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Massive parallel sequencing
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<h2 id="history">History</h2> <p>In the 1990s, <a href="/facts/Applied_Biosystems/G4RbzyoX">Applied Biosystems</a> dominated DNA sequencing technology with their automated <a href="/facts/Capillary_electrophoresis/pMEuQD8Y">capillary electrophoresis</a> <a href="/facts/Sanger_sequencing/PzqMpaaM">Sanger sequencing</a> machines. However, the early 2000s saw many new companies entering the market, driven by the goal of reducing <a href="/facts/$1,000_genome/sKdbMDCg">genome sequencing costs below $1000</a> following the enthusiasm generated by the <a href="/facts/Human_Genome_Project/eQDHgHTP">Human Genome Project</a>.<a class="footnote-ref" id="fnref:8" href="#fn:8"><sup>8</sup></a> Many of these new methods were first developed with support from the <a href="/facts/National_Institutes_of_Health/G3Eytfcc"> National Institutes of Health (NIH)</a> funding under the 'Technology Development for the $1,000 Genome' program,<a class="footnote-ref" id="fnref:9" href="#fn:9"><sup>9</sup></a> launched during <a href="/facts/Francis_Collins/bSXu9hxM">Francis Collins</a>’ tenure as director of the <a href="/facts/National_Human_Genome_Research_Institute/t007yxfS">National Human Genome Research Institute</a>.<a class="footnote-ref" id="fnref:10" href="#fn:10"><sup>10</sup></a> </p><p>The first next-generation sequencers were based on <a href="/facts/Pyrosequencing/r7py9uUR">pyrosequencing</a>, originally developed by Pyrosequencing AB and later commercialized by <a href="/facts/454_Life_Sciences/uJckH5GR">454 Life Sciences</a>. In 2003, 454 Life Sciences launched the GS20, the first NGS DNA sequencer. This system provided reads approximately 400–500 bp long with 99% accuracy, enabling sequencing of about 25 million bases in a four-hour run at significantly lower costs compared to Sanger sequencing.<a class="footnote-ref" id="fnref:11" href="#fn:11"><sup>11</sup></a> The sequencing machines developed by 454 represented a paradigm shift by enabling the mass parallelisation of sequencing reactions, which significantly boosted the amount of DNA sequenced per run, making 454 Life Sciences the first major success in commercial NGS technology.<a class="footnote-ref" id="fnref:12" href="#fn:12"><sup>12</sup></a> </p><p>Also in 2003, Solexa began developing a competing method known as <a href="/facts/Illumina_dye_sequencing/MNPnNxAK">Sequencing by Synthesis (SBS)</a>. In 2004, Solexa acquired colony sequencing (bridge amplification) technology from <a href="/facts/Manteia_Predictive_Medicine/XjWcclev">Manteia</a>, producing densely clustered DNA fragments ("polonies") immobilized on flow cells. These dense clusters generated stronger fluorescent signals, improving accuracy and reducing optical costs. In 2005, Solexa integrated an engineered <a href="/facts/DNA_polymerase/xtNLncBu">DNA polymerase</a> and reversible terminator nucleotides, allowing repeated cycles of sequencing and imaging. The first commercial sequencer based on this technology, Genome Analyzer, was launched in 2006, providing shorter reads (about 35 bp) but higher throughput (up to 1 Gbp per run) and paired-end sequencing capability (i.e. both DNA strands were sequenced simultaneously).<a class="footnote-ref" id="fnref:13" href="#fn:13"><sup>13</sup></a> </p><p>in 2007, 454 Life Sciences was acquired by <a href="/facts/Roche/hHVcLCXb">Roche</a><a class="footnote-ref" id="fnref:14" href="#fn:14"><sup>14</sup></a> and Solexa by <a href="/facts/Illumina,_Inc./0nofTkVp">Illumina</a>,<a class="footnote-ref" id="fnref:15" href="#fn:15"><sup>15</sup></a> the same year Applied Biosystems introduced <a href="/facts/ABI_Solid_Sequencing/YU0hJq6f">SOLiD</a>, a <a href="/facts/Sequencing_by_ligation/6kBdWw2S">ligation-based sequencing</a> platform.<a class="footnote-ref" id="fnref:16" href="#fn:16"><sup>16</sup></a> However, SOLiD encountered issues sequencing palindromic regions<a class="footnote-ref" id="fnref:17" href="#fn:17"><sup>17</sup></a> and was eventually discontinued.<a class="footnote-ref" id="fnref:18" href="#fn:18"><sup>18</sup></a> In 2011, <a href="/facts/Ion_Torrent/AoCD9HKa">Ion Torrent</a> introduced another alternative, measuring <a href="/facts/Proton/clCjepXP">proton</a> (<a href="/facts/PH/RnSv8D7y">pH</a>) changes during nucleotide incorporation using <a href="/facts/Semiconductor/zFf3mGTE">semiconductor</a>-based sensors.<a class="footnote-ref" id="fnref:19" href="#fn:19"><sup>19</sup></a> Ion Torrent systems rapidly produced 100 bp reads but frequently struggled with accurately sequencing homopolymers,<a class="footnote-ref" id="fnref:20" href="#fn:20"><sup>20</sup></a> ultimately leading to their abandonment.<a class="footnote-ref" id="fnref:21" href="#fn:21"><sup>21</sup></a> </p><p>Due to limitations in competing methods, Illumina’s SBS technology eventually dominated the sequencing market.<a class="footnote-ref" id="fnref:22" href="#fn:22"><sup>22</sup></a> By 2012, expectations that 454 would gain a substantial share of the sequencing market had not been realized, and Roche’s 2007 acquisition was increasingly viewed as underperforming; that same year, Roche made an unsuccessful attempt to acquire Illumina.<a class="footnote-ref" id="fnref:23" href="#fn:23"><sup>23</sup></a> In October 2013, Roche announced that it would shut down 454, and stop supporting the platform by mid-2016.<a class="footnote-ref" id="fnref:24" href="#fn:24"><sup>24</sup></a> By 2014, Illumina controlled approximately 70% of DNA sequencer sales and generated over 90% of sequencing data.<a class="footnote-ref" id="fnref:25" href="#fn:25"><sup>25</sup></a><a class="footnote-ref" id="fnref:26" href="#fn:26"><sup>26</sup></a> That year, Illumina introduced the HiSeq X Ten platform, significantly increasing throughput and claiming the long-targeted goal of sequencing human genomes at roughly $1000 each.<a class="footnote-ref" id="fnref:27" href="#fn:27"><sup>27</sup></a> Illumina surpassed this milestone in 2017 with the release of NovaSeq, a system capable of generating over 3000 Gbp per run.<a class="footnote-ref" id="fnref:28" href="#fn:28"><sup>28</sup></a> </p> <h2 id="ngs-platforms">NGS platforms</h2> <p>DNA sequencing with commercially available NGS platforms is generally conducted with the following steps. First, DNA sequencing libraries are generated by clonal amplification by <a href="/facts/Polymerase_chain_reaction/G0jWzJy1">PCR</a> <a href="/facts/In_vitro/evp3hAX6">in vitro</a>. Second, the DNA is sequenced by <a href="/facts/DNA_replication/wrU6RuR3">synthesis</a>, such that the DNA sequence is determined by the addition of <a href="/facts/Nucleotides/pQKEwlJI">nucleotides</a> to the complementary strand rather than through chain-termination chemistry. Third, the spatially segregated, amplified DNA templates are sequenced simultaneously in a massively parallel fashion without the requirement for a physical separation step. These steps are followed in most NGS platforms, but each utilizes a different strategy.