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

Doug Brutlag 2011

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

Doug Brutlag 2011

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

Doug Brutlag 2011

Processive Synthesis

Doug Brutlag 2011

Synthesis of Long Duplex DNA

Doug Brutlag 2011

Highly Parallel Optics System

Doug Brutlag 2011

Circular Templates Gives Redundant

Sequencing and Accuracy

Doug Brutlag 2011

Circular Templates Gives Redundant

Sequencing and Accuracy

Doug Brutlag 2011

Ion Torrent Sequencing

Doug Brutlag 2011

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