Sequencing and analysis of gene expression Jiří Fajkus CG080 – Methods of Genomics and Proteomics 220px-RNA-codons 220px-RNA_chemical_structure 500px-Aminoacids_table A = adenine C = cytosine G = guanine T = thymine R = G A (purine) Y = T C (pyrimidine) K = G T (keto) M = A C (amino) S = G C (strong bonds) W = A T (weak bonds) B = G T C (all but A) D = G A T (all but C) H = A C T (all but G) V = G C A (all but T) N = A G C T (any) http://www.wiley.com/college/pratt/0471393878/student/animations/dna_sequencing/index.html Historical methods: 1975 - Sanger and Coulson – plus-minus sequencing (4 pairs of plus (1 dNTP) and minus (3 other dNTPs) reactions, restriction enzyme fragments as primers for elongation by DNA polymerase I. – used for sequencing of 5,386 nucleotides of the single-stranded bacteriophage φX174 Chemical sequencing / Maxam-Gilbert Sequencing (Nobel Prize 1980) 1976–1977- Allan Maxam and Walter Gilbert (Maxam AM, Gilbert W (Feb 1977). "A new method for sequencing DNA". Proc. Natl. Acad. Sci. U.S.A. 74 (2): 560–4). 1) radioactive labeling at one 5' end of the DNA (typically by a kinase reaction using gamma-32P ATP) and purification of the DNA fragment to be sequenced. 2) Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). purines (A+G) are depurinated using formic acid, guanines (G) (and to some extent the adenines) are methylated by dimethyl sulfate pyrimidines (C+T) are methylated using hydrazine. The addition of salt (NaCl) to the hydrazine reaction inhibits the methylation of thymine for the C-only reaction. 3) The modified DNAs are then cleaved by hot piperidine at the position of the modified base. Thus a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule. 4) The fragments in the four reactions are electrophoresed side by side in denaturing PAGE for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabeled DNA fragment, from which the sequence may be inferred. Maxam ChemSeq 1-s2 2_1_5c 220px-Frederick_Sanger2 Nobel Prize in Chemistry (1958) – amino acid sequence of insulin Nobel Prize in Chemistry (1980) – dideoxy method of DNA sequencing Frederick Sanger Sanger F, Nicklen S, Coulson AR (December 1977). "DNA sequencing with chain-terminating inhibitors". Proc. Natl. Acad. Sci. U.S.A. 74 (12): 5463–7. Chain-termination sequencing (Sanger sequencing, dideoxy-sequencing) - the use of dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators. - Requirements: ss (heat-denatured) DNA template, a DNA primer, a DNA polymerase, normal deoxynucleotidetriphosphates (dNTPs), and modified nucleotides (dideoxyNTPs, ddNTPs) that terminate DNA strand elongation. -The DNA sample is divided into 4 separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is added only one of the four ddNTPs (ddATP, ddGTP, ddCTP, or ddTTP) which are the chain-terminating nucleotides, lacking a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, thus terminating DNA strand extension and resulting in DNA fragments of varying length. - The newly synthesized and labelled DNA fragments are heat denatured, and separated by size (with a resolution of just one nucleotide) by gel electrophoresis on a denaturing polyacrylamide-urea gel with each of the four reactions run in one of four individual lanes (lanes A, T, G, C); - the DNA bands are then visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel image. Sequencing -In the image on the right, X-ray film was exposed to the gel, and the dark bands correspond to DNA fragments of different lengths. A dark band in a lane indicates a DNA fragment that is the result of chain termination after incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP). The relative positions of the different bands among the four lanes are then used to read (from bottom to top) the DNA sequence. dnaseq1 One of the four sequencing reactions File:DNA Sequencin 3 labeling methods.jpg File:CE Basic.jpg File:Radioactive Fluorescent Seq.jpg File:Sanger sequencing read display.gif File:DNA Sequencing gDNA libraries.jpg General sequencing strategy Next generation sequencing star-trek-tng1 454 pyrosequencing A parallelized version of pyrosequencing was developed by 454 Life Sciences, (now part of Roche Diagnostics). Amplification of DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony. The sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes. Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs. This technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other. Sequencing1_02 see ANIMATION: http://www.roche-applied-science.com/publications/multimedia/genome_sequencer/presentation/wbt.htm Fragments_05 The Roche 454/GS FLX Sequencing Technology The GS FLX sequencer supports sequencing of various different nucleic acid starting materials such as genomic DNA, PCR products, BACs and cDNA. Samples consisting of longer sequences are first sheared into a random library of 300-800 base-pair long fragments. Adaptors essential for purification, amplification and sequencing are added to both ends of the fragments. If the sample is double stranded one strand is removed and the remaining single strandes are used in the following steps. SS_05 Bead_02 Aided by the adaptors individual fragments are captured on their own unique beads. A bead and the bound fragment together with a water-in-oil emulsion form a microreactor so that each fragment can be amplified without contamination via the so called emulsion PCR (emPCR). The entire fragment collection is amplified in parallel. BeadPolySeq_02 The emPCR amplifies each fragment several million times. After amplification the emulsion shell is broken and the clonally amplified beads are ready for loading onto the fibre-optic PicoTiterDevice for sequencing. Wells_02 The PicoTiterPlate is loaded with one fragment carrying bead per well and smaller beads with the enzymes necessary for sequencing. Sequencing is accomplished by synthesizing the complementary strands of the bead attached templates. In a number of cycles the four bases (ATGC) are sequentially washed over the PicoTiterPlate. The incorporation of a new base is associated with the release of inorganic pyrophosphate starting a chemical cascade. This results in the generation of a light signal which is captured by a CCD camera. Sequencing1_02 Pyrosequencing technology step3.gif ATP sulfurylase 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) chip and seen as a peak in the raw data output (Pyrogram). The height of each peak (light signal) is proportional to the number of nucleotides incorporated. Addition of dNTPs is performed sequentially. It should be noted that deoxyadenosine alfa-thio triphosphate (dATP·S) is used as a substitute for the natural dATP since it is efficiently used by the DNA polymerase, but not recognized by the luciferase. As the process continues, the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peaks in the Pyrogram trace. Pyrosequencing technology step5.gif llumina (Solexa) sequencing Solexa, now part of Illumina, developed a sequencing technology based on reversible dye-terminators. DNA molecules are first attached to primers on a slide and amplified so that local clonal colonies are formed (isothermal bridge amplification). Four types of reversible terminator bases (RT-bases) are added, and non-incorporated nucleotides are washed away. Unlike pyrosequencing, the DNA can only be extended one nucleotide at a time. A camera takes images of the fluorescently labelled nucleotides, then the dye along with the terminal 3' blocker is chemically removed from the DNA, allowing the next cycle. SEE ANIMATION: http://www.illumina.com/media/flash_player.ilmn?dirname=systems&swfname=GA_workflow_vid&width=780&h eight=485&iframe llumina llumina llumina llumina SOLiD sequencing Applied Biosystems' SOLiD technology employs sequencing by ligation. Here, a pool of all possible oligonucleotides of a fixed length are labeled according to the sequenced position. Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position. Before sequencing, the DNA is amplified by emulsion PCR. The resulting bead, each containing only copies of the same DNA molecule, are deposited on a glass slide. The result is sequences of quantities and lengths comparable to Illumina sequencing. SEE ANIMATION: http://www.youtube.com/watch?v=nlvyF8bFDwM abi-fig5 SOLiD sequencing http://www.appliedbiosystems.com/etc/medialib/appliedbio-media-library/images/application-and-techn ology/solid-next-generation-sequencing/data-images.Par.33471.Image.-1.0.1.gif Library Preparation Prepare one of the two types of libraries for SOLiD™ System sequencing-fragment or mate-paired. Your choice of library depends on the application you're performing and the information you desire from your experiments. SOLiD sequencing http://www.appliedbiosystems.com/etc/medialib/appliedbio-media-library/images/application-and-techn ology/solid-next-generation-sequencing/data-images.Par.62718.Image.