<a class="footnote-ref" id="fnref:29" href="#fn:29"><sup>29</sup></a> </p><p>NGS parallelization of the sequencing reactions generates hundreds of megabases to gigabases of nucleotide sequence reads in a single instrument run. This has enabled a drastic increase in available sequence data and fundamentally changed genome sequencing approaches in the biomedical sciences.<a class="footnote-ref" id="fnref:30" href="#fn:30"><sup>30</sup></a> Newly emerging NGS technologies and instruments have further contributed to a significant decrease in the cost of sequencing nearing the mark of $1000 per <a href="/facts/Genome_sequencing/WNuHaU2K">genome sequencing</a>.<a class="footnote-ref" id="fnref:31" href="#fn:31"><sup>31</sup></a><a class="footnote-ref" id="fnref:32" href="#fn:32"><sup>32</sup></a> </p><p>As of 2014, massively parallel sequencing platforms are commercially available and their features are summarized in the table. As the pace of NGS technologies is advancing rapidly, technical specifications and pricing are in flux. </p> NGS platforms<table><tbody><tr><th>Platform</th><th>Template preparation</th><th>Chemistry</th><th>Max read length (bases)</th><th>Run times (days)</th><th>Max Gb per Run</th></tr><tr><th>Roche 454</th><td>Clonal-emPCR</td><td>Pyrosequencing</td><td>400‡</td><td>0.42</td><td>0.40-0.60</td></tr><tr><th>GS FLX Titanium</th><td>Clonal-emPCR</td><td>Pyrosequencing</td><td>400‡</td><td>0.42</td><td>0.035</td></tr><tr><th>Illumina MiSeq</th><td>Clonal Bridge Amplification</td><td>Reversible Dye Terminator</td><td>2x300</td><td>0.17-2.7</td><td>15</td></tr><tr><th>Illumina HiSeq</th><td>Clonal Bridge Amplification</td><td>Reversible Dye Terminator</td><td>2x150</td><td>0.3-11<a class="footnote-ref" id="fnref:33" href="#fn:33"><sup>33</sup></a></td><td>1000<a class="footnote-ref" id="fnref:34" href="#fn:34"><sup>34</sup></a></td></tr><tr><th>Illumina Genome Analyzer IIX</th><td>Clonal Bridge Amplification</td><td>Reversible Dye Terminator<a class="footnote-ref" id="fnref:35" href="#fn:35"><sup>35</sup></a><a class="footnote-ref" id="fnref:36" href="#fn:36"><sup>36</sup></a></td><td>2x150</td><td>2-14</td><td>95</td></tr><tr><th>Life Technologies SOLiD4</th><td>Clonal-emPCR</td><td>Oligonucleotide 8-mer Chained Ligation<a class="footnote-ref" id="fnref:37" href="#fn:37"><sup>37</sup></a></td><td>20-45</td><td>4-7</td><td>35-50</td></tr><tr><th>Life Technologies Ion Proton<a class="footnote-ref" id="fnref:38" href="#fn:38"><sup>38</sup></a></th><td>Clonal-emPCR</td><td>Native dNTPs, proton detection</td><td>200</td><td>0.5</td><td>100</td></tr><tr><th>Complete Genomics</th><td>Gridded DNA-nanoballs</td><td>Oligonucleotide 9-mer Unchained Ligation<a class="footnote-ref" id="fnref:39" href="#fn:39"><sup>39</sup></a><a class="footnote-ref" id="fnref:40" href="#fn:40"><sup>40</sup></a><a class="footnote-ref" id="fnref:41" href="#fn:41"><sup>41</sup></a></td><td>7x10</td><td>11</td><td>3000</td></tr><tr><th>Helicos Biosciences Heliscope</th><td>Single Molecule</td><td>Reversible Dye Terminator</td><td>35‡</td><td>8</td><td>25</td></tr><tr><th><a href="/facts/Pacific_Biosciences/eTsyIunH">Pacific Biosciences</a> SMRT</th><td>Single Molecule</td><td>Phospholinked Fluorescent Nucleotides</td><td>10,000 (<a href="/facts/N50_statistic/WqNw7xCN">N50</a>); 30,000+ (max)<a class="footnote-ref" id="fnref:42" href="#fn:42"><sup>42</sup></a></td><td>0.08</td><td>0.5<a class="footnote-ref" id="fnref:43" href="#fn:43"><sup>43</sup></a></td></tr></tbody></table> <p>Run times and gigabase (Gb) output per run for single-end sequencing are noted. Run times and outputs approximately double when performing paired-end sequencing. ‡Average read lengths for the Roche 454 and Helicos Biosciences platforms.<a class="footnote-ref" id="fnref:44" href="#fn:44"><sup>44</sup></a> </p> <h2 id="template-preparation-methods-for-ngs">Template preparation methods for NGS</h2> <p>Two methods are used in preparing templates for NGS reactions: amplified templates originating from single DNA molecules, and single DNA molecule templates. For imaging systems which cannot detect single fluorescence events, amplification of DNA templates is required. The three most common amplification methods are emulsion PCR (emPCR), rolling circle and solid-phase amplification. The final distribution of templates can be spatially random or on a grid. </p> <h3>Emulsion PCR</h3> <p>In <a href="/facts/Emulsion/YMSqSCuH">emulsion</a> <a href="/facts/Polymerase_chain_reaction/G0jWzJy1">PCR</a> methods, a <a href="/facts/DNA_library/0AJpQmBT">DNA library</a> is first generated through random fragmentation of genomic DNA. Single-stranded DNA fragments (templates) are attached to the surface of beads with adaptors or linkers, and one bead is attached to a single DNA fragment from the DNA library. The surface of the beads contains <a href="/facts/Oligonucleotide/Gc99fPnC">oligonucleotide</a> probes with sequences that are complementary to the adaptors binding the DNA fragments. The beads are then compartmentalized into water-oil emulsion droplets. In the aqueous water-oil emulsion, each of the droplets capturing one bead is a PCR <a href="/facts/Microreactor/uokqUU9j">microreactor</a> that produces amplified copies of the single DNA template.<a class="footnote-ref" id="fnref:45" href="#fn:45"><sup>45</sup></a><a class="footnote-ref" id="fnref:46" href="#fn:46"><sup>46</sup></a><a class="footnote-ref" id="fnref:47" href="#fn:47"><sup>47</sup></a> </p> <h3>Gridded rolling circle nanoballs</h3> <p>Amplification of a population of single DNA molecules by <a href="/facts/Rolling_circle_amplification/czQsJ8b2">rolling circle amplification</a> in solution is followed by capture on a grid of spots sized to be smaller than the DNAs to be immobilized.<a class="footnote-ref" id="fnref:48" href="#fn:48"><sup>48</sup></a><a class="footnote-ref" id="fnref:49" href="#fn:49"><sup>49</sup></a><a class="footnote-ref" id="fnref:50" href="#fn:50"><sup>50</sup></a><a class="footnote-ref" id="fnref:51" href="#fn:51"><sup>51</sup></a> Second-generation sequencing technologies like MGI Tech's DNBSEQ or Element Biosciences' AVITI use this approach for the preparation of the sample on the flow cell that is then imaged cycle by cycle. </p> <h3>DNA colony generation (Bridge amplification)</h3> <p>Forward and reverse primers are covalently attached at high-density to the slide in a flow cell. The ratio of the primers to the template on the support defines the surface density of the amplified clusters. The flow cell is exposed to reagents for <a href="/facts/Polymerase/hN2ECr7B">polymerase</a>-based extension, and priming occurs as the free/distal end of a ligated fragment "bridges" to a complementary <a href="/facts/Oligonucleotide/Gc99fPnC">oligo</a> on the surface. Repeated <a href="/facts/Denaturation_(biochemistry)/Eh1hc3Bg">denaturation</a> and extension results in localized amplification of DNA fragments in millions of separate locations across the flow cell surface. Solid-phase amplification produces 100–200 million spatially separated template clusters, providing free ends to which a universal sequencing primer is then hybridized to initiate the sequencing reaction.