Goals of the Human Genome Project (1990 ~) Map and sequence the 3,000 Mb human genome
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Transcript of Goals of the Human Genome Project (1990 ~) Map and sequence the 3,000 Mb human genome
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Genomics Databases and Bioinformatics Applications
Wailap Victor NgInstitute of Biotechnology in Medicine
Institute of BioinformaticsDept Biotechnology and Lab Science in Medicine
National Yang Ming University
[email protected] 22, 2005
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Goals of the Human Genome Project (1990 ~)• Map and sequence the 3,000 Mb human genome
• Map and sequence the genomes of model organism
- The bacterium E. coli (4.6 Mb) - The yeast S. cerevisiae (12 Mb) - The roundworm C. elegans (100 Mb) - The fruit fly D. melanogaster (180 Mb) - The mouse M. musculus (3,000 Mb)
• Collect and distribute data
• Study the ethical, legal, and social implications of genetic research
• Train researchers
• Develop technologies
• Transfer technology to the private sector
http://www.genome.gov/Pages/EducationKit/online.htm
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What is the total number of human genes? (Science, 2000)
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Milestones of Genome Projects
1995 Haemophilus influenzae (1.83 Mb; 1,742 genes) 1996 Saccharomyces cerevisae (12 Mb; 6,000 genes) 1998 Caenorhabditis elegans (97 Mb; 19,000 genes) 2000 Arabidopsis thaliana (115/125 Mb; 25,000 genes) 2000 Drosophila melanogaster (~120 Mb; 13,600 genes) 2001 Homo sapiens (90%; 2,900 Mb; ~30k genes) 2002 Mus musculus (96%; 2,500 Mb; ~30K genes) 2002 Oryza sativa L. ssp. indica (92%; 466 Mb; 46-56k genes) 2002 Fugu rubripes (95%; 365 Mb; 33,609 genes) 2004 H. sapiens (99% euchromatin; 2,850 Mb; 20,000-25,000
genes)
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Homo sapiens
Number of cells: ~1x1014
Number of genes encoded by the genome: 20,000 – 25,000
Number of Expressed genes per cell type: 10,000-15,000
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Genome Transcriptome(mRNAs)
Proteome(Proteins)
Alternative splicing
Post-translational modifications
25-30K genes
(Human)
Complexity
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Genome Sequencing Strategies
• Top-down approach - Clone large genomic DNA fragments into special vector, e.g. BAC (bacterial artificial chromosome)
- Create an ordered array of BAC clones
- Carry out full-length BAC clone sequencing
- Assemble the BAC insert sequences
- Identify the next BAC for full length sequencing (Hybridization method or searching BAC end sequence library)
• Bottom-up approach
- Whole genome shotgun sequencing
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Top-down genome sequencing method
Method I. Systematic sequencing of ordered clones- Construct shotgun genomic library in YAC (yeast artificial chromosome) or BAC vector
- Use the YAC or BAC clone DNAs to construct smaller insert shotgun cosmid DNA library (~45 kb inserts)
- Multiple Complete Digest (MCD) mapping of cosmid DNAs ordered cosmid clone library
- Choose the minimal overlap set of cosmid DNA to construct shotgun libraries in M13 or plasmid vector DNA sequencing Assembly
Genomic DNA Large insert library in BAC/YAC
Medium insert library in cosmid
ordered cosmid library
Small insert library
in plasmid
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Flow chart of wet bench procedures for YAC → cosmid and BAC → cosmid MCD mapping. The main difference is that, while BAC DNA can readily be purified from bacterial chromosomal DNA, there is no good preparative method to separate YAC DNA from yeast chromosomal DNA. In the YAC case, the few percent of the cosmids that are derived from the YAC are identified by a hybridization-based colony-screening protocol. With BAC-derived cosmids, this step is unnecessary because the mapping software can readily eliminate the small number of cosmids that do not originate from the BAC.
Proc Natl Acad Sci U S A. 94: 5225 (1997)
(YAC DNA)
Multiple Complete Digest Mapping
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Schematic representation of MCD mapping process.
(a) Gel image.
(b) List of fragment sizes for each enzyme domain in each clone. Lanes labeled with a number identify the clone as c01 or c02. Lanes labeled with the letter M identify size markers.
(c) Three single-enzyme maps are independently constructed (Right). Synchronization across enzyme domains results in a composite map (Left). Long tick marks indicate boundaries between ordered groups of fragments; short tick marks demarcate unordered fragments within a group, arbitrarily drawn in order of decreasing size.
