Todd D. Taylor, Ph.D.Genome Annotation and Comparative Analysis TeamComputational and Experimental Systems Biology GroupRIKEN Genomic Sciences Centertaylor@gsc.riken.jp

Bioinformatics and Comparative Genome Analysis CourseInstitut Pasteur Tunis - TunisiaApril 2, 2007

Human Chromosome 21 (Nature, May 2000)

17 of 33.5 Mb Chromosome 18p (Nature, September 2005)

16 Mb Chromosome 11q (Nature, March 2006)

81 Mb ~4-5 % contribution to the Human Genome

Project Chimpanzee

Chromosome 22q (Nature, May 2004) 33.5 Mb (syntenic to human chr21)

Chromosome Y (Nature Genetics, January 2006) Development of novel methods for gene and promoter

prediction Identifying genes missed by other high-throughput

methods Identification of unique regulatory mechanisms

Looking for similarities Compare with distant species, like mouse Regions that are conserved may be important

Looking for differences Compare with close species, like primates Regions that are different may be important

Of course, there are exceptions to every rule!

Homo

Pan

Gorilla

Gibbons

Old world monkeys

New world monkeys

Prosimians

Hom

inid

ae

Cat

arrh

ini

Hom

inoi

dea

Ant

hrop

oide

a

Pri

mat

es

Eut

heri

a (p

lace

ntal

ia)

Mam

mal

ia

Lagomorpha

Rodents

Sauropsida

Am

niot

a (a

mni

otes

)

Pongo

5 MYa

Hom

inoi

dea

Pri

mat

es

Mam

mal

ia

Reptilia + Aves

~35

0MY

a

~２

５0M

Ya

Metatheria

Prototheria

Hom

inid

ae

HeterodontyMammary glandsHomoeothermicHairPlacentation (in most), amnion, internal fertilizationSweat and sebaceous glandsAnucleate red blood cells

34% maps to identical sequence in human genome

Hiram Clawson and Kate Rosenbloom (UCSC). 09 June 2006

95% maps to identical sequence in human genome

Hiram Clawson and Kate Rosenbloom (UCSC). 09 June 2006

Nobrega, et al. Science 302, 413 (2003)

Size Intelligence Language Ageing Disease susceptibility

Cancer Schizophrenia Autism Triplet expansion

diseases AIDS Hepatitis

Newton,2002 年４月号

Science 295, 131-134 (2002)

1.23% substitution

Number of simple repetitive sequences

Insertion of Alu and L1 elements Unique sequences Local duplications Translocations Inversions Fewer CpG Islands predicted in chimp

Compare with small ‘representative’ human chromosome (21)

Clone-based sequencing strategy Map chimp BAC-end sequences to human chr. 21 Screen libraries for additional clones to fill gap

regions 3 gaps, over 99% coverage

Human Chr21 q-arm

Chi

mp

Chr

22 q

-arm

100%

85%

5Mb

Iden

tity

Human Chr21 q-arm

Chi

mp

Chr

22 q

-arm

100%

85%

1Mb

Iden

tity

HSA21q

0.00

0.05

0.10

0.15

0.20

0.25

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32

Bas

e ch

ange

s or

inse

rtio

n si

ze p

erbp

0.0000

0.0010

0.0020

0.0030

0.0040

0.0050

Inse

rtio

n fr

eque

ncy

per

bp

PTR22q

0.00

0.05

0.10

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0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32Position (Mb)

Bas

e ch

ange

s or

inse

rtio

nsi

ze p

er b

p

0.0000

0.0010

0.0020

0.0030

0.0040

0.0050

Inse

rtio

n fr

eque

ncy

per

bpBase change

Insertion size (bp)