<a class="footnote-ref" id="fnref:52" href="#fn:52"><sup>52</sup></a><a class="footnote-ref" id="fnref:53" href="#fn:53"><sup>53</sup></a> This technology was filed for a patent in 1997 from Glaxo-Welcome's Geneva Biomedical Research Institute (GBRI), by <a href="/facts/Pascal_Mayer/pDYY1aN3">Pascal Mayer</a>, Eric Kawashima, and Laurent Farinelli,<a class="footnote-ref" id="fnref:54" href="#fn:54"><sup>54</sup></a><a class="footnote-ref" id="fnref:55" href="#fn:55"><sup>55</sup></a> and was publicly presented for the first time in 1998.<a class="footnote-ref" id="fnref:56" href="#fn:56"><sup>56</sup></a> In 1994 <a href="/facts/Christopher_Adams_(scientist)/uGXodBQn">Chris Adams</a> and Steve Kron filed a patent on a similar, but non-clonal, surface amplification method, named “bridge amplification”<a class="footnote-ref" id="fnref:57" href="#fn:57"><sup>57</sup></a> adapted for clonal amplification in 1997 by Church and Mitra.<a class="footnote-ref" id="fnref:58" href="#fn:58"><sup>58</sup></a><a class="footnote-ref" id="fnref:59" href="#fn:59"><sup>59</sup></a> </p> <h3>Single-molecule templates</h3> <p>Protocols requiring DNA amplification are often cumbersome to implement and may introduce sequencing errors. The preparation of single-molecule templates is more straightforward and does not require PCR, which can introduce errors in the amplified templates. AT-rich and GC-rich target sequences often show amplification bias, which results in their underrepresentation in genome alignments and assemblies. Single molecule templates are usually immobilized on solid supports using one of at least three different approaches. In the first approach, spatially distributed individual primer molecules are covalently attached to the solid support. The template, which is prepared by randomly fragmenting the starting material into small sizes (for example,~200–250 bp) and adding common adapters to the fragment ends, is then hybridized to the immobilized primer. In the second approach, spatially distributed single-molecule templates are covalently attached to the solid support by priming and extending single-stranded, single-molecule templates from immobilized primers. A common primer is then hybridized to the template. In either approach, DNA polymerase can bind to the immobilized primed template configuration to initiate the NGS reaction. Both of the above approaches are used by Helicos BioSciences. In a third approach, spatially distributed single polymerase molecules are attached to the solid support, to which a primed template molecule is bound. This approach is used by Pacific Biosciences. Larger DNA molecules (up to tens of thousands of base pairs) can be used with this technique and, unlike the first two approaches, the third approach can be used with real-time methods, resulting in potentially longer read lengths. </p> <h2 id="sequencing-approaches">Sequencing approaches</h2> <h3>Sequencing by synthesis</h3> <p>The objective for sequential sequencing by synthesis (SBS) is to determine the sequencing of a <a href="/facts/DNA/nB89Iauz">DNA</a> sample by detecting the incorporation of a <a href="/facts/Nucleotide/pQKEwlJI">nucleotide</a> by a <a href="/facts/DNA_polymerase/xtNLncBu">DNA polymerase</a>. An engineered polymerase is used to synthesize a copy of a single strand of DNA and the incorporation of each nucleotide is monitored. The principle of sequencing by synthesis was first described in 1993 <a class="footnote-ref" id="fnref:60" href="#fn:60"><sup>60</sup></a> with improvements published some years later.<a class="footnote-ref" id="fnref:61" href="#fn:61"><sup>61</sup></a> The key parts are highly similar for all embodiments of SBS and include (1) <a href="/facts/Solid_phase_sequencing/o9qzHvTX">amplification of DNA</a> to enhance the subsequent signal and to attach the DNA to be sequenced to a solid support, (2) generation of single stranded DNA on the solid support, (3) incorporation of nucleotides using an engineered polymerase and (4) detection of the incorporation of nucleotide. Then steps 3-4 are repeated and the sequence is assembled from the signals obtained in step 4. This principle of sequencing-by-synthesis has been used for almost all massive parallel sequencing instruments, including <a href="/facts/454_Life_Sciences/uJckH5GR">454</a>, <a href="/facts/Pacific_Biosciences/eTsyIunH">PacBio</a>, <a href="/facts/Ion_Torrent/AoCD9HKa">IonTorrent</a>, <a href="/facts/Illumina,_Inc./0nofTkVp">Illumina</a> and <a href="/facts/MGI_(company)/LHLtYhTz">MGI</a>. </p> <h3>Pyrosequencing</h3> <p>The principle of <a href="/facts/Pyrosequencing/r7py9uUR">Pyrosequencing</a> was first described in 1993 <a class="footnote-ref" id="fnref:62" href="#fn:62"><sup>62</sup></a> by combining a solid support with an engineered DNA polymerase lacking <a href="/facts/Directionality_(molecular_biology)/TIAXEQmY">3´to 5´exonuclease</a> activity (proof-reading) and <a href="/facts/Luminescence/1QkIWyUj">luminescence</a> real-time detection using the <a href="/facts/Firefly/wyRUbc1w">firefly</a> <a href="/facts/Luciferase/7yl8dytU">luciferase</a>. All the key concepts of sequencing by synthesis were introduced, including (1) amplification of DNA to enhance the subsequent signal and attach the DNA to be sequenced (template) to a solid support, (2) generation of single stranded DNA on the solid support (3) incorporation of nucleotides using an engineered polymerase and (4) detection of the incorporated nucleotide by light detection in real-time. In a follow-up article,<a class="footnote-ref" id="fnref:63" href="#fn:63"><sup>63</sup></a> the concept was further developed and in 1998, an article <a class="footnote-ref" id="fnref:64" href="#fn:64"><sup>64</sup></a> was published in which the authors showed that non-incorporated nucleotides could be removed with a fourth enzyme (<a href="/facts/Apyrase/cMh8Ua7M">apyrase</a>) allowing sequencing by synthesis to be performed without the need for washing away non-incorporated nucleotides. </p> <h3>Sequencing by reversible terminator chemistry</h3> <p>This approach uses reversible terminator-bound dNTPs in a cyclic method that comprises nucleotide incorporation, fluorescence imaging and cleavage. A fluorescently-labeled terminator is imaged as each dNTP is added and then cleaved