Visualization and analysis of RNA-Seq assembly graphs.

RNA-Seq is a powerful transcriptome profiling technology enabling transcript discovery and quantification. Whilst most commonly used for gene-level quantification, the data can be used for the analysis of transcript isoforms. However, when the underlying transcript assemblies are complex, current visualization approaches can be limiting, with splicing events a challenge to interpret. Here, we report on the development of a graph-based visualization method as a complementary approach to understanding transcript diversity from short-read RNA-Seq data. Following the mapping of reads to a reference genome, a read-to-read comparison is performed on all reads mapping to a given gene, producing a weighted similarity matrix between reads. This is used to produce an RNA assembly graph, where nodes represent reads and edges similarity scores between them. The resulting graphs are visualized in 3D space to better appreciate their sometimes large and complex topology, with other information being overlaid on to nodes, e.g. transcript models. Here we demonstrate the utility of this approach, including the unusual structure of these graphs and how they can be used to identify issues in assembly, repetitive sequences within transcripts and splice variants. We believe this approach has the potential to significantly improve our understanding of transcript complexity.

Modelling the Structure and Dynamics of Biological Pathways.

There is a need for formalised diagrams that both summarise current biological pathway knowledge and support modelling approaches that explain and predict their behaviour. Here, we present a new, freely available modelling framework that includes a biologist-friendly pathway modelling language (mEPN), a simple but sophisticated method to support model parameterisation using available biological information; a stochastic flow algorithm that simulates the dynamics of pathway activity; and a 3-D visualisation engine that aids understanding of the complexities of a system's dynamics. We present example pathway models that illustrate of the power of approach to depict a diverse range of systems.

A method of layer-by-layer gold nanoparticle hybridization in a quartz crystal microbalance DNA sensing system used to detect dengue virus.

Dengue virus (DENV) is nowadays the most important arthropod-spread virus affecting humans existing in more than 100 countries worldwide. A rapid and sensitive detection method for the early diagnosis of infectious dengue virus urgently needs to be developed. In the present study, a circulating-flow quartz crystal microbalance (QCM) biosensing method combining oligonucleotide-functionalized gold nanoparticles (i.e. AuNP probes) used to detect DENV has been established. In the DNA-QCM method, two kinds of specific AuNP probes were linked by the target sequences onto the QCM chip to amplify the detection signal, i.e. oscillatory frequency change (DeltaF) of the QCM sensor. The target sequences amplified from the DENV genome act as a bridge for the layer-by-layer AuNP probes' hybridization in the method. Besides being amplifiers of the detection signal, the specific AuNP probes used in the DNA-QCM method also play the role of verifiers to specifically recognize their target sequences in the detection. The effect of four AuNP sizes on the layer-by-layer hybridization has been evaluated and it is found that 13 nm AuNPs collocated with 13 nm AuNPs showed the best hybridization efficiency. According to the nanoparticle application, the DNA-QCM biosensing method was able to detect dengue viral RNA in virus-contaminated serum as plaque titers being 2 PFU ml(-1) and a linear correlation (R(2) = 0.987) of DeltaF versus virus titration from 2 x 10(0) to 2 x 10(6) PFU ml(-1) was found. The sensitivity and specificity of the present DNA-QCM method with nanoparticle technology showed it to be comparable to the fluorescent real-time PCR methods. Moreover, the method described herein was shown to not require expensive equipment, was label-free and highly sensitive.

Assembly of a parts list of the human mitotic cell cycle machinery.

The set of proteins required for mitotic division remains poorly characterized. Here, an extensive series of correlation analyses of human and mouse transcriptomics data were performed to identify genes strongly and reproducibly associated with cells undergoing S/G2-M phases of the cell cycle. In so doing, 701 cell cycle-associated genes were defined and while it was shown that many are only expressed during these phases, the expression of others is also driven by alternative promoters. Of this list, 496 genes have known cell cycle functions, whereas 205 were assigned as putative cell cycle genes, 53 of which are functionally uncharacterized. Among these, 27 were screened for subcellular localization revealing many to be nuclear localized and at least three to be novel centrosomal proteins. Furthermore, 10 others inhibited cell proliferation upon siRNA knockdown. This study presents the first comprehensive list of human cell cycle proteins, identifying many new candidate proteins.

Disposable amperometric immunosensing strips fabricated by Au nanoparticles-modified screen-printed carbon electrodes for the detection of foodborne pathogen Escherichia coli O157:H7.

A disposable amperometric immunosensing strip was fabricated for rapid detection of Escherichia coli O157:H7. The method uses an indirect sandwich enzyme-linked immunoassay with double antibodies. Screen-printed carbon electrodes (SPCEs) were framed by commercial silver and carbon inks. For electrochemical characterization the carbon electrodes were coupled with the first E. coli O157:H7-specific antibody, E. coli O157:H7 intact cells and the second E. coli O157:H7-specific antibody conjugated with horseradish peroxidase (HRP). Hydrogen peroxide and ferrocenedicarboxylic acid (FeDC) were used as the substrate for HRP and mediator, respectively, at a potential +300 mV vs. counter/reference electrode. The response current (RC) of the immunosensing strips could be amplified significantly by 13-nm diameter Au nanoparticles (AuNPs) attached to the working electrode. The results show that the combined effects of AuNPs and FeDC enhanced RC by 13.1-fold. The SPCE immunosensing strips were used to detect E. coli O157:H7 specifically. Concentrations of E. coli O157:H7 from 10(2) to 10(7)CFU/ml could be detected. The detection limit was approximately 6CFU/strip in PBS buffer and 50CFU/strip in milk. The SPCE modified with AuNPs and FeDC has the potential for further applications and provides the basis for incorporating the method into an integrated system for rapid pathogen detection.