-1.0.1.gif Emulsion PCR/Bead Enrichment Prepare clonal bead populations in microreactors containing template, PCR reaction components, beads, and primers. After PCR, denature the templates and perform bead enrichment to separate beads with extended templates from undesired beads. The template on the selected beads undergoes a 3’ modification to allow covalent attachment to the slide. SOLiD sequencing http://www.appliedbiosystems.com/etc/medialib/appliedbio-media-library/images/application-and-techn ology/solid-next-generation-sequencing/data-images.Par.67475.Image.-1.0.1.gif Bead Deposition Deposit 3’ modified beads onto a glass slide (Figure 3). During bead loading, deposition chambers enable you to segment a slide into one, four, or eight sections. A key advantage of the system is the ability to accommodate increasing densities of beads per slide, resulting in a higher level of throughput from the same system. SOLiD sequencing data-images Sequencing by Ligation 1. Primers hybridize to the P1 adapter sequence on the templated beads. 2. A set of four fluorescently labeled di-base probes compete for ligation to the sequencing primer. Specificity of the di-base probe is achieved by interrogating every 1st and 2nd base in each ligation reaction. 3. Multiple cycles of ligation, detection and cleavage are performed with the number of cycles determining the eventual read length. 4. Following a series of ligation cycles, the extension product is removed and the template is reset with a primer complementary to the n-1 position for a second round of ligation cycles. http://www.appliedbiosystems.com/etc/medialib/appliedbio-media-library/images/application-and-techn ology/solid-next-generation-sequencing/data-images.Par.57418.Image.-1.0.1.gif Primer Reset Five rounds of primer reset are completed for each sequence tag (Figure 5). Through the primer reset process, virtually every base is interrogated in two independent ligation reactions by two different primers. For example, the base at read position 5 is assayed by primer number 2 in ligation cycle 2 and by primer number 3 in ligation cycle 1. This dual interrogation is fundamental to the unmatched accuracy characterized by the SOLiD™ System. SOLiD sequencing a | A four-colour sequencing by ligation method using Life/APG's support oligonucleotide ligation detection (SOLiD) platform is shown. Upon the annealing of a universal primer, a library of 1,2-probes is added. Unlike polymerization, the ligation of a probe to the primer can be performed bi-directionally from either its 5′-PO4 or 3′-OH end. Appropriate conditions enable the selective hybridization and ligation of probes to complementary positions. Following four-colour imaging, the ligated 1,2-probes are chemically cleaved with silver ions to generate a 5′-PO4 group. The SOLiD cycle is repeated nine more times. The extended primer is then stripped and four more ligation rounds are performed, each with ten ligation cycles. The 1,2-probes are designed to interrogate the first (x) and second (y) positions adjacent to the hybridized primer, such that the 16 dinucleotides are encoded by four dyes (coloured stars). The probes also contain inosine bases (z) to reduce the complexity of the 1,2-probe library and a phosphorothiolate linkage between the fifth and six nucleotides of the probe sequence, which is cleaved with silver ions106. Other cleavable probe designs include RNA nucleotides107, 108 and internucleosidic phosphoramidates107, which are cleaved by ribonucleases and acid, respectively. b | A two-base encoding scheme in which four dinucleotide sequences are associated with one colour (for example, AA, CC, GG and TT are coded with a blue dye). Each template base is interrogated twice and compiled into a string of colour-space data bits. The colour-space reads are aligned to a colour-space reference sequence to decode the DNA sequence. c | Pyrosequencing using Roche/454's Titanium platform. Following loading of the DNA-amplified beads into individual PicoTiterPlate (PTP) wells, additional beads, coupled with sulphurylase and luciferase, are added. In this example, a single type of 2′-deoxyribonucleoside triphosphate (dNTP) — cytosine — is shown flowing across the PTP wells. The fibre-optic slide is mounted in a flow chamber, enabling the delivery of sequencing reagents to the bead-packed wells. The underneath of the fibre-optic slide is directly attached to a high-resolution charge-coupled device (CCD) camera, which allows detection of the light generated