Final Activity Report Summary - FORMAPLEX (The Exploitation of Formalin Fixed Tissues for High-Throughput Genetic Analyses: An HIV-1-HLA Cointeraction Case Study)
Over the two years of my Marie Curie Intra-European Fellowship my research has focused heavily on the development and adoption of new analytical and laboratory techniques, to expand the power of analyses performed on degraded sources of DNA. I have undertaken a series of parallel, related experiments and analyses that have produced the following outcomes.
1) Through a large scale, comprehensive comparison and assessment of published techniques that deal with the recovery of nucleic acids (DNA and RNA) from formalin fixed materials, I have developed and published guidelines that will help future researchers identify exactly which methods should be used in what situations.
2) I have investigated the use of multiplex PCR with minisequencing as a tool for the rapid simultaneous typing of up to 52 nuclear DNA SNPs in a single reaction. The results indicate that the success of the tool correlates strongly with nucleic acid quality, and that while useful on recently fixed tissues, the method is not suitable for using on older samples.
3) I have used archival frozen HIV-1 infected blood samples dating to the early 1980s to investigate the origin and spread of HIV-1 out of Africa and into the Americas, Europe and Australia. The data indicates that Haiti played a key stepping stone in the spread of this disease, and that HIV-1 was present in the United States over 10 years before it was first identified.
4) I have investigated the use of new high-throughput sequencing-by-synthesis platforms, Roche's FLX and GS20, in the analysis of degraded sources of DNA. In particular I have undertaken analysis to (a) develop the tools required to work with samples containing extremely low levels of DNA, (b) analysed the quality of the data to investigate what problems may exist with it and (c) developed methods to couple it with PCR. This research has been extremely fruitful. In particular, I have demonstrated the feasibility of working with very DNA-poor tissues in this way, using keratinous tissues such as hair shaft to rapidly generate 15 complete ancient mammoth mtDNA genomes. I have also developed a 'primer-barcoding' methodology that enables the large scale pooling of PCR amplicons into single reaction, economising the process considerably. Furthermore, I have performed data analyses that both characterise the inherent sequencing errors present in the system and provide insights into the DNA damage problems suffered by old sources of DNA.
The funding and valuable experience provided by this fellowship, as well as the wide media coverage of the results of my research has enabled me to successfully be awarded the position of Associate Professor at the University of Copenhagen's Biological Institute.
1) Through a large scale, comprehensive comparison and assessment of published techniques that deal with the recovery of nucleic acids (DNA and RNA) from formalin fixed materials, I have developed and published guidelines that will help future researchers identify exactly which methods should be used in what situations.
2) I have investigated the use of multiplex PCR with minisequencing as a tool for the rapid simultaneous typing of up to 52 nuclear DNA SNPs in a single reaction. The results indicate that the success of the tool correlates strongly with nucleic acid quality, and that while useful on recently fixed tissues, the method is not suitable for using on older samples.
3) I have used archival frozen HIV-1 infected blood samples dating to the early 1980s to investigate the origin and spread of HIV-1 out of Africa and into the Americas, Europe and Australia. The data indicates that Haiti played a key stepping stone in the spread of this disease, and that HIV-1 was present in the United States over 10 years before it was first identified.
4) I have investigated the use of new high-throughput sequencing-by-synthesis platforms, Roche's FLX and GS20, in the analysis of degraded sources of DNA. In particular I have undertaken analysis to (a) develop the tools required to work with samples containing extremely low levels of DNA, (b) analysed the quality of the data to investigate what problems may exist with it and (c) developed methods to couple it with PCR. This research has been extremely fruitful. In particular, I have demonstrated the feasibility of working with very DNA-poor tissues in this way, using keratinous tissues such as hair shaft to rapidly generate 15 complete ancient mammoth mtDNA genomes. I have also developed a 'primer-barcoding' methodology that enables the large scale pooling of PCR amplicons into single reaction, economising the process considerably. Furthermore, I have performed data analyses that both characterise the inherent sequencing errors present in the system and provide insights into the DNA damage problems suffered by old sources of DNA.
The funding and valuable experience provided by this fellowship, as well as the wide media coverage of the results of my research has enabled me to successfully be awarded the position of Associate Professor at the University of Copenhagen's Biological Institute.