Introduction to Genomics and the Tree of Life Chapter 13.

Introduction to Genomics and the Tree of Life

Chapter 13

Extra-Reading

• Next generation sequencer– What next generation sequencer can do for

genetics/genomics research?

• Compar_genomics– What can we learn from comparative

genomics?

Outline of today’s lecture

Introduction: 5 perspectives, history of life

Genome-sequencing projects: chronology

Genome analysis: criteria, resequencing, metagenomics

DNA sequencing technologies: Sanger, 454, Solexa

Process of genome sequencing: centers, repositories

Genome annotation: features, prokaryotes, eukaryotes

Five approaches to genomics

As we survey the tree of life, consider these perspectives:

Approach I: cataloguing genomic informationGenome size; number of chromosomes; GC

content; isochores; number of genes; repetitive DNA; unique features of each genome

Approach V: Bioinformatics aspectsAlgorithms, databases, websites

Approach IV: Human disease relevance

Approach III: function; biological principles; evolutionHow genome size is regulated; polyploidization; birth and death of genes; neutral theory of

evolution; positive and negative selection; speciation

Approach II: cataloguing comparative genomic informationOrthologs and paralogs; COGs; lateral gene transfer

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IntroductionLessons learned form comparative genomics What have we learned about genes by comparing genomic

sequences? What have we learned about regulation? About 5% of the human genome is under purifying selection Positively regulated regions Mechanisms and history of mammalian evolution Nonuniformity of neutral evolutionary rates within species Nonuniformity of evolution along the branches of phylogenyLearning more form existing data Choice of species Choice of toolsFuture of comparative genomics

Levels of analysis in genomics

level topics databasesDNA genes, chromosomes GenBankRNA ESTs, ncRNA UniGene, GEOprotein ORFs, composition UniProtcomplexes binary, multimeric BINDpathways COGs, KEGGorganellesorgansindividuals variation and disease HapMapspecies speciation TaxBrowser; SGDgenus JAX mouse phylum FishBasekingdom TOL

Definitions of terms

Genomics is the study of genomes (the DNA comprising an organism) using the tools of bioinformatics.

Bioinformatics is the study protein, genes, and genomes using computer algorithms and databases.

Systematics is the scientific study of the kinds and diversity of organisms and of any and all relationships among them.

Classification is the ordering of organisms into groups on the basis of their relationships. The relationships may be evolutionary (phylogenetic) or may refer to similarities of phenotype (phenetic).

Taxonomy is the theory and practice of classifying organisms.

Fig. 13.1Page 521

Pace (2001) described a tree of life based on small subunit rRNA sequences.

This tree shows the mainthree branches describedby Woese and colleagues.

Historically, trees were generated primarily usingcharacters provided by morphological data. Molecularsequence data are now commonly used, includingsequences (such as small-subunit RNAs) that arehighly conserved.

Visit the European Small Subunit Ribosomal RNAdatabase for 20,000 SSU rRNA sequences.

Molecular sequences as basis of trees

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http://www.zo.utexas.edu/faculty/antisense/Download.html

Tree of life from David Hillis’ lab (based on ~3000 rRNAs)

animalsplants

fungi

protists

bacteriaarchaea

you are here

http://www.zo.utexas.edu/faculty/antisense/Download.html

you are here

Tree of life from David Hillis’ lab (based on ~3000 rRNAs)

Ribosomal RNA Database

Ribosomal Database Projecthttp://rdp.cme.msu.edu/index.jsp

Santos, S. R. and Ochman H. Identification and phylogenetic sorting of bacterial lineages with universally conserved genes and proteins. Environmental Microbiology. 2004. Jul(6)7:754-9.

