Apicomplexan Genome Sequencing in Sanger Arnab Pain, The Pathogen - - PowerPoint PPT Presentation

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Apicomplexan Genome Sequencing in Sanger

Arnab Pain, The Pathogen Sequencing Unit (PSU) 2nd November, 2005; ICGEB, New Delhi

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Overview

• Overview of Apicomplexan genome

sequencing projects in Sanger

• Update on Plasmodium genome projects
• Theileria and Tropical Theileriosis
• Sequencing & annotation strategies
• Genome architecture
• Gene families
• Metabolic reconstruction
• Comparative genomics (eg. dN/dS analysis,

synteny)

• SNPs/ INDELs

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Apicomplexans and ongoing genome seuencing projects

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Overview of Eukaryotic Pathogen Sequencing in PSU

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The World of Apicomplexans!

Intestinal disease in mammals Parasitise brain/ kidney of rodent Malaria Tropical theileriosis Babesiosis

• Periph. eosinophilia (30%)

Brain infection (Chinchila) Parasitises heart muscle/brain Toxoplasmosis parasitic disease of falcolns bowel diease in human Coccidiosis Insect gut parasites Cryptosporidiosis Insect gut parasites Dog parasites Oyster parasite

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Update on Plasmodium genome projects

• P. falciparum (Gardner et al 2002)
• P. reichenowi (3x shotgun in progress)
• P. knowlesi (3x shotgun)
• P. berghei (3x shotgun complete)
• P. yoelii (5.6x, Carlton et al 2002)
• P. chabaudi (3x shotgun complete)
• P. gallinaceum (3x shotgun in progress)
• P. vivax (complete sequencing)

3D7: 3 gaps Clinical isolate: 8x IT strain: 31,000 reads (0.8x) 8x complete, prefinishing, annotation 8x complete, some finishing 8x complete, prefinish

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Theileria and Tropical Theileriosis

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Genus-Theileria

Theileria parva Parasite of cattle: East/Central Africa

‘East-Coast Fever’

Theileria annulata Parasite of cattle: S. Europe, North Africa, Middle East, Asia

‘Tropical Theileriosis’

Theileria hirci Parasite of sheep/goats:

• S. Europe, North Africa,

Middle East & Asia

(B. Shiels)

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Theileria annulata

Disease; Tropical Theileriosis 250 million cattle are at risk Pathogenic in exotic animals, up to 70% mortality Mild to moderate pathogenicity in indigenous breeds but productivity loss

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Theileria parva

Disease: East Coast Fever 50 million cattle at risk Highly pathogenic in naïve animals 97-100 % mortality rates

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Clonal expansion of infected cells

H.nuc

Macroschizont Merozoite production merozoites Piroplasm infected erythrocytes

Theileria Life Cycle

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Clinical Pathology Tropical Theileriosis (T. annulata) Following lymph node enlargement get fever Marked anaemia - pale mucous membranes which may become jaundiced, diarrhoea/blood stained faeces common Sub acute/chronic cases show intermittent fever, anaemia and Jaundice can be seen Poor condition and convalescence is protracted

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Sequencing & annotation strategies

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Shotgun sequencing

DNA Contiguous sequence

STS-1 STS-2 STS-3 STS-4

pUC clone end sequence physical gap sequence gap large clone end sequence “scaffold”

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Strategy

• Separate chromosomes by PFGE.
• Shotgun sequence individual

chromosomes

• Align Contigs to map and close

gaps using PCR/primer walking.

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Map Resources

• Mapped STS markers.

– Short sequence markers, mapped genetically.

• Mapped YAC clones.

– reads from mapped YAC clones align with contigs and thus position.

• Optical map

– DNA fragments of partial digestion of genome are sized

• ptically and tiled, providing ordered restriction fragments.
• HAPPY MAP

– Fragmented DNA diluted and replicated. STS markers detected by PCR.