Insertion frequency

Chimpanzee Sequencing & Analysis Consortium. Nature (205) 437:69-87

Overall : 1.44%

SINE/Alu 1.81% LINE/L1 1.38% CpG islands 2.26% Simple repeats 4.06%

Base change

Insertion frequency

Base change 1.000 -

Insertion frequency 0.907 1.000

Insertion size 0.051 0.013

Size (bp)# of Sequence gapsEstimated total size ofclone gaps# of clone gaps

Base content G+C%CG dinucleotideCpG islandsRepeats bp # ID# bp # ID#

SINEs 3,647,427 15,574 15,131 3,614,185 15,481 9,551

Young Alus *1 21,798 75 75 3,122 12 12

LINEs 5,848,427 13,758 8,731 5,737,082 13,671 6,223

Young L1s *2 92,171 59 52 78,653 64 53

LTRs 3,612,930 9,975 7,269 3,551,044 9,838 5,324

DNA elements 949,215 4,169 3,363 943,348 4,187 2,887

RNAs 8,625 98 97 8,672 99 98

Satellite 17,246 23 20 14,773 20 17

Others 30,452 41 38 34,852 49 42

Total 14,114,322 43,638 34,649 13,903,956 43,345 24,142

42.6% 42.4%

*1 AluYa5, AluYa8, AluYb8 and AluYb9

*2 L1HS and L1PA2

241.01%

PTR22q32,799,845

2274,311

358,450885

HSA21q33,102,702

1473,108

340.94%361,259

950

Family Subfamily HS21 PTR22

LINE/L1 L1HS 11 2

LTR/ERV1 HERVIP10FH 14 5

MER41A-int 10 2

MER4A1-int 5 0

MER83B-int 11 0

MER87 32 12

SINE/Alu AluYa5 23 3

AluYb8 37 2

AluYb9 7 1

DNA/MER2 Tigger3 42 67

LTR/ERV1 LTR49-int 11 23

LTR/MaLR MLT1E-int 0 5

Human-specific characteristics have been acquired during the 5 million years since the divergence between Pan and Homo.

Phylogeny of Hominidae

Time

Gorilla

Pan(Chimpanzee)

Homo (Human)

Pongo(Orangutan)

5 〜６ MYa

Human(?)Chimpanzee

Gorilla

Orangutan

Homo ACGTGTTTGAAATATTACTGATTGTAAPan ACGAGTTTGAAATATTATTGATTGTAAGorilla ACGTGTTTGAATCATTATTGATTGTAAOrangutan ACGTGTTTAAATTATTATTGGTTGCAALCA ACGTGTTTGAAATATTATTGATTGTAA

Gorilla

Pan(Chimpanzee)

Homo

Pongo(Orangutan)

Time

LCA

Outgroup

(LCA: The Last Common Ancestor)

HSA21q

0.00

0.05

0.10

0.15

0.20

0.25

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32

Bas

e ch

ange

s or

inse

rtio

n si

ze p

erbp

0.0000

0.0010

0.0020

0.0030

0.0040

0.0050

Inse

rtio

n fr

eque

ncy

per

bp

Human

Chimpanzee

Gorilla

Orangutan

IN/DEL examination based on 10,292,002 finished sequences RIKEN

totalPCR primers designable

good amplification for both*

insertion to the human sequence

267 158 139

insertion to the chimp sequence

222 147 128

489 305 267

* positive amplification found for both chimp and human template DNA

106

1 2 3 4 1 2 3 4 1 1 2

Example 1 Deletion in Human Lineage

Example 2 Insertion in Human Lineage

1 2 3 4 1 2 3 4 1 1 2

Pt Hs Gg Pp

117

129

Example 3 Deletion in Chimp Lineage

1 2 3 4 1 2 3 4 1 1 2

Pt Hs Gg Pp

Pt Hs Gg Pp

1900

9802900

4200

1300

4200

106

1 2 3 4 1 2 3 4 1 1 2

154

Example 4Allelic Deletion in Chimp Lineage

1 2 3 4 1 2 3 4 1 1 2 Pt Hs Gg Pp

1200

2400

284 genes 223 known 19 novel CDS 25 novel transcripts 12 putative 5 predicted

85 pseudogenes

We lacked information for 6 genes located in sequencing gaps

6 hsa21 genes are absent from the ptr22 sequence (H2BFS, 5 KAP genes from the 21q22.1 cluster)

4 hsa21 genes appear to be pseudogenes in chimp

3 ptr22 pseudogenes are absent from the hsa21 sequence

1 hsa21 pseudogene has a complete ORF in ptr22

83% of genes have at least one amino acid replacement

10% of the potential ptr22 proteins are predicted to have a different length Amino acid insertion or deletion Different start codon Different stop codon Other, more complex rearrangement

Shorter in chimp: ADAMTS5

Longer in chimp: C21orf30

•17 bp deletion in chimpanzee•Human and chimpanzee splice sites are different•Splice-site diversity

C21orf71

C21orf9

TCP10L

C21orf96

FLJ32835

The human chr21 genes ordered according to their chromosomal position

Se

qu

en

ce

id

en

tity

Human-specific replacements

1. KIAA0184 2. COL6A2 3. HUNK 4. AGPAT3 5. DSCR3 6. PWP2H 7. STCH 8. SLC5A3 9. CHAF1B 10.SIM2 11.KCNE2 12.APP 13.C21orf98 14.C21orf61 15.IFNAR1 16.UBASH3A 17.TMPRSS3 18.DSCR1 19.C21orf7 20.ADARB1 21.TSGA2 22.IFNAR2 23.C21orf63 24.KCNE1 25.C21orf2 26.C21orf55 27.ATP5A 28.CLDN8 29.C21orf5630.DNMTA1

Chimp-specific replacements

1. BACE2 2. TIAM1 3. BACH1 4. FAM3B 5. C21orf33 6. ADAMTS1 7. C21orf103 8. ITGB2 9. HLCS 10.DNMT3L 11.IFNGR2 12.PPIA3L 13.C21orf59 14.MRPL39 15.CLDN17 16.KRTAP11-117.CCT818.DSCR219.TFF220.BTG321.HSF2BP22.C21orf115

0.0

0.5

1.0

1.5

2.0

2.5

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34

0%

5%

10%

15%

20%

25%

30%

35%

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 >1.1

Ka / Ks

Gen

e F

requ

ency

Chimpanzee Sequencing & Analysis Consortium. Nature (205) 437:69-87

Correralate phenotype with genotype

Using Affymetrix arrays it could be shown thatthe amount of transcript/gene varies in a species-specific manner (Enard et al. 2001).

