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Next Generation Sequencing andHuman Genome DatabasesGenomics, Bioinformatics & Medicinehttp://biochem158.stanford.edu/Doug BrutlagProfessor Emeritus of Biochemistry & MedicineStanford University School of Medicine Doug Brutlag 2014

Illumina Solexa Sequencing Technology Doug Brutlag 2014

Life Sciences 454 Process Overview1) Prepare Adapter Ligated ssDNA Library2) Clonal Amplif icationon 28 µ beads3) Load beads and enzymesin PicoTiter Plate 4) Perform Sequencing by synthesison the 454 Instrument Doug Brutlag 2014

Pacifc Biosciences SMRT Sequencing Doug Brutlag 2014

New PacBio Sequencing Technology t-technology/ Doug Brutlag 2014

Pacifc Biosciences SMRT Sequencing Doug Brutlag 2014

Phospholinked Fluorophores Doug Brutlag 2014

Processive Synthesis Doug Brutlag 2014

Synthesis of Long Duplex DNA Doug Brutlag 2014

Highly Parallel Optics System Doug Brutlag 2014

Circular Templates Gives RedundantSequencing and Accuracy Doug Brutlag 2014

Ion Torrent Sequencing Doug Brutlag 2014

The Human GenomeHow fast is the cost going down? 2006: 50 million2008: 500,0002009: 50,0002010: 20,0002011: 5,0002012: 4,0002013: 3,0002014 1,700Thanks to SerafimBrutlagBatzoglou Doug2014

The Human GenomeHow fast is the cost going down?http://www.genome.gov/sequencingcosts/ Doug Brutlag 2014

Components of a Typical Human GeneTFBS Promoter ExonIntronExonIntronExonTerminatorGene Doug Brutlag 2014

Active Genes are Transcribed into rimaryTranscript Doug Brutlag 2014

Splicing Transcript Yields Mature mRNAPromoter licing5’3’mRNA Doug Brutlag 2014

Mature mRNA contains Coding Regionand 5’ and 3’ Untranslated RegionsPromoter anscriptSplicing5’3’5’UTRCoding RegionGenemRNA3’UTR Doug Brutlag 2014

Mature mRNA contains7-Methyl-Guanosine 5’ Cap and 3’ Poly A TailPromoter neTranscriptSplicingmRNA7-Me-G-Cap5’UTRCoding Region3’UTR3’ Poly A Tail Doug Brutlag 2014

ESTs, Full Length cDNAUniGene & RefSeq DatabasesPromoter licing5’5’ ESTs3’mRNA3’ ESTsFull Length cDNA Doug Brutlag 2014

ESTs, Full Length cDNAUniGene & RefSeq DatabasesPromoter ExonIntronExonIntronTerminatorExonGene5’ UTR3’ UTRTranscriptSplicing3’5’mRNA3’ ESTs5’ ESTs5’ UTR3’ UTRProteinFull Length cDNAProteins Doug Brutlag 2014

GENSCAN Gene Modelhttp://genes.mit.edu/GENSCAN.htmlHidden Markovmodels of genestructure Doug Brutlag 2014

Genome DatabasesA MappingB Gene PredictionC Expression DataGrailEXPGenScandbSNPFGENESHFGENESH UniGeneHumanRefSeqHumanMouseESTsEntrez GeneMouseRefSeqMousenrPROE Additional DataGenomic DNAdbSTSHumanESTsD Protein SimilarityF SummaryAssembledcontigsGeneMarkEnsemblcDNApFAM MotifsPromotersEntrez GeneUCSC BrowserEnsembl Doug Brutlag 2014

Entrez Gene db geneNR-ProUniGeneESTsGrailEXPFGENESHGenscanEntrez Gene Doug Brutlag 2014

Alternative Splicing GeneratesDistinct Proteins in Different eneTranscriptSplicing3’5’Alternate Splicing5’mRNA-1Transcript3’mRNA-2 Doug Brutlag 2014

NCBI Genomeshttp://www.ncbi.nlm.nih.gov/genome Doug Brutlag 2014

Eukaryote Genome shttp://www.ncbi.nlm.nih.gov/genomes/leuks.cgi Doug Brutlag 2014

Canis lupus familiaris Genomehttp://www.ncbi.nlm.nih.gov/sites/entrez?db bioproject&cmd Retrieve&dopt Ov Doug Brutlag 2014

NCBI Entrez Genehttp://www.ncbi.nlm.nih.gov/sites/entrez?db gene Doug Brutlag 2014

NCBI Entrez Gene: Human Opsinhttp://www.ncbi.nlm.nih.gov/gene?term human%20opsin Doug Brutlag 2014