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Curating gene models in Artemis

Use of multiple lines of evidence

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• T. annulata Genome

From Karyotype:

• Four chromosomes

– 2.6 Mb (3 gaps) – 2 Mb (Finished) – 1.9 Mb (2 small gaps) – 1.8 Mb (Finished)

From Sequencing:

• Number of bases: 8,351,610
• Gene number: 3792
• Genes with orthologues in T. parva: 3265
• GC percentage: 32.5
• Unique T. annulata genes: 34 (60 in T.

parva)

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Gene finding & Annotation

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e.g. Theileria annulata (~ 8.4 Mb)

Total contig length vs No of reads. Contig no. vs No. of reads

4X 8X 6X 3X 4X 8X 6X 3X

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Genome architecture

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The Chromosomes of P. falciparum

Rep20

TARE2-5

telomere Subtelomeric repeats

VAR genes

Rifin/stevor genes Other gene families

antigenic variation House-keeping antigenic variation

VAR genes Rifin

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Chromosome Structure (Theileria)

Other families (0-2) Family_3 (0-3) Family_1 (up to 28), Family_3 (0-3) Repeats Family_5 (1-3)

(T)TTAGGG telomere

Secreted antigens House-keeping genes Secreted antigens Putative centromere

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Telomeres

• T. parva
• T. parva
• T. annulata
• T. parva

e e

• T. annulata
• T. annulata

A) B) C)

Other fam (0-2) [Fam-1 (up to 28), Fam-3 (0 to 4)] [Fam-3 (0 to 3)]

Subtelomeric repeats (species-specific)

Fam-5 (1-3)

[(T)TTTAGGG]n

m

TaSR3 [TaSrpt2,TaSrpt1] TpSR3 TpSR2 TpSrpt1

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• P. falciparum & P. vivax: sub-telomeric

species-specific gene families

• P. falciparum
• P. vivax

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Centromeres

• P. falciparum

Chromosome 3 P falciparum Chromosome 2

• T. annulata

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Synteny: TA & TP ACT comparison

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Comparative Genomics: synteny

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Comparing genomes with Artemis Comparison Tool (ACT): Chr 02

BlastN & TBlastX

• T. annulata
• T. parva

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Pain et al. Science (2005)

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• T. parva TPR loci

T PARVA T ANN Chr_02

TPR_related family shown in pink

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ACT comparison: 3 malaria species

P P

• P. knowlesi

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“species-specific” genes at interruptuions in synteny

Plasmodium falciparum Plasmodium yoelii Plasmodium knowlesi

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Plasmodium core proteome and “species- specific” genes

Hall et al. Science (2005)

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ACT Comparison: 3D7 vs PFCLIN

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Gene Families

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Clustering Theileria proteins

• All peptide sets from TA and TP combined
• BLASTed against itself with a cutoff of E=10-5.
• TRIBE-MCL run with an inflation value of 5 (quite stringent).
• Each checked for numbers of peptides from each organism.

To identify which gene families have expanded in which

• rganisms.
• Clusters annotated using predicted products in TA & TP

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Theileria-specific gene families: Family 1 (SVSP)

Exclusively Sub-telomeric Contain 1 or more DUF529 (now called FAINT) domains Majority contain signal peptides and conserved C-termini Unequally expanded (48 in TA, 85 in TP) Expressed during macroschizont stage (EST evidence)

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DUF529 domain containing proteins Frequently Appears IN Theileria: FAINT

• Only found in Theileria proteins
• Highly diverged ~70 residue domain
• Majority of FAINT- domain containing proteins have signal peptides
• > 900 copies per genome (in at least 166 Theileria annulata proteins)
• Many are expressed at least at the macroschizont stage

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Comparative Genomics: protein domains

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Architecture of Theileria proteins with FAINT domain

[TA03125 / TP01_0608, Tash1] [TA20095 / TP01_0602, TashAT2] [TA20082, TashAT3] [TA17375 / TP03_0861, Polymorphic antigen precursor / P150] [TA20085 / TP01_0604, TashAT1] [TA08425 / TP04_0437, Microneme-rhoptry protein] [TA17505, SfiI-fragment-related hypothetical protein, family 3] [TA20090 / TP01_0603, TashHN]

• FAINT
• AT-hook
• PEST
• PT
• Signal peptide

(332 aa) (416 aa) (465 aa) (994 aa) (1163 aa) (893 aa) (1338 aa) (2732 aa) [TA18865, Subtelomeric hypothetical protein (SVSP), family 1] [TA18950, Subtelomeric hypothetical protein (SVSP), family 1] (502 aa) (605 aa) Domain keys:

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Whole genome domain

• rganisation of pfEMP1 proteins
• 59 var genes in total.
• Expressed on red

cell surface and involved in sequestration

• 3 types of domain.

