DNA as a general information readout platform
Target
Converting to DNA
DNA
--
RNA
Reverse transcription
Protein
Proximity ligation (PLA)
Ecology
28S ribosomal RNA->cDNA
Chromosomal aberration
Shotgun sequencing of
maternal blood
Counterfeit products
Tracer DNA
Small molecule binding
Tag with DNA
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Before DNA
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What is the physical basis of the gene?
The ”aperiodic crystal” (Schrödinger 1944)
Evolution occurs by natural selection operating on
inherited but variable characters
Charles Darwin (1858)
Gregor Mendel (1865)
Cells consist of lipids, sugars, proteins and nucleic acids
Friedrich Miescher discovered nucleic acids in 1869
Erwin Schrödinger
Chromosomes carry the genetic information
Weisman (germ plasm theory)
Walter Sutton, Theodor Boveri 1902
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The transforming principle (Griffith 1928)
Genes have physical location on the chromosome
Thomas Hunt Morgan (1916)
Frederick Griffith (with Bobby)
Thomas Hunt Morgan
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Namn Efternamn
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The gene is made of DNA
(Avery, McLeod, McCarty 1944)
Transformation in vitro
The Waring Blender Experiment
(Hershey & Chase 1952)
Isolate the ”transforming principle”
and determine its character
Martha Chase & Alfred Hershey
Oswald Avery (Christmas, with egg-nog)
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The double helix
(Watson & Crick 1953)
Chargaff’s rules (1952)
Francis Crick and James Watson
Erwin Chargaff
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DNA is a triplet code for proteins
(Khorana, Nirenberg, Holley; also Crick & Brenner)
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RNA sequencing of bacteriophage MS2
Robert Holley, Marshall Nirenberg, Har Gobind Khorana
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Maxam-Gilbert Sequencing
DNA Sequencing (early days)
”Wandering spot analysis” (Maxam & Gilbert 1973)
Plus/minus method (Sanger 1975)
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Sanger (dideoxy) sequencing (1977)
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Scaling it up (2nd generation)
Hood/Smith 1986: fluorescent sequencing
Kary Mullis 1986: PCR
Human genome project proposed (1986)
1990: Lloyd Smith capillary sequencer
Hideki Kambara sheath-flow cell
Leroy Hood
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Strategies
How to sequence longer pieces of DNA
Money
Hierarchical shotgun
Whole-genome shotgun
Colony picker
Liquid handler
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Craig Venter
The mighty Perkin-Elmer 3700
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The end of the beginning
Next-generation sequencing
Sanger sequencing is based on physical separation of subfragments
’Next-generation sequencing’:
Clonal display of DNA fragments
Stepwise sequencing with optical detection
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HiSeq sample prep
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HiSeq cluster generation
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HiSeq sequencing
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Reversible terminators (Illumina)
Sample input requirements
Concentration (nM)
Volume (µL)
Fragment length (bp)
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HiSeq
FLX
2-10
1
SOLiD
1
2
1.2
5-10
100-600
200-600
150-200
2-3 billion 300 bp molecules
~1-10 nanograms
~700 diploid human genomes
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454 Genome Sequencer
Illumina HiSeq 2000
(first of the ’next’ generation)
(current leader)
Feature
Specification
Feature
Specification (per flowcell*)
Read length
400 – 600 bp
Read length
50 - 100 bp (selectable)
Reads/run
1 million
Reads/run
1.5 billion
Paired-end
YES (long)
Sequence yield per run
300 Gbp
Run time
10 hours
Paired-end
YES (short and long)
Raw error rate
<1%
Run time
Reagent cost
35,000 SEK/run
2 days (50 bp)
11 days (2x100 bp)
Raw error rate
<1%
Reagent cost
35,000 SEK/run (1x50 bp)
(*) Can run two flowcells in parallel
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Impact of the ’next generation’
Current sequencing instruments
HiSeq
MiSeq
SOLiD
IonTorrent
454 GS
PacBio RS
Dec 2012: $1000 genome
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RNA-Seq
Applications
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Isolate RNA
Convert to cDNA
Fragment and ligate adapters
Sequence
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RNA from single cells can be sequenced
RNA-Seq is already superior to microarrays
Cost ~2500 SEK/sample
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ChIP-Seq
ChIP-Seq
ChIP-Seq reveals sites where
proteins bind chromatin
Not same as transcriptional
activation/repression!
Elaine Mardis
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Mikkelsen et al.
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Human gut microflora
Metagenomics
Shotgun sequencing of microbial ecosystems
Soil, water, air
Human microflora (saliva, lung, intestine, skin, …)
Direct discovery of microbes without preconceptions
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Genome sequencing
Human skin microbiome
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Glossary of terms
Read
Read length
Mate-pair
Paired-end read
Assembly
Contig
Scaffold
Quality score
Error rate
Coverage
N50 length
Sequencing depth
Metagenomics
Colorspace
Raw error
Consensus error
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