►Download fusA (translation elongation factor 2 [EF-2])►Obtain DNA in the fasta format►Align by ClustalW in MEGA►Create a neighbor-joining tree

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European Small Subunit Ribosomal RNA database(http://www.psb.ugent.be/rRNA/ssu/)

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Lactococ lactis Il1403 fusA

Strep agalactiae NEM316 fusA

Strep agalactiae 2603VR fusA

Strep pneumoniae R6 fusA

Strep pneumoniae TIGR4 fusA

Strep pyogenes M1 GAS fusA

Strep pyogenes MGAS8232 fusA

Strep pyogenes MGAS315 fusAStrep pyogenes SSI1 fusAOnion yel phytoplasm OYM fusAMycoplas mobile 163K fusAMycoplas pulmonis UAB CTIP fusAMycoplas mycoides PG1 fusA

Mycoplas penetrans HF2 fusA

Ureaplasma parvum 700970 fusA

Mycoplas galli R fusA

Mycoplas genita G37 fusA

Mycoplas pneumon M129 fusA

Thermoanaero tengcongensis fusA

Fuso nucleatum ATCC25586 fusA

Clost perfringens 13 fusA

Clost acetobutylicum 824 fusA

Clost tetani E88 fusA

Parachlamydia UWE25 fusA

Chlamy muridarum fusA

Chlamy tracho DUW3CX fusA

Chlamydo caviae GPIC fusA

Chlamydo pneumon J138 fusA

Chlamydo pneumon CWL029 fusA

Chlamydo pneumon AR39 fusA

Chlamydo pneum

on TW183 fusA

Prochloro marinus CCM

P1375 fusA

Prochloro marinus CCM

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Nostoc PCC7120 fusA

Synechocystis PCC6803 fusA

Gloeo violaceus PC

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Thermosynecho elongatus BP1 fusA

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E coli CFT073 fusA

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Shigella flexneri 2457T fusA

Shigella flexneri 301 fusA

Lepto inter lai 56601 fusA

Lepto inter Copen Fio L1130 fusA

Pirellula 1 fusA

Aquifex aeolicus fusA

Thermotoga maritima MSB8 fusA

Bacteroides thetaio VPI5482 fusA

Porphyro gingiv W83 fusA

Geo sulfur PCA fusAChloro tepidum TLS fusA

Bordet bronchi RB50 fusABordet pertussis TohamaI fusABordet parapert 12822 fusARalstonia solan GMI1000 fusA

Chromo violaceum 12472 fusA

Xanthomonas axonopodis 306 fusA

Xanthomonas campestris 33913 fusA

Pseudo aeruginosa PA01 fusA

Pseudo putida KT2440 fusA

Pseudo syringae DC3000 fusA

Desulfo vulgaris Hilden fusA

Agro tumefaciens C58 fusA

Sinorhiz meliloti 1021 fusA

Mesorhiz loti MAFF303099 fusA

Bruc suis 1330 fusA

Caulo crescentus CB15 fusA

Bradyrhiz japonicum USDA110 fusA

Rhodopseudo palustris CGA009 fusA

Deino radiodurans R1 fusA

Thermus therm

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Coryne efficiens YS314 fusA

Coryne gluta 13032 fusA

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Yersinia pestis

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Neighbor-joining tree of ~150 fusA (GTPase) DNA sequences

History of life on earth

4.55 BYA formation of earth (violent 100 MY period)4.4-3.8 BYA last ocean-evaporating impacts3.9 BYA oldest dated rocks3.8 BYA sun brightened to 70% of today’s luminosity

Ammonia, methane, or carbon dioxide atmosphere.Earliest life: RNA, protein

Source: Schopf J.W. (ed.), Life’s Origins (U. Calif. Press, 2002)

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1000 100 0500

InsectsCambrianexplosion

Age of Reptiles ends

Land plants

Proterozoic eon Phanerozoic eon

deuterostome/protostome

echinoderm/chordate

Millions of years ago (MYA)

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Dinosaurs extinct;Mammalian radiation

Human/chimpdivergence

100 10 050

Mass extinction

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Homo sapiens/Chimp divergence

Emergence ofHomo erectus

Earlieststone tools

10 1 05

AustralepithecusLucy

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Homo erectusemerges in Africa

MitochondrialEve

1,000,000 100,000 0500,000

Years ago

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Years ago

Neanderthal and Homo erectus disappear

Emergence ofanatomically

modern H. sapiens

100,000 10,000 050,000

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Years ago

“Ice Man”from Alps Aristotle

10,000 1,000 05,000

Earliestpyramids

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Years ago

algebra calculusDarwin,MendelGutenberg

1,000 100 0500

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We will next summarize the major achievements ingenome sequencing projects from a chronologicalperspective.

