Repetitive and Duplicitous Structure of Genomes

Jeff BaileyS5-432

Human Genome Structure

Hetrochromatic Sequence (tandem satellite repeats) Centromeric alpha-satellite, telomere CAGGG, acrocentric rRNA and beta-

satellite Euchromatic sequence ~3.1 gigabases

Genes (35%) ~25,000 Exons (1%) (transcription more ubiquitous ENCODE) Repetitive Sequences

3% Simple Sequence Repeats (poly A runs, dinucleotide and trinucleotide repeats)

45% Interspersed Repetitive Elements Repetitive Element Size Copies Fraction LINE elements (retrotransposon) up to 8 kb 850,000 21% Alu elements (retrotransposon) 300 bp 1,500,000 13% LTR-retrovirus-like 6-11 kb 450,000 8% DNA transposons 1-3 kb 300,000 3%

(International Human Genome Sequencing Consortium. Science 2001

Vast majority of sequence is non-coding and repetitive.Vast majority of sequence is non-coding and repetitive.

Human Satellites

Centromeric Sequence Human:

171 bp alpha-satellite in array of 2-5 Mb

higher order structure (only in Great Apes) 4-20

4-30 k-mer (A-B-C-D-A-B-C-D-A-B-C-D) A-B-C-D to A-B-C-D (2-5%) A-D- 20-40% Further flanked by other satellites (beta satellite)

Mouse:

234 bp major satellite (6 Mb) an 120 bp (600 kb) minor satellite at centromeric constriction

Arabibdopsis

178 bp satellite in 3 Mb array

Drosophilia:

5 bp simple arrays of AATAT and AAGAG

C. elegans:

Holocentric – entire chromosome acts as centromere

Yeast:

CEN3 1-2 kb of 83 bp repeat

Simple sequence repeats (SSRs) ATGATGATGATG

• SSR: perfect or slightly imperfect tandem repeats of a particular k-mer• About 3% of the human genome (~0.5% by dinucleotide)• Derived from slippage during DNA replication

Microsatellites: n=1-13 basesMinisatellites: n=14-500 bases

Repeat unit Number of SSRs per Mb

Interspersed Repeats

DNA transposons “extinct” in primate lineage (~40 mya). Quiescent in mammalian lineages.

Genome Variability

Annu Rev Genet. 2007; 41: 331–368.

Sc: Saccharomyces cerevisiae; Sp: Schizosaccharomyces pombe; Hs: Homo sapiens; Mm: Mus musculus; Os: Oryza sativa; Ce: Caenorhabditis elegans; Dm: Drosophila melanogaster; Ag: Anopheles gambiae, malaria mosquito; Aa: Aedes aegypti, yellow fever mosquito; Eh: Entamoeba histolytica; Ei: Entamoeba invadens; Tv: Trichomonas vaginalis.

Variation in Relative Content

DNA Transposons

Copy / pastel

Human Retrotransposons

Serial evolution of master elements

L1: 80-100 active L1s (6 hot L1-Ta)

Alu 143 active elements

Alu Yb (puncuated)

– 2000 copies; only handufl in other primates.

SVA (~25 mya)

– pol II, 3000 copies

New integration: L1 and Alu ~ 1 in 20 meioses; SVA 1 in 90

Pol II

Pol III

Pol III

L1 “master” elements

Mouse vs. Human

MGSC Nature, Volume 420, Issue 6915, pp. 520-562 (2002).

Biological Impact of Retrotransposons

Cordaux and batzer Nature Reviews Genetics 10, 691-703 (October 2009)

Biological Importance (cont.)

Boundary / Insulator Elements Alternative splicing / novel

exons / novel genes Role in suppression of poly II

transcription in cellular stress What accounts for long-

term maintenance?

Human Genome Structure

Hetrochromatic Sequence (tandem satellite repeats) Centromeric alpha-satellite, telomere CAGGG, acrocentric rRNA and beta-

satellite Euchromatic sequence ~3.1 gigabases

Genes (35%) ~25,000 Exons (1%) (transcription more ubiquitous ENCODE) Repetitive Sequences

3% Simple Sequence Repeats (poly A runs, dinucleotide and trinucleotide repeats)

45% Interspersed Repetitive Elements Repetitive Element Size Copies Fraction LINE elements (retrotransposon) up to 8 kb 850,000 21% Alu elements (retrotransposon) 300 bp 1,500,000 13% LTR-retrovirus-like 6-11 kb 450,000 8% DNA transposons 1-3 kb 300,000 3%

(International Human Genome Sequencing Consortium. Science 2001

Vast majority of sequence is non-coding and repetitive.Vast majority of sequence is non-coding and repetitive.

• Whole Genome Duplication– Ancient 4N 2N

• Segmental Duplications– Tandem– Interspersed

• Interchromosomal• intrachromosomal

Types of Duplications

Susumu Ohno

• Whole Genome Duplication

• Vertebrate Paradigm: ancient whole genome duplications and recent tandem duplications– (review: Panopoulou (2005) TIG 10:560)

• KEY CONCEPT: New genes usually derived from copies

2n 4n rearrangement 2n

Paralogy--two genes/proteins in the same species which share sequence similarity due to duplication.

2b. Orthology--two genes/proteins in different species which share sequence similarity and are descended from a common ancestor.

