sedaDNA Flashcards
(32 cards)
What is sedimentary
ancient DNA (sedaDNA)?
Ancient DNA recovered from a substrate lacking apparent macroscopic remains and usually originating from many different taxa.
What is the biggest advantage of sedaDNA?
The biggest advantage is that sedaDNA is so abundant compared to bones and other macroremains - easier to find. This allows for sampling for a lack of an organism, which gives high resolution data of the LAD. It also provides high temporal resolution from sediment core, which allows for identifying for example ecosystem changes much closed in time. sedaDNA also allows you to go back and sample with really close proximity in a specific time you’re interested in.
Overall the strengths of sedaDNA is high temoporal resolution, high taxonomic breadth and prescision.
What are the disadvantages of sedaDNA?
The main disadvantages of sedaDNA is:
- that it can not be used for individuals, as the DNA is “loose” from the organism itself.
- Due to leeching risks it is much weaker evidence than bone for example, which can be radiocarbon dated.
What are the six main sources of sedaDNA?
The main sources of sedaDNA is permafrost, lake sediments, caves, archeologcal sites, rodent. middens and marine sediments.
Compare bone/tissue aDNA samples with sedaDNA samples, what are the biggest differences?
With sedaDNA from a lake, the area from which the DNA originates is MUCH bigger, It represent a much bigger part of the local biota and has higher complexity.
Name the seven steps of a typical sedaDNA workflow.
- Fieldwork: coring/excavation.
- Sediment subsampling.
- DNA extraction.
- Data generation strategy, by either:
1. Metabarcoding + library preparation
2. Library preparation only (shotgun metagenomics)
3. Library preparation + targeted enrichment - Illumina sequencing.
- Data filtering, identification, and authentication.
- Analysis and inference.
How does subsampling of sedaDNA differ from that of fossil samples? What consequences does this have?
Since sedaDNA is present in soft sediments, it contains a lot of. different DNA and can not be “cleaned” as that would destroy the DNA. Fossil samples on the other hand, can be pretreated with for example bleach to remove surface contaminants. Because of this, sedaDNA subsampling neeed to be done in clean rooms, and only unexposed parts can be sampled.
Also, the sediment cores are not homogenous, so there layers might need to be handled. differently.
There are two ways of sedaDNA preservation, which?
sedaDNA can be:
- intracellular: preserved in microscopic and cellular detritus (dead organisms), also pollen.
- extracellular: DNA that have leaket out of cells and is bound to clay, or other inorganic, minerals.
Which three metods can be used for sedaDNA amplification?
- Metabarcoding
- Shotgun metagenomics
- Targeted enrichment/hybridiization capture.
How does metabarcoding PCR work, briefly?
When performing metabarcoding, you amplify a short section of DNA (locus) that is shared, such as an essential gene (or e.g. 16S rRNA region) but is variable (has a different sequence and/or length) between taxa. The locus is flanked by conserved regions, which allows the same primer set to be used across broad taxonomic groups.
This makes it possible to detect many different taxa at the same time, and the primers can be customized according to your research question.
Why is robust reference databases so crucial to sedaDNA studies?
When you are working with sedaDNA you have a lot of unknowns, you never really know what you’ll find if you use universal detection methods. Because of this, it is so important that there is good and broad reference data available.
Today most of the reference material is biased towards the northern hemisphere, but sedaDNA has legacy potential - meaning that it can be retroactively identified with a growing reference database.
Does the degree of DNA degradation affect which methods you can use for amplification?
Yes! Metabarcoding requires relatively long ancient DNA molecules, since both forward and reverse primer need to be able to bind to the same molecule (conserved flanking sites) in order for the target to be amplified, so if the DNA is too fragmented it’s not possible to use. An alternative is shotgun sequencing, which can be used on highly fragmented DNA (which is was developed for)
Can PMD authentication be used for metabarcoded samples? Why/why not?
No. When doing metabarcoding, the primers bind to the ends, where the conserved sequences are, and thus the ends of the amplified sequences will be based on the synthetic primers and therefor cannot be used for authentication through deamination/PMD.