– DBL- duffy binding like – CIDR- cystine rich interdomain region – C2 - constant2

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Comparative Genomics: metabolic reconstruction

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KEGG: Phospholipid metabolism

X8

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IMP AMP XMP GMP GTP GDP

glutamate N-acetyl-glutamate glutamine

Amino Compounds

aspartate methionine salvage pathway NOVEL INHIBITORS glycine serine cysteine alanine proline asparagine

• rnithine
• rnithine

spermidine putrescine

Purine salvage, Pyrimidine synthesis

PEP

pyruvate

GLYCEROL GLUCOSE

fructose-6P fructose-1,6-bisP dihydroxyacetone-P + glyceraldehyde-3P

Glycolysis Glycolysis

myo-inositol-1P L-LACTATE 6-phospho- gluconate ribulose-5P ribose-5P

Pentose Phosphate Pentose Phosphate Pathway Pathway

PRPP glucosamine-6P glucosamine glucosamine-1P dihydroorotate

• rotate

dUMP Methylene THF DHF THF xylulose-5P + erythrose-4P CDP dCDP DNA RNA chorismate ?

Shikimic Acid Shikimic Acid Pathway Pathway

CoA dephosphoCoA riboflavin FMN FAD CO2 pABA Folate biosynthesis glycosyl phophatidylinositol (GPI anchors)

NOVEL INHIBITORS

Glycerolipid Metabolism Glycerolipid Metabolism

3-deoxyarabino- heptulosanate- 7-phosphate dTMP Pyrimethamine Cycloguanyl

• xaloacetate

glycerol triacylglycerol phosphatidylcholine choline ethanolamine phosphatidylethanolamine Protohaem (FPIX2+) porpho- bilinogen acetoacetyl-ACP + malonyl-ACP

APICOPLAST

isopentenyl-PP 3-oxoacyl-ACP 3-hydroxy- acyl-ACP enoyl-ACP acyl-ACP acetyl-CoA

Haem Haem Biosynthesis Biosynthesis Fatty acid Fatty acid elongation elongation

Glycerolipids Triclosan Thiolactomycin ALA Haem A Haem C Ubiquinone (UQ) acetate glycine NAD+ NADH modified tRNAs ? malonyl-CoA pyruvate glucose-6P malate

NOVEL INHIBITORS

2C-methylerythrose-4P deoxyxylulose-5P

NOVEL INHIBITORS

O2 FPIX2+ Large peptides Small peptides Amino Amino acids acids Haemoglobin FPIX3+ O2- Haemozoin

FOOD VACUOLE

Chloroquine Artemesinin Quinine

PROTEASE INHIBITORS PROTEASE INHIBITORS F, V, & P-type ATPases

ADP ATP

H+

V

ADP ATP P-lipids, Cu2+,

• ther cations?

(16)

P

Na+ H+ Ca2+ H+ Zn2+ H+ Mn2+ H+

water/ glycerol nucleotide

• r nt-sugar?

(2)

nucleo- side/base ADP ATP

H+

F ?

