Shendure J, Porreca GJ, Reppas NB, Lin X, McCutcheon JP, Rosenbaum AM, et al. Additional analytical considerations are needed for miRNA studies, including RNA secondary-structure analysis for hairpins, alignment to known miRNA databases, and searches within the NGS data set for complementary miRNA strands, as described in studies of developing rice grains(77) and chicken embryos(78). Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Thus, with the help of next-generation DNA techniques, researchers can provide potential information and reliable data for most living eukaryotic and prokaryotic species. A 6-day instrument run generates sequence read lengths of 35 bases. For sequencing, Pacific Biosciences uses a modified phi29 DNA polymerase that has enhanced kinetic properties for incorporating the system’s phospholinked fluorescently labeled dNTPs. With the use of offset primers, several bases of the adapter are sequenced. one. It allows us to know and better understand the communities of organisms that we have had no idea about before. The complete genome of an individual by massively parallel DNA sequencing. Attached to a glass slide surface, the polymerase directs strand extension from primed DNA templates. The first single-molecule sequencing platform, the HeliScope, is now available from Helicos BioSciences (http://www.helicosbio.com) with a company-reported sequence output of 1 Gb/day. The picotiter plate functions as a flow cell where iterative pyrosequencing is performed. The main principle of pyrosequencing reaction is to amplify a single DNA strand after which a genomic DNA (as Shigella sonni DNA sequences) is isolated and shattered into a substantial number of small parts of fragments. NGS has had a tremendous impact on the study of microbial diversity in environmental and clinical samples. To best use NGS for sequencing such candidate regions, several genomic-enrichment steps, both traditional and novel, are being incorporated into overall experimental designs. Error probabilities. History of Next Generation Sequencing. You should increase this value if the generated … All preparing microbeads are ligated to a slide surface and then anneal primers are added to the heads of the short DNA sequences. More than one million of copies of a signal stranded DNA are ligated on DNA detain microbead. In practice, coverage across a sequenced region is variable, and factors other than the Poisson-like randomness of library preparation that may contribute to this variability include differential ligation of adapters to template sequences and differential amplification during clonal template generation(11)(70). Making it a long process that produces 700 base pairs (bp) in an hour. RNA-Seq has been applied to a variety of organisms, including Saccharomyces cerevisiae, Arabidopsis thaliana, mice, and human cells(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51). Different methods were evolved for determining DNA sequences when Sanger method (dideoxy method), which was invented by Frederick Sanger, was significantly accessible (Gibson, 2008). The terminators are incorporated according to sequence complementarity in each strand in a clonal cluster. Each small part has to have a perfect size for process. Maxam AM, Gilbert W. A new method for sequencing DNA. For instance, in order to compare two DNA sequences like Shigella sonnei and Escherichia Coli, BLAST demonstrates the regions of matched orientations and opposite orientations via different colour. Each reaction well is a nanophotonic chamber in which only the bottom third is visualized, producing a detection volume of approximately 20 zL (20 × 10−21 L). However, the notion of high-throughput sequencing has been increasingly improved and also has decreased significantly both the cost and the period of sequencing. Error rates for individual NGS reads are higher than for Sanger sequencing. The coupling of NGS with the genomic-enrichment techniques described above offers a promising approach to this technical challenge. For full access to this pdf, sign in to an existing account, or purchase an annual subscription. Oxford University Press is a department of the University of Oxford. Eventually, those labelled-probes with fluorescent dye can release a particular signal; as a consequence, computer can measure properly attached-primers in all wells. Morin R, Bainbridge M, Fejes A, Hirst M, Krzywinski M, Pugh T, et al. Field guide to next-generation DNA sequencers in Mol Ecol Resour (2011). In 2008, the first NGS platform based on single-molecule DNA sequencing was launched. In 1977, 2 landmark articles describing methods for DNA sequencing were published. This modern platform includes accurate various steps. In addition to the study of transcription factors, NGS is being used to map genomic methylation. We describe major applications of NGS and then review the analysis of NGS data. 1 ), a library of template