Studying
Modern Human Origins
from Neandertal DNA
Max Planck Institute for Evolutionary Anthropology
Evolutionary Genetics
Martin Kircher
September 9 2010, Berlin
Studying Modern Human Origins
from Neandertal DNA
• Closely related species
and our own history
• Ancient DNA
• Importance of how highthroughput sequencing
• Insights from the
Neandertal genome
2
Closest relatives
• Tell us:
– How evolution shaped our
• Genome
Apes
• Transcriptome
• Proteome
– When things changed
New world
monkeys
Old world
monkeys
~ Lemurs
¾ How we adapted to
environmental changes
¾ What makes us human!
Steiper and Young (2006) Mol. Phyl. & Evol. 41(2):384
3
Closest relatives
•
Chimpanzees and Bonobos
are our closest living relatives
•
~98.8% genome identical
Apes
Bonobo
New world
monkeys
Old world
monkeys
~ Lemurs
Chimpanzee
Steiper and Young (2006) Mol. Phyl. & Evol. 41(2):384
4
Closest relatives
http://www.mnh.si.edu/anthro/humanorigins/index2.htm
5
Neandertals
•
Closest extinct relative
•
Lived in Europe and West Asia
~400 – 30ka
•
Neandertal
Went extinct when modern
humans spread in Europe & Asia
modern human
•
Share ~99.9% of genome
¾ Allow to distinguish recent
changes from shared evolutionary
history
6
Recent changes ...
world-wide dispersion
morphological changes
appearance of art
agricultural and
technological
revolution
7
Ancient DNA
•
DNA can be extracted from
blood, soft tissues as well as
bones, hair and teeth
•
Successful for up to 100ka
old samples
~ 500 mg
1 cm
DNA extract
8
Two types of DNA?
Modern DNA
Contamination
~1μg DNA
per gram tissue
Ancient DNA
~0.0000001-0.001μg DNA
per gram tissue
9
Contamination avoidance
Minimizing contamination
from handling (e.g. Sidron 1253)
EL SIDRÓN (ASTURIAS, SPAIN)
10
Quantifying human contamination
•
Neandertal mitochondrial genomes
fall outside of human variation
– 133 fixed differences can be used
as informative sites
Green et al. Cell 2008 / en.wikipedia.org
11
Quantifying human contamination
•
Neandertal nuclear genome falls within human genomic variation
•
No known fixed differences, other measures possible:
– Triallic sites
– X homozygosity
– Y chromosomal coverage
Green et al. Science 2010
12
Sampling ancient DNA
4.17% 4.49% 2.17% 4.10%
2.5% 1.77% 1.60% 0.86% 1.7%
0.27% 0.40% 0.41%
13
Neandertal libraries
Screened ~200 DNA extracts from at least 70 fossils from 16 sites
Vi33.16
Vi33.25
Vi33.26
Feld1
Mez1
Sid1253
3
2
0
1
% of reads
4
•
Vindija
El Sidron
Neander Valley
Mezmaiskaya
0.2 – 4.0%
0.1 - 0.4%
0.2 - 0.5%
0.8 - 1.5%
0
20
40
60
80
100
120
140
Length
14
Feasibility: Neandertal genome
1997
2000
2005
Sanger sequencing of hypervariable region
of first Neandertal mitochondrial genome
Two additional mitochondrial sequences
454 platform becomes available
Neandertal 1x genome project idea is born
Bone (grams)
Nov. 2006
Aug. 2007
May. 2007
Aug. 2007
Sep. 2007
Feb. 2008
Jul. 2008
(“Proof-of-Principle”)
Improved library prep
454 FLX upgrade
Library amplification
Library enrichment
Titanium upgrade
Illumina/Solexa
20
2
2
0.2
0.2
0.2
0.2
Sequencing (runs)
6.000 (454 GS20)
6.000 (454 GS20)
4.000 (454 FLX)
4.000 (454 FLX)
700 (454 FLX)
300 (454 Titanium)
20 (Illumina GAII)
15
