1GenomicsThe Human Genome Project Mapping and Sequencing the Genomes of Model Organisms  Data Collection and Distribution Ethical, Legal, and Social Considerations Research Training  Technology Development  Technology TransferA Few Genome Resources NCBI Genome Resources UCSC Human Genome Browser Ensembl Human Genome ServerGenome Sequencing Progress NCBI Genome Sequence Repository All organisms Eukaryotic genomes Prokaryotic genomes Archaea genomes VirusesGenome SequencingFrom NCBI, 5/2001Human Genome Sequencing 2/11/2001 From NCBI2Human Genome Progress 2/11/2001Total sequence (kb)Non-redundant sequence (kb)Percentage of genomeFinished 1,140,365 1,040,372 32.50%Unfinished 3,547,899 1,951,344 61.00%Total 4,688,264 2,991,716 93.50%From NCBIMicrobial Genomes Published complete microbial genomes Microbial genomes and chromosomes in progress Genome Informatics Annotation and Analysis Data Handling Metabolic Reconstruction Comparative Genomics Functional GenomicsGenome Project Organization Cloning Mapping Sequencing Annotation AnalysisCloning and MappingCloning Large YAC’s 1 Mb BAC’s 100 - 200 KbIntermediate Cosmids Lambda clones Small Plasmids; M133Mapping Establishment of Guideposts Aids in Assembly Error Checking Useful in mapping of genetic disordersGenetic Maps Cytogenetic markers Linkage maps Polymorphic loci screened by PCR to determine inheritence patterns Produce linkage map with nearby lociPhysical Maps Radiation Hybrid/YACs/Cosmids Restriction Sites Sequence Tagged Sites 100 Kb resolution needed 30,000 STS’s Expressed Sequence Tags Detection PCR Hybridization FISH Fluoresecent in situ HybridizationHuman Genome STS Mapping Strategy STS Content Mapping Screen YAC’s by PCR Radiation Hybrid Mapping Screen RH Cell lines by PCR Genetic Mapping PCR Screening of polymorphic loci Combine above to produce an integrated mapMapping Resolution YAC mapping 1 Mb Radiation hybrid mapping 10 Mb Genetic map 30 MbGeneMap’98 Integrated Human Genetic Map Over 30,000 unique gene-based markers 100 Kb resolution http://www.ncbi.nlm.nih.gov/genemap98/4Map IntegrationHuman Chromosome 1 Genetic MapHuman Chromosome 1 Combination MapSequencingSequencing Methods Random Shotgun Ordered Shotgun Directed Primer Walking Direct genomic sequencingRandom Shotgun Sequencing Randomly shear or cut DNA into small pieces 2-4 Kb Clone into M13, pUC or some other sequencing vector Sequence the clones from both ends Rely on the computer to assemble the sequences into one (or as few as possible) contigs5Shotgun Sequencing Statistics Lander and Waterman equation poisson distribution Po = e-m probability that a base is not sequenced where m=sequence coverageH. influenza Sequencing For 1X random sequence coverage = 1.8 Mb P = 0.37 (63% of the bases are sequenced) To get > 99% of the bases sequenced 5X coverage = 8.74 Mb of sequence Po = e-5 = 0.0067 This coverage would leave approx. 128 gaps of about 100 bp in size From Science 269:496-512. 1995Ordered Sequencing Generate a set of large sequence clones in lambda phage May be subcloned from YACs or BACs as necessary End sequence the lambda clones and order the clones to produce a map of the genome Choose a minimal tiling path of the genome from the ordered lambda clonesOrdered Sequencing... Shear and subclone the lambda inserts that comprise the minimal tiling set into sequencing vectors Shotgun sequence and assemble each of these lambda inserts individually Assemble all sequences into one, contiguous genomeDirected Sequencing Process used for finishing following the shotgun sequencing phase Gap closure Use specific sequencing primers to extend appropriate clones into gap regions Use specific sequencing primers to sequence directly from genomic DNASequence Assembly6Assembly of Shotgun Fragments For H. influenzae (TIGR) 1.8 Mb 24,304 Sequence fragments were generated for the random assembly phase 11,631,485 bases Generated 140 contigs Assembled using the TIGR Assembler 30 hours of cpu timephred/phrap/consed Widely used programs for sequence: base calling (phred) assembly (phrap) editing (consed) Developed at the University of Washington Phil Green (phrap) Brent Ewing (phred) David Gordon (consed)Genome Annotation and AnalysisPattern MatchingSequence Annotation ORF identification Frameshift resolution Genome map construction Functional assignments Metabolic pathway assignment Metabolic pathway Reconstruction Comparative analysisAnnotation Tools Semi-automated Manual7MAGPIE Multipurpose Automated Genome Project Investigation Environment Terry Gaasterland et. al.  http://genomes.rockefeller.edu/magpie/magpie.htmlAutomated  Semi-automated analysis tool for microbial genome projectsMAGPIE ExampleNon-Automated Analysis and Prediction The Ureaplasma urealyticum genome database Run analysis tool Parse results Dump results into the database View results Manually annotateGenomic Sequence Database Data Storage Sequence Gene Map Annotation User Interface Web browser CustomizableThe Ureaplasma urealyticum Genome Project Uu - 751,719 bp http://genome.microbio.uab.edu/uu/uugen.htm Web-based genome analysis tool8Annotation Problems Problems with existing sequence databases Incomplete datasets Skewed datasets Incorrectly annotated records Annotations based on experimental vs. predicted data Nomenclature differences Transitive errors in gene function predictions Functional predictions for “hypothetical” genesMetabolic Pathway ReconstructionMetabolic Pathway Reconstruction Role assignment Extract metabolic pathways from genomes Navigation and analysis Pathway editing9Metabolic Assignments Amino acid Biosynthesis Biosynthesis of cofactors, prosthetic groups, and carriers Cell envelope Cellular processes Central intermediary metabolism Energy metabolism Fatty acid and phospholipid metabolism Purines, pyrimidines, nucleosides, and nucleotides Regulatory functions Replication Transcription Translation Transport and binding proteins Other categories, Unassigned