Chronology of genome sequencing projects

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1976: first viral genomeFiers et al. sequence bacteriophage MS2 (3,569 base pairs,Accession NC_001417).

1977:Sanger et al. sequence bacteriophage X174.This virus is 5,386 base pairs (encoding 11 genes).See accession J02482; NC_001422.


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1981Human mitochondrial genome16,500 base pairs (encodes 13 proteins, 2 rRNA, 22 tRNA)Today (10/09), over 1800 mitochondrial genomes sequenced

1986Chloroplast genome 156,000 base pairs (most are 120 kb to 200 kb)


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mitochondrion

chloroplast

Lackmitochondria (?)

http://www.ncbi.nlm.nih.gov/genomes/ORGANELLES/organelles.html

Entrez Genomes organelle resource at NCBI

There are >2100 eukaryotic organelles (10/09)

http://megasun.bch.umontreal.ca/gobase/

GOBASE: resource for organelle genomes

http://www-lecb.ncifcrf.gov/mitoDat/

MitoDat: resource for organelle genomes

“This database is dedicated to the nuclear genes specifying the enzymes, structural proteins, and other proteins, many still not identified, involved in mitochondrial biogenesis and function. MitoDat highlights predominantly human nuclear-encoded mitochondrial proteins.”

Not updated recently.

http://www.mitomap.org/

MitoMap: resource for organelle genomes

It is possible to map mutations in human mitochondrial DNA that are responsible for disease

1995: first genome of a free-living organism, the bacterium Haemophilus influenzae


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1996: first eukaryotic genome

The complete genome sequence of the budding yeastSaccharomyces cerevisiae was reported. We willdescribe this genome soon.

Also in 1996, TIGR reported the sequence of the firstarchaeal genome, Methanococcus jannaschii.


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1997:More bacteria and archaeaEscherichia coli4.6 megabases, 4200 proteins (38% of unknown function)

1998: first multicellular organismNematode Caenorhabditis elegans 97 Mb; 19,000 genes.

1999: first human chromosomeChromosome 22 (49 Mb, 673 genes)


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1999: Human chromosome 22 sequenced

2000:Fruitfly Drosophila melanogaster (13,000 genes)

Plant Arabidopsis thaliana

Human chromosome 21

2001: draft sequence of the human genome(public consortium and Celera Genomics)


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• Selection of genomes for sequencing

• Sequence one individual genome, or several?

• How big are genomes?

• Genome sequencing centers

• Sequencing genomes: strategies

• When has a genome been fully sequenced?

• Repository for genome sequence data

• Genome annotation

Overview of genome analysis

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Applications of Genome Sequencing

Purpose Template Example

De novo sequencing

Genome sequencing Sequencing >1000 influenza genomes

Ancient DNA Extinct Neanderthal genome

Metagenomics Human gut

Resequencing Whole genomes Individual humans

Genomic regions Assessment of genomic rearrangements or disease-associated regions

Somatic mutations Sequencing mutations in cancer

Transcriptome Full-length transcripts Defining regulated messenger RNA transcriptsSerial Analysis of

Gene Expression (SAGE)

Noncoding RNAs Identifying and quantifying microRNAs in samples

Epigenetics Methylation changes Measuring methylation changes in cancer

Table 13.15 p.538

Fig. 13.8p.539


Criteria include:

• genome size (some plants are >>>human genome)• cost• relevance to human disease (or other disease)• relevance to basic biological questions• relevance to agriculture

Criteria for selecting genomes for sequencing

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Criteria include:

• genome size (some plants are >>>human genome)• cost• relevance to human disease (or other disease)• relevance to basic biological questions• relevance to agriculture

Recent projects:Chicken Fungi (many)Chimpanzee Honey beeCow Sea urchinDog Rhesus macaque

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Selection of genomes for sequencing is basedon specific criteria.