from each PTP well undergoing the pyrosequencing reaction. d | The light generated by the enzymatic cascade is recorded as a series of peaks called a flowgram. PPi, inorganic pyrophosphate. nrg2626-f3 Applications De novo sequencing: 454 (longer reads and high coverage - preferred for genome assembly) Resequencing: Solexa, SOLiD (shorter, but reliable reads) – identification of differences compared to reference sequence – e.g. molecular diagnostics,... Transcriptome sequencing – substitutes for cDNA/RNA microarrays, - representation of reads transcripts (normalized to reference transcript) reflects abundance of the transcript - more precise than microarrays -possibility of detection of unknown transcripts Whole genome sequencing, 1000 genome project (human genome variation) Metagenomics – analysis of complete genetic material from environmental samples without cultivation – real diversity (e.g. microbial composition of certain habitats, human organs - only about 1 out of 10 cells in the human body is actually a human cell: most of the cells that make up our bodies are microbes!) Epigenetics / epigenomics - DNA methylation, ChIP-Seq Ion semiconductor sequencing Ion Torrent Systems Inc. (now owned by Life Technologies) developed a system based on using standard sequencing chemistry, but with a novel, semiconductor based detection system. This method of sequencing is based on the detection of hydrogen ions that are released during the polymerisation of DNA, as opposed to the optical methods used in other sequencing systems. A microwell containing a template DNA strand to be sequenced is flooded with a single type of nucleotide. If the introduced nucleotide is complementary to the leading template nucleotide it is incorporated into the growing complementary strand. This causes the release of a hydrogen ion that triggers a hypersensitive ion sensor, which indicates that a reaction has occurred. If homopolymer repeats are present in the template sequence multiple nucleotides will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal. http://www.iontorrent.com/ Ion-Torrent In nature, when a nucleotide is incorporated into a strand of DNA by a polymerase, a hydrogen ion is released as a byproduct. Step One Ion Torrent cm400726f1_online Ion-Torrent Step Two Ion Torrent™ uses a high-density array of micro-machined wells to perform this biochemical process in a massively parallel way. Each well holds a different DNA template. Beneath the wells is an ion-sensitive layer and beneath that a proprietary Ion sensor. Ion Torrent Ion-Torrent Step Three If a nucleotide, for example a C, is added to a DNA template and is then incorporated into a strand of DNA, a hydrogen ion will be released. The charge from that ion will change the pH of the solution, which can be detected by our proprietary ion sensor. The sequencer - essentially the world's smallest solid-state pH meter - will call the base, going directly from chemical information to digital information. Ion Torrent Ion-Torrent The Ion Personal Genome Machine™ (PGM™) sequencer then sequentially floods the chip with one nucleotide after another. If the next nucleotide that floods the chip is not a match, no voltage change will be recorded and no base will be called. Step Four Ion Torrent Ion-Torrent Step Five If there are two identical bases on the DNA strand, the voltage will be double, and the chip will record two identical bases called. Because this is direct detection - no scanning, no cameras, no light - each nucleotide incorporation is recorded in seconds. The semiconductor will inevitably transform the life sciences, just as it has transformed every other industry it has touched. By creating a direct connection between chemical and digital information, Ion Torrent™ will democratize research, providing a fast, simple, scalable sequencing solution that every lab can afford. Eventually, Ion Torrent Technology™ will also be able to provide diagnostics that are less expensive and more reliable, improving human health around the world. Ion Torrent HelioscopeTM single molecule sequencing Based on "true single molecule sequencing" technology, Helioscope sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface. The next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides (one nucleotide type at a time, as with the Sanger method). The reads are performed by the Helioscope sequencer. The reads are short, up to 55 bases per run, but recent improvement of the methodology