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= Sedimentary ancient DNA = Over a century of paleoecological investigations have been dedicated to study the preserved hard parts of organisms contained in geological archives. Although the fossil record has revealed valuable insights into past ecosystems, the vast majority of past life has remained undetected due to a lack of preservation. Sedimentary ancient DNA (sedaDNA), DNA sourced from proximal organisms and preserved in coeval sediments, can provide important information about environmental and life changes through time but, due to its limits, it  has been possible to reach “just” 2 million years back in time, age at which sediments from Greenland have been able to provide seda(DNA) for paleoclimate reconstruction.

Ancient DNA sources
The more common sources of ancient DNA are: tissues, calculus/dung, permafrost and sediments coming from seafloor, cave or lake. Most of the ancient DNA investigations have concentrated on sites that are generally characterized by low temperatures, dark, stable, dry and anoxic conditions. These environments provide the better preservation chances possible. Remote, high latitude sites are ideal to track environmental changes that are not directly induced by human activity. On the other hand, sites from temperate and tropical regions have been studied to a lesser extent but are important for the analysis of human history and anthropogenic ecosystem modifications. These places are usually described by hot temperature, generally wet, high UV impact and high oxidation potential, all of them factors enabling weathering of the organic matter. More in general, good preservation conditions coupled with adequate precautions to ensure clean subsampling of the inside of sediment cores and for work with ancient DNA, a high diversity of authentic taxa can be retrieved.

Information provided by Sedimentary Ancient DNA
There are many information that can be provided through seda(DNA) analysis. It is possible to assess and determine genes hybridization (above all in human being), which is impossible to detect just using fossils, rebuild migration events, populations size, past environments and the respective biota and define the phylogeny of a species. For instance, analysis of marine sedimentary ancient DNA (sedaDNA) allows identification of deceased organisms that have sunk from the upper water layers to the bottom of the ocean and become preserved. As a result of the sedimentation process, the remains of deceased organisms accumulate over time, forming a continuous record of past communities that have inhabited the ocean. Marine sedaDNA can be used to study a broad variety of taxa, including viruses, archaea, prokaryotes (bacteria), and eukaryotes (phytoplankton to larger predators). Eukaryotic planktonic organisms, such as diatoms, dinoflagellates, coccolithophores, and foraminifers, are particularly interesting targets for sedaDNA studies because of their established reliability as environmental indicators.

Differences between ancient and modern DNA
Ancient DNA is highly fragmented and degraded. Once an organism dies, cellular processes such as DNA repair mechanisms are no longer active, and the unmaintained DNA degrades over time. Previous research has shown that ancient DNA is usually <100 base pairs (bp) long, and, for instance, marine sedaDNA fragments tend to be even shorter, about 69 bp.

In contrast to ancient DNA, DNA from living organisms (modern DNA) is highly intact and overwhelmingly abundant in the environment. Its study, as regards modern marine settings, continues to generate invaluable reference sequences of living marine organisms to which ancient sequences can be compared.

Sedimentary ancient DNA workflow
In order to get ancient DNA from sediments there are my steps that have to be followed.


 * 1)      Identification of a good site where organic matter could be highly preserved and then starting with samples collection through excavation or   coring, depending on rocks and deepness of the target.
 * 2)      Sediment subsampling is required to get a more precise and punctual specimen from the rough sample.
 * 3)      DNA extraction through specific acids that don’t affect the organic matter.
 * 4)      Selecting the right sequencing approach among metabarcoding, metagenomic (shotgun) and target capture (targeted enrichment) method (see sequencing approaches).
 * 5)      Illumina sequencing.
 * 6)      Data filtering, identification and authentication (see Sedimentary ancient DNA data validation process).
 * 7)      Analysis and inference.

·       Metabarcoding (fig.1A)
Most sedaDNA studies have used a metabarcoding approach to investigate paleocommunities. This method targets a specific DNA region used as a taxonomic marker to identify different species that are aggregated in a sediment sample. These genetic markers are amplified using primers (short sequences matching the start and end of the target gene) in a polymerase chain reaction (PCR) and are subsequently sequenced. This technique has been shown to be unsuitable for the study of ancient DNA (e.g., ) for the following reasons: i) ancient DNA is typically highly damaged so that primers may not bind; ii) the DNA segments to be amplified are usually longer (>100 bp) than most ancient DNA fragments (<100 bp ), introducing biases toward longer sequences that favour modern contaminants where present, and skew the taxonomic composition; iii) PCRs are prone to inherent biases such as random amplification in the first few amplification cycles and PCR drift; hence, biases become more severe with increasing numbers of amplification cycles as necessitated with sedaDNA protocols; iv) the characteristic DNA damage patterns described above are no longer detectable in metabarcoding data as polymerases correct these patterns during amplification, preventing this mode of authenticity testing. Studies that have applied metabarcoding should therefore be interpreted with caution unless they have shown authenticity of the sedaDNA through complementary analyses (e.g., fossils, biomarkers).

·       Metagenomics (fig.1B)
With ongoing increases in sequencing power and decreases in cost, metagenomics is becoming a viable alternative to metabarcoding. Metagenomics studies extract and amplify the “total” DNA in a sample (potentially all species), thereby facilitating the recovery of DNA sequences proportionate to their original representation in that sample and independent of DNA fragment-size (“shotgun sequencing”). Thus, meta­genomics approaches are better suited to studying sedaDNA, as they permit detection of bacteria, archaea, and eukaryotes, and they recover DNA damage patterns and fragment size variability without the biases inherent to metabarcoding. Community composition can then be reconstructed from this large pool of metagenomic data by screening for the occurrence of taxonomic marker genes. ­­If the representation of the target organisms/genes is relatively low in the pool of total DNA data, very deep sequencing is required to recover sufficient genetic information to perform meaningful statistical analyses.

·       Target capture (fig.1C)
Another possibility, that combines the specificity of the PCR approach avoiding its biases, is the targeted enrichment of specific genetic sequences via hybridization-capture techniques. This approach uses short RNA probes (also called “baits”) that are designed to be complementary to any DNA sequences the researchers may choose. By binding to the target sequence, these genetic baits “capture” DNA fragments in a manner that is akin to the PCR targeting, but independent of fragment size and with the preservation of damage patterns, allowing detailed authenticity testing.

Sedimentary ancient DNA data validation process
An important step to validate seda(DNA) analysis is the data authenticity demonstration (i.e., the DNA recovered is ancient and free from modern contamination, ). Validation protocols for ancient DNA include DNA damage analysis, using for instance mapDamage software, which has been specifically developed to detect nucleotide misincorporations and fragmentation patterns that characterize ancient DNA. The identification and authentication of ancient sequences, above all in highly complex metagenomic data of very rare organisms, remains challenging, partly due to the lack of high-quality reference sequences and as the threshold of ~250 reads per species required to analyze and plot DNA damage patterns in mapDamage is often not reached. To overcome this issue, recent studies have focused on developing new bioinformatic techniques as HOPS (Heuristic Operations for Pathogen Screening ) to screen for ancient pathogens in meta­genomic samples, and a new way for processing and analyzing ancient metagenomic shotgun data focusing on the conservation of rare reads. Less precise but simpler is to assess the authenticity through DNA fragment size analysis (fragmentation is expected to increase with age of the sediment sample), which should be the minimum authenticity analysis undertaken in any marine sedaDNA study.