Lectures 14+15: Molecular Ecology and Ancient DNA

Based on lectures by Burbano and Sumner

What are the two main approaches to investigating how genotypes change over time

Real-time monitoring approach and retrospective approach

Describe a real-time monitoring approach to investigating evolution

Use organisms with a short life span

“Experimental evolution” putting different selective pressures on the organisms

Observe phenotypes and sequence genotypes through time

What is a retrospective approch to investigating evolution

Sequencing DNA from eg. sediments, bones, archaeological remains, old specimens

What are some of the problems associated with ancient DNA?

Degredation- hydrolytic and oxidolytic attack

Fragments are much shorter than fresh DNA

Contamination (microbial DNA and from modern human DNA)

Outline the three basic steps involved in aDNA sequencing

DNA isolation, creation of genomic libraries, bioinformation processing

What is the median fragment length in aDNA

About 60bp

How do aDNA researchers know which nucleotides have been misincorporated

Aligning to a reference genome

What is the most common nucleotide misincorortation in aDNA?

Ts in the place of Cs

Describe the process of cytosine deamination

Hydrolytic attack (spontaneous, post-mortem) on cytosine turns it into uracil, then uracil becomes thymine during sequencing

C-to-T substitutions accumulate with time

At which point in the DNA fragment are C to T substitutions most common?

5’ end of the molecule

What is a solution to C-to-T substitutions?

Library repairs: the enzyme UDG/endoVIII to restore original base. HOW???

How does DNA break organically into fragments?

DNA depurination- hydrolysis where the purine base is lost and then the backbone breaks

β-N-glycosidic bond is hydrolytically cleaved releasing a nucleic base, adenine or guanine, respectively (Wikipedia)

What is evidence that depurination is the process breaking DNA?

The base found before a read starts in the reference genome is almost always Adenine or Guanine (purine bases)

Give an example of how contaminated aDNA samples can become

The amount of Neanderthal DNA that could be aligned with a human reference genome was 3.5%

The rest was non-endogenous microbial DNA

How can plant aDNA be a “time capsule of ecological interactions”?

Samples may contain aphids, microbes in the rhizosphere (around the roots), DNA from bacteria/viruses/fungi that infected the plant

How has method of aDNA sequencing changed since the 1990s?

From direct PCR to library-based methods

What are the advantages of using DNA library methods instead of PCR to sequence ancient DNA?

PCR primers miss out ends of the molecules, which is particularly bad when they are so short anyway

Won’t see the level of C to T misincorporation

How is positive evidence for the authenticity of the aDNA given?

Level of C to T nucleotide misincorporation, which depends not only on age but where the sample is found

What is the criterion for a sample to be considered aDNA?

Needs to be significantly degraded: depends on biochemical condition rather than age eg. a plant sample a couple of hundred years old can be considered aDNA if it is degraded enough

How is it inferred when a particular genetic change was accrued?

If a change happens in lineage A but is not present in B and C, it is inferred that it happened after species A diverged from the others. Can narrow down the window that the change happened in by adding more species to the phylogenetic tree.

Paleogenomics can be used for “calibration of a molecular clock.” What is meant by this?

To find out how fast mutations are accruing in a particular lineage

What are DNA microsatellites?

Selectively neutral short repeat DNA sequences

They are polymorphic (many alleles per locus)

They are rapidly evolving

How can DNA microsatellites be useful to molecular ecologists?

Can infer kinship relationships

How many mates are contributing to the next generation

Is there inbreeding

Genetic diversity

Size of population

How far individuals are dispersing

How does is genotyping with microsatellites done?

DNA is extracted

PCR is used to amplify the fragment

Capillary electrophoresis (fragment analysis) is used to sequence

Why does a little peak before the big peak

“Stutter peak” caused by PCR slippage.

What is PCR slippage?

Something that can happen during PCR or natural DNA replication of repeat sequences

When the last few bases parent and daughter strand detach and the strands repair incorrectly, leading to a higher (or lower) number of repeats in the daughter cell

This is thought to be the basis of variable numbers of microsatellites

What are SNPs? Why are they useful?

Single nucleotide polymorphisms arising from point mutations

Used in GWAS

Not selectively neutral, unlike STNs

How many SNPs are there in the human genome? What is the criterion for a point mutation to be considered a SNP?

