Archaic & Environmental DNA

Challenges Problems

1. Bacteria contamination and degradation.

2. Chemical degradation.

3. Human contamination.

4. Small amounts of target.

Solutions

• Extract DNA from deep in tissue

• Frozen samples

Samples

Rarely have soft tissue: Most samples are bone and teeth.

Bone

Advantage: Most samples are bone.

Disadvantage: Bone is porous, so marrow is contaminated.

Teeth

Advantage: Enamel protects tissues.

Disadvantage: Few teeth, very little tissue in tooth

Human contamination soln

suit up

small amts of target soln

PCR

PCR

Is imperfect

• Polymerase extension can still occur with imperfect pairing.

amplification equation

P(amplification)*fraction in sample = fraction amplified

Solution: Enrichment

• Step 1: Affinity purify target.

• Step 2: Amplify purified DNA.

  • Given prior example, you will still have off-targets, BUT:

target/ off target

jump 1: 10²

jump 2: 10⁴

Amplification challenge problem

Problem:

• Enrichment works IF you know a target sequence to amplify!

• Often, we don’t know the target sequences!

Amplification challenge Solutions:

• Educated guesses based on similar organisms.

• Many off-target sequences are BACTERIA contamination.

  • Amplify genes NOT found in bacteria.

Ancient Genomes

Most sequences come from mitochondria because this DNA is the most protected and is very conserved. Nuclear DNA decays at twice the rate.

Stability of DNA puts a hard limit on this work. For mtDNA, the per nucleotide decay rate is ~5.5 x 10^-6 /year. This means that after ~6 million years, the DNA is completely fragmented.

Contamination is also a problem. Often ~99% of DNA extracted from ancient tissue is microbial.

Oldest genome sequenced directly from ancient DNA is ~700,000 year old horse.

Oldest DNA sequenced ~ 1 million years mammoth:

Others include:

Denisovans

Neanderthals

Mastodons (mitochondrial)

Polar bear

Denisovans

• Ancient humans that lived in Eurasia

• Lived ~300,000 – 30,000 years ago. 

• Denisovans are more closely related to Neanderthals.

Denisovans 2

• When PCA analysis is used to compare modern human sequences to Denisovan sequences, Melanesian samples appear to have more introgression.

Denisovans 3

• Introgression from Denisovan to modern humans likely occurred in Southeast Asia.

• Due to admixture, Denisovan SNPs can be found in most non-African populations.

Introgression was widespread

• Denisovans and Neanderthals also introgressed.

• “Denny”: F1 Denisovan – Neanderthal hybrid.

Denny is

50% Denisovan, 50% Neanderthal\

If Denny is an F1, why aren’t ALL of her SNPs heterozygous?

She is likely descended from individuals who had introgressions in their past as well.

Dire Wolf

• Native to the Americas

• Distantly related to wolves.

• All samples from bone or teeth.

Claim: Dire wolves were cloned!

Facts: Not really.

• They used Grey Wolf donor cells.

• They edited 14 genes (out of ~30,000).

• ORFs (open reading frame) are ~3% of the genome.

• Exons are ~10% of ORF.

• Dire wolf is 99.7% Grey wolf.

Woolly Mouse

Woolly Mammoth?

• Replaced three mouse genes with three woolly mammoth genes.

• Mice are 99.99% mouse, and 0.01% woolly mammoth.

Environmental DNA

• You are covered and surrounded with DNA. 

• Every surface is covered in bacteria DNA.

• Can also detect remnants of DNA from organisms that shed cells in the vicinity.

Environmental DNA 2

• Cloned DNA from ice cores.

• Why did they amplify 18S (eukaryotic) ribosomal DNA?

1. All eukaryotes have ribosomes.

2. Ribosome sequences evolve SLOWLY and have many conserved sequences.

3. This is referred to as “barcoding”

   1. Amplify several key genes that can help you distinguish different organisms in the sample.

Environmental DNA 3

• Cloned DNA from 2 million year old sediment.

  • Sediment was cold and dry.

    • High clay composition appears preferable for DNA preservation

• Identified several species.

• Identified changes in species diversity over time based on age of samples.