Entrez Gene: Human Opsin OPN1MWhttp://www.ncbi.nlm.nih.gov/gene/2652 Doug Brutlag 2014

MapViewer: Human Opsin OPN1MWhttp://www.ncbi.nlm.nih.gov/gene/2652 Doug Brutlag 2014

Evidence Viewer for axid 9606&contig NT 167198.1&gene OPN1MW Doug Brutlag 2014

OMIM Home db OMIM Doug Brutlag 2014

Colorblindness in OMIM Doug Brutlag 2014

Colorblindness in OMIMhttp://omim.org/entry/303800 Doug Brutlag 2014

Human Genome human/ Doug Brutlag 2014

RefSeqhttp://www.ncbi.nlm.nih.gov/RefSeq/ Doug Brutlag 2014

RefSeq Genehttp://www.ncbi.nlm.nih.gov/refseq/rsg/ Doug Brutlag 2014

NCBI UniGenehttp://www.ncbi.nlm.nih.gov/sites/entrez?db unigene Doug Brutlag 2014

NCBI Homologene Databasehttp://www.ncbi.nlm.nih.gov/homologene Doug Brutlag 2014

Comparative Genomics Doug Brutlag 2014

Ensembl Home Pagehttp://www.ensembl.org/ Doug Brutlag 2014

EBI Genomes Home Pagehttp://www.ensembl.org/ Doug Brutlag 2014

Ensembl Human Genomehttp://www.ensembl.org/Homo sapiens/ Doug Brutlag 2014

Ensembl Human Opsin Searchhttp://uswest.ensembl.org/Homo sapiens/Search/Results?species Homo sapiens;idx ;q Doug Brutlag 2014

Ensembl Human Opsin Geneshttp://uswest.ensembl.org/Homo sapiens/Search/Results?species Homo sapiens;idx ;q Doug Brutlag 2014

Ensembl Human OPN1MW Genehttp://uswest.ensembl.org/Homo sapiens/Gene/Summary?g ENSG000001473 Doug Brutlag 2014

Ensembl Opsin OPN1MW Gene Locationhttp://uswest.ensembl.org/Homo sapiens/Location/View?h Havana%20gene;r X Doug Brutlag 2014

Ensembl OPN1MW Transcriptshttp://uswest.ensembl.org/Homo sapiens/Location/View?h Havana%20gene;r X Doug Brutlag 2014

Ensembl OPN1MW Opsin Proteinhttp://uswest.ensembl.org/Homo sapiens/Transcript/ProteinSummary?db core;g ENSG0000 Doug Brutlag 2014

Ensembl torials/index.html Doug Brutlag 2014

UCSC Genome Home Pagehttp://genome.ucsc.edu/ Doug Brutlag 2014

UCSC Genome Browserhttp://genome.ucsc.edu/cgi-bin/hgGateway Doug Brutlag 2014

UCSC Genome ition chrX:153485203-153499469&hgsid 216983641&knownG Doug Brutlag 2014

UCSC Proteome Browserhttp://genome.ucsc.edu/cgi-bin/pbGateway Doug Brutlag 2014

UCSC Help sHelp.html Doug Brutlag 2014

Doug Brutlag 2014

Emulsion Based Clonal AmplifcationSingle test tube generation of millions of clonally amplif ied sequencing templatesNo cloning and colony pickingA PCR Reagents Emulsion OilBMicro-reactorsAdapter carryinglibrary DNAAnneal DNA templateto capture beadsBreak micro-reactorsIsolate DNA containingbeads“Water-in-oil”emulsionPerform emulsionPCR Doug Brutlag 2014

Depositing DNA Beads into thePicoTiter PlateLoad Enzyme BeadsLoad beads into PicoTiter PlateCentrifuge Step44 m 70x75mm array of fused optical f ibers with etched wells 1.6 million wells monitored optically through f iber Doug Brutlag 2014

Sequencing By SynthesisSequencing-By-Synthesis Simultaneous sequencing of the entire genome inhundreds of thousands of picoliter-size wells Pyrophosphate signal generation Doug Brutlag 2014

Flowgrams and BaseCallingFlow Order4-merTACG3-mer2-mer1-mer Doug Brutlag 2014

Next Generation Sequencing and Human Genome Databases Doug Brutlag Professor Emeritus of Biochemistry & Medicine Stanford University School of Medicine . Single test tube generation of millions of clonally amplif ed sequencing templates No cloning and colony picking "Water-in-oil" emulsion PCR Reagents Emulsion Oil Perform emulsion .