allows more accurate reads of homopolymers (stretches of one type of nucleotides) and RNA sequencing. Advantages: High number of reads, no sequencing bias (no amplification step) a | The four-colour cyclic reversible termination (CRT) method uses Illumina/Solexa's 3′-O-azidomethyl reversible terminator chemistry23, 101 (Box 1) using solid-phase-amplified template clusters (FIG. 1b, shown as single templates for illustrative purposes). Following imaging, a cleavage step removes the fluorescent dyes and regenerates the 3′-OH group using the reducing agent tris(2-carboxyethyl)phosphine (TCEP)23. b | The four-colour images highlight the sequencing data from two clonally amplified templates. c | Unlike Illumina/Solexa's terminators, the Helicos Virtual Terminators33 are labelled with the same dye and dispensed individually in a predetermined order, analogous to a single-nucleotide addition method. Following total internal reflection fluorescence imaging, a cleavage step removes the fluorescent dye and inhibitory groups using TCEP to permit the addition of the next Cy5-2′-deoxyribonucleoside triphosphate (dNTP) analogue. The free sulphhydryl groups are then capped with iodoacetamide before the next nucleotide addition33 (step not shown). d | The one-colour images highlight the sequencing data from two single-molecule templates. nrg2626-f2 How tSMS Works Within two flow cells, billions of single molecules of sample DNA are captured on an application-specific proprietary surface. These captured strands serve as templates for the sequencing-by-synthesis process: 1. Polymerase and one fluorescently labeled nucleotide (C, G, A or T) are added. 2. The polymerase catalyzes the sequence-specific incorporation of fluorescent nucleotides into nascent complementary strands on all the templates. 3. After a wash step, which removes all free nucleotides, the incorporated nucleotides are imaged and their positions recorded. 4. The fluorescent group is removed in a highly efficient cleavage process, leaving behind the incorporated nucleotide. 5. The process continues through each of the other three bases. Multiple four-base cycles result in complementary strands greater than 25 bases in length synthesized on billions of templates—providing a greater than 25-base read from each of those individual templates. http://www.helicosbio.com/Technology/TrueSingleMoleculeSequencing/tabid/64/Default.aspx Single molecule SMRTTM sequencing SMRT sequencing is based on the sequencing by synthesis approach. The DNA is synthesized in zero-mode wave-guides (ZMWs) - small well-like containers with the capturing tools located at the bottom of the well. The sequencing is performed with use of unmodified polymerase (attached to the ZMW bottom) and fluorescently labelled nucleotides flowing freely in the solution. The wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected. The fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand. According to Pacific Biosciences, the SMRT technology developer, this methodology allows detection of nucleotide modifications (such as cytosine methylation). This happens through the observation of polymerase kinetics. This approach allows reads of up to 15,000 nucleotides, with mean read lengths of 2.5 to 2.9 kilobases smrt_Technology The SMRT Cell One of the fundamental challenges with observing a DNA polymerase working in real time is the ability to detect the incorporation of a single nucleotide, taken from a large pool of potential nucleotides, during DNA synthesis. We applied the same principle that operates in the metallic screen of a microwave oven door. In a microwave oven, the screen is perforated with holes that are much smaller than the wavelength of the microwaves. Because of their relative size, the holes prevent the much longer microwaves from passing through and penetrating the glass. However, the much smaller wavelength visible light is able to pass through the holes in the screen, allowing food to be visible. We have reduced this same principle to the nanoscale and we call our innovation a zero-mode waveguide, or ZMW. With an active polymerase immobilized at the bottom of each ZMW, nucleotides diffuse into the ZMW chamber. In order to detect incorporation events and identify the base, each of the four nucleotides A, C, G and T are labeled with a different fluorescent dye having a distinct emission spectrum. Since the excitation illumination is directed to the bottom of the ZMW, nucleotides