to allow incorporation of the next base. These nucleotides are chemically blocked such that each incorporation is a unique event. An imaging step follows each base incorporation step, then the blocked group is chemically removed to prepare each strand for the next incorporation by DNA polymerase. This series of steps continues for a specific number of cycles, as determined by user-defined instrument settings. The 3' blocking groups were originally conceived as either enzymatic<a class="footnote-ref" id="fnref:65" href="#fn:65"><sup>65</sup></a> or chemical reversal<a class="footnote-ref" id="fnref:66" href="#fn:66"><sup>66</sup></a><a class="footnote-ref" id="fnref:67" href="#fn:67"><sup>67</sup></a> The chemical method has been the basis for the Solexa and Illumina machines. Sequencing by reversible terminator chemistry can be a four-colour cycle such as used by Illumina/Solexa, or a one-colour cycle such as used by Helicos BioSciences. Helicos BioSciences used “virtual Terminators”, which are unblocked terminators with a second nucleoside analogue that acts as an inhibitor. These terminators have the appropriate modifications for terminating or inhibiting groups so that DNA synthesis is terminated after a single base addition.<a class="footnote-ref" id="fnref:68" href="#fn:68"><sup>68</sup></a><a class="footnote-ref" id="fnref:69" href="#fn:69"><sup>69</sup></a><a class="footnote-ref" id="fnref:70" href="#fn:70"><sup>70</sup></a> </p> <h3>Sequencing-by-ligation mediated by ligase enzymes</h3> <p>In this approach, the sequence extension reaction is not carried out by polymerases but rather by DNA <a href="/facts/Ligase/cnwMmUln">ligase</a> and either one-base-encoded probes or two-base-encoded probes. In its simplest form, a fluorescently labelled probe hybridizes to its complementary sequence adjacent to the primed template. DNA ligase is then added to join the dye-labelled probe to the primer. Non-ligated probes are washed away, followed by <a href="/facts/Fluorescence_imaging/lAaeQEdg">fluorescence imaging</a> to determine the identity of the ligated probe. The cycle can be repeated either by using cleavable probes to remove the fluorescent dye and regenerate a 5′-PO4 group for subsequent ligation cycles (chained ligation<a class="footnote-ref" id="fnref:71" href="#fn:71"><sup>71</sup></a><a class="footnote-ref" id="fnref:72" href="#fn:72"><sup>72</sup></a>) or by removing and hybridizing a new primer to the template (unchained ligation<a class="footnote-ref" id="fnref:73" href="#fn:73"><sup>73</sup></a><a class="footnote-ref" id="fnref:74" href="#fn:74"><sup>74</sup></a>). </p> <h3>Phospholinked Fluorescent Nucleotides or Real-time sequencing</h3> <p><a href="/facts/Pacific_Biosciences/eTsyIunH">Pacific Biosciences</a> is currently leading this method. The method of real-time sequencing involves imaging the continuous incorporation of dye-labelled nucleotides during DNA synthesis: single DNA polymerase molecules are attached to the bottom surface of individual zero-mode waveguide detectors (Zmw detectors) that can obtain sequence information while phospholinked nucleotides are being incorporated into the growing primer strand. Pacific Biosciences uses a unique DNA polymerase which better incorporates phospholinked nucleotides and enables the resequencing of closed circular templates. While single-read accuracy is 87%, consensus accuracy has been demonstrated at 99.999% with multi-kilobase read lengths.<a class="footnote-ref" id="fnref:75" href="#fn:75"><sup>75</sup></a><a class="footnote-ref" id="fnref:76" href="#fn:76"><sup>76</sup></a> In 2015, Pacific Biosciences released a new sequencing instrument called the Sequel System, which increases capacity approximately 6.5-fold.<a class="footnote-ref" id="fnref:77" href="#fn:77"><sup>77</sup></a><a class="footnote-ref" id="fnref:78" href="#fn:78"><sup>78</sup></a> </p> <h2 id="see-also">See also</h2> <ul><li><a href="/facts/Clinical_metagenomic_sequencing/XRwUMDng">Clinical metagenomic sequencing</a></li> <li><a href="/facts/First-generation_sequencing/PzqMpaaM">First-generation sequencing</a></li> <li><a href="/facts/Third-generation_sequencing/WySXm5Bx">Third-generation sequencing</a></li> <li>RNA Velocity</li></ul> <h2 id="references">References</h2> <ol> <li id="fn:1"><p>Nyren, P.; Pettersson, B.; Uhlen, M. (January 1993). "Solid Phase DNA Minisequencing by an Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay". Analytical Biochemistry. 208 (1): 171–175. doi:10.1006/abio.1993.1024. PMID 8382019. <a href="https://linkinghub.elsevier.com/retrieve/pii/S0003269783710249" target="_blank">https://linkinghub.elsevier.com/retrieve/pii/S0003269783710249</a> <a href="#fnref:1" class="footnote-back-ref">↩</a></p></li> <li id="fn:2"><p>Ronaghi M, Karamohamed S, Pettersson B, Uhlén M, Nyrén P (November 1996). "Real-time DNA sequencing using detection of pyrophosphate release". Analytical Biochemistry. 242 (1): 84–89. doi:10.1006/abio.1996.0432. PMID 8923969. <a href="/wiki/Doi_(identifier)" target="_blank">/wiki/Doi_(identifier)</a> <a href="#fnref:2" class="footnote-back-ref">↩</a></p></li> <li id="fn:3"><p>US 5641658, Adams CP, Kron SJ, "Method for performing amplification of nucleic acid with two primers bound to a single solid support", published 1997-06-24, assigned to Mosaic Technologies Inc. and Whitehead Institute for Biomedical Research <a href="https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=US5641658" target="_blank">https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=US5641658</a> <a href="#fnref:3" class="footnote-back-ref">↩</a></p></li> <li id="fn:4"><p>EP 0972081, Farinelli L, Kawashima E, Mayer P ), "Method of nucleic acid amplification", published 2007-06-13, assigned to Solexa Ltd. <a href="https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=EP0972081" target="_blank">https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=EP0972081</a> <a href="#fnref:4" class="footnote-back-ref">↩</a></p></li> <li id="fn:5"><p>EP 0975802, Kawashima E, Farinellit L, Mayer P, "Method of nucleic acid sequencing", published 2004-06-23 <a href="https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=EP0975802" target="_blank">https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=EP0975802</a> <a href="#fnref:5" class="footnote-back-ref">↩</a></p></li> <li id="fn:6"><p>Voelkerding KV, Dames SA, Durtschi JD (April 2009). "Next-generation sequencing: from basic research to diagnostics". Clinical Chemistry. 55 (4): 641–658. doi:10.1373/clinchem.2008.112789. PMID 19246620. <a href="https://doi.org/10.1373%2Fclinchem.2008.112789" target="_blank">https://doi.org/10.1373%2Fclinchem.2008.112789</a> <a href="#fnref:6" class="footnote-back-ref">↩</a></p></li> <li id="fn:7"><p>Ballard D, Winkler-Galicki J, Wesoły J (July 2020). "Massive parallel sequencing in forensics: advantages, issues, technicalities, and prospects". International Journal of Legal Medicine. 