Behind the scenes: working in an ancient DNA lab - part 2
In a previous blog post, we explored the daily routine of an ancient DNA laboratory, and the challenges scientists encounter working under sterile conditions to preserve the integrity of fossil DNA. Such careful procedures are necessary to obtain reliable data for interpreting genetic patterns of past individuals, populations, and species.
Credit: Marina Buffoli. Work in progress, UV-light decontaminating lab material.
The successful extraction of ancient DNA from various sample types, such as bones, teeth, shells, fossils, herbarium specimens, and pollen, has enabled the reconstruction of the genome of individual species from the past. DNA obtained from coprolites (preserved feces) and dental plaque (calculus) instead has provided an unprecedented view into the complex microbial communities that existed in the gut or oral cavity of ancient humans and animals. Moreover, the analysis of ancient DNA from ice and sediment cores has allowed for the reconstruction of entire paleo ecosystems at a larger scale.
None of these achievements would have been possible without the recent advances in omics technologies – such as metagenomics.
The study of ancient DNA requires suitable sequencing techniques. To date many studies aiming at paleo-environmental and paleo-community reconstructions have relied on the metabarcoding approach, which uses targeted DNA regions, like genes or gene fragments, as taxonomic markers to differentiate between species. This involves amplifying genetic markers using primers that match the start and end of the target gene through a process known as polymerase chain reaction (PCR), followed by sequencing. The sequences are then compared to a reference database to determine the identity of the species present. Despite being cost-effective and sensitive, due to the degraded and fragmented nature of ancient DNA this approach has limitations in the field of paleo-genomics. The use of longer PCR cycles to amplify degraded DNA can lead to amplification bias towards modern contaminants, and primers may not bind effectively because DNA is highly fragmented. Additionally, the characteristic damage pattern of fossil DNA is completely removed by the amplification process, thus preventing any authentication of what is truly ancient and what is not.
Credit: Marina Buffoli. Gel to check what PCR reactions have worked. Each band represents a sample where aDNA was amplified successfully.
As sequencing power increases and costs decrease, metagenomics is becoming an increasingly attractive option for studying ancient DNA. Metagenomics studies extract and amplify all DNA in a sample, allowing for the recovery of DNA sequences that are independent of DNA fragment size. This makes metagenomics better suited to studying ancient DNA because it enables detection of the organisms of the past, as well as recovery of DNA damage patterns and fragment size variability without the biases associated to metabarcoding. Additionally, metagenomics provides functional information, revealing not only which organisms were present in the past, but also what they were doing.
Credit: Marina Buffoli. Marina processing samples in the aDNA lab.
While omics approaches such as metabarcoding and metagenomics hold great promise for studying the organisms and ecosystems of the past, there is still a lot of work to be done to improve the availability and quality of taxonomic database. This can be accomplished by combining both morphological and molecular characterization of individual species. By generating modern reference genomes from these profiles, sequences of ancient specimens can be compared and accurately identified. This will be fundament for the future of ancient DNA research, which is still in its early stages but has huge potential to provide new and valuable insights into the evolution and dynamics of past organisms and ecosystems.