For an overview, see a series of white papers posted on the National Human Genome Research Institute (NHGRI) website: http://www.genome.gov/10002154

For a description of NHGRI selection criteria, visit:http://www.genome.gov/10001495

Selection criteria

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Sequence one individual genome, or several?

Try one…

--Each genome center may study one

chromosome from an organism

--It is necessary to measure polymorphisms

(e.g. SNPs) in large populations

For viruses, thousands of isolates may be sequenced.

For the human genome, cost is the impediment.

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How big are genomes?

Viral genomes: 1 kb to 350 kb (Mimivirus: 1181 kb)

Bacterial genomes: 0.5 Mb to 13 Mb

Eukaryotic genomes: 8 Mb to 686 Gb (human: ~3 Gb)

Diversity of genome sizes

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viruses

plasmids

bacteria

fungi

plants

algae

insects

mollusks

reptiles

birds

mammals

Genome sizes in nucleotide base pairs

104 108105 106 107 10111010109

The size of the humangenome is ~ 3 X 109 bp;almost all of its complexityis in single-copy DNA.

The human genome is thoughtto contain ~30,000-40,000 genes.

bony fish

amphibians

http://www3.kumc.edu/jcalvet/PowerPoint/bioc801b.ppt

Genus, species Subgroup Size (Mb) #chr common name

Macropus eugenii Mammals 3800 8 tammar wallaby

Oryctolagus cuniculus Mammals 3500 22 rabbit

Cavia porcellus Mammals 3400 31 guinea pig

Pan troglodytes Mammals 3100 24 chimpanzee

Homo sapiens Mammals 3038 23 human

Bos taurus Mammals 3000 30 cow

Dasypus novemcinctus Mammals 3000 32 nine-banded armadillo

Loxodonta africana Mammals 3000 28 African savanna elephant

Sorex araneus Mammals 3000 European shrew

Rattus norvegicus Mammals 2750 21 rat

Canis familiaris Mammals 2400 39 dog

Zea mays Land Plants 2365 10 corn

Aplysia californicaOther Animals 1800 17 California sea hare

Danio rerio Fishes 1700 25 zebrafish

Gallus gallus Birds 1200 40 chicken

Triphysaria versicolor Land Plants 1200 plant parasite

16 eukaryotic genome projects > 1000 megabases

Ancient DNA projects

Special challenges:

• Ancient DNA is degraded by nucleases• The majority of DNA in samples derives from unrelated organisms such as bacteria that invaded after death• The majority of DNA in samples is contaminated by human DNA• Determination of authenticity requires special controls, and analysis of multiple independent extracts

2

Metagenomics projects

Two broad areas:

• Environmental (ecological) e.g. hot spring, ocean, sludge, soil

• Organismal e.g. human gut, feces, lung

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Introduction: 5 perspectives, history of life: time lines






20 Genome sequencing centers contributedto the public sequencing of the human genome.

Many of these are listed at the Entrez genomes site.(Or see Table 19.3, page 803.)


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Whole genome shotgun sequencing (Celera)

Hierarchical shotgun sequencing (public consortium)

Two approaches to genome sequencing

Whole Genome Shotgun (from the NCBI website)

An approach used to decode an organism's genome by shredding it into smaller fragments of DNA which can be sequenced individually. The sequences of thesefragments are then ordered, based on overlaps in the genetic code, and finally reassembled into the complete sequence. The 'whole genome shotgun' (WGS) method isapplied to the entire genome all at once, while the 'hierarchical shotgun' method is applied to large, overlapping DNA fragments of known location in the genome.

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Human genome project: strategies

Whole genome shotgun sequencing (Celera)

-- given the computational capacity, this approach is far faster than hierarchical shotgun sequencing-- the approach was validated using Drosophila

Hierarchical shotgun methodAssemble contigs from various chromosomes, then sequence and assemble them. A contig is a set of overlapping clones or sequences from which a sequence can be obtained. The sequence may be draft or finished.