held by the polymerase prior to incorporation emit an extended signal that identifies the base being incorporated. A ZMW is a hole, tens of nanometers in diameter, fabricated in a 100nm metal film deposited on a glass substrate. The small size of the ZMW prevents visible laser light, which has a wavelength of approximately 600nm, from passing entirely through the ZMW. Rather than passing through, the light exponentially decays as it enters the ZMW. Therefore, by shining a laser through the glass into the ZMW, only the bottom 30nm of the ZMW becomes illuminated. Within each ZMW, a single DNA polymerase molecule is anchored to the bottom glass surface using a proprietary technique. Nucleotides, each type labeled with a different colored fluorophore, are then flooded above an array of ZMWs at the required concentration. Diffusion at the nanoscale is incredibly fast. Within microseconds, labeled nucleotides travel down into the ZMW, surround the DNA polymerase, then diffuse back up and exit the hole. As no laser light penetrates up through the holes to excite the fluorescent labels, the labeled nucleotides above the ZMWs are dark. Only when they diffuse through the bottom 30nm of the ZMW do they fluoresce. When the correct nucleotide is detected by the polymerase, it is incorporated into the growing DNA strand in a process that takes milliseconds in contrast to simple diffusion which takes microseconds. This difference in time results in higher signal intensity for incorporated versus unincorporated nucleotides, which creates a high signal-to-noise ratio. Thus, the ZMW has the ability to detect a single incorporation event against the background of fluorescently labeled nucleotides at biologically relevant concentrations. smrt_Technology DNA sequencing is performed on proprietary SMRT Cells, each having an array of approximately 75,000 ZMWs. Each ZMW is capable of containing a DNA polymerase loaded with a different strand of DNA sample. As a result, the SMRT Cell enables the potential detection of approximately 75,000 single molecule sequencing reactions in parallel. Phospholinked Nucleotides Previous labeling technologies attach a fluorescent label to the base of the nucleotide, which is incorporated into the DNA strand. This is problematic for any system to observe DNA synthesis in real time because the dye’s large size can interfere with the activity of the DNA polymerase. Typically, a DNA polymerase can incorporate only a few base-labeled nucleotides before it halts. Our proprietary phospholinked nucleotides have a fluorescent dye attached to the phosphate chain of the nucleotide rather than to the base. The phosphate chain is cleaved when the nucleotide is incorporated into the DNA. Thus, upon incorporation of a phospholinked nucleotide, the DNA polymerase naturally frees the dye molecule from the nucleotide when it cleaves the phosphate chain. Upon cleaving, the label quickly diffuses away, with no evidence of labeling remaining. 11.3.2013 > > Schematic of PacBio’s real-time single molecule sequencing. (A) The side view of a single ZMW nanostructure containing a single DNA polymerase (Φ29) bound to the bottom glass surface. The ZMW and the confocal imaging system allow fluorescence detection only at the bottom surface of each ZMW. (B) Representation of fluorescently labeled nucleotide substrate incorporation on to a sequencing template. The corresponding temporal fluorescence detection with respect to each of the five incorporation steps is shown below. Reprinted with permission from ref 39. Copyright 2009 American Association for the Advancement of Science. Published in: Thomas P. Niedringhaus; Denitsa Milanova; Matthew B. Kerby; Michael P. Snyder; Annelise E. Barron; Anal. Chem. 2011, 83, 4327-4341. DOI: 10.1021/ac2010857 Copyright © 2011 American Chemical Society Nanopore DNA sequencing This method is based on the readout of electrical signal occurring at nucleotides passing by alpha-hemolysin pores covalently bound with cyclodextrin. The DNA passing through the nanopore changes its ion current. This change is dependent on the shape, size and length of the DNA sequence. Each type of the nucleotide blocks the ion flow through the pore for a different period of time. http://www.nanoporetech.com/technology/analytes-and-applications-dna-rna-proteins/dna-an-introducti on-to-nanopore-sequencing Nanopore_sensing_101_0_rs Nanopore_sensing_101_0_rs This diagram shows a protein nanopore set in an electrically