Overview | Culturing | DNA extraction | DNA quantification | Library preparation | Data analysis | Laboratory Manual | Costs
Purpose
Oxford Nanopore (ONT) sequencing, unlike Illumina, utilises native DNA (there are no PCR amplification steps). This is because it's difficult to successfully amplify the long strands needed for ONT. Whilst there are some papers that have used straight-from-sputum DNA extractions, the minimum input for the rapid barcoding kit (SBK110.96) is 50 ng (which is around 5 ng/ul). Unless the patient has a very high number of bacteria in their sputum, it is extremely unlikely DNA extraction from sputum will yield this amount. Therefore, culturing the bacteria is necessary to obtain the concentration of DNA needed.
Principle
The Mycobacteria can be cultured in a number of different ways, but it is important to note that it is vital to do all culturing in the appropriate biosafety laboratory (some Mycobacterial species, such as M. tuberculosis, are classed as a hazard group 3 (HG3) organism, whilst others are classed as hazard group 2). You can read more about biosafety levels on our health and safety page (whilst the page relates to SARS-CoV-2, the same biosafety rules will apply to all HG3 organisms).
There are a number of methods for TB culture, including LJ slopes, 7H9 (liquid) or 7H11 (solid) medias and the BD’s automated system Mycobacterial Growth Indicator Tubes (MGIT). More about the BACTEC MGIT system can be found here.