Spring 2007 Bioinformatiatics Ch. 6 - Genomics. Completed genomes Spring 2009 Bioinformatiatics .

Spring 2007 BioinformatiaticsBioinformatiatics

Ch. 6 - Genomics

Completed genomesSpring 2009 BioinformatiaticsBioinformatiatics

• http://www.genomesonline.org


•Avg. genome = 5 mb•Typical sequence coverage = 20X, therefore approx. 100 mb of DNA

•Avg. English word size = 5 letters•Avg. words per page = 250, therefore 1250 letters per page•Avg. book size = 200 pages, therefore 250,000 letters per book•Approximately 400 books per genome

•958 completed genomes as of January 1, 2009•Approximately 383,200 books worth of genomic information

•MSU library holdings: 182,000


• Whole Genome Sequencing• Shotgun Sequencing• Expressed Sequence Tags• Comparative Genomics• Metagenomics

Approaches to Genome Sequencing

Overview of Genome SequencingOverview of Genome Sequencing

Genomic DNA

Create Genomic LibraryCreate Genomic Library

BAC Clones

Construction of Genome MapConstruction of Genome Map

DNA Sequencing and AssemblyDNA Sequencing and Assembly

Isolate Genomic DNAIsolate Genomic DNA

Isolating Genomic DNAIsolating Genomic DNAala, Qiagen’s DNeasy kit

Lysis:• Proteinase K digestion• Lysis by chaotropic salt

Purification:• DNA negatively charged• Bind positively charged column• Wash (EtOH) away impurities

Elution:• Removal of DNA• Disrupt ionic interaction with high salt buffer

Preservation:• Store at -20°C to -160°C• Tris•EDTA buffer [pH 8.0]

Sephadex StructureSephadex Structure

Creating a Genomic LibraryCreating a Genomic Library

BAC Clones

Cut Genomic DNA:

• Partial Restriction Digest•EcoRI & EcoRI methylase

• Mechanical Shearing

• Determine Avg. fragment size

Clone Fragments into BAC vectors:• Proporties of BACs

Transform E. coli:• Electroporation

Pulse Field Gel ElectrophoresisPulse Field Gel Electrophoresis

Average Insert Size by Pulse Field Gel Electrophoresis

Average Insert Size in Human BACs


BAC Clones

Cut Genomic DNA:






Bacterial Artificial ChromosomeBacterial Artificial Chromosome

• Derived from F plasmids • Multiple cloning site• Selectable Marker

• Antibiotic Resistance Gene - ie, cm• Ori S - unidirectional• Par genes

• partitioning genes• maintain single copy of BAC


BAC Clones

Cut Genomic DNA:







BAC Clones



• BAC end sequencing• Identify overlapping BACs• Subclone BACs into plasmids

Transformed E. coli:

Plasmid MiniprepPlasmid Miniprep

Genome Assembly and

Annotation

Overview of Shotgun SequencingOverview of Shotgun Sequencing

Genomic DNA


Plasmid Clones



Isolate Genomic DNAIsolate Genomic DNA

Overview of EST SequencingOverview of EST Sequencing


DNA SequencingDNA Sequencing

Isolate mRNAIsolate mRNA

Create cDNACreate cDNA

Comparative GenomicsComparative Genomics


BAC Clones



Isolate mRNA and create cDNAIsolate mRNA and create cDNA

Synteny - same gene order preserved between species

Comparative Genomics BAC array

Comparative Genome Hybridization


Bordetella phylogenyBordetella phylogeny

Metagenomic analysisMetagenomic analysis

• What is metagenomics?– Metagenomics is the genomic analysis of the collective

genomes of an assemblage of organisms from a defined environment.

» Handelsman, et al, 2002– a.k.a., community genomics, environmental genomics– Derived from tools, techniques and models used in genomics.

• Why do metagenomic analysis?– Genomic content of all eucaryotes, bacteria, archaea and

viruses in an evironment.– Provides a picture of genetic/functional potential of the

community.

MetagenomicsMetagenomics

Venter’s Trip

Yooseph, et al, PLOS biology, 2007

Creation of Fosmid Libraries

Preliminary Categorization of 263 ORFsfrom a Fosmid Library of Subgingival Plaque

Preliminary Categorization of 263 ORFsfrom a Fosmid Library of Subgingival Plaque

Category Percentage of library

Eucaryotic 34%

Bacterial 21%

Archaeal 1.1%

Viral1 0.8%

Bacteriophage 2%

Unidentified 41% 1not bacteriophage


Genome Annotation

Genome Assembly and

Annotation

RefSeq db

Caveats

• Finding genes involves computational methods as well as experimental validation

• Computational methods are often inadequate, and often generate erroneous ‘gene’ (false positive) sequences which:– Are missing exons– Have incorrect exons– Over predict genes– Where the 5’ and 3’ UTR are missing

Things we are looking to annotate?