-> What DNA sequence differences are responsible for the observed differences in transcript-levels?

Transcription start site

(TSS)

Promoter

Enhancer

3‘UTR5‘UTR

•Transcriptional control

• RNA stability

ANNOTATED GENES

DETECTED GENES

UPREGULATED (IN HUMAN)

DOWNREGULATED (IN HUMAN)

237 genes annotated for chromosome 21

189 represented on the affymetrix A-E arrays

brain liverIFNAR2IFNGR2ETS2ITSNC21orf97DSCR1LSSTTC3CXADR

higher in chimphigher in human

189 annotated genes represented on the Affymetrix A-E arrays (Hellmann, Pääbo)

Identifying cis-regulatory elements in the human genome is a major challenge of the post-genomic era Promoters and enhancers that regulate gene expression in

normal and diseased cells and tissues Inter-species sequence comparisons have emerged as a

major technique for identifying human regulatory elements Particularly those to the sequenced mouse, chicken and fish

genomes A significant fraction of empirically defined human

regulatory modules Too weakly conserved in other mammalian genomes, such as

the mouse, to distinguish them from nonfunctional DNA Completely undetectable in nonmammalian genomes

Identification of such significantly divergent functional sequences will require complementary methods in order to complete the functional annotation of the human genome Deep intra-primate sequence comparison is a novel

alternative to the commonly used distant species comparisons

Non-coding sequences with primate-specific conservation include three

regulatory elements

Nature (2003) 424:788-793

• Transcript A-B combines at least one exon (complete or partial overlap) from both Gene A & Gene B– Usually only supported by a few mRNA/EST sequences, and

rarely by a CCDS• Currently, about 32 known cases found by searching NCBI

Entrez (including 8 from chr 11 recently submitted by our group)

• Transcript A-B combines at least one exon (complete or partial overlap) from both Gene A & Gene B– Usually only supported by a few mRNA/EST sequences, and

rarely by a CCDS• Currently, about 32 known cases found by searching NCBI

Entrez (including 8 from chr 11 recently submitted by our group)

Child gene AChild gene A

Child gene BChild gene B

Conjoined Gene A – BConjoined Gene A – B

Fused transcript formed by combining the exons of two or more distinct genes (child genes)

Fused transcript formed by combining the exons of two or more distinct genes (child genes)

ExonExon IntronIntron

Chr1 SRP9 – EPHX1 fusion (1 EST evidence-DA417873)Chr1 SRP9 – EPHX1 fusion (1 EST evidence-DA417873)Alternate splicing and novel exons observed in fused mRNAAlternate splicing and novel exons observed in fused mRNA

Number of mRNAs examinedNumber of mRNAs examined 456 (326 conjoined genes)456 (326 conjoined genes)

At least one exon* from both child genes At least one exon* from both child genes conserved in conserved in NumberNumber

Chimpanzee mRNAsChimpanzee mRNAs 125 (69 conjoined genes)125 (69 conjoined genes)

Mouse mRNAsMouse mRNAs 30 (15 conjoined genes)30 (15 conjoined genes)

Both Chimpanzee and Mouse mRNAsBoth Chimpanzee and Mouse mRNAs 25 (11 conjoined genes)25 (11 conjoined genes)

27%27% Conjoined Conjoined genes genes conserved conserved in in ChimpanzeeChimpanzee

6.5%6.5% Conjoined Conjoined genes genes conserved conserved in Mousein Mouse

* Exons considered were part of conjoined gene mRNAs* Exons considered were part of conjoined gene mRNAs

• RIKEN• Yoshiyuki Sakaki• Tulika P. Srivastava• Vineet K. Sharma• Asao Fujiyama• Masahira Hattori• Atsushi Toyoda• Yoko Kuroki• Yasushi Totoki• Hideki Noguchi• Hidemi Watanabe• Takehiko Itoh (MRI)

• Chimpanzee Chr 22 Sequencing Consortium• Chinese National Human Genome

Center at Shanghai, China• KRIBB Genome Research Center,

Daejeon, Korea• National Yang Ming University Genome

Research Center, Taipei, Taiwan• National Institute of Genetics, Mishima,

Japan• RIKEN Genomic Sciences Center,

Yokohama, Japan• GBF, Dept. of Genome Analysis,

Braunschweig, Germany• Institute for Molecular Biotechnology,

Jena, Germany• Max-Planck Institute for Molecular

Genetics, Berlin, Germany