10 million

What is KASP genotyping?

Kompetitive Allele Specific PCR genotyping

Relies on allele specific SNP-primers, designed based on idnetified SNPs

1:11:54

1. Sample DNA is added to mix containing allele specific SNP-primer, Taq polymerase, and fluorescent reporting system. 2. During real time PCR (Polymerase Chain Reaction) denaturation, heat separates DNA into single strands. SNP-primer binds to target SNP region and amplifies. 3. Polymerase amplifies (i.e. replicates) region marked by SNPprimer (PCR cycles repeated = exponential replication). 4. Fluorescence system binds to amplified regions, releasing a signal.

What is a DNA barcode?

A highly-conserved, short standardized DNA sequencing which can be used to identify species

Can be used by non-experts in taxonomy to identify species

What region in the genome is used as the ‘genetic barcode’ for most animal species?

A 648-bp region in the mitochondrial CO1 gene (cytochrome oxidase 1 gene)

Varies a lot between species but not within species

Why is CO1 not a reliable genetic barcode to identify plants? What is used instead?

It evolves too slowly

Instead two chloroplast genes (MATTK??? and RBCL) are used

Why is Sanger sequencing used instead of NGS to identify species from barcodes?

Cheaper

Doesn’t matter that it is low throughput because not a lot of DNA is used

What is metabarcoding?

A branch of metagenomics

The process by which millions of copies of a specific target region are sequenced from a mixed slurry of materials eg. earth or seawater

What is metabarcoding useful for? What is it not useful for?

Good for uncovering one gene in many genomes

Good for uncovering extent of biodiversity but not abundance of any guven species

What are some considerations when designing a metabarcoding experiment?

Might use more than one primer to capture eg. plant AND animal diversity

Contamination of sample means you might have to do more than one repeat

Avoiding false positives and negatives that can arise from having species with simialr barcodes

What are the typical fragment sizes used in barcoding vs metabarcoding?

Barcoding - >500bp

Metabarcoding - <400bp

Describe how molecular ecology can tell us about mating and breeding systems?

Female haplodiploids (bees, wasps and ants) share the same 50% of her genome with all sisters

If all sisters share the same STN at a given locus only one breeding male contributed to that generation of the family

Describe how molecular biology can tell us about the effects of land-use change on populations: case study

Microsatellites tell us about the origin and relatedness of bee colonies

Found that colonies are more dense where there are is diverse flora

Evidence for the efficacy of “Bee corridors” on farmland

Describe how molecular biology can tell us about invasive species

The Argentine ant displaces European ant population and destroys leaves/buds

Argentine ant has low genetic diversity at recognition sites, acting as a unicolony across Europe: this was worked out using microsatellite markers

They do not show aggression between colonies which gives them a competitive advantage

Describe genetic pest management

Pest management strategies for invasive species eg. Asian hornets preying on Western honeybee

Genetically engineered DNA sequences ‘transgenes’ introduced into wild populations

Usually into males because they have multiple mates

eg. bisex-lethal transgene (kills 2nd-gen individuals of either sex), destroy sex ratios by making transgenic males only make Y-sperm or transgene females die

Describe how molecular biology can tell us about biodiversity and species ID: case study

Helpful for identifying new species (especially morphologically-similar species)

eg. 186 new species of parasitoid wasps identified in a 1200km² area in Costa Rica as part of a 35 year ongoing project (Frenandez-Triana et al, 2014)

Describe nanopore barcoding

A technique that allows genetic techniques to be used on remote locations

A drop of sample is placed onto the flow cell

Disruption of the ionic current as the DNA molecule moves through the nanopore allows the computer to sequence the bases

What are the advantages of nanopore barcoding?

Allows in-situ analyses of fauna

Equipment fits into a backpack

Sequence data returned within 24 hours

>99% accuracy

What is meant by eDNA?

Environmental DNA

Give a case study for the efficacy of metabarcoding as opposed to visual survey for detecting species number

In Japan, using universal fish primers, evidence of 128 fish species was found in a bay in 6 hours

Only 62% had been detected by visual surveys over the past 14 years… however the visual surveys had also picked up species not found by metabarcoding

So metabarcoding is best used alongside, not instead of, visual surveys