Doug Brutlag 2011
Finishing the Human Genomehttp://biochem158.stanford.edu/
Doug BrutlagProfessor Emeritus of Biochemistry & Medicine
Stanford University School of Medicine
Genomics, Bioinformatics & Medicine
Doug Brutlag 2011
Chromosome 21:Public vs Celera Assemblies
Doug Brutlag 2011
Chromosome 8:Public vs. Celera
Doug Brutlag 2011
Finishing
Strategy for the Public
Genome Project
Doug Brutlag 2011
Polymerase Chain Reaction Overview:Exponential Amplification of DNA
Doug Brutlag 2011
After N cycles, amount of target DNA is 2N-2N
Original DNA After Cycle 1
After Cycle 2
After Cycle 3
The First Three Cycles
Doug Brutlag 2011
DNA•Need to know at least the beginning and end of DNA sequence•These flanking regions have to be unique to strand interested in amplifying•Region of interest can be present in as little as one copy•Enough DNA in 0.1 microliter of human saliva to use PCR
DNA Polymerase Enzyme•DNA polymerase from Thermus aquaticus--Yellowstone•Alternatives: Thermococcus litoralis, Pyrococcus furiosus
Thermocycler
PCR Requirements
Doug Brutlag 2011
TAQ polymerase optimum at 72° C
Temperature Cycling
Doug Brutlag 2011
PCR on a Chip
Uses: Reaction complete in 2-20 minutesExtremely portable
Doug Brutlag 2011
Fluidigm PCR Arrayshttp://www.fluidigm.com/access-array-system.html
Doug Brutlag 2011
Real-Time PCR
Uses: •Portable means to diagnose bacteria: epidemics•Bioterrorism detection•Military, medical, and municipal applications•Fast: Results in less than seven minutes
Doug Brutlag 2011
Quantitative PCR
Doug Brutlag 2011
QuantaLifehttp://www.quantalife.com/
Doug Brutlag 2011
PCR Applications
Forensics•assessment/reassessment of crimes
Archaeology•determine gene sequences of ancient organisms•rethinking the past, human origins
Molecular Biology•Cloning genes•Sequencing genes•Finishing genome sequences•Amplification of DNA or RNA
•Medicine•Diagnostics for inherited disease•Diagnostics for gene expression•Diagnostics for gene methylation
Doug Brutlag 2011
Finishing
Strategy for the Public
Genome Project
Doug Brutlag 2011
Finished Sequence in 2004 (Build 35)
Doug Brutlag 2011
Comparison of Chromosome 7Draft versus Finished Sequence
Doug Brutlag 2011
Substitutions in BAC Overlaps withBACs from Same or Different Libraries
Doug Brutlag 2011
Gaps in BAC Overlaps withBACs from Same or Different Libraries
Doug Brutlag 2011
Duplications and Deletionsin the Human Genome
Doug Brutlag 2011
Percentage of Chromosomes Duplicated
Doug Brutlag 2011
Duplications near Centromeres
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Duplications near Telomeres
Doug Brutlag 2011
• Crossing over between maternal and paternal chromosomes
• Unequal crossing over between maternal and paternal chromosomes
Deletions and Duplications can Arise from Unequal Crossing Over in Repeated
RegionsMaternal
Maternal
Paternal
Paternal
Offspring
Offspring
Offspring
Offspring
Doug Brutlag 2011
The Diploid Sequence of anIndividual Human (HuRef)
Doug Brutlag 2011
Karyotype of J.Craig Venter
Giemsa Stain FISH Stain
Doug Brutlag 2011
Comparing NCBI Assembly to HuRef Assembly
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SNPs & InDels in HuRef Autosomes
Doug Brutlag 2011
Illumina Solexa Sequencing Technology
Doug Brutlag 2011
Illumina Solexa Sequencing Technology
Doug Brutlag 2011
Illumina Solexa Sequencing Technology
Doug Brutlag 2011
Illumina Solexa Sequencing Technology
Doug Brutlag 2011
Illumina Solexa Sequencing Technology
Doug Brutlag 2011
Illumina Solexa Sequencing Technology
Doug Brutlag 2011
Illumina Solexa Sequencing Technology
Doug Brutlag 2011
Illumina Solexa Sequencing Technology
Doug Brutlag 2011
Illumina Solexa Sequencing Technology
Doug Brutlag 2011
Illumina Solexa Sequencing Technology
Doug Brutlag 2011
Illumina Solexa Sequencing Technology
Doug Brutlag 2011
Illumina Solexa Sequencing Technology
Doug Brutlag 2011
Life Sciences 454 Process Overview
4) Perform Sequencing by synthesison the 454 Instrument
1) Prepare Adapter Ligated ssDNA Library
2) Clonal Amplification on 28 µ beads
3) Load beads and enzymes in PicoTiter Plate™
Doug Brutlag 2011
Emulsion Based Clonal Amplification
Micro-reactors Adapter carrying
library DNAAnneal DNA template
to capture beads
Break micro-reactors Isolate DNA
containing beads
Single test tube generation of millions of clonally amplified sequencing templatesNo cloning and colony picking
“Water-in-oil” emulsion
+ PCR Reagents
+ Emulsion Oil
Perform emulsion PCR
A
B
Doug Brutlag 2011
Depositing DNA Beads into thePicoTiter™Plate
Centrifuge Step
Load Enzyme Beads
Load beads into PicoTiter™Plate
44 μm
• 70x75mm array of fused optical fibers with etched wells
• 1.6 million wells monitored optically through fiber
Doug Brutlag 2011
Sequencing By SynthesisSequencing-By-Synthesis
Simultaneous sequencing of the entire genome in hundreds of thousands of picoliter-size wells
Pyrophosphate signal generation
Doug Brutlag 2011
TACG
Flow Order
Flowgrams and BaseCalling
1-mer
2-mer
3-mer
4-mer
Doug Brutlag 2011
Pacific Biosciences SMRT Sequencing
Doug Brutlag 2011
Pacific Biosciences SMRT Sequencing
Doug Brutlag 2011
Phospholinked Fluorophores
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Processive Synthesis
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Synthesis of Long Duplex DNA
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Highly Parallel Optics System
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Circular Templates Gives Redundant
Sequencing and Accuracy
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Circular Templates Gives Redundant
Sequencing and Accuracy
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Ion Torrent Sequencing
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Ion Torrent Sequencing
Doug Brutlag 2011
Ion Torrent Sequencing
Doug Brutlag 2011
The Human GenomeHow fast is the cost going down?
• 2006: $ 50 million• 2008: $500,000• 2009: $50,000• 2010: $20,000• 2011: $5,000• 2012:??? $1,000
Thanks to Serafim Batzoglou
Doug Brutlag 2011
Archon Genomics X-Prize
Doug Brutlag 2011
Archon Genomics X-Prize