• CDS• mRNA• Alternative RNA• Promoter and Poly-A Signal• Pseudogenes• ncRNA• Repeat elements• G+C content

Pseudogenes• Could be as high as 20-30% of all Genomic sequence

predictions could be pseudogene• Non-functional copy of a gene

– Processed pseudogene• Retro-transposon derived• No 5’ promoters• No introns• Often includes poly-A tail

– Non-processed pseudogene• Gene duplication derived

– Both include events that make the gene non-functional• Frameshift• Stop codons

• We assume pseudogenes have no function, but we really don’t know!

Noncoding RNA (ncRNA)

• tRNA – transfer RNA: involved in translation• rRNA – ribosomal RNA: structural component

of ribosome, where translation takes place• snRNA – small nuclear RNA:

functional/catalytic in RNA maturation• Antisense RNA - gene regulation• siRNA - gene silencing

Noncoding RNA (ncRNA)

• ncRNA represent 80-98% of all transcripts in cell• ncRNA have not been taken into account in gene

counts• cDNA• ORF computational prediction• Comparative genomics looking at ORF

• ncRNA can be:– Structural– Catalytic– Regulatory

GenBank Features

-10_signal-35_signal3'clip3'UTR5'clip5'UTRattenuatorCAAT_signalCDSconflictC_regionD-loopD_segmentenhancerexon

GC_signalgeneiDNAintronJ_segmentLTRmat_peptidemisc_bindingmisc_differencemisc_featuremisc_recombmisc_RNAmisc_signalmisc_structuremodified_base

mRNAN_regionold_sequencepolyA_signalpolyA_siteprecursor_RNAprimer_bindprim_transcriptpromoterprotein_bindRBSrepeat_regionrepeat_unitrep_originrRNA

satellitescRNAsig_peptidesnoRNAsnRNAS_regionstem_loopSTSTATA_signalterminatortransit_peptidetRNAunsurevariationV_regionV_segment

LOCUS NG_005487 1850 bp DNA linear ROD 14-FEB-2006DEFINITION Mus musculus ubiquitin-conjugating enzyme E2 variant 2 pseudogene (LOC625221) on chromosome 6.ACCESSION NG_005487VERSION NG_005487.1 GI:87239965KEYWORDS .SOURCE Mus musculus (house mouse) ORGANISM Mus musculus Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Sciurognathi; Muroidea; Muridae; Murinae; Mus.REFERENCE 1 (bases 1 to 1850) AUTHORS Wilson,R. TITLE Mus musculus BAC clone RP24-201D17 from 6 JOURNAL Unpublished (2003)COMMENT PROVISIONAL REFSEQ: This record has not yet been subject to final NCBI review. The reference sequence was derived from AC121925.2.FEATURES Location/Qualifiers source 1..1850 /organism="Mus musculus" /mol_type="genomic DNA" /db_xref="taxon:10090" /chromosome="6" /note="AC121925.2 32277..34126" gene 101..1750 /gene="LOC625221" /pseudo /db_xref="GeneID:625221" repeat_region 1792..1827 /rpt_family="ID"ORIGIN 1 tcttctgcct caattcctca agtgctagta tcatatgccc atgccattat ttttaactcc 61 cctttttcat gctaagaatt gaacacacgg ccctgcgtgc ggtggtgcgt ctggtagcag 121 gagaagatgg cggtctccac aggagttaaa gttcctcgta attttcgctt gttggaagaa

LOCUS NG_005487 1850 bp DNA linear ROD 14-FEB-2006DEFINITION Mus musculus ubiquitin-conjugating enzyme E2 variant 2 pseudogene (LOC625221) on chromosome 6.ACCESSION NG_005487VERSION NG_005487.1 GI:87239965KEYWORDS .SOURCE Mus musculus (house mouse) ORGANISM Mus musculus Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Sciurognathi; Muroidea; Muridae; Murinae; Mus.REFERENCE 1 (bases 1 to 1850) AUTHORS Wilson,R. TITLE Mus musculus BAC clone RP24-201D17 from 6 JOURNAL Unpublished (2003)COMMENT PROVISIONAL REFSEQ: This record has not yet been subject to final NCBI review. The reference sequence was derived from AC121925.2.FEATURES Location/Qualifiers source 1..1850 /organism="Mus musculus" /mol_type="genomic DNA" /db_xref="taxon:10090" /chromosome="6" /note="AC121925.2 32277..34126" gene 101..1750 /gene="LOC625221" /pseudo /db_xref="GeneID:625221" repeat_region 1792..1827 /rpt_family="ID"ORIGIN 1 tcttctgcct caattcctca agtgctagta tcatatgccc atgccattat ttttaactcc 61 cctttttcat gctaagaatt gaacacacgg ccctgcgtgc ggtggtgcgt ctggtagcag 121 gagaagatgg cggtctccac aggagttaaa gttcctcgta attttcgctt gttggaagaa

The ideal annotation of “MyGene”

MyGene

All mRNAs

All proteins

All structures

All SNPs

All clones

• All protein modifications• Ontologies • Interactions (complexes, pathways, networks)•Expression (where and when, and how much)•Evolutionary relationships

Promoter(s)

Spring 2007 Bioinformatiatics Ch. 6 - Genomics. Completed genomes Spring 2009 Bioinformatiatics .

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