134 (4): 1291–1303. doi:10.1007/s00414-020-02294-0. PMC 7295846. PMID 32451905. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295846" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295846</a> <a href="#fnref:7" class="footnote-back-ref">↩</a></p></li> <li id="fn:8"><p>Giani AM, Gallo GR, Gianfranceschi L, Formenti G (2020). "Long walk to genomics: History and current approaches to genome sequencing and assembly". Computational and Structural Biotechnology Journal. 18: 9–19. doi:10.1016/j.csbj.2019.11.002. PMC 6926122. PMID 31890139. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926122" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926122</a> <a href="#fnref:8" class="footnote-back-ref">↩</a></p></li> <li id="fn:9"><p>"Program Announcement Concept: Technology Development for the $1000 Genome". Archived from the original on March 1, 2025. Retrieved April 1, 2025. <a href="https://web.archive.org/web/20250301060733/https://www.genome.gov/11008124/concept-papers-for-two-DNA-sequencing-technology-development-programs-2003#al-4" target="_blank">https://web.archive.org/web/20250301060733/https://www.genome.gov/11008124/concept-papers-for-two-DNA-sequencing-technology-development-programs-2003#al-4</a> <a href="#fnref:9" class="footnote-back-ref">↩</a></p></li> <li id="fn:10"><p>Mardis, E. (2011). "A decade's perspective on DNA sequencing technology". Nature. 470: 198–203. doi:10.1038/nature09796. <a href="/wiki/Doi_(identifier)" target="_blank">/wiki/Doi_(identifier)</a> <a href="#fnref:10" class="footnote-back-ref">↩</a></p></li> <li id="fn:11"><p>Margulies M, Egholm M, Altman W, et al. (2005). "Genome sequencing in microfabricated high-density picolitre reactors". Nature. 437 (7057): 376–380. Bibcode:2005Natur.437..376M. doi:10.1038/nature03959. PMC 1464427. PMID 16056220. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1464427" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1464427</a> <a href="#fnref:11" class="footnote-back-ref">↩</a></p></li> <li id="fn:12"><p>Heather, James M.; Chain, Benjamin (2016). "The sequence of sequencers: The history of sequencing DNA". Genomics. 107 (1): 1–8. doi:10.1016/j.ygeno.2015.11.003. PMC 4727787. PMID 26554401. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4727787" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4727787</a> <a href="#fnref:12" class="footnote-back-ref">↩</a></p></li> <li id="fn:13"><p>Bentley D, Balasubramanian S, Swerdlow H, et al. (2008). "Accurate whole human genome sequencing using reversible terminator chemistry". Nature. 456 (7218): 53–59. Bibcode:2008Natur.456...53B. doi:10.1038/nature07517. PMC 2581791. PMID 18987734. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2581791" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2581791</a> <a href="#fnref:13" class="footnote-back-ref">↩</a></p></li> <li id="fn:14"><p>Check Hayden, E. (2012). "Roche chases stake in medical sequencing". Nature. 484: 152. doi:10.1038/484152a. <a href="/wiki/Doi_(identifier)" target="_blank">/wiki/Doi_(identifier)</a> <a href="#fnref:14" class="footnote-back-ref">↩</a></p></li> <li id="fn:15"><p>Balasubramanian, Shankar (2015). "Solexa Sequencing: Decoding Genomes on a Population Scale". Clinical Chemistry. 61 (1): 21–24. doi:10.1373/clinchem.2014.221747. <a href="/wiki/Doi_(identifier)" target="_blank">/wiki/Doi_(identifier)</a> <a href="#fnref:15" class="footnote-back-ref">↩</a></p></li> <li id="fn:16"><p>McKernan KJ, Peckham HE, Costa GL, et al. (2009). "Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding". Genome Res. 19 (9): 1527–1541. doi:10.1101/gr.091868.109. PMC 2752135. PMID 19546169. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752135" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752135</a> <a href="#fnref:16" class="footnote-back-ref">↩</a></p></li> <li id="fn:17"><p>Huang YF, Chen SC, Chiang YS, et al. (2012). "Palindromic sequence impedes sequencing-by-ligation mechanism". BMC Syst Biol. 6 (Suppl 2): S10. doi:10.1186/1752-0509-6-S2-S10. PMC 3521181. PMID 23281822. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3521181" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3521181</a> <a href="#fnref:17" class="footnote-back-ref">↩</a></p></li> <li id="fn:18"><p>Giani AM, Gallo GR, Gianfranceschi L, Formenti G (2020). "Long walk to genomics: History and current approaches to genome sequencing and assembly". Computational and Structural Biotechnology Journal. 18: 9–19. doi:10.1016/j.csbj.2019.11.002. PMC 6926122. PMID 31890139. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926122" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926122</a> <a href="#fnref:18" class="footnote-back-ref">↩</a></p></li> <li id="fn:19"><p>Rothberg JM, Hinz H, Rearick TM, et al. (2011). "An integrated semiconductor device enabling non-optical genome sequencing". Nature. 475 (7356): 348–352. doi:10.1038/nature10242. PMID 21776081. <a href="https://doi.org/10.1038%2Fnature10242" target="_blank">https://doi.org/10.1038%2Fnature10242</a> <a href="#fnref:19" class="footnote-back-ref">↩</a></p></li> <li id="fn:20"><p>Loman NJ, Misra RV, Dallman TJ, et al. (2012). "Performance comparison of benchtop high-throughput sequencing platforms". Nat Biotechnol. 30 (5): 434–439. doi:10.1038/nbt.2198. PMID 22522955. <a href="/wiki/Doi_(identifier)" target="_blank">/wiki/Doi_(identifier)</a> <a href="#fnref:20" class="footnote-back-ref">↩</a></p></li> <li id="fn:21"><p>Giani AM, Gallo GR, Gianfranceschi L, Formenti G (2020). "Long walk to genomics: History and current approaches to genome sequencing and assembly". Computational and Structural Biotechnology Journal. 18: 9–19. doi:10.1016/j.csbj.2019.11.002. PMC 6926122. PMID 31890139. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926122" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926122</a> <a href="#fnref:21" class="footnote-back-ref">↩</a></p></li> <li id="fn:22"><p>Giani AM, Gallo GR, Gianfranceschi L, Formenti G (2020). "Long walk to genomics: History and current approaches to genome sequencing and assembly". Computational and Structural Biotechnology Journal. 18: 9–19. doi:10.1016/j.csbj.2019.11.002. PMC 6926122. PMID 31890139. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926122" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926122</a> <a href="#fnref:22" class="footnote-back-ref">↩</a></p></li> <li id="fn:23"><p>Check Hayden, E. (2012). "Roche chases stake in medical sequencing". Nature. 