software and tutorial are available at https://github.com/sd2e/CDM and the configurable differential expression analysis tools and tutorials are available at https://github.com/SD2E/omics_tools. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

An intelligent bioreactor system for the cultivation of a bioartificial vascular graft.

Cardiovascular disease is the most common cause of death, accounting for 31% of deaths worldwide. As purely synthetic grafts implicate concomitant anticoagulation and autologous veins are rare, tissue-engineered vascular grafts are urgently needed. For successful in vitro cultivation of a bioartificial vascular graft, the suitable bioreactor should provide conditions comparable to vasculogenesis in the body. Such a system has been developed and characterized under continuous and pulsatile flow, and a variety of sensors has been integrated into the bioreactor to control parameters such as temperature, pressure up to 500 mbar, glucose up to 4.5 g/L, lactate, oxygen up to 150 mbar, and flow rate. Wireless data transfer (using the ZigBee specification based on the IEEE 802.15.4 standard) and multiple corresponding sensor signal processing platforms have been implemented as well. Ultrasound is used for touchless monitoring of the growing vascular structure as a quality control before implantation (maximally achieved ultrasound resolution 65 µm at 15 MHz). To withstand the harsh conditions of steam sterilization (120°C for 20 min), all electronics were encapsulated. With such a comprehensive physiologically conditioning, sensing, and imaging bioreactor system, all the requirements for a successful cultivation of vascular grafts are available now.

Greater fibroblast proliferation on an ultrasonicated ZnO/PVC nanocomposite material.

There has been a significant and growing concern over nosocomial medical device infections. Previous studies have demonstrated that embedding nanoparticles alone (specifically, zinc oxide [ZnO]) in conventional polymers (eg, polyvinyl chloride [PVC]) can decrease bacteria growth and may have the potential to prevent or disrupt bacterial processes that lead to infection. However, little to no studies have been conducted to determine mammalian cell functions on such a nanocomposite material. Clearly, for certain medical device applications, maintaining healthy mammalian cell functions while decreasing bacteria growth is imperative (yet uncommon). For this reason, in the presented study, ZnO nanoparticles of varying sizes (from 10 nm to >200 nm in diameter) and functionalization (including no functionalization to doping with aluminum oxide and functionalizing with a silane coupling agent KH550) were incorporated into PVC either with or without ultrasonication. Results of this study provided the first evidence of greater fibroblast density after 18 hours of culture on the smallest ZnO nanoparticle incorporated PVC samples with dispersion aided by ultrasonication. Specifically, the greatest amount of fibroblast proliferation was measured on ZnO nanoparticles functionalized with a silane coupling agent KH550; this sample exhibited the greatest dispersion of ZnO nanoparticles. Water droplet tests showed a general trend of decreased hydrophilicity when adding any of the ZnO nanoparticles to PVC, but an increase in hydrophilicity (albeit still below controls or pure PVC) when using ultrasonication to increase ZnO nanoparticle dispersion. Future studies will have to correlate this change in wettability to initial protein adsorption events that may explain fibroblast behavior. Mechanical tests also provided evidence of the ability to tailor mechanical properties of the ZnO/PVC nanocomposites through the use of the different ZnO nanoparticles. Coupled with previous antibacterial studies, the present study demonstrated that highly dispersed ZnO/PVC nanocomposite materials should be further studied for numerous medical device applications.