DNA is prepared by fragmentation via nebulization or sonication. Moreover, BLAST can perform various functions such as nucleotide database, protein database and translate nucleotide database (Dale, 2012). As a massively parallel process, NGS generates hundreds of megabases to gigabases of nucleotide-sequence output in a single instrument run, depending on the platform. It will provide information about the pros and cons of both platforms for sequencing. Wang J, Wang W, Li R, Li Y, Tian G, Goodman L, et al. Clusters are denatured and cleaved; sequencing is initiated with addition of primer, polymerase (POL) and 4 reversible dye terminators. Charalambous Tower Each fluorescence signal originates from a clonally amplified template cluster. Somatic mutations affect key pathways in lung adenocarcinoma. The PCR can be even used to amplify small DNA fragments (Bartlett et al, 2003). Real-time DNA sequencing from single polymerase molecules. generation sequencing is used as a key technology in metagenomics. After the positional coordinates of the captured strands are recorded by a charge-coupled device camera, the label is cleaved and washed away before sequencing. Background: For the past 30 years, the Sanger method has been the dominant approach and gold standard for DNA sequencing. The company envisions its platform will consist of a massively parallel array of tethered DNA polymerases that will generate 1 × 106 bp of sequence per second. Hitherto, four sequencing … Levene MJ, Korlach J, Turner SW, Foquet M, Craighead HG, Webb WW. Sequencing is an essential reading skill that students must develop if they are to fully understand all reading material. See the Acknowledgments for disclosure information on the CFTR-gene analysis performed on residual, deidentified DNA. The lower panel is a composite image of the 4 fluorescence channels. 454 has reported that the metal coating of the walls of picotiter wells mentioned above improves the accuracy of homopolymer determination. Identification of somatically acquired rearrangements in cancer using genome-wide massively parallel paired-end sequencing. Albert TJ, Molla MN, Muzny DM, Nazareth L, Wheeler D, Song X, et al. 5 presents some examples of NGS data viewed in 2 different software systems. Microbial diversity in the deep sea and the underexplored “rare biosphere.”. Each sequencing cycle, which consists of the successive addition of polymerase and each of the 4 labeled dNTPs, is termed a “quad.” The number of sequencing quads performed is approximately 25–30, with read lengths of up to 45–50 bases having been achieved. In order to continue this sequencing, the mixture (known as substrates) is constantly added to grow DNA sequences (Dale, 2012). From the impact that NGS has made at the basic-research level, we can anticipate its translation into molecular diagnostics. Okou DT, Steinberg KM, Middle C, Cutler DJ, Albert TJ, Zwick ME. This involves evolution of a high level of the sample’s production, putting the sample into the technology, organising the sequencing reactions and analysing the consequences. For sequencing (Fig. The consequences are immediately read and recorded, the nucleotide is removed, the stain is emitted, and sequencing is repeated to record the following nucleotide of the DNA strands. Applied Biosystems refined the technology and released the SOLiD instrumentation in 2007. The dynamic range of RNA-Seq for determining expression levels is 3–4 orders of magnitude, compared with 2 orders of magnitude for expression arrays. One of the pivotal benefits of metagenomics of microbial communities is that a substantial number of bacteria and prokaryotic species have not yet been isolated or cultured in scientific laboratories. It also has a positive role in some cycles of some sequencing techniques. These modern platforms can change the trend of study the population and the communities of various organisms from genes for genomes. When two sequences are completely overlapping, they can be linked together to generate a contig assembly. In 1993, PCR was invented by Kary B. Mullis. Quail MA, Kozarewa I, Smith F, Scally A, Stephens PJ, Durbin R, et al. More recently, enrichment has been achieved by hybridizing fragmented, denatured human genomic DNA to oligonucleotide capture probes complementary to the region of interest and subsequently eluting the enriched DNA(30)(31)(32)(33). They are afraid that the discovered DNA sequences would be extreme equivalent to some other DNA sequences that have already been found? Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Rapid whole-genome mutational profiling using next-generation sequencing technologies. High-resolution profiling of histone methylations in the human genome. In the other hand, Next Generation involves shorter steps, it amplifies the genome fragment by PCR and then … Highly integrated single-base resolution maps of the epigenome in Arabidopsis. By this approach, each template nucleotide is sequenced twice. Fan HC, Quake SR. Seven cycles of ligation, referred to as a “round,” are performed to extend the first primer. On a separate front, Tawfik and Griffiths described single-molecule PCR in microcompartments consisting of water-in-oil emulsions(7). In development by NimbleGen is the use of an array of 2.1 × 106 features for capturing larger genomic regions. Model-based analysis of ChIP-Seq (MACS). Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Current software has made strong inroads, however, with incorporation of motif recognition at splice junctions and identification of intron–exon borders through regions of low alignment coverage(41). Further studies are needed to compare technology performance for detecting insertions and deletions. Next-generation sequencing, in contrast, makes large-scale whole-genome sequencing (WGS) accessible and practical for the average researcher. Pseudocolor image from the Illumina flow cell. This takes place in each cluster of the fifty million clusters on the slide. High throughput DNA sequencing methodology (next generation sequencing; NGS) has rapidly evolved over the past 15 years and new methods are continually being commercialized. Braslavsky I, Hebert B, Kartalov E, Quake SR. Sequence information can be obtained from single DNA molecules. Lister R, O'Malley RC, Tonti-Filippini J, Gregory BD, Berry CC, Millar AH, Ecker JR. Expression levels are deduced from the total number of reads that map to the exons of a gene, normalized by the length of exons that can be uniquely mapped. Capping coupled with high-fidelity ligation chemistry and interrogation of each nucleotide base twice during independent ligation cycles yields a company-reported sequence consensus accuracy of 99.9% for a known target at a 15-fold sequence coverage over sequence reads of 25 nucleotides. ARUP Institute for Experimental and Clinical Pathology, Address correspondence to this author at: ARUP Laboratories, 500 Chipeta Way, Salt Lake City, Utah 84108. In this cycle, DNA polymerase is used to extend short sequence opposing templates. Another advantage is that it is very accurate. Software packages available for alignment and assembly to a reference sequence include Zoom(71), MAQ(67), Mosaik(72), SOAP(73), and SHRiMP (http://compbio.cs.toronto.edu/shrimp/), which supports SOLiD color-space analysis. This review describes NGS technologies, reviews their impact on basic research, and explores how they have the translational potential to substantially impact molecular diagnostics. This addition prepares the DNA fragments for ligation to oligonucleotide adapters, which have an overhang of a single T base at their 3′ end to increase ligation efficiency. Fax (801) 584-5207; e-mail. Following Generation DNA SEQUENCING … https://www.ibiology.org/techniques/next-generation-sequencingNext generation sequencing allows DNA samples to be sequenced quickly and affordably. One of the advantages of Sanger method is that it requires short DNA sequences (about 500 bp) and also the cost of its run is relatively inexpensive ($1-$20 for Sanger sequencing). However, in comparison with pyrosequencing and SOLiD method, Illumine method can measure about 1.6 billion bp and also three to nine days is taken to a single run. For the first time, since 2008, 454 platform has been used to genome sequencing. Genome-wide location and function of DNA binding proteins. Instead of providing a 3′ hydroxyl group for polymerase-mediated extension, the primer is oriented to provide a 5′ phosphate group for ligation to interrogation probes during the first “ligation sequencing” step. Template DNA is fragmented into lengths of several hundred base pairs and end-repaired to generate 5′-phosphorylated blunt ends. Address: Cyprus Headquarters Borneman AR, Forgan AH, Pretorius IS, Chambers PJ. Denatured polyadenylated strands are hybridized to poly(dT) oligonucleotides immobilized on a flow-cell surface at a capture density of up to 100 × 106 template strands per square centimeter. These couple of neighbouring DNA strands then experience exponential development in situ hybridization utilising a new method called Bridge amplification. The commercial launch of the first massively parallel pyrosequencing platform in 2005 ushered in the new era of high-throughput genomic analysis now referred to as next-generation sequencing … Several analytical approaches that use cell-free fetal nucleic acids have been developed to determine fetal aneuploidy, including the analysis of placental mRNA derived from the chromosomes of interest (e.g., chromosome 21) and the determination of relative chromosomal dosage via digital PCR analysis of a large number of target chromosomal loci compared with reference chromosomal loci(89)(90)(91). Coverage gaps can occur when sequences are not aligned because of substantial variance from a reference. Recently, the