resistant membrane bilayer. An ionic current is passed through the nanopore by setting a voltage across this membrane. If an analyte passes through the pore or near its aperture, this event creates a characteristic disruption in current. By measuring that current it is possible to identify the molecule in question. For example, this system can be used to distinguish the four standard DNA bases and G, A, T and C, and also modified bases. It can be used to identify target proteins, small molecules, or to gain rich molecular information for example to distinguish the enantiomers of ibuprofen or molecular binding dynamics. Strand sequencing Exonuclease sequencing 11.3.2013 > > Nanopore DNA sequencing using electronic measurements and optical readout as detection methods. (A) In electronic nanopore schemes, signal is obtained through ionic current, tunneling current, and voltage difference measurements. Each method must produce a characteristic signal to differentiate the four DNA bases. (B) In the optical readout nanopore design, each nucleotide is converted to a preset oligonucleotide sequence and hybridized with labeled markers that are detected during translocation of the DNA fragment through the nanopore. Published in: Thomas P. Niedringhaus; Denitsa Milanova; Matthew B. Kerby; Michael P. Snyder; Annelise E. Barron; Anal. Chem. 2011, 83, 4327-4341. DOI: 10.1021/ac2010857 Copyright © 2011 American Chemical Society 11.3.2013 > > Biological nanopore scheme employed by Oxford Nanopore. (A) Schematic of αHL protein nanopore mutant depicting the positions of the cyclodextrin (at residue 135) and glutamines (at residue 139). (B) A detailed view of the β barrel of the mutant nanopore shows the locations of the arginines (at residue 113) and the cysteines. (C) Exonuclease sequencing: A processive enzyme is attached to the top of the nanopore to cleave single nucleotides from the target DNA strand and pass them through the nanopore. (D) A residual current-vs-time signal trace from an αHL protein nanopore that shows a clear discrimination between single bases (dGMP, dTMP, dAMP, and dCMP). (E) Strand sequencing: ssDNA is threaded through a protein nanopore and individual bases are identified, as the strand remains intact. Published in: Thomas P. Niedringhaus; Denitsa Milanova; Matthew B. Kerby; Michael P. Snyder; Annelise E. Barron; Anal. Chem. 2011, 83, 4327-4341. DOI: 10.1021/ac2010857 Copyright © 2011 American Chemical Society 11.3.2013 > > Several synthetic nanopore sequencing device designs. (A) The device consists of 1–5 nm thick graphene membrane which is suspended in a Si chip coated with 5 μm SiO2 layer. It is placed in a PDMS cell with microfluidic channels on both sides of the chip. (B) A nanopore (shown in the inset to the figure) is drilled through a graphene membrane, which is suspended in SiNx across a Si frame. The graphene membrane separates two ionic solutions and is in contact with Ag/AgCl electrodes. (C) IBM DNA transistor setup. A nanometer sized pore is fabricated using an electron beam. Electric field is created between the gated regions allowing for charge trapping. The substrate is composed of metal and dielectric regions, labeled with ‘‘M’’ and ‘‘D’’, respectively. (D) HANS method adopted by NABsys for electronic readout of DNA fragments through solid-state nanopores. 6-mer oligonucleotide probes are hybridized to ssDNA fragments, and current-verses-time trace is detected. Published in: Thomas P. Niedringhaus; Denitsa Milanova; Matthew B. Kerby; Michael P. Snyder; Annelise E. Barron; Anal. Chem. 2011, 83, 4327-4341. DOI: 10.1021/ac2010857 Copyright © 2011 American Chemical Society VisiGen Biotechnologies approach VisiGen Biotechnologies introduced a specially engineered DNA polymerase for use in their sequencing. This polymerase acts as a sensor - having incorporated a donor fluorescent dye by its active centre. This donor dye acts by FRET (fluorescent resonant energy transfer), inducing fluorescence of differently labeled nucleotides. This approach allows reads performed at the speed at which polymerase incorporates nucleotides into the sequence (several hundred per second). The nucleotide fluorochrome is released after the incorporation into the DNA strand. The expected read lengths in this approach should reach 1000 nucleotides. Future methods Sequencing by hybridization is a non-enzymatic method that uses a DNA microarray. A single pool of DNA whose sequence is to be determined is