Presentation - Final Draft

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“Great” Grandma and You: Methods of Analyzing Human MtDNA Substitution Rate BY SETH NELSONTHURSDAY, OCTOBER 8TH, 2015

2Outline

I. Mitochondria and DNAII. MtDNA anatomy and replicationIII. Methods of finding substitution rateIV. Improvement on current findingsV. Using the rate

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I. Mitochondria and DNA

I. MITOCHONDRIA AND DNA

II. MTDNA ANATOMYIII. METHODS OF

FINDING SUBSTITUTION RATE

IV. IMPROVEMENT ON CURRENT FINDINGS

V. USING THE RATE

Mitochondria

Endosymbionts with proto-eukaryotes (Andersson et al., 2003)

Applications in forensics and evolutionary relatedness Need to know

mutation rates for accurate judgment

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Figures: Sadava et al. (2005)

5Using DNA for phylogenetic inference

Figure: García et al. (2011)

6Why use mtDNA

More mtDNA copies than nDNA (Robin and Wong, 1988)

Mitochondria are inherited from mother (Schwartz and Vissing, 2003)

High mutation rate, good for closely related individuals (Butler and Levin, 1998)

Image from https://www.thermofisher.com/us/en/home/technical-resources/research-tools/image-gallery/image-gallery-detail.2643.html

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II. MtDNA Anatomy and Replication


II. MTDNA ANATOMY AND REPLICATION

III. METHODS OF FINDING SUBSTITUTION RATE


V. USING THE RATE

8Anatomy of mtDNA

Figure: Pakendorf and Stoneking (2005)

Transfer RNAs

NADH Dehydrogenase subunits

Cytochrome c Oxidase subunits

Cytochrome bRibosomal RNAs

ATP Synthase subunits

9Control region

Controls replication

No protein product Two hypervariable

regions

Figure: Pakendorf and Stoneking (2005)

10Beginning of replication

Initiates at heavy strand origin

Light strand synthesis follows

Figure: Clayton (2000)

11Mutations in replication of DNA

Insertion Deletion Frameshift Substitution

Transition Transversion

Figure: Sadava et al., 2011

12Substitutions happen at specific rates

Substitutions per site per million years Numerator: Number of sequence differences,

only counting substitutions That is, no insertions, deletions, etc.

Denominator: Time since last common ancestor between sequences of comparison

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III. Methods of Finding Substitution Rate

I. MITOCHONDRIA AND DNAII. MTDNA ANATOMYIII. METHODS OF FINDING

SUBSTITUTION RATEI. ConsiderationsII. PedigreeIII. Phylogenetic


V. USING THE RATE

14Secondary structure forms

Light strand forms loop structure (Pereira et al., 2008)

Selective pressure on control region

Figure: Pereira et al. (2008)

15MtDNA can recombine

Mitochondria possess recombinase activity (Thyagarajan et al., 1996)

Does not affect substitution rate (Kraytsberg et al., 2004)

Figure: Thyagarajan et al. (1996)

16Some paternal inheritance

Single case of paternal inheritance in man (Kraytsberg et al., 2004)

Figure: Kraytsberg et al. (2004)

17Pedigree analysis is direct observation

Analyze mtDNA from closely related individuals English family with

Leber’s hereditary optic neuropathy

Age of last common ancestor is known with certainty

Figure: Howell et al. (2003)

18Less time means fewer mutations

Pedigree analysis tends to count fast mutations Potentially overestimate substitution rate

19Phylogenetic analysis uses equations

Analyze mtDNA from distantly related individuals Primates, back to

chimp and human CA

Figure: Hasegawa et al. (1993)

20Equations as estimates

Use of equations for rate Transition rate: Transversion rate: Substitution rate:

21More time means more uncertainty

Denominator more uncertain Phylogenetic analysis counts all substitutions

since last CA Reversions will cause undercount in mutations

Need methods of calibration

22Nodes vs. tips

Figure: Rieux et al. (2014)

23Calibration affects rates

Figure: Rieux et al. (2014)

24Noncoding region is higher than coding region

Pedigree rate is higher by order of magnitude Rates are in substitutions per site per million

years

Method

Noncoding

RegionCoding Region

Pedigree (99.5% CI)0.475 (0.265-

0.785)a0.15 (0.02-0.49)a

Phylogenetic (±1 Std

Error)

0.033 (0.027-

0.039)b0.0170 (--)c

Pedigree rates from Howell et al. (2003)Phylogenetic noncoding from Hasegawa et al. (1993)Phylogenetic coding from Ingman et al. (2000)

25Pedigree is higher than phylogenetic

Method Weighted ratePedigree 0.17Phylogenetic, Tip 0.021Phylogenetic, Node 0.018

Pedigree rate from Howell et al. (2003)Phylogenetic, tip-calibrated rate from Rieux et al. (2014)Phylogenetic, node-calibrated rate from Hasegawa et al. (1993) & Ingman et al. (2000)

*Rates are in substitutions per site per million years

26Context is everything (Pääbo, 1996)

Phylogenetic rate: Common ancestor is >100,000 years ago

Pedigree rate: Common ancestor in <10,000 years ago

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IV. Improvement on Current Findings





V. USING THE RATE

28Bringing the rates together

Figure: Ho et al. (2005)

29Bringing the rates together


30A better outgroup is in the nucleus

MtDNA integrated into nucleus

540 bp segment Identical in all

tested genomes (Zischler et al., 1995)

Figure: Zischler et al. (1995)

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V. Using the Rate





V. USING THE RATE

32Use in forensics

Forensic applications focus on HV1 and HV2 Romanov identification (Butler and Levin,

1998) Tsarina, her daughters, Prince Philip were exact

matches One mismatch for Tsar Nicholas II and relatives “Anastasia” did not match

33Dating divergence

Estimating common ancestor of Neanderthals and Humans


34Unrelated to “Great” Grandma

How far back in time do we need to go to be “unrelated” to our ancestors?

1.1% (12 bp) difference in unrelated control sequences (Piercy et al., 1993)

Roughly 1000 generations before we are unrelated to our ancestors

35Knowing this, we look deeper

Substitution rate is effectively variable Temporally and spacially

Allows a second look at archeological dates Could help us understand relationships

better Methods used in mtDNA could be extended

36Acknowledgements

Thank you! Friends Family Chris Cole and rest of biology faculty Everyone else here

37Questions?

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