3. Xenology--introduction of a new sequence into the genome by horizontal transfer between two species

Segmental Duplication (SD)

Segmental Duplications

Repetitive Element Exon

Time (100s mya)

Key raw material for the evolution of novel genes

Time (1-50 mya)

`

Segmental Duplications (SD)

Bailey and Eichler (2006) Nat Rev Genet

Properties:•Clustered•Complex regions•Dynamic regions

99.1% identical over 180 kb (VCF/DiGeorge Syndrome in 1 in 3000 births)

5.4% of the genome (>90% identity and >1 kb)chr22

SDs Underlie Recurrent Germline Deletions and Duplications

Cen TelI

D D’

CenI D’D

Tel

Tel

Cen

Cen

GAMETES

D D’I I

Change in Dosage Sensitive Genes → phenotype or disease

Dynamic Regions – predisposed to further rearrangements

Non-allelic Homologous Recombination (Lupski, 1999)

D’- D

D - D’

Figure 1identify high-copy repeats

splice out

Analyze alignments (>1 KB; >90% identity)

blast comparisons--allowing for large gaps

reinsert repeats

heuristic end trimming

global alignments

Detection of Segmental Duplications:Whole genome assembly comparison

Human Draft: Regions of SD poorly assembled (collapsed) and many unique regions with unmerged overlaps (allelic) (Bailey et al. Genome Res 2001)

Genome Wide Detection

Assembly % finished 90-98% >98%July 2000 20% 3.6% 12.9%

January 2001 23% 3.6% 10.6%August 2001 44% 4.1% 15.3%

Problem:

Allelic/True Overlap vs.

Duplication

Shotgun Sequence: assembly-independentdetection of high-identity SD

Whole Genome Shotgun Sequence: random sample

Bailey et al. Science 2002

Combined with whole-genome assembly comparison:5.4% of the human genome composed of SDs >1 kb and >90% identity

99.8%

False Positive SD Absent SD (collapsed or missing)

Examine All Public Sequence

Publicsequence

Align Reads: >96% identity

Celera(27.1 M reads)

REPEATS

47

100

200

# R

ead

s / 5

kb

Public

Celera

223

Xq28 donor

Celera Read Depth Across Chr. 22

Covera

ge

Nu

mb

er o

f Read

s/5

kb

w

ind

ow

Diploid Copy # of Duplication

Depth of Coverage vs. Copy Number

R2=0.96

0

200

400

600

800

1000

1200

1400

1600

1800

2000

0 10 20 30 40 50 60

Global Alignments filtered with SDD

5.7%

3.2%

3.2%

3.4%

2.8%

3.4%

7.8%

3.0%

8.2%

5.7% 4.4% 3.3%

3.4% 2.1%

8.2%

9.8% 8.5%

3.1%

8.1%

2.1%

5.2%

10.9%

5.5%

8.8%

40

.7%

0%

5%

10%

15%

20%

25%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

Chromosome

INITIAL

FILTERED

68

.6.%

0%

5%

10%

15%

20%

25%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

Du

plicated

Bases (%

To

tal Ch

rom

oso

me)

INITIAL

FILTERED

•130 candidate regions (298 Mb) •23 associated with genetic disease

SD “Hotspot”Map of Human

Genome

Bailey et al. Science 2002

Interrogation of these regions has lead to detection of 16 additional pathogenic rearrangements including new microdeletions on 1q21.1, 15q13, 15q24 and 17q12. (Sharp et al. Nat Genet 2006; Mefford et al. Am J Hum Genet 2007; Mefford et al. N Engl J Med 2008)

Genetic Distance Finished Sequence

Sept 2000 NT data set(>2KB; >90%; no X—Y)

0200400

600800

1000

12001400

1600

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0100200300400500600700800900

1000

0.0

1

0.0

2

0.0

3

0.0

4

0.0

5

0.0

6

0.0

7

0.0

8

0.0

9

0.1

0Tota

l A

lig

ned

b

ases (

kb

p)

Genetic distance (K)

Intrachromosomal Interchromosomal

Species SDs

Marques-bonet et al. TIG 2009

Duplicated Bases FLY WORM Chrom 22> 1 KB 1.20% 4.25% 9.50%> 5 KB 0.37% 1.50% 7.90%>10 KB 0.08% 0.66% 6.40%

Duplicated Genes

Johnson et al 2001 Nature

Gene Enrichments Immunological Environmental

response Reproduction:

sperm-egg interactions

Morpheus

Duplicon Structure Chr 22

Organizing the MESS

Jiang et al. 2007 Nat Gen:39:1361-8

437 Hubs

Jiang et al. 2007 Nat Gen:39:1361-8

Mechanism: Junction Content

Control +/- 1 kb

Junction (50 bp)

•Duplications >95% and < 99.5%•Only finished sequence•Enrichment for Alu elements

Alu Proximity to Junctions

5%

15%

25%

-500 -400 -300 -200 -100 0 100 200 300 400 500

10 bp window

DUPLICATED UNIQUE

Center of Window (bp from Junction)

Av

era

ge

Alu

Co

nte

nt

(bp

)

Alu Simulation

0

50

100

150

200

250

300

350

0 5 10 15 20 25

Proportion Alu (%)

Nu

mb

er o

f replica

tes

23.8%

Computer simulations to determine significance.

Subfamily Enrichment

20,000

40,000

60,000

80,000

100,000AluY

AluS

AluJ

20

humanchimp

orangutanOld World

New World

ProsimianMammal

gorilla

AluJAluSAluY

40 60 80 mya

≥90% 1.8 1.9 1.1

≥95% 2.2 1.8 1.1

0

Nu

mb

er o

f Ele

me

nts

Whole Genome Duplication

Whole Genome Duplication Yeast

Kellis and Lander (Nature 428:617-24 2004)

Explore Resources

REMINDER OF CLASSExercises for analysis of repetitive elements and segmental duplications