Proc Natl Acad Sci U S A. 94: 5225 (1997)
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Gray scale image of a typical mapping gel poststained with SYBR–green I. There are five marker lanes, at positions 1, 8, 15, 22, and 29. Two clones, each independently digested with EcoRI, HindIII, and NsiI (and loaded in that order) are placed between every pair of marker lanes.
Proc Natl Acad Sci U S A. 94: 5225 (1997)
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Representative MCD map from chromosome 7
Proc Natl Acad Sci U S A. 94: 5225 (1997)
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Method II. BAC by BAC sequencing• Choose BAC clone seeds • Construct BAC shotgun library in plasmid vector• Sequence the shotgun plasmid DNAs• Assemble the shotgun reads• Look for adjacent BAC clones for sequencing –
- By colony array hybridization or- BAC end sequence library
Genomic DNA Large insert library in BAC
Small insert shotgun library
in plasmid
DNS sequencin
g and assembly
Identify neighborin
g BAC clones for sequencin
g
Top-down genome sequencing method
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Genomic DNA Large insert library in BAC
Small insert shotgun library
in plasmid vector
E. coli transformants
Array E. coli on nylon
membrane and grow cells agar
plate
25x25 cm2
BAC colony array hybridization assay
Lyze E. coli colonies on nylon mem
brane
Fix the DNA onto
nylon membrane
Hybridize with PCR amplified
BAC end probes
Autoradiogram
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BAC clone genomic DNA insert (sequenced)PCR-1 PCR-2
BAC colony array hybridization
Restriction fingerprinting
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How many reads is needed to How many reads is needed to determine a genome determine a genome
sequence?sequence? Usually ~8X coverage of each base pair
# reads = ( 8 x genome_size ) / (av._read_length)
e.g. Haloarcula marismortui (4,274,315 bp)
# reads = (8 x 4,274,315 bp) / (550 bp) = 62,172 sequencing reactions
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Principle of Sanger Dideoxy DNA sequencingPrinciple of Sanger Dideoxy DNA sequencing
http://genetics.nbii.gov/basic2.htmlUSB
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Simple one step fluorescent dye-Simple one step fluorescent dye-terminator DNA cycle sequencingterminator DNA cycle sequencing
-ddCTP-ddATP
-ddGTP-ddTTP
DNAPrimerTaq DNA Pol
Reaction buffer
Thermocycling
2-propanol precipitation
DNA analyzer
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Applied Biosystems Capillary DNA SequencerApplied Biosystems Capillary DNA Sequencer
ABI 3730 xlGATCAGGGTTACATGCTACGGCTTCACACGTCGACCCATATTAC...................
Electropherogram (chromatogram)
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> vtrace HM023_0188.y1_096.ab1
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phred
Function – base calling and quality assignment
chromat files (input) phd files (output)
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Example of phd fileExample of phd file
q value: numbers in middle column
q = -10 log (P)
q, quality value
P, estimated error rates
q20 1 error in 100 bases (p=0.01)
q40 1 error in 10,000 bases
(p=0.0001)
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Sequence Assembly SoftwareSequence Assembly Software
phredPhrap (Phil Green) cap3 (Xiaoqiu Huang) TIGR Assembler (TIGR)
ATLAS (BCM) SPS phrap (Geospiza) Genome Assembler (Paracel) Celera Assembler (Celera) BGI Assembler (BGI)
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Basic Functional Genomic Analysis
• Gene Prediction (P: Prokaryotes; E: Eukaryotes)- Glimmer (P)
- GenMark (PE)
- Genscan (E)
- X-grail (E)
- Fgenes (E)
- est2genome (E; EST driven prediction)
* others (http://www.cs.jhu.edu/~salzberg/appendixa.html#Gene_finders)
• Gene Functional Analysis- Blast searches
- Motif analysis
- Structure prediction and homology searches
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Sources of genomics databases and bioinformatics applications
• Public Data Banks - NCBI, EMBL-EBI, and DDBJ
• Genome Centers- DOE Joint Genome Institute
- Baylor College of Med. Human Genome Sequencing Center
- The Welcome Trust Sanger Institute
- Washington Univ. School of Med. Genome Sequencing Center
- Whitehead Institute/MIT Center for Genome Research
- Others (www.ornl.gov/sci/techresources/Human_Genome/research/centers.shtml)
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NCBI Genome Resources
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NCBI Map ViewerH
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Full-
leng
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Animal genome projects
Completed (3)Homo sapiensCaenorhabditis elegansDrosophila melanogaster Draft assembly (17)Canis familiaris (dog)Bos taurus (cattle)Gallus gallus (chicken)Monodelphis domestica Mus musculus (mouse)Rattus norvegicus (rat)Aedes aegypti (mosquito)Anopheles gambiae (mosquito)Apis mellifera (honey bee)Bombyx mori (silk worm)Caenorhabditis briggsae Caenorhabditis remaneiCiona intestinalis Ciona savignyiDrosophila pseudoobscura Drosophila yakubaTakifugu rubripes Tetraodon nigroviridis
