Finishing the Human Genome http://biochem158.stanford.edu/

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Finishing the Human Genomehttp//biochem158.stanf
ord.edu/
Genomics, Bioinformatics Medicine
Doug Brutlag Professor Emeritus of Biochemistry
Medicine Stanford University School of Medicine
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Chromosome 21Public vs Celera Assemblies
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Chromosome 8Public vs. Celera
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Finishing Strategy for the Public Genome
Project
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Polymerase Chain Reaction Overview Exponential
Amplification of DNA
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The First Three Cycles
Original DNA
After N cycles, amount of target DNA is 2N-2N
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PCR Requirements
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
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Temperature Cycling
TAQ polymerase optimum at 72 C
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PCR on a Chip
Uses Reaction complete in 2-20
minutes Extremely portable
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Fluidigm PCR Arrayshttp//www.fluidigm.com/access
-array-system.html
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Real-Time PCR

• Uses Portable means to diagnose bacteria
  epidemics
• Bioterrorism detection
• Military, medical, and municipal applications
• Fast Results in less than seven minutes

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Quantitative PCR
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QuantaLifehttp//www.quantalife.com/
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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

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Finishing Strategy for the Public Genome
Project
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Finished Sequence in 2004 (Build 35)
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Comparison of Chromosome 7Draft versus Finished
Sequence
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Substitutions in BAC Overlaps withBACs from Same
or Different Libraries
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Gaps in BAC Overlaps withBACs from Same or
Different Libraries
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Duplications and Deletionsin the Human Genome
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Percentage of Chromosomes Duplicated
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Duplications near Centromeres
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Duplications near Telomeres
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Deletions and Duplications can Arise from Unequal
Crossing Over in Repeated Regions

• Crossing over between maternal and paternal
  chromosomes
• Unequal crossing over between maternal and
  paternal chromosomes

Maternal
Paternal
Offspring
Offspring
Maternal
Paternal
Offspring
Offspring
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The Diploid Sequence of anIndividual Human
(HuRef)
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Karyotype of J.Craig Venter
Giemsa Stain
FISH Stain
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Comparing NCBI Assembly to HuRef Assembly
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SNPs InDels in HuRef Autosomes
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Illumina Solexa Sequencing Technology
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Illumina Solexa Sequencing Technology
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Illumina Solexa Sequencing Technology
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Illumina Solexa Sequencing Technology
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Illumina Solexa Sequencing Technology
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Illumina Solexa Sequencing Technology
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Illumina Solexa Sequencing Technology
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Illumina Solexa Sequencing Technology
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Illumina Solexa Sequencing Technology
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Illumina Solexa Sequencing Technology
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Illumina Solexa Sequencing Technology
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Illumina Solexa Sequencing Technology
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Life Sciences 454 Process Overview
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Emulsion Based Clonal Amplification
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Depositing DNA Beads into thePicoTiterPlate
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Sequencing By Synthesis
Sequencing-By-Synthesis
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Flowgrams and BaseCalling
Flow Order
TACG
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Pacific Biosciences SMRT Sequencing
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Pacific Biosciences SMRT Sequencing
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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 RedundantSequencing and
Accuracy
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Circular Templates Gives RedundantSequencing and
Accuracy
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Ion Torrent Sequencing
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Ion Torrent Sequencing
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Ion Torrent Sequencing
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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
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Archon Genomics X-Prize
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Archon Genomics X-Prize
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PCR Primer Design
Primer sequence Length 20-30 base pairs long
(1/4N) 50 15 G-C Avoid repeating sequences
and poly-A sequences Avoid inverted repeating
sequences Two primers should have little self
complementarity 3 end of primer should be G/C
Melting Temperature Tm (number of AT
residues) x 2 C (number of GC residues) x 4
C Both primers should have comparable melting
temperatures Annealing temperature is about 5 C
lower than melting temp
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GeneFisher
1 ccgtaacgga ggatgttttt cagaatgtgg ttgggattga
tggatgggag gtagaacgag 61 ttcgagttga aaaggttttg
tgtgccatgt gaaaaggtta gcatctatta cgtagacgag 121
agaaattcat tggaaatttg agaaggagat tgagcataat
gaaacttgtt ttggaaaaat 181 atgttgttat taatgtggag
gtgggcaaga atgagaataa tcagtagcaa tgaggtgtca 241
ataatttgat actgtctaca tggaagacgg cgaccagagc
catggaagtc agaatgaaaa 301 atgataaatg tgaaaacatt
ctagagaaga aatgaatacg cgaaggcccg tggtgggtga 361
tgacatgatg tgatttctgc ccagtgctct gaatgtcaaa
gtgaagaaat tcaatgaagg 421 acgggtaaac ggcgggagta
actatgactc tcttaaggta gccaaatgca tagtcatcta 481
attagtgacg ttcatgaatg gatgaacgag attcccactg
tccctaccta ctatccagcg 541 aaaccacagc caaggtaacg
ggcttggtgg aatccgcggg gaaagaagac cctgttgagc 601
ttgactctag tctggcacgg tgaagagcca tgagaagtgt
agaataagtg ggaggcccct 661 gggcccccct gcccagcaag
gggacagagt ggggcaaggc cagaggtgaa ataccactac 721
tctgattgtt tattcactga cccgtgaggt gccccaaggg
gctcttgctt ctggcgccga 781 gtgcccggcc acatgcacat
gccaaattgt aaagaccatc gat
http//bibiserv.techfak.uni-bielefeld.de/genefishe
r/
Forward Primer Data Reverse
Primer Data Sequence GATTGATGGATGGGAGGTA
CTGTGGTTTCGCTGGA GC Content 47
56 Position
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533 Degeneracy 0
0 3' GC
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50 3' Degeneracy
0
0 Tm 52.7996

51.349 Location 34
533