484: 152. doi:10.1038/484152a. <a href="/wiki/Doi_(identifier)" target="_blank">/wiki/Doi_(identifier)</a> <a href="#fnref:23" class="footnote-back-ref">↩</a></p></li> <li id="fn:24"><p>"Following Roche's Decision to Shut Down 454, Customers Make Plans to Move to Other Platforms". October 22, 2013. <a href="http://www.genomeweb.com/sequencing/following-roches-decision-shut-down-454-customers-make-plans-move-other-platform" target="_blank">http://www.genomeweb.com/sequencing/following-roches-decision-shut-down-454-customers-make-plans-move-other-platform</a> <a href="#fnref:24" class="footnote-back-ref">↩</a></p></li> <li id="fn:25"><p>Zimmerman, Eilene (18 February 2014). "50 Smartest Companies: Illumina". MIT Technology Review. Massachusetts Institute of Technology. Archived from the original on 11 December 2015. Retrieved 25 August 2014. <a href="https://web.archive.org/web/20151211020908/http://www.technologyreview.com/featuredstory/524531/why-illumina-is-no-1/" target="_blank">https://web.archive.org/web/20151211020908/http://www.technologyreview.com/featuredstory/524531/why-illumina-is-no-1/</a> <a href="#fnref:25" class="footnote-back-ref">↩</a></p></li> <li id="fn:26"><p>Regalado, Antonio. "EmTech: Illumina Says 228,000 Human Genomes Will Be Sequenced This Year". MIT Technology Review. Massachusetts Institute of Technology. Archived from the original on 26 December 2015. Retrieved 26 September 2014. <a href="https://web.archive.org/web/20151226182622/http://www.technologyreview.com/news/531091/emtech-illumina-says-228000-human-genomes-will-be-sequenced-this-year/" target="_blank">https://web.archive.org/web/20151226182622/http://www.technologyreview.com/news/531091/emtech-illumina-says-228000-human-genomes-will-be-sequenced-this-year/</a> <a href="#fnref:26" class="footnote-back-ref">↩</a></p></li> <li id="fn:27"><p>Clark, Liat (15 Jan 2014). "Illumina announces landmark $1,000 human genome sequencing". Wired. Archived from the original on 4 November 2019. Retrieved 4 Nov 2019. <a href="https://www.wired.co.uk/article/1000-dollar-genome" target="_blank">https://www.wired.co.uk/article/1000-dollar-genome</a> <a href="#fnref:27" class="footnote-back-ref">↩</a></p></li> <li id="fn:28"><p>Giani AM, Gallo GR, Gianfranceschi L, Formenti G (2020). "Long walk to genomics: History and current approaches to genome sequencing and assembly". Computational and Structural Biotechnology Journal. 18: 9–19. doi:10.1016/j.csbj.2019.11.002. PMC 6926122. PMID 31890139. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926122" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926122</a> <a href="#fnref:28" class="footnote-back-ref">↩</a></p></li> <li id="fn:29"><p>Anderson MW, Schrijver I (May 2010). "Next generation DNA sequencing and the future of genomic medicine". Genes. 1 (1): 38–69. doi:10.3390/genes1010038. PMC 3960862. PMID 24710010. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3960862" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3960862</a> <a href="#fnref:29" class="footnote-back-ref">↩</a></p></li> <li id="fn:30"><p>Tucker T, Marra M, Friedman JM (August 2009). "Massively parallel sequencing: the next big thing in genetic medicine". American Journal of Human Genetics. 85 (2): 142–154. doi:10.1016/j.ajhg.2009.06.022. PMC 2725244. PMID 19679224. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2725244" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2725244</a> <a href="#fnref:30" class="footnote-back-ref">↩</a></p></li> <li id="fn:31"><p>von Bubnoff A (March 2008). "Next-generation sequencing: the race is on". Cell. 132 (5): 721–723. doi:10.1016/j.cell.2008.02.028. PMID 18329356. S2CID 8413828. <a href="https://doi.org/10.1016%2Fj.cell.2008.02.028" target="_blank">https://doi.org/10.1016%2Fj.cell.2008.02.028</a> <a href="#fnref:31" class="footnote-back-ref">↩</a></p></li> <li id="fn:32"><p>"2008 Release: NHGRI Seeks DNA Sequencing Technologies Fit for Routine Laboratory and Medical Use". Genome.gov. Retrieved 2012-08-05. <a href="http://www.genome.gov/27527585" target="_blank">http://www.genome.gov/27527585</a> <a href="#fnref:32" class="footnote-back-ref">↩</a></p></li> <li id="fn:33"><p>"Specifications for HiSeq 2500". Archived from the original on 2014-12-06. Retrieved 2014-11-06. <a href="https://web.archive.org/web/20141206044410/http://systems.illumina.com/systems/hiseq_2500_1500/performance_specifications.html" target="_blank">https://web.archive.org/web/20141206044410/http://systems.illumina.com/systems/hiseq_2500_1500/performance_specifications.html</a> <a href="#fnref:33" class="footnote-back-ref">↩</a></p></li> <li id="fn:34"><p>"HiSeq v4 is here… and it delivers | Edinburgh Genomics". Archived from the original on 2014-11-06. Retrieved 2014-11-06. <a href="https://web.archive.org/web/20141106114253/http://genomics.ed.ac.uk/blog/hiseq-v4-here-and-it-delivers" target="_blank">https://web.archive.org/web/20141106114253/http://genomics.ed.ac.uk/blog/hiseq-v4-here-and-it-delivers</a> <a href="#fnref:34" class="footnote-back-ref">↩</a></p></li> <li id="fn:35"><p>US patent 7790869, Ju J, Li Z, Edwards JR, Itagaki Y, "Massive parallel method for decoding DNA and RNA", published 2010-09-07, assigned to The Trustees of Columbia University in the City of New York <a href="https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=US7790869" target="_blank">https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=US7790869</a> <a href="#fnref:35" class="footnote-back-ref">↩</a></p></li> <li id="fn:36"><p>Bentley DR, Balasubramanian S, Swerdlow HP, Smith GP, Milton J, Brown CG, et al. (November 2008). "Accurate whole human genome sequencing using reversible terminator chemistry". Nature. 456 (7218): 53–59. Bibcode:2008Natur.456...53B. doi:10.1038/nature07517. PMC 2581791. PMID 18987734. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2581791" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2581791</a> <a href="#fnref:36" class="footnote-back-ref">↩</a></p></li> <li id="fn:37"><p>McKernan KJ, Peckham HE, Costa GL, McLaughlin SF, Fu Y, Tsung EF, et al. (September 2009). "Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding". Genome Research. 19 (9): 1527–1541. doi:10.1101/gr.091868.109. PMC 2752135. PMID 19546169. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752135" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752135</a> <a href="#fnref:37" class="footnote-back-ref">↩</a></p></li> <li id="fn:38"><p>"Ion Torrent". Archived from the original on 30 December 2013. Retrieved 1 Jan 2014. <a href="https://web.archive.org/web/20131230044834/http://www.allseq.com/knowledgebank/sequencing-platforms/life-technologies-ion-torrent" target="_blank">https://web.archive.org/web/20131230044834/http://www.allseq.com/knowledgebank/sequencing-platforms/life-technologies-ion-torrent</a> <a href="#fnref:38" class="footnote-back-ref">↩</a></p></li> <li id="fn:39"><p>Drmanac R, Sparks AB, Callow MJ, Halpern AL, Burns NL, Kermani BG, et al. (January 2010). "Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays". Science. 