2 Illumina (solexa) sequencing by synthesis technology. a Clonal amplification of adaptor flanked fragments is performed by bridge PCR and then single-stranded DNA is produced for sequencing step. b Base detection is conducted by the addition of four labeled reversible blocked nucleotides, primers and a DNA polymerase enzyme. After each nucleotide incorporation, the image of emitted fluorescence is captured and then the 3 0 blocked nucleotide and the fluorescent dye are removed to permit initiating another cycle of nucleotide detection [7]

Contexts in source publication

Context 1

... this method, a DNA library can be constructed by any method that generates adaptor-flanked fragments up to several hundred base pairs in length (Fig. 2.2a) [6]. Template amplification is performed with a method called ''bridge PCR,'' in which both forward and reverse primers that are complementary to the adaptors' sequences are attached to a solid surface called ''flow cell,'' containing eight independent channels that allow performing PCR amplification on its surface. The name ''bridge ...

Context 2

... forms a bridge to the other bound primer and its complementary strand is synthesized. Since each initial single fragment has been tethered to a distinct channel on the surface, the amplified fragments form clusters, each of which contains approximately one million copies of the initial fragment that would be sufficient for sequencing reaction (Fig. 2.2b). Moreover, each cluster contains both forward as well as reverse strands of the original sequences, but to have homogenous populations of strands in each cluster, which can be sequenced precisely without the interference of the complementary strand, one of the strands must be removed before initiating the sequencing process [12]. The ...

Context 3

... matched with a template then can be hybridized, which will be followed by the process of ligation in the presence of enzyme ligase. (2) In this step, an image is taken (the specific fluor is detected, which corresponds to the specific dinucleotide composition; in fact, each fluorescent dye is in correlation with four dinucleotide combinations-see Fig. 2.4). The unextended fragments are capped in the presence of the same mixture of nonfluorescent dinucleotides. (3) In the next step, the last three bases of octamer (i.e., the bases 6, 7 and 8) along with the fluorescent dye are chemically removed, so five bases of octamer are left behind and the above steps are repeated for base 6 and 7. ...

Context 4

... In the next step, the last three bases of octamer (i.e., the bases 6, 7 and 8) along with the fluorescent dye are chemically removed, so five bases of octamer are left behind and the above steps are repeated for base 6 and 7. This step is typically repeated 10 times [12] and at the end, the fluorescent colors, corresponding to 10 dinucleotides ( Fig. ...

Context 5

... there are 16 possible combinations of dinucleotides (4 2 ) and only four fluorescent dyes ( Fig. 2.3b), nucleotide identity detection would not be possible only from data color obtained from the incorporation of dinucleotides. The dinucleotide composition of the first dinucleotide, i.e., nucleotides 0/1, is readily deduced from its fluorescent signal because the first base of newly synthesized strand corresponds to the last base of the ...

Context 6

... (Life technologies). This technology, which is one of the least expensive methods, has been introduced to research and clinical laboratories as a personal genomic machine. The biochemical basis of this system is very simple and involves the release of hydrogen ion following the incorporation of a nucleotide into a strand of DNA by DNA polymerase (Fig. 2.4a). To carry out this biochemical process in a highly parallel way, Ion Torrent uses a high-density array of micro-machined wells that is provided on the Ion Torrent proprietary microchips. There is an Ion-sensitive field effect transistor (ISFET) below the wells that detects the change in pH as a result of hydrogen release. This change ...

Context 7

... 2.4a). To carry out this biochemical process in a highly parallel way, Ion Torrent uses a high-density array of micro-machined wells that is provided on the Ion Torrent proprietary microchips. There is an Ion-sensitive field effect transistor (ISFET) below the wells that detects the change in pH as a result of hydrogen release. This change is b Fig. 2.3 Schematic representation of the steps involved in AB-SOLiD sequencing. a After initial primer (n-nucleotides) annealing, seven cycles of oligonucleotide hybridization and ligation, imaging and cutting the 5 0 three nucleotides along with fluorescent dye is performed. Then the initial primer and extended strand are removed, which is ...

Context 8

... by four fluorescent dyes and decoding of each base using two dinucleotides. Alignment of the color-space reads of the particular sequence with color-space reads of the reference genome will result in SNP identification [1,7] recorded as a potential change (DV) by an ion sensor layer that indicates the nucleotide incorporation event [15] ( Fig. ...

Context 9

... the bases by this method goes like this: Following the addition of a certain base to a DNA template (''A,'' for example), if it is incorporated in to the DNA strand, a hydrogen ion will be released and consequently the charge of that ion will change the pH of the solution that can be detected by the ion sensor ( Fig. 2.4c). The chip will be flooded by introducing one nucleotide after another. If no incorporation occurs following the addition of a nucleotide, no voltage change is recorded, and as a result no base will be called. In the case of the presence of two consecutive identical bases on the DNA strand, the voltage will be doubled, and as a result, ...

Context 10

... sequencing technology was first introduced by Dr. George Church's group at the Harvard Medical School. Unlike other sequencing technologies, it has opensource software and free downloadable protocols. The polony sequencing method is begun by paired-end-tag library construction ( Fig. 2.5). The Target DNA sequence is randomly sheared, and then the fragments-about 1 kb in size-are selected. After making the ends of these fragments blunt and A-tailing, in which an A is added to the 3 0 ends of fragments, the fragments are circularized, using 30 bpsynthesized oligonucleotides (T30) with two outward facing recognition sites ...