In progress (139)
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Plant genome projects
Completed (2)
Arabidopsis thaliana Oryza sativa (rice)
Draft assembly (0)
None listed
In progress (52)
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Fungus genome projectsCompleted (9)
Candida glabrataFilobasidiella neoformansDebaryomyces hanseniiEncephalitozoon cuniculi@Eremothecium gossypii #Kluyveromyces lactis Saccharomyces cerevisiaeSchizosaccharomyces pombe Yarrowia lipolytica Draft assembly (18)Aspergillus terreus ( lovastatin)Candida albicans Coccidioides immitisCoprinopsis cinereaEmericella nidulans Gibberella zeae Kluyveromyces waltiiMagnaporthe griseaNaumovia castellii Neurospora crassa Phanerochaete chrysosporiumPichia angustaSaccharomyces bayanusSaccharomyces kluyveriSaccharomyces kudriavzevii Saccharomyces mikatae Saccharomyces paradoxus Ustilago maydis In progress (27)
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Completed (3)Cyanidioschyzon merolae str
ain 10D (unicellular red alga)
Entamoeba histolytica (amoeba)
Leishmania major
Draft assembly (8)Cryptosporidium hominis Cryptosporidium parvum Dictyostelium discoideumGiardia intestinalis Plasmodium bergheiPlasmodium chabaudiPlasmodium yoelii Thalassiosira pseudonana
In progress - 38 organisms
Protist genome projects
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Microbial genome projects
November 2003
November 2004
March 2005
Archaea (completed)
16 20 21
Bacteria (completed)
127 177 204
Total completed 143 197 225
Total number of listed projects
- - 562
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6767
Genome Browsers Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGateway)
Ensembl Genome Browser (http://www.ensembl.org/)
[Vega Genome Browser (http://vega.sanger.ac.uk/Homo_sapiens/)]
Generic Genome Browser (http://www.gmod.org/index.php)
VISTA Genome Browser VISTA Genome Browser (http://pipeline.lbl.gov/cgi-bin/gateway2)(http://pipeline.lbl.gov/cgi-bin/gateway2)
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Genom
e B
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ser
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7171
Genom
e B
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7272
Genom
e B
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7373
Genom
e B
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ser
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7474
Genom
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Psa gene
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7676
Gene S
ort
er
Gene Sorter - This program displays a sorted table of genes that are related to one another. The relationship can be one of several types, including protein-level homology, similarity of gene expression profiles, or genomic proximity.
psa
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7777
Gene S
ort
er
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7878
Gene S
ort
er
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Pro
teom
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PSA
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Pro
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PSA continued
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8282
Pro
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Brca1
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Pro
teom
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Brca1 continued
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Pro
teom
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Brca1 continued
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Pro
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Brca1 continued
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8686
Ensembl Genome Browser
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Ens
embl
Gen
ome
Bro
wse
r
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Ens
embl
Gen
ome
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Ens
embl
Gen
ome
Bro
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r
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A workbench for analysis of large-scale genomic sequence data, with strong emphasis on the production of enriched graphical representation of the analysed data. The GESTALT Workbench (GEnomic Sequence Total Analysis and Lookup Tool) can execute a variety of external analysis programmes (e.g. for gene recognition) as well as internal analyses (e.g. compositional complexity analysis); the resulting analysis output files are stored in an internal database. Integrating the analysis results, a Gestalt* is created for each sequence.
http://bioinformatics.weizmann.ac.il/GESTALT
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9191http://bioinformatics.weizmann.ac.il/GESTALT/
Example of a large-scale GESTALT map of the Familial Mediterranean Fever region on human chromosome 16 (GenBank locus HSAJ03147).
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