Neandertal Illumina sequencing
• Improved base caller (Ibis)
• Ancient DNA aware
aligner (ANFO)
• Paired End sequencing:
reconstruction of original molecule
• Deep sequencing: PCR duplicate consensus
16
Improved base calling: Ibis
Ibis
Perfect
Error
Perfect
Error
GA II
(51nt, v1)
39.8%
2.0%
60.2%
1.1%
GA II
(77nt, v2)
9.19%
2.74%
36.58%
0.73%
GA II
GA IIx
(76nt, v3)
(101nt, v4)
51.52%
62.60%
0.89%
0.41%
58.90%
65.05%
0.65%
0.33%
0.03
Bustard
GA I
(26nt)
11.3%
7.1%
23.4%
5.4%
Estimated error rate
0.02
0.01
Bustard
Ibis
20
40
60
Position in read
80
100
0.00
0
17
Ancient DNA aligner: ANFO
•
Short, erroneous and
damaged reads are
difficult to align
•
Ancient DNA damage
model, substitutions
and indel aware aligner
•
Modified from Briggs et al. NAR 2010
Highr resolution mapping quality: search for second best alignment
¾ Correct alignments: important for downstream analyses
18
Error profile
Paired End read merging
Reverse read
0
20
Forward read
40
60
80
100
Position in read
19
Paired End read merging
Forward read
20
40
60
Position in read
80
100
0.1
0.2
0
21x error reduction
Average of raw reads (no merging)
Error-informative scores
0.0
Sequencing error on reads [%]
0.3
Reverse read
0
50
100
Insert size
150
20
Neandertal Genome Consortium
Max Planck Institute for
Evolutionary Anthropology
* Adrian Briggs
* Anne Fischer
* Jeffrey Good
* Ed Green
* Janet Kelso
* Johannes Krause
* Martin Kircher
* Michael Lachmann
* Tomislav Maricic
* Matthias Meyer
* Svante Pääbo
* Kay Prüfer
* Susan Ptak
* Qiaomei Fu
* Susanna Rankin
* Rigo Schultz
* Udo Stenzel
* Johann Visagie
* Hernan Burbano
Sequencing group at MPI EVA:
* Aximu Ayinuer-Petri
* Anne Butthof
* Barbara Höber
* Barbara Höffner
* Madlen Siegemund
* Antje Weihmann
Museo Nacional de Ciencias Naturales, Madrid
* Javier Fortea
* Carles LaLueza-Fox
* Marco de la Rasilla
* Antonio Rosas
Rheinisches Museum/University of Tübingen
* Ralf Schmitz
Broad Institute/ MIT
* David Reich
* Nick Patterson
* Chad Nussbaum
* Eric Lander
Whitehead Institute
* Steve Rozen
* Jen Hughes
* Helen Skaletsky
Slatkin Lab: UC Berkeley
* Hua Chen
* Philip Johnson
* Anna-Sapfo Malaspinas
* Josh Pollack
* Montgomery Slatkin
* Rasmus Nielsen
U. of Washington, Seattle
* Evan Eichler
LBL
* Gavin Crookes
EMBL, Heidelberg
* Peer Bork
NIH/NHGRI
* Jim Mullikin
CSHL
* Greg Hannon
* Emily Hodges
* Zhenyu Xuan
* Michelle Rooks
Uppsala University
* Siv Andersson
Oxford University
* Daniel Falush
European Bioinformatics Institute (EBI)
* Ewan Birney
* Paul Flicek
* Ben Paten
* Michael Hoffmann
* Daniel Zerbino
Croatian Academy of Sciences and Arts
* Maja Paunovic
* Dejana Brajkovic
* Jadranka Mauch Lenardic
* Zeljko Kucan
* Ivan Gusic
* Pavao Rudan
Cornell University
* Andy Clark
* Kirk Lohmueller
* Carlos Bustamante
454 Life Sciences Inc
* Jan Berka
* Brian Desany
* Lei Du
* Michael Egholm
* Xavier Gomes
* Jerry Irzyk
* Clotilde Perbost
* Jason Affourtit
21
Science 328, May 2010
22
Neandertal genome
•
454 data from Vindija extracts (206 million reads; 1.4 Gb hominid)
•
Illumina data (214 lanes; 2.5 billion raw reads; 4.1Gb hominid)
•
~ 1.5x: coverage for ~63% bases of human genome
23
Browse the genome...
