Molecular techniques for the characterisation of pathogens: target enrichment, second generation sequencing and mass spectrometry.
4. October 2013, at 13:00.
Pathogens have always had a major interest to humans due to their central role in sickness and death. Influenza A annually kills at least 250,000 humans, and has been the cause of millions of further deaths during pandemic years in the past.
Plague (Yersinia pestis) has been the cause of the Black Death that was leading to the desertion of whole cities, and as a result was for centuries one of the most feared events in human life. For both of these organisms we generally have situations with only very small amounts of pathogen nucleic acids available, usually because many interesting samples are degraded, as these samples are historic or generally patient samples.
In this thesis we have principally explored methods to extract genetic information from such degraded samples, using the modern ‘high-throughput’ techniques of second generation sequencing (SGS) and mass spectrometry (MS), coupled with target enrichment. Using these techniques we are able to investigate the nucleic acids from these pathogens despite the poor quality or the small amounts.
We have developed methods for the SGS of influenza A and employed this technique for patient samples to show intra-host variation together with investigation of the transmission chain.
Furthermore, we have developed a new method for the subtyping of influenza A using proteins as these are not as fast degradable as nucleic acids and the method therefore is well-suited for old and degraded samples.
Lastly, we have used SGS together with enrichment capture for the detection of Y. pestis in samples from the Justinian plague (600 AD) as an attempt to detect this pathogen as a cause of death in the victims.