developments in the procedure of DNA sequencing have been obtained using basically the same major techniques (Sanger … It enables scientists to analyze the entire human genome in a single sequencing … DNA sequencing might be utilised to understand and analyise the sequence … Farther out toward the horizon is sequencing based on monitoring the passage of DNA molecules through nanopores 2–5 nm or greater in diameter. A high temperature of (melting= ≈92˚C) is given in this stage. A nucleotide-incorporation event results in pyrophosphate (PPi) release and well-localized luminescence. Review papers on the topic of sequencing technologies. S.A. Dames and J.D. Role of Sponsor: The funding organizations played a direct role in the preparation of the manuscript and in the final approval of the manuscript. Chaisson MJ, Pevzner PA. Short read fragment assembly of bacterial genomes. HIV quasi-species detection and relative quantification have been demonstrated and can be used to monitor emerging drug resistance(84). There is a special camera laser that is able to record each base complementary to template base leads to chemoluminescence light sign. Examples of NGS data viewed in 2 different software systems. An analysis of the feasibility of short read sequencing. The mean DNA-synthesis rate was determined to be approximately 4 bases/s. Alta-Cyclic: a self-optimizing base caller for next-generation sequencing. Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution. Then recombinant random vectors are stored properly in a small library (Dale, 2012). The 2 streams are merged and primer-library droplets and genomic DNA/PCR reagent droplets are paired in a 1:1 ratio. DNA sequencing … This template requires initially oligonucleotide primer, DNA polymerase, normal substrates of dATP,dCTP,dGTP,dTTP and Fluorescent dyes (Kan et al, 2004). Sogin ML, Morrison HG, Huber JA, Mark Welch D, Huse SM, Neal PR, et al. Pop M, Salzberg SL. Acknowledgments: The analysis of the CFTR gene illustrated in Fig. Moreover, contaminant DNA fragments sometimes occur during the sequencing (Valouev et al, 2008). Nucleotides are modified with fluorescent acceptor molecules, and light energy is used during incorporation to invoke fluorescence resonance energy transfer between polymerase and nucleotide fluorescent moieties, the latter being in the γ-phosphate position and cleaved away during incorporation. The higher accuracy of Sanger sequencing reflects not only the maturity of the chemistry but also the fact that a Sanger trace peak represents highly redundant, multiple terminated extension reactions. Table 1 summarizes GS FLX, Genome Analyzer, and SOLiD platform features. Practical accuracy in 454 pyrosequencing and illumine sequencing are nearly similar (approximately 99.99%). Loaded into the GS FLX, the picotiter plate functions as a flow cell wherein iterative pyrosequencing is performed by successive flow addition of the 4 dNTPs. With the newest chemistry, termed “Titanium,” a single GS FLX run generates approximately 1 × 106 sequence reads, with read lengths of ≥400 bases yielding up to 500 million base pairs (Mb) of sequence. The d… Analysis of copy number variants and segmental duplications in the human genome: evidence for a change in the process of formation in recent evolutionary history. The main processing feature of the data pipeline is the computationally intensive conversion of image data into sequence reads, known as base calling. Indeed, oligonucleotide primers are later washed to remove unattached DNA sequences. Multiplex amplification of large sets of human exons. In this example, the 2 specific bases complementary to the template are AT. II. Cokus SJ, Feng S, Zhang X, Chen Z, Merriman B, Haudenschild CD, et al. Genome sequencing in microfabricated high-density picolitre reactors. Research with ChIP-Seq has led to analysis methods and software that exploit the advantages over ChIP-in-chip, namely a larger, more information-rich data set. On the other hand, in order to compare and match two genomes of two microbial strains or two species, researchers can use ORFs. Other bases in red may be errors. Finally, all of those complementary sequences will be made appropriate contigs (Anderson, 1981). In this approach, proteins in contact with genomic DNA are chemically cross-linked (typically with mild formaldehyde treatment) to their binding sites, and the DNA is fragmented by sonication or digestion with micrococcal nuclease. Upper pane shows percent variation from reference (vertical bars) and coverage (pale blue line). One limitation of short-read alignment and assembly is the inability to uniquely align large portions of a read set when the read length becomes too short. The SOLiD (Supported Oligonucleotide Ligation and Detection) System 2.0 platform, which is distributed by Applied Biosystems (http://www.solid.appliedbiosystems.com), is a short-read sequencing technology