http://neandertal.ensemblgenomes.org
http://genome.ucsc.edu/Neandertal
24
Time of last common ancestor
x 6.5 ma
Neandertal: 12.7%
French:
8.0%
Han:
8.4%
Papua:
9.3%
Yoruba:
9.4%
San: 10.3%
825,000 yrs
520,000 yrs
550,000 yrs
605,000 yrs
610,000 yrs
670,000 yrs
~6.5 Myr
12.7%
5 human HGDP samples,
sequenced to ~6-8x
(Illumina GAII)
Reference
human
25
Catalog of novel features
fixed in the human genome
•
78 amino acid substitutions
•
45 fixed changes in 5‘ UTRs and
223 fixed changes in 3‘ UTRs of
protein-coding genes
•
1 fixed change in seed region of
hsa-mir-1304
....
Burbano et al. Science 2010
5 genes with two amino acid changes since Neandertal split:
RPTN
SPG17
CAN15
TTF1
PCD16
Epidermal matrix protein
Sperm axoneme
Optic lobe homolog
RNA pol. I termination factor
Ca-dep. fibroblast adhesion
26
Adaptive evolution
• Adaptive changes spread fast in a population
• Regions will show recent SNPs not known to Neandertal
Haplogroups before selection
After selection
Regaining diversity
27
Positive selection
Top 20 candidate regions:
Type II diabetes
Down syndrome
Schizophrenia
Cleidocranial dysplasia
Autism
Green et al. Science 2010
28
Cleidocranial dysplasia (CCD)
•
RUNX2 only gene associated with CCD, a skeletal dysplasia
Ancestral state
CCD patient
Normal rib cage
29
Neandertals interbreed
with modern humans?
• Neandertal mitochondrial DNA outside of known human
variation: No maternal decendents of Neandertals
• Arguments for Neandertal admixture in Europe for
morphological and geographical/temporal reasons: Is
there gene flow detectable in the nuclear genome?
30
Neandertals interbreed
with modern humans?
13 non-african
haplotypes: 10 are
shared with Neandertal
Green et al. Science 2010
31
Neandertals interbreed
with modern humans?
1-4% admixture in
all (tested) out-of-Africa
populations
7
6
Z-Score
5
4
% Neandertal matching to H2 –
% Neandertal matching to H1
HGDP01029 (San) HGDP01029 (Yoruba)
HGDP01029 (San) HGDP00521 (French)
HGDP01029 (San) HGDP00542 (Papuan)
HGDP01029 (San) HGDP00778 (Han)
HGDP01029 (Yoruba) HGDP00521 (French)
3
HGDP01029 (Yoruba) HGDP00542 (Papuan)
2
HGDP01029 (Yoruba) HGDP00778 (Han)
1
0
-1
HGDP00521 (French) HGDP00542 (Papuan)
HGDP00521 (French) HGDP00778 (Han)
HGDP00542 (Papuan) HGDP00778 (Han)
32
What next?
• Higher coverage genome (~20x?)
• Targeted analyses of genomic
regions and candidates
• Functional characterization
of changes!
• Other human forms?
33
Denisovans ...
34
Contact
Max Planck Institute for evolutionary Anthropology
Evolutionary Genetics
Deutscher Platz 6
D-04103 Leipzig
PhD or PostDoc at MPI EVA?
– Viola Mittag (Assistant to
Svante Pääbo)
[email protected]
– Bioinformatics (Janet Kelso)
[email protected]