? H+ NOVEL INHIBITORS

Sulfonamides ATP hypoxanthine xanthine guanine

Folate Biosynthesis Folate Biosynthesis

7,8-dihydropteroate dihydrofolate (DHF) tetrahydrofolate (THF) pABA Pyrimethamine Cycloguanyl

Purines and Purines and Pyrimidines Pyrimidines

H2O Cytc Fe3+ Cytc Fe2+ UQ UQH2 O2

• r

Atovaquone CO2 + aspartate

MITOCHONDRION

acetyl-CoA glucose-1P

DOXP Pathway DOXP Pathway

Fosmidomycin aminolevulinic acid (ALA)

• xoglutarate

citrate

Tricarboxylic acid Tricarboxylic acid cycle cycle

• xaloacetate

malate fumarate succinate succinyl-CoA isocitrate cis-aconitate

Fatty Acid Fatty Acid Biosynthesis Biosynthesis

NOVEL INHIBITORS

PPi

H+

(2)

Pi ADP ATP

ABC transporters

(13)

drugs?

NOVEL INHIBITORS NOVEL INHIBITORS

? malate

• xaloacetate
• r

ubiquinone pool

Gardner et al. Nature (2002)

? ++

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Transcription-Associated Proteins (TAPs)

Ta Pf Sp Sc At Ce Ag Dm Hs Ta Pf

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Transcription Associated Proteins (TAPs)

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Comparative Genomics: genes under selection

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The dN/dS ratio is higher on average in genes with signal peptides

dN/dS ratio of genes with and without signal peptides in T.parva and T. annulata

0.05 0.1 0.15 0.2 0.25 0.3 0.35

• .

4 9 . 1

• .

1 4 9 . 2

• .

2 4 9 . 3

• .

3 4 9 . 4

• .

4 4 9 . 5

• .

5 4 9 . 6

• .

6 4 9 . 7

• .

7 4 9 . 8

• .

8 4 9 dN/dS Proportion of genes sigp non_sigp

The dN/dS distribution is different. Possibly proteins with signal peptide are interacting with the host immune system hence dN/dS may be an indicator of antigenicity

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Orthologous genes under ‘selection’

Pb vs Py Pb vs Pc Pc vs Py Pb vs Pf Pc vs Pf All rodent All

• Av. Protein

Identity 88 % 83 % 85 % 63 % 62%

• Av Nucleotide

Identity 91 % 87 % 88 % 70 % 70 %

• Median dN

4.2% 7% 6.7% 27% 26.9%

• Median dS

26 % 48% 53% 26% 26.5%

• Mean dN/dS

0.16 0.13 0.11 0.009 0.0092

• Number of
• rthologous gene

pairs detected 3153 4678 3318 3890 3842 2528 2125

Most orthologues are easily identifiable, so where do the differences lie?

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Comparative Genomics: genes under selection

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Comparing P. falciparum strains / isolates

• Genes not present in 3D7
• Genes unique to 3D7
• Rapidly evolving genes
• SNP detection
• Indels (insertions, deletions)
• Any other noteworthy differences from

3D7

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Data

• Plasmodium falciparum (3D7) genome

– Laboratory-adapted isolate, completed 2002 – 23 Mb, 14 chromosomes

• Sanger Pathogen Group projects

–

• P. falciparum Ghanaian Isolate (PFCLIN)

– 266K Plasmodium reads (~8x) – Only just completed – all prelim. analysis to date on 5X

–

• P. falciparum IT strain (IT)

–

• Ex. Brazil, laboratory-adapted

– 36K Plasmodium reads (~1X) –

• P. riechenowi (REICH)

– chimp parasite – 51K Plasmodium reads (~1x)

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Analysis

• Genome reconstruction by mapping

PFCLIN (X5 assembly) contigs onto 3D7 scaffold (ACT)

• Comparative annotation
• Mapping WGS reads onto 3D7 scaffold

(SSAHA2-Sequence Search and Alignment by Hashing Algorithm )

– SNPs – INDELs

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Contig ordering & annotation of PFCLIN

PF3D7 (chromosome)

NNNNNNNNNN NNN NNN NNN

PFCLIN (ordered contigs) PFCLIN (annotated contigs)

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Contig ordering I: Chr 1

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Mapping reads onto 3D7 assembly using SSAHA

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SNP detection (SSAHA2): Chr1 & Chr2

(1135) (738, 633) (576, 559) (446) (1140) (963, 789) (809, 601) (464)

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Acknowledgements