DNA Next Generation Sequencing Methods Applications
DNA sequencing: the Maxam-Gilbert method G A/G C/T C
The Sanger Method uses "stop" nucleotides for sequence
The Sanger Method
DNA-SequenzierungDNA-Sequenzierung nach der Kettenabbruchmethode (Sanger 1977) Didesoxynucleotide: werden von DNA Polymerasen eingebaut, führen aber anschließend zum Kettenabbruch ddctp Primer viele Moleküle statistisch Kettenabbrüche an jeder Position Vier parallele Reaktionsansätze: GATC H
DNA-Sequenzierung:Fluoreszenz Automatisierte nicht-radioaktive DNA-Sequenzierung Dye Terminator Technologie: jedes der vier Didesoxynucleotide ist chemisch mit einem individuellen Fluoreszenzfarbstoff gekoppelt. für jede Position wird ein Kettenabbruchfragment erzeugt, dessen Fluoreszenz-Lichtfarbe die entsprechende Base anzeigt alle 4 Sequenzierreaktionen werden in EINER Gelspur getrennt
Improvements in the rate of DNA sequencing over the past 30 years and into the future. MR Stratton et al. Nature 458, 719-724 (2009)
Highly parallel sequencing on clonal arrays. polony Fan et al. Nature Reviews Genetics 7, 632 644 (August 2006)
General concepts for clonal-array generation and sequencing are illustrated. Details of implementation vary slightly from platform to platform. a Genomic DNA is fragmented and adaptors are ligated to create an insert library that is flanked by two universal priming sites, A and B. Because of the random fragmentation, the complexity of this signature sequence library is equivalent to the genome. This library is cloned on beads using emulsion PCR technology. A water-in-oil emulsion is created from a PCR mix that contains a limiting dilution of DNA and beads. The emulsion creates micro-compartments with, on average, a single bead and single DNA template each. After PCR, beads with clones are affinity selected and assembled onto a planar substrate. A subsequent cycle-sequencing reaction is used to read out the sequence on the clones. b Sequencing by synthesis (SBS). A common anchor primer is annealed to a constant sequence (universal priming site) that is contained within the library clones that are located on the polony (clonal bead) array (the orientation of the immobilized target might vary depending on the platform that is used). The sequence is read out by polymerase extension in a base-by-base fashion using either reversible terminators or sequential nucleotide addition (pyrosequencing). After incorporation of a single base or base type, the incorporated base is identified by fluorescence (laser) or chemiluminescence (no laser required). c Sequencing by ligation. The polony array set-up is similar to SBS in which a common primer is annealed to an arrayed polony library and used to read out the sequence through a stepwise ligation of random oligomers. The labelled oligomers are designed to have random bases inserted at every site except the query site. The query site has one of four base substitutions, each matched to a particular fluorescent label on the oligonucleotide. After read-out of each ligation event, the primer and the ligated oligomer are stripped, a new primer reannealed and the process repeated with an oligomer that contains a query base at a different position.
Pyrosequencing "454"
Nature 437, 376-380 (2005)
a, Genomic DNA is isolated, fragmented, ligated to adapters and separated into single strands (top left). Fragments are bound to beads under conditions that favour one fragment per bead, the beads are captured in the droplets of a PCR-reaction-mixture-in-oil emulsion and PCR amplification occurs within each droplet, resulting in beads each carrying ten million copies of a unique DNA template (top right). The emulsion is broken, the DNA strands are denatured, and beads carrying single-stranded DNA clones are deposited into wells of a fibre-optic slide (bottom right). Smaller beads carrying immobilized enzymes required for pyrophosphate sequencing are deposited into each well (bottom left). b, Microscope photograph of emulsion showing droplets containing a bead and empty droplets. The thin arrow points to a 28- microm bead; the thick arrow points to an approximately 100-microm droplet. c, Scanning electron micrograph of a portion of a fibre-optic slide, showing fibre-optic cladding and wells before bead deposition.
The sequencing instrument consists of the following major subsystems: a fluidic assembly (a), a flow chamber that includes the well-containing fibre-optic slide (b), a CCD camerabased imaging assembly (c), and a computer that provides the necessary user interface and instrument control.
PyrosequencingTM is an established genetic analysis method based on the principle of sequencing by synthesis. It is the only genetic analysis method capable of delivering explicit sequence information within minutes. Pyrosequencing is an ideal choice for genetic analysis in clinical research. The output data from Pyrosequencing is the optimal standard of genetic information and the best possible assurance of correct genetic test. Sequences up to ca. 400 nucleotides.