Context 11

... the Heliscope sequencer and uses the sequencing by synthesis technique. The DNA sample is first sheared into small pieces (100-200 bp) and then becomes adaptor-flanked. This adaptor is usually a poly-A tail, and adaptor-flanked fragments can consequently be tethered to a surface of flow cells on which poly-T oligonucleotides have been bound ( Fig. 2.6). After the attachment of fragments, a mixture of a single-labeled nucleotide and polymerase are streamed into the surface and the polymerase will add nucleotide wherever it is complementary to the first positions of the attached fragments. Then the image is taken from all bound fragments that contain incorporated labeled nucleotides. ...

Context 12

... is no need for any DNA fragment shearing, amplification step, fluorescently labeled nucleotides and optical instrumentation for detecting fluorescent labels. This technology has its origin in threading a single-stranded DNA molecule through a nanoscale protein pore (staphylococcal a-haemolysin) created in a membrane under an applied potential ( Fig. 2.7). Passing a DNA strand through the pore results in fluctuation of the ionic current. The translocation of each base through the pore causes a decrease in current intensification which is specific for each kind of base. In fact, while each of the four bases passes through the pore, a different amount of current can translocate, and this ...

Context 13

... whose fluorescence is restored when they bind to a target nucleic acid [19]. For a two-color readout (i.e., two types of fluorophores), the four sequences are combinations of two predefined unique sequences, bit ''0'' and bit ''1,'' such that an A would be ''1 1,'', a G would be ''1 0,'' a T would be ''0 1'' and finally a C would be ''0 0'' ( Fig. 2.8a). Two types of molecular beacons, carrying two types of fluorophores, hybridize specifically to the ''0'' and ''1'' sequences. The converted DNA and hybridized molecular beacons are electrophoretically passed through a solid-state pore where the beacons are sequentially removed. Each time a beacon is removed, a new fluorophore is ...

Context 14

... hybridize specifically to the ''0'' and ''1'' sequences. The converted DNA and hybridized molecular beacons are electrophoretically passed through a solid-state pore where the beacons are sequentially removed. Each time a beacon is removed, a new fluorophore is lighted, which results into a burst of photons recorded at the location of the pore (Fig. 2.8b). This method allows wide field imaging and spatially fixed pores that make possible the simultaneous detection of several pores using a special camera, Electron multiplying charged coupled device (EM-CCD) [17]. (4) The adaptor is basically used to reduce the speed of passing DNA through the pore, which is necessary for the exact ...

Context 15

... possible the simultaneous detection of several pores using a special camera, Electron multiplying charged coupled device (EM-CCD) [17]. (4) The adaptor is basically used to reduce the speed of passing DNA through the pore, which is necessary for the exact identification of the DNA strand base composition (picture from MIT's Technology Review) Fig. 2.8 Another version of nanopore sequencing methods introduced by McNally. a The biochemical preparation step involving conversion of each base of the sequence into an oligonucleotide that can be hybridized with a molecular beacon. b The threading of the beacon hybridized oligonucleotides through a nanopore makes it possible to detect ...

Context 16

... detection of several pores using an EM-CCD camera [17] platform, SMRT cells are used, with each cell having thousands of zero-mode waveguides (ZMWs), which are holes in a surface that acts as a nanoscale chamber. In each ZMW (which is tens of nanometers in diameter), a single molecule of DNA polymerase is attached to the bottom surface ( Fig. ...

Context 17

... result of the physical bulk of several dye molecules, which in turn leads to the limitation of enzyme activity. In SMRT technology, the fluorescent label is attached to the phosphate chain, and as a result of nucleotide incorporation the pentaphosphate-label couple will be removed from the nucleotides and will diffuse out of the reaction volume ( Fig. 2.9b) [20], (Pacific Biosciences, 2009. Single Molecule Real Time (SMRT TM ) DNA Sequencing) [1]). b Each incorporated phospholinked nucleotide will reside on the enzyme's active site for a few milliseconds, which is enough time for a fluorescent signal to be recorded. The released labled pentaphosphates will diffuse quickly [1] ...

Context 18

... assembly algorithm works in overlap detection and contig generation phases. As previously explained, paired-end read libraries are other available data for assemblers. This data can be useful to extend contigs and also resolve repeat areas. The task of ordering and orienting contigs along a chromosome using paired-reads is called scaffolding (Fig. 4.2). If one end of a paired-read is assembled in a contig and the other end in a second contig, it can be inferred that these contigs are adjacent in the final ...

Context 19

... the graph After the graph is constructed, some simplification is applied to it in order to remove fragmentations in the graph structure (fragmentation is the source memory usage). The simplification uses a simple idea: whenever a node A has only one outgoing arc that points to another node B that has only one ingoing arc, the two nodes are merged (Fig. ...

Context 20

... EULER-USR algorithm is based on the notion of repeat graphs. A repeat graph of a genome (or reads) is a simplified version of the de Burijn graph with small bulges and whirls removed (Fig. 4.22e, f). The key point in this algorithm is based on this observation, that a repeat graph of a whole genome can be approximated from a repeat graph generated from reads. Reads may be corrected by mapping them to repeat graphs of the genome, if the graph is known. The idea of EULER-USR is to construct repeat graph from accurate reads, and then ...