Laboratory techniques

Event Date: 
Wednesday, January 28, 2015 - 19:00 - 19:45
University of Sydney

The use of genomics in diagnostic and public health microbiology


Since 2004 technological advances have enabled us to sequence more nucleic acid and generate more data in a shorter amount of time. Decreases in cost per nucleotide sequenced, the initial price of sequencing machines and the complexity of library construction means that whole genome sequencing (WGS) is available in many research labs and an increasing number of public health microbiology labs. I will examine the use of WGS in public health microbiology, particularly the possibility of investigating organisms without culture, the interrogation of genomes where PCR may be unavailable, outbreak investigation, tracking resistance mutations and novel pathogen discovery.

You are invited to the Sydney Next Generation Sequencing Special Interest Group Meeting,  which will be held at the University of Technology, Sydney.
A/Prof Aaron Darling (ithree Institute, UTS), MinIONs & Hi-C: short vignettes on the state of nanopore sequencing and application of Hi-C to metagenomic sequencing. 
Dr Fabian Buske (Garvan Institute of Medical Research), Title to be advised.
When: Thursday 9th October, 4.00pm – 5.00pm followed by drinks and nibbles. 
Where: Room 5.01, Level 5, Building 4, University of Technology, Sydney.


In September, JAMS was back into top gear, with a bigger audience, and a room with a view. Kent Lim from Macquarie University led off with a talk on his PhD work on the biocontrol agent Pseudomonas strain Pf5. As is often the case in science, things didn’t work out as expected, and Kent found that knocking out suspected pyochelin transporters led to an increase rather than a decrease in efflux of this siderophore and its metabolic precursors. Kent valiantly soldiered on, applying qRT-PCR and Biolog phenotype microarrays to untangle the problem, but unfortunately, this released even more worms from the seemingly-bottomless can provided by strain Pf5. It seems that these transporters may in fact also be regulatory proteins, explaining the unexpected pleiotropic effects of the knockouts.

Kerensa McElroy (UNSW) started us off immersing the audience in deep sequencing in order to understand pathogen evolution in biofilms. Two model pathogens, Phaeobacter gallaeciensis and Pseudomonas aeruginosa, were used to grow biofilms under conditions that select for reproducible phenotypic diversification. Variations in the genetic structure were revealed addressing different stages of biofilm development. Kerensa could describe genetic variation accurately and comprehensively within evolving populations using her established approach in genome-wide deep sequencing.

Event Date: 
Wednesday, June 27, 2012 - 18:15 - 18:30
University of Western Sydney

The Taguchi methods, or how to quickly and efficiently optimise PCR conditions.


Originally, the Taguchi methods were formulated for the optimisation of industrial processes, where several factors (3 to 50) of complex multifactorial experiments were tested at different levels (Taguchi, 1986). The Taguchi methods use orthogonal arrays to organise the ‘control’ parameters/factors affecting a process and the levels at which they should vary. A particular algorithm (quadratic loss function) is then applied in order to predict the optimum conditions of a process, whilst accounting for performance variations due to ‘noise’ factors beyond the control of the design. In a normal factorial strategy, every parameter should be individually tested at several levels, thus becoming extremely time-consuming, labour-intensive and expensive. The Taguchi methodology allows for testing only a few combinations, therefore dramatically decreasing the total number of experiments and simultaneously identifying the optimum condition of several factors.
Because some functional genes are present only in small fractions of microbial communities, and only few copies can be present in each genome, their detection by classical PCR methods can be challenging. Optimisation of the experimental conditions of a PCR includes the different components of the reaction mix (concentrations of salt, primers, enzyme, DNA template, etc.) as well as the cycling features (time and temperature of the denaturation, annealing and extension steps, number of cycles, etc.). We used this approach for the optimisation of the detection by PCR of functional genes of non-cultivable microorganisms present in environmental samples. In particular, we tested the different parameters involved in a (touchdown/nested) PCR and estimated the optimum settings for the detection of the functional gene pmoA, coding for the putative active site of the particulate methane monooxygenase, involved in the oxidation of methane by methanotrophic bacteria. The application of the Taguchi method allowed the suppression of a nesting step and thus a significant reduction in the amplification time, as well as reagent cost.

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