A contig is thus a chromosome map showing the locations of those regions of a chromosome where contiguous DNA segments overlap. Contig maps are important because they provide the ability to study a complete, and often large segment of the genome by examining a series of overlapping clones which then provide an unbroken succession of information about that region.


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Hierarchical shotgun sequencing (public consortium)

-- 29,000 BAC clones-- 4.3 billion base pairs-- it is helpful to assign chromosomal loci to sequenced fragments, especially in light of the large amount of repetitive DNA in the genome-- individual chromosomes assigned to centers


Source: IHGSC (2001)

Fig. 19.8Page 804Source: IHGSC (2001)

Sequenced-clone contigs are merged to form scaffolds of known order and orientation

A typical goal is to obtain five to ten-fold coverage.

Finished sequence: a clone insert is contiguouslysequenced with high quality standard of error rate0.01%. There are usually no gaps in the sequence.

Draft sequence: clone sequences may contain severalregions separated by gaps. The true order andorientation of the pieces may not be known.

When has a genome been fully sequenced?

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Fold coverage % sequenced0.25 220.5 390.75 531 632 87.53 954 98.25 99.46 99.757 99.918 99.979 99.9910 99.995


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Raw data from many genome sequencing projectsare stored at the trace archive at NCBI or EBI

(main NCBI page, bottom right).

Also visit: http://trace.ensembl.org/

As of October 2008, the Trace Archive had ~2b traces.

As of October 2009 it has ~2,108,000,000 traces.

Trace repository for genome sequence data

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Fig. 13.12Page 553

http://www.jgi.doe.gov/education/

http://www.youtube.com/watch?v=RLsb0pMx_oU&feature=channel_page

A Howard Hughes Medical Institute (HHMI) video production describing the Whole Genome Shotgun Sequencing process at the JGI. This video is viewable on YouTube in three parts: Part1(chapters 1-5), Part 2 (chapters 6-8), Part 3 (chapters 9-14).

http://www.jgi.doe.gov/education/




http://www.youtube.com/watch?v=eJ3WvQsPLUk&feature=channel

http://www.youtube.com/watch?v=VhH-SJKGAPo&feature=channel

Role of comparative genomics

Phylogenetic footprinting

Phylogenetic shadowing

Population shadowing

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Fig. 13.13Page 554


Introduction: 5 perspectives, history of life: time lines






Fig. 13.14Page 555

Information content in genomic DNA includes:

-- nucleotide composition (GC content)

-- repetitive DNA elements

-- protein-coding genes, other genes

Genome annotation

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20 30 40 50 60 70 80

GC content (%)

Vertebrates

Invertebrates

Plants

Bacteria

3

5

10

Nu

mb

er o

f sp

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sin

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h G

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5

GC content varies across genomes

Fig. 13.15Page 556

Gene prediction tools• http://bioinformatics.ca/links_directory/?subcategory_i

d=39• http://www.geneprediction.org/

Common tools

GenScan: http://genes.mit.edu/GENSCAN.html

HMMgene: http://www.cbs.dtu.dk/services/HMMgene/

Microbial: http://www.ncbi.nlm.nih.gov/genomes/MICROBES/glimmer_3.cgi

Fungal:

http://www.cbcb.umd.edu/software/GlimmerHMM/

http://bioinformatics.ca/links_directory/?subcategory_id=39

http://bioinformatics.ca/links_directory/?subcategory_id=39

http://www.geneprediction.org/

http://genes.mit.edu/GENSCAN.html

http://www.ncbi.nlm.nih.gov/genomes/MICROBES/glimmer_3.cgi

http://www.ncbi.nlm.nih.gov/genomes/MICROBES/glimmer_3.cgi

http://www.cbcb.umd.edu/software/GlimmerHMM/

Introduction to Genomics and the Tree of Life Chapter 13.

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