based on ligation. Using quality scores and longer reads improves accuracy of Solexa read mapping. Nanopores are being fabricated in inorganic membranes (solid-state nanopores), assembled from protein channels in lipid membranes, or configured in polymer-based nanofluidic channels. First cycle, free adapter is added to the flow cell surface (slide), followed by dye-labelled nucleotides are individually added one by one to adaptor. Leveraging the high-throughput capacity of NGS platforms can be facilitated by analyzing multiple samples with separate flow-cell lanes or compartments. Green and red arrows show direction of sequencing; base calls at variance with reference are indicated (red). However, the computer programs are appropriate systems to compare DNA molecules. In 2000, Jonathan Rothberg founded 454 Life Sciences, which developed the first commercially available NGS platform, the GS 20, launched in 2005. Each preparing bead (ligated its DNA fragment) loads onto the well (known as Pico TiterPlate device), Where the surface of wells permit only one specific microbead because one bead is equal to one read in the eventual cycle. NGS has had a substantial impact on basic genomics research in terms of scale and feasibility. For sequencing, polymerase and one of 4 Cy5-labeled dNTPs are added to the flow cell, which is imaged to determine incorporation into individual strands. Thirdly, extension cycle will be starting when the temperature rises again to (extention =≈72˚C). This approach was developed in the laboratory of George Church and reported in 2005 along with the resequencing of the Escherichia coli genome(17). Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. The complete genome sequence of Campylobacter jejuni strain 81116 (NCTC11828). The range of data volumes, hardware, software packages, and settings leads to processing times from a few minutes to multiple hours, emphasizing the need for sufficient computational power. To assemble, align, and analyze NGS data requires an adequate number of overlapping reads, or coverage. The single-base resolution of the data allows improved estimation of binding-site positions in the programs QuEST(79) and MACS(80). ChIP-on-chip technology is now being supplanted in a variety of experimental settings with ChIP-Seq, in which the DNA harvested from the immunoprecipitate is converted into a library for NGS. In each cycle of SOLiD sequencing, the mixture of Labelled-probes and Ligase enzyme will be added to repeat the sequencing several times. In contrast to emulsion PCR, DNA templates are amplified in the flow cell by “bridge” amplification, which relies on captured DNA strands “arching” over and hybridizing to an adjacent anchor oligonucleotide. This expectation was confirmed in trisomy 21 pregnancies, with additional supporting evidence obtained for trisomy 18 and 13 pregnancies. In order to compare and identify positions of two DNA strands that match each other, BLAST search is a very powerful tool to compare a query sequence with databases in databanks or genomic library. Such “paired-end” sequencing provides positional information that facilitates alignment and assembly, especially for short reads(12)(13). Human genome project was then sequenced by contemporary second generation sequencing (wheeler et al., 2008). The released single-nucleotide bases enter the nanopore and alter the electrical current, creating a characteristic current change for each individual base(95)(96). Investigators at the Wellcome Trust Sanger Institute, who have extensive experience with the Illumina platform, have published a series of technical improvements for library preparation, including methods for increasing the reproducibility of fragmentation by adaptive focused acoustic wave sonication, enhanced efficiency of adapter ligation by use of an alternate ligase, and reducing the G+C bias that has been observed in Illumina reads via a modified gel-extraction protocol(16). By all these measures, NGS platforms will benefit from continued process streamlining, automation, chemistry refinements, cost reductions, and improved data handling. These make the small DNA fragments to be significantly amplified. Sequence is inferred by interpreting the ligation results for the 16 possible 2 base–combination interrogation probes. In one comparative study, the 454, Illumina, and SOLiD technologies all accurately detected single-nucleotide variations when coverage depth was ≥15-fold per allele(20) (the critical issue of coverage depth is discussed further in the NGS Data Analysis section). Sanger F, Nicklen S, Coulson AR. Three columns in agreement (red) indicate presumptive SNPs. Recently, several significant developments in sequencing techniques have been made to know and better understand the population and the communities of various plants, animals and microbes. Most use the Linux operating system, and a few are available for Windows. Top: SOLiD color-space coding. After attaching of the adapters with both microheads of DNA fragments, each sequence is linked to a microbead. The 454 technology (http://www.454.com) is derived from the technological convergence of pyrosequencing and emulsion PCR. With the use of progressively offset primers, in this example (n − 1), adapter bases are sequenced, and this known sequence is used in conjunction with the color-space coding for determining the template sequence by deconvolution (see Fig. In contrast, the template ends with opposing base pairs (Adenine, Cytosine) due to the ligation. Our staff work as … New single-molecule sequencing technologies in development may decrease sequencing time, reduce costs, and streamline sample preparation. These advantages, however, must be weighed against the substantial computer resources required by the large volumes of image data. NEXT GENERATION SEQUENCING Introduction DNA sequencing is the method of determining the order of nucleotides in DNA. With 50 × 106 clusters per flow cell, the overall sequence output is >1 billion base pairs (Gb) per analytical run(11). Single-molecule DNA sequencing of a viral genome. After sequencing, sequences of individual samples are derived by data deconvolution(81)(82)(83). The sequence output is aligned to known reference sequences for microorganisms that are predicted to be present in the sample. The synthesized strand is then denatured, and a new sequencing primer offset by 1 base in the adapter sequence (n − 1) is annealed. Multiple amplification cycles convert the single-molecule DNA template to a clonally amplified arching “cluster,” with each cluster containing approximately 1000 clonal molecules. This design is a paradigm shift from that of Sanger sequencing, which is based on the electrophoretic separation of chain-termination products produced in individual sequencing reactions. Fragments several hundred base pairs in length are end-repaired and ligated to adapter oligonucleotides. Essential elements to make this transition successful will be the requirement of streamlining the processes, especially sample preparation, coupled with improvements in technology robustness and characterization of accuracy through validation studies. DNA sequencing with chain-terminating inhibitors. (A), Roche Amplicon Variant Analyzer software displaying GS FLX data from the CFTR gene [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)]. Valouev A, Johnson DS, Sundquist A, Medina C, Anton E, Batzoglou S, et al. The intensity of each integration at each well is measured by a 454 pyrosequencing program. You can chose a new technology involve genome sequencing and answering these … Meyer M, Stenzel U, Myles S, Prufer K, Hofreiter M. Targeted high-throughput sequencing of tagged nucleic acid samples. Rehears and experts are ceaseless to produce and develop modern sequencing techniques which will cause the amplification of DNA sequences. In this essay throughput, cost and other technological and biological aspects will be summarized for the next generation of sequencing … Furthermore, the level of its accuracy is 99.94% which is a leading benefit of this technique and also each cycle may receive approximately 60 Gb (Dale, 2012). Unextended strands are capped before the ligation to mitigate signal deterioration due to dephasing. Porreca GJ, Zhang K, Li JB, Xie B, Austin D, Vassallo SL, et al. Quality scores and SNP detection in sequencing-by-synthesis systems. Software for de novo assembly includes Edina(70), EULER-SR(74), SHARCGS(75), SSAKE(69), Velvet(76), and SOAPdenovo (http://soap.genomics.org.cn/). Gene discovery and annotation using LCM-454 transcriptome sequencing. Eventually the machine is available to read all those sequences are stained with Fluorescent dye. Gupta PK. Nanopore sequencing technology: research trends and applications. In this technology, template bases are interrogated twice. Next Generation DNA SEQUENCING TECHNOLOGIES Recently, there has been a major boom in commercially available software for genome sequencing. Wells and progressively digested by exonuclease iterative pyrosequencing is performed, followed by wash removal of unbound.. Generation sequencing Introduction DNA sequencing techniques reads assembled on a separate front, Tawfik Griffiths. Second generation sequencing ( NGS ) technologies are similar theoretically yield twice the luminescence of a target sequence also. The coupling of NGS data requires a resource-intensive data-pipeline system for data storage management... Region of human genomic loci by microarray hybridization suitable recombinant vectors can be inferred its associated length... Of clonally amplified template DNA is hybridized with cRNA sequences for 24 H in solution then... Be starting when the temperature rises again to ( extention =≈72˚C ) disclosure information on the are! 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