327 (5961): 78–81. Bibcode:2010Sci...327...78D. doi:10.1126/science.1181498. PMID 19892942. S2CID 17309571. <a href="https://doi.org/10.1126%2Fscience.1181498" target="_blank">https://doi.org/10.1126%2Fscience.1181498</a> <a href="#fnref:39" class="footnote-back-ref">↩</a></p></li> <li id="fn:40"><p>Shendure J, Porreca GJ, Reppas NB, Lin X, McCutcheon JP, Rosenbaum AM, et al. (September 2005). "Accurate multiplex polony sequencing of an evolved bacterial genome". Science. 309 (5741): 1728–1732. Bibcode:2005Sci...309.1728S. doi:10.1126/science.1117389. PMID 16081699. S2CID 11405973. <a href="https://doi.org/10.1126%2Fscience.1117389" target="_blank">https://doi.org/10.1126%2Fscience.1117389</a> <a href="#fnref:40" class="footnote-back-ref">↩</a></p></li> <li id="fn:41"><p>Peters BA, Kermani BG, Sparks AB, Alferov O, Hong P, Alexeev A, et al. (July 2012). "Accurate whole-genome sequencing and haplotyping from 10 to 20 human cells". Nature. 487 (7406): 190–195. Bibcode:2012Natur.487..190P. doi:10.1038/nature11236. PMC 3397394. PMID 22785314. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3397394" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3397394</a> <a href="#fnref:41" class="footnote-back-ref">↩</a></p></li> <li id="fn:42"><p>Inc, Pacific Biosciences of California (October 3, 2013). "Pacific Biosciences Introduces New Chemistry With Longer Read Lengths to Detect Novel Features in DNA Sequence and Advance Genome Studies of Large Organisms". GlobeNewswire News Room (Press release). {{cite press release}}: |last= has generic name (help) <a href="https://www.globenewswire.com/news-release/2013/10/03/577891/16261/en/Pacific-Biosciences-Introduces-New-Chemistry-With-Longer-Read-Lengths-to-Detect-Novel-Features-in-DNA-Sequence-and-Advance-Genome-Studies-of-Large-Organisms.html" target="_blank">https://www.globenewswire.com/news-release/2013/10/03/577891/16261/en/Pacific-Biosciences-Introduces-New-Chemistry-With-Longer-Read-Lengths-to-Detect-Novel-Features-in-DNA-Sequence-and-Advance-Genome-Studies-of-Large-Organisms.html</a> <a href="#fnref:42" class="footnote-back-ref">↩</a></p></li> <li id="fn:43"><p>Nederbragt L (2013-07-05). "De novo bacterial genome assembly: a solved problem?". <a href="http://flxlexblog.wordpress.com/2013/07/05/de-novo-bacterial-genome-assembly-a-solved-problem/" target="_blank">http://flxlexblog.wordpress.com/2013/07/05/de-novo-bacterial-genome-assembly-a-solved-problem/</a> <a href="#fnref:43" class="footnote-back-ref">↩</a></p></li> <li id="fn:44"><p>Voelkerding KV, Dames S, Durtschi JD (September 2010). "Next generation sequencing for clinical diagnostics-principles and application to targeted resequencing for hypertrophic cardiomyopathy: a paper from the 2009 William Beaumont Hospital Symposium on Molecular Pathology". The Journal of Molecular Diagnostics. 12 (5): 539–551. doi:10.2353/jmoldx.2010.100043. PMC 2928417. PMID 20805560. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2928417" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2928417</a> <a href="#fnref:44" class="footnote-back-ref">↩</a></p></li> <li id="fn:45"><p>Chee-Seng K, Yun LE, Yudi P, Kee-Seng C (April 2010). "Next Generation Sequencing Technologies and Their Applications.". Encyclopedia of Life Sciences (ELS). Chichester: John Wiley & Sons, Ltd. <a href="#fnref:45" class="footnote-back-ref">↩</a></p></li> <li id="fn:46"><p>Metzker ML (January 2010). "Sequencing technologies - the next generation". Nature Reviews. Genetics. 11 (1): 31–46. doi:10.1038/nrg2626. PMID 19997069. S2CID 205484500. <a href="/wiki/Doi_(identifier)" target="_blank">/wiki/Doi_(identifier)</a> <a href="#fnref:46" class="footnote-back-ref">↩</a></p></li> <li id="fn:47"><p>Dressman D, Yan H, Traverso G, Kinzler KW, Vogelstein B (July 2003). "Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations". Proceedings of the National Academy of Sciences of the United States of America. 100 (15): 8817–8822. Bibcode:2003PNAS..100.8817D. doi:10.1073/pnas.1133470100. PMC 166396. PMID 12857956. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC166396" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC166396</a> <a href="#fnref:47" class="footnote-back-ref">↩</a></p></li> <li id="fn:48"><p>US patent 6485944, Church GM, Mitra R, "Replica amplification of nucleic acid arrays", published 2002-11-26, assigned to President and Fellows of Harvard College <a href="https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=US6485944" target="_blank">https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=US6485944</a> <a href="#fnref:48" class="footnote-back-ref">↩</a></p></li> <li id="fn:49"><p>Mitra RD, Church GM (December 1999). "In situ localized amplification and contact replication of many individual DNA molecules". Nucleic Acids Research. 27 (24): 34e–34. doi:10.1093/nar/27.24.e34. PMC 148757. PMID 10572186. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC148757" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC148757</a> <a href="#fnref:49" class="footnote-back-ref">↩</a></p></li> <li id="fn:50"><p>US 9624538, Church GM, Porreca GJ, Shendure J, Rosenbaum AM, "Nanogrid rolling circle DNA sequencing", published 2017-04-18, assigned to President and Fellows of Harvard College <a href="https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=US9624538" target="_blank">https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=US9624538</a> <a href="#fnref:50" class="footnote-back-ref">↩</a></p></li> <li id="fn:51"><p>US patent 8445194, Drmanac R, Callow MJ, Drmanac S, Hauser BK, Yeung G, "Single molecule arrays for genetic and chemical analysis", published 2013-05-21, assigned to Callida Genomics Inc. <a href="https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=US8445194" target="_blank">https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=US8445194</a> <a href="#fnref:51" class="footnote-back-ref">↩</a></p></li> <li id="fn:52"><p>Chee-Seng K, Yun LE, Yudi P, Kee-Seng C (April 2010). "Next Generation Sequencing Technologies and Their Applications.". Encyclopedia of Life Sciences (ELS). Chichester: John Wiley & Sons, Ltd. <a href="#fnref:52" class="footnote-back-ref">↩</a></p></li> <li id="fn:53"><p>Metzker ML (January 2010). "Sequencing technologies - the next generation". Nature Reviews. Genetics. 