Using oligonucleotide-functionalized Au nanoparticles to rapidly detect foodborne pathogens on a piezoelectric biosensor.

A circulating-flow piezoelectric biosensor, based on an Au nanoparticle amplification and verification method, was used for real-time detection of a foodborne pathogen, Escherichia coli O157:H7. A synthesized thiolated probe (Probe 1; 30-mer) specific to E. coli O157:H7 eaeA gene was immobilized onto the piezoelectric biosensor surface. Hybridization was induced by exposing the immobilized probe to the E. coli O157:H7 eaeA gene fragment (104-bp) amplified by PCR, resulting in a mass change and a consequent frequency shift of the piezoelectric biosensor. A second thiolated probe (Probe 2), complementary to the target sequence, was conjugated to the Au nanoparticles and used as a "mass enhancer" and "sequence verifier" to amplify the frequency change of the piezoelectric biosensor. The PCR products amplified from concentrations of 1.2 x 10(2) CFU/ml of E. coli O157:H7 were detectable by the piezoelectric biosensor. A linear correlation was found when the E. coli O157:H7 detected from 10(2) to 10(6) CFU/ml. The piezoelectric biosensor was able to detect targets from real food samples.

Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance.

A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5'-monophosphate (12-dT)] for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104 mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5'-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P<0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.

A piezoelectric immunosensor for specific capture and enrichment of viable pathogens by quartz crystal microbalance sensor, followed by detection with antibody-functionalized gold nanoparticles.

A sensitive bacteria enrichment and detection system for viable Escherichia coli O157:H7 was developed using a piezoelectric biosensor-quartz crystal microbalance (QCM) with antibody-functionalized gold nanoparticles (AuNPs) used as detection verifiers and amplifiers. In the circulating-flow QCM system, capture antibodies for E. coli O157:H7 were first immobilized onto the QCM chip. The sample containing E. coli O157:H7 was circulated through the system in the presence of 10 ml of brain heart infusion (BHI) broth for 18 h. The cells of E. coli O157:H7 specifically captured and enriched on the chip surface of the QCM were identified by QCM frequency changes. Listeria monocytogenes and Salmonella Typhimurium were used as negative controls. After bacterial enrichment, detection antibody-functionalized AuNPs were added to enhance the changes in detection signal. The use of BHI enrichment further enhanced the sensitivity of the developed system, achieving a detection limit of 0-1 log CFU/ml or g. The real-time monitoring method for viable E. coli O157:H7 developed in this study can be used to enrich and detect viable cells simultaneously within 24h. The unique advantages of the system developed offer great potential in the microbial analysis of food samples in routine settings.

An optical biosensing platform for proteinase activity using gold nanoparticles.

The surface plasmon resonance (SPR) wavelength of colloidal gold nanoparticles (AuNPs) can vary when the AuNPs aggregate, have different sizes or shapes, or are modified with chemical molecules. In this study, an optical biosensing platform for a proteinase activity assay was established based on the SPR property of AuNPs. The 13-nm AuNPs were modified with gelatin (AuNPs-gelatin) as a proteinase substrate and subsequently modified with 6-mercaptohexan-1-ol (MCH) (AuNPs/MCH-gelatin). After proteinase (trypsin or gelatinase) digestion, the AuNPs lose shelter, and MCH increases the attractive force between the modified AuNPs. Therefore, the AuNPs gradually move closer to each other, resulting in AuNPs aggregation. The AuNPs aggregation can be monitored by the red shift of surface plasmon absorption and a visible color change of the AuNPs is from red to blue. Such a color change can be observed with the naked eye. For detection, the absorption ratio, A(625)/A(525), of the reacted AuNPs solution can be used to estimate quantitatively the proteinase activity. A linear correlation has been established with trypsin activity at concentrations from 1.25 x 10(-1) to 1.25 x 10(2) U and matrix metalloproteinase-2 activity at concentrations from 50 ng/mL to 600 ng/mL.

Optical detection of human papillomavirus type 16 and type 18 by sequence sandwich hybridization with oligonucleotide-functionalized Au nanoparticles.

The importance of detecting and subtyping human papillomaviruses (HPVs) in clinical and epidemiological studies has been well addressed. In detecting the most common types of HPV, type 16 (HPV-16) and type 18 (HPV-18), in the cervical mucous of patients in a simple and rapid manner, the assay of a label-free colorimetric DNA sensing method based on sequence sandwich hybridization with oligonucleotide-functionalized Au nanoparticles (AuNPs) was fabricated in this study. Specific oligonucleotide probes were designed for the sequence detection within the L1 gene of HPV-16 and HPV-18, and the probes were capped onto AuNPs, as AuNP probes. The target HPV sequences in clinical specimens were obtained by an asymmetric polymerase chain reaction (PCR) with universal primers, which can amplify the target sequences from several HPV serotypes, including HPV-16 and HPV-18. The DNA sandwich hybridization between the target sequences and the specific AuNP probes was performed at a temperature closer to the theoretical melting temperature of the DNA hybridization. Next, the procedure of increasing salt concentration and cooling the hybridizing solution was immediately utilized to discriminate the target sequences of HPV-16 or HPV-18. If the target sequences were not complementary to sequences of AuNP probes, the AuNPs would aggregate because no duplex DNA formation occurred such that the color of the reaction solution changed from red to purple. If the AuNP probes were a perfect match to the target sequences and a full DNA sandwich hybridization occurred, the reaction solution maintained its red color. A total of 70 mucous specimens from patients with cervical intraepithelial neoplasia were tested by the AuNP probes sandwich hybridization. The results show that there were 33, 16, 5, and 16 cases detected with HPV-16, HPV-18, both HPV-16 and HPV-18 (HPV-16/HPV-18), and neither HPV-16 nor HPV-18, respectively.