Give two examples of methods that can be used for sedaDNA data analysis.
- Kmer-based. e.g. KRAKEN2: Searches for exact k-mer (e.g. k = 30 bp), Very fast but memory intensive
- Alignment-based. e.g. BWA/Bowtie: End-to-end/local alignment; mismatches allowed, slow
- BLAST-based: you get % identity; % covered but very slow…so not used in sedaDNA analysis anymore.
For all three of the amplification methods for sedaDNA, what are their respecitive pros and cons?
Metabarcoding:
+ sensitivity; cheap
- unable to authenticate; intact template needed
Shotgun metagenomics
+ authentication; unbiased - inefficient; false negatives
Targeted enrichment
+ authentication
- expensive
All require reference database availability; false positives can occur if references are not robus and checked.
Name two possible uses for sedaDNA.
There are many use cases for sedaDNA:
- Past ecosystem reconstructions
- single taxon reconstructions
- community reconstructions: for example, you can look at sedaDNA appearance date for different data at different locations to show where they appeared first and how they spread over time.
What are the potential pitfalls of sedaDNA data? How can one handle them?
- Reference database availability: One way to confidently identify that the DNA maps to the correct species/reference, is to map the reads to an unrelated genome. Fr example, if you want to idntify mammoth DNA, it should map to both mammoth and elephant, but not sloth. If the data maps equally well to the sloth, the DNA you have extracted is probably not mammoth DNA.
- Authentication of aDNA data.
If you hav used shotgun or targeted sequencing you can use the smiley plot to evaluate PMD. If it has the smiley shape, you can be confident that the DNA you have extracted is ancient and not modern contamination. Also look at the read lengths, if short, this is also good confirmation of no modern contamination. - Use of appropriate negative controls: If you are for example dating the sample, you can date modern samples the same way, to show that i dates much younger.
- Ensure data are corroborated by other proxies and sediments that are directly dated: For example, date bones found in the same layer of sediment that the sedaDNA was taken from, if they date to the time of your sample, you can be more confident in th age determination.
SedaDNA can also be used for population genetics in some cases, what does it depend on?
In order to use
Which of the sedaDNA sources can be used for population genomics? Which type of organism(s) is best suited for population genomics for each type?
- Caves: best for sedaDNA based pop. gen. for vertebrates, predators bring in prey into caves for further “handling”, which is why cave sediments are often solid sources for sedaDNA samples with high enough DNA content to perform pop. gen.
- Lakes, lake sediment: Great for algae, aquatic plants and surrounding plants.
- Permafrost: good for any terrestrial organisms, mainly vertebrates and plants.
- Archeological sites: great for domesticated taxa, both plants and animals.
- Sea floor: very unexplored but could potentially be good for whales.
Which type of material in sediments has the best DNA preservation?
Bone micro-fragments are the best for sedaDNA preservation.
Define the term “Environmental palaeogenome”
Environmental palaeogenome/Environmental genome/Palaeoenvironmental genome (these all mean the same thing) is defined as: Genome-wide data derived from a single taxon in sedaDNA.
What is important to do when assembling an environmental palaeogenome?
Before assembly, you need to perform filtering to remove “bycatch” sedaDNA from other related taxa that map to the reference genome. As some reads may have been incorrectly mapped before, it is possible that there are contaminating sequences that are mapped into the reference genomes, so this need to be checked and filtered out.
What can we do with an “environmental palaeogenome”? What can we not do?
Genome-wide data derived from a single taxon and sedaDNA is a composite of MULTIPLE individuals (population sample).
- Cannot perform: individual genome pop. gen. analyses e.g. D/F-stats, heterozygosity, runs of homozygosity, PSMC
- Can perform: phylogenetic placements, principal component analysis, population histories, genomic sexing - so useful still!
Can we use metabarcoding in sedaDNA population genetics?
No, as we need data from multiple loci for the pop. genomics analyses. Metabarcoding only amplifies specific regions.