fluorescently labeled and hybridized to an array containing known sequences. Strong hybridization signals from a given spot on the array identifies its sequence in the DNA being sequenced. Mass spectrometry may be used to determine mass differences between DNA fragments produced in chain-termination reactions. DNA sequencing methods currently under development include labeling the DNA polymerase, reading the sequence as a DNA strand transits through nanopores,and microscopy-based techniques, such as AFM or transmission electron microscopy that are used to identify the positions of individual nucleotides within long DNA fragments (>5,000 bp) by nucleotide labeling with heavier elements (e.g., halogens) for visual detection and recording. Third generation technologies aim to increase throughput and decrease the time to result and cost by eliminating the need for excessive reagents and harnessing the processivity of DNA polymerase. In microfluidic Sanger sequencing the entire thermocycling amplification of DNA fragments as well as their separation by electrophoresis is done on a single glass wafer (approximately 10 cm in diameter) thus reducing the reagent usage as well as cost. In some instances researchers have shown that they can increase the throughput of conventional sequencing through the use of microchips. Research will still need to be done in order to make this use of technology effective. In October 2006, the X Prize Foundation established an initiative to promote the development of full genome sequencing technologies, called the Archon X Prize, intending to award $10 million to "the first Team that can build a device and use it to sequence 100 human genomes within 10 days or less, with an accuracy of no more than one error in every 100,000 bases sequenced, with sequences accurately covering at least 98% of the genome, and at a recurring cost of no more than $10,000 (US) per genome." Each year NHGRI promotes grants for new research and developments in genomics. 2010 grants and 2011 candidates include continuing work in microfluidic, polony and base-heavy sequencing methodologies. Major landmarks in DNA sequencing 1953 Discovery of the structure of the DNA double helix. 1972 Development of recombinant DNA technology, which permits isolation of defined fragments of DNA; prior to this, the only accessible samples for sequencing were from bacteriophage or virus DNA. 1977 The first complete DNA genome to be sequenced is that of bacteriophage φX174. 1977 Allan Maxam and Walter Gilbert publish "DNA sequencing by chemical degradation". Frederick Sanger, independently, publishes "DNA sequencing with chain-terminating inhibitors". 1984 Medical Research Council scientists decipher the complete DNA sequence of the Epstein-Barr virus, 170 kb. 1986 Leroy E. Hood's laboratory at the California Institute of Technology and Smith announce the first semi- automated DNA sequencing machine. 1987 Applied Biosystems markets first automated sequencing machine, the model ABI 370. 1990 The U.S. National Institutes of Health (NIH) begins large-scale sequencing trials on Mycoplasma capricolum, Escherichia coli, Caenorhabditis elegans, and Saccharomyces cerevisiae (at US$0.75/base). 1991 Sequencing of human expressed sequence tags begins in Craig Venter's lab, an attempt to capture the coding fraction of the human genome. 1995 Craig Venter, Hamilton Smith, and colleagues at The Institute for Genomic Research (TIGR) publish the first complete genome of a free-living organism, the bacterium Haemophilus influenzae. The circular chromosome contains 1,830,137 bases and its publication in the journal Science marks the first use of whole-genome shotgun sequencing, eliminating the need for initial mapping efforts. 1996 Pål Nyrén and his student Mostafa Ronaghi at the Royal Institute of Technology in Stockholm publish their method of pyrosequencing 1998 Phil Green and Brent Ewing of the University of Washington publish "pyrex" for sequencer data analysis. 2000 Lynx Therapeutics publishes and markets "MPSS" - a parallelized, adapter/ligation-mediated, bead-based sequencing technology, launching "next-generation" sequencing. 2001 A draft sequence of the human genome is published. 2004 454 Life Science markets a parallelized version of pyrosequencing. The first version of their machine reduced sequencing costs 6-fold compared to automated Sanger sequencing, and was the second of a new generation of sequencing technologies, after MPSS.