Pyrosequencing chemistry, step by step STEP 1: A sequencing primer is hybridized to a single stranded, PCR-amplified, DNA template, and incubated with the enzymes DNA-Polymerase, ATP-sulfurylase, luciferase and apyrase, and the substrates adenosine-5 -phosphosulfate (APS) and luciferin. STEP2: The first of four deoxyribonucleotide triphosphates (dntp) is added to the reaction. DNA polymerase catalyzes the incorporation of the deoxyribonucleotide triphosphate into the DNA strand, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated nucleotide. STEP3: ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine-5 -phosphosulfate (APS). This ATP drives the luciferase mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a charge coupled device (CCD) camera and seen as a peak in a Pyrogramm TM. The height of each peak (light signal) is proportional to the number of nucleotides incorporated. STEP4: Apyrase, a nucleotide degrading enzyme, continuously degrades ATP and unincorporated dntps. This switches off the light and regenerates the reaction solution. The next dntp is then added. STEP5: Addition of dntps is performed once at a time. It should be noted that deoxyadenosine alfa-thio triphosphate (datpalpha5) is used as a substitute for the natural deoxyadenosine triphosphate (datp) since it is efficiently used by the DNA polymerase, but not recognized by the luciferase. Nucleotide sequence G C - A GG CC T As the process continues, the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peaks in the Pyrogramm. G C T A G C T Nucleotide added
DNA Sequencing with Solexa Illumina Genome Analyser Flow Cell
1. Prepare genomic DNA sample 2. attach DNA to surface 3. bridge amplification
4. fragments become double stranded 5. denature the double stranded molecules 6. complete amplification
7. determine first base 8. image first base 9. determine second base
10. image second chemistry cycle 11. sequence reads over multiple chemistry cycles 12. align data
Sequencing by Ligation The SOLiD 3 System
Systemvergleich Roche 454 Solexa Solid Prinzip Pyrosequencing Seq. by Synthesis Seq. by Ligation Reads pro Lauf 10 6 2x10 8 3x10 8 Leselänge 400 100 50 Sequenzinfo 500 MB 27 GB 32 GB Laufzeit 10h 9d 10d Durchsatz pro Tag 1 GB 3 GB 3,2 GB Größe Rohdaten 40 Gigabyte 4 Terabyte 7 Terabyte
PacBio s technology www.pacificbiosciences.com
SMRT Synthesis of Long DNA ZMW has a volume of 20 zeptoliters (10-21 liters) and contains DNA polymerase, DNA, and phospholinked nucleotides
Base-labeled nucleotide.
Phospholinked nucleotides
Applications for genome analysis Genome sequencing and polymorphism discovery Mutation mapping by deep sequencing Analysis of DNA protein interactions through ChIP-seq Genome-wide detection of sites of DNA methylation Applications for transcriptome sequencing Deep sequencing of small RNA populations mrna sequencing for transcript discovery and profiling
Mutation mapping by deep sequencing
Identification of mutations by deep sequencing. A plant with Col-0 background that harbors a recessive mutation leading to a mutant phenotype is crossed to a wild-type Ler-1 plant. The heterozygous F1 hybrid plant is allowed to self-fertilize to produce a large pool of F2 plants that are segregating for the mutation. A large number of F2 plants that display the mutant phenotype are pooled and their gdna subjected to deep sequencing. The density of single nucleotide polymorphisms (SNPs) inherent in the Ler-1 strain is subtracted from the density of SNPs indicative of the Col-0 background, identifying a discrete region on the chromosome in which only Col-0 marker SNPs are present. The deep sequencing data in this interval are then scoured for the potential causative mutation.
Analysis of DNA protein interactions through ChIP-seq
Massively parallel interrogation of all pairwise protein interactions for all proteins encoded by a genome by bait prey recombination and deep-sequencing. Interaction of bait and prey constructs results in the activation of the CRE recombination system and expression of a selective marker gene. Recombination at loxp sites located at the end of each gene forms a chimeric DNA molecule containing the two genes that encode the interacting proteins. Digestion to release the chimeric ORFs followed by paired-end sequencing of its two ends will produce one sequence tag from each of the genes, thus identifying the two proteins that directly interacted. Two complex pools of yeast cells, each one containing the full complement of an organism's genes fused to either the bait or the prey domain, would be mixed and allowed to mate. Sequencing of the complex pool of chimeric ORFs would reveal all pairwise interaction that occurred, interrogating the hundreds of millions of possible interactions between any two proteins encoded in a eukaryotic genome.