11 (1): 31–46. doi:10.1038/nrg2626. PMID 19997069. S2CID 205484500. <a href="/wiki/Doi_(identifier)" target="_blank">/wiki/Doi_(identifier)</a> <a href="#fnref:53" class="footnote-back-ref">↩</a></p></li> <li id="fn:54"><p>EP 0972081, Farinelli L, Kawashima E, Mayer P ), "Method of nucleic acid amplification", published 2007-06-13, assigned to Solexa Ltd. <a href="https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=EP0972081" target="_blank">https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=EP0972081</a> <a href="#fnref:54" class="footnote-back-ref">↩</a></p></li> <li id="fn:55"><p>EP 0975802, Kawashima E, Farinellit L, Mayer P, "Method of nucleic acid sequencing", published 2004-06-23 <a href="https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=EP0975802" target="_blank">https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=EP0975802</a> <a href="#fnref:55" class="footnote-back-ref">↩</a></p></li> <li id="fn:56"><p>Mayer P, Matton G, Adessi C, Turcatti G, Mermod JJ, Kawashima E (October 7–10, 1998). A very large scale, high throughput and low cost DNA sequencing method based on a new 2-dimensional DNA auto-patterning process. Fifth International Automation in Mapping and DNA Sequencing Conference. St. Louis, MO, USA. DNA colony massively parallel sequencing ams98 presentation <a href="https://www.slideshare.net/pascalmayer/dna-colony-massively-parrallel-sequencing-ams98-presentation" target="_blank">https://www.slideshare.net/pascalmayer/dna-colony-massively-parrallel-sequencing-ams98-presentation</a> <a href="#fnref:56" class="footnote-back-ref">↩</a></p></li> <li id="fn:57"><p>US 5641658, Adams CP, Kron SJ, "Method for performing amplification of nucleic acid with two primers bound to a single solid support", published 1997-06-24, assigned to Mosaic Technologies Inc. and Whitehead Institute for Biomedical Research <a href="https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=US5641658" target="_blank">https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=US5641658</a> <a href="#fnref:57" class="footnote-back-ref">↩</a></p></li> <li id="fn:58"><p>US patent 6485944, Church GM, Mitra R, "Replica amplification of nucleic acid arrays", published 2002-11-26, assigned to President and Fellows of Harvard College <a href="https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=US6485944" target="_blank">https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=US6485944</a> <a href="#fnref:58" class="footnote-back-ref">↩</a></p></li> <li id="fn:59"><p>Mitra RD, Church GM (December 1999). "In situ localized amplification and contact replication of many individual DNA molecules". Nucleic Acids Research. 27 (24): 34e–34. doi:10.1093/nar/27.24.e34. PMC 148757. PMID 10572186. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC148757" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC148757</a> <a href="#fnref:59" class="footnote-back-ref">↩</a></p></li> <li id="fn:60"><p>Nyren, P.; Pettersson, B.; Uhlen, M. (January 1993). "Solid Phase DNA Minisequencing by an Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay". Analytical Biochemistry. 208 (1): 171–175. doi:10.1006/abio.1993.1024. PMID 8382019. <a href="https://linkinghub.elsevier.com/retrieve/pii/S0003269783710249" target="_blank">https://linkinghub.elsevier.com/retrieve/pii/S0003269783710249</a> <a href="#fnref:60" class="footnote-back-ref">↩</a></p></li> <li id="fn:61"><p>Ronaghi, Mostafa; Uhlén, Mathias; Nyrén, Pål (1998-07-17). "A Sequencing Method Based on Real-Time Pyrophosphate". Science. 281 (5375): 363–365. doi:10.1126/science.281.5375.363. ISSN 0036-8075. PMID 9705713. S2CID 26331871. <a href="https://www.science.org/doi/10.1126/science.281.5375.363" target="_blank">https://www.science.org/doi/10.1126/science.281.5375.363</a> <a href="#fnref:61" class="footnote-back-ref">↩</a></p></li> <li id="fn:62"><p>Nyren, P.; Pettersson, B.; Uhlen, M. (January 1993). "Solid Phase DNA Minisequencing by an Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay". Analytical Biochemistry. 208 (1): 171–175. doi:10.1006/abio.1993.1024. PMID 8382019. <a href="https://linkinghub.elsevier.com/retrieve/pii/S0003269783710249" target="_blank">https://linkinghub.elsevier.com/retrieve/pii/S0003269783710249</a> <a href="#fnref:62" class="footnote-back-ref">↩</a></p></li> <li id="fn:63"><p>Ronaghi M, Karamohamed S, Pettersson B, Uhlén M, Nyrén P (November 1996). "Real-time DNA sequencing using detection of pyrophosphate release". Analytical Biochemistry. 242 (1): 84–89. doi:10.1006/abio.1996.0432. PMID 8923969. <a href="/wiki/Doi_(identifier)" target="_blank">/wiki/Doi_(identifier)</a> <a href="#fnref:63" class="footnote-back-ref">↩</a></p></li> <li id="fn:64"><p>Ronaghi, Mostafa; Uhlén, Mathias; Nyrén, Pål (1998-07-17). "A Sequencing Method Based on Real-Time Pyrophosphate". Science. 281 (5375): 363–365. doi:10.1126/science.281.5375.363. ISSN 0036-8075. PMID 9705713. S2CID 26331871. <a href="https://www.science.org/doi/10.1126/science.281.5375.363" target="_blank">https://www.science.org/doi/10.1126/science.281.5375.363</a> <a href="#fnref:64" class="footnote-back-ref">↩</a></p></li> <li id="fn:65"><p>US 6833246, Balasubramanian S, "Polynucleotide sequencing", published 2004-12-21, assigned to Solexa Ltd. <a href="https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=US6833246" target="_blank">https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=US6833246</a> <a href="#fnref:65" class="footnote-back-ref">↩</a></p></li> <li id="fn:66"><p>US patent 7790869, Ju J, Li Z, Edwards JR, Itagaki Y, "Massive parallel method for decoding DNA and RNA", published 2010-09-07, assigned to The Trustees of Columbia University in the City of New York <a href="https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=US7790869" target="_blank">https://worldwide.espacenet.com/textdoc?DB=EPODOC&IDX=US7790869</a> <a href="#fnref:66" class="footnote-back-ref">↩</a></p></li> <li id="fn:67"><p>Bentley DR, Balasubramanian S, Swerdlow HP, Smith GP, Milton J, Brown CG, et al. (November 2008). "Accurate whole human genome sequencing using reversible terminator chemistry". Nature. 456 (7218): 53–59. Bibcode:2008Natur.456...53B. doi:10.1038/nature07517. PMC 2581791. PMID 18987734. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2581791" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2581791</a> <a href="#fnref:67" class="footnote-back-ref">↩</a></p></li> <li id="fn:68"><p>Metzker ML (January 2010). "Sequencing technologies - the next generation". Nature Reviews. Genetics. 11 (1): 31–46. doi:10.1038/nrg2626. PMID 19997069. S2CID 205484500. <a href="/wiki/Doi_(identifier)" target="_blank">/wiki/Doi_(identifier)</a> <a href="#fnref:68" class="footnote-back-ref">↩</a></p></li> <li id="fn:69"><p>"Assay Technology". Illumina. Archived from the original on 2012-08-26. Retrieved 2012-08-05. <a href="https://web.archive.org/web/20120826030201/http://www.illumina.com/company/assay_technology.ilmn" target="_blank">https://web.archive.org/web/20120826030201/http://www.illumina.com/company/assay_technology.ilmn</a> <a href="#fnref:69" class="footnote-back-ref">↩</a></p></li> <li id="fn:70"><p>"True Single Molecule Sequencing (tSMS™): Helicos BioSciences". Helicosbio.com. Archived from the original on 2012-03-11. Retrieved 2012-08-05. <a href="https://web.archive.org/web/20120311112120/http://www.helicosbio.com/Technology/tabid/64/Default.aspx" target="_blank">https://web.archive.org/web/20120311112120/http://www.helicosbio.com/Technology/tabid/64/Default.aspx</a> <a href="#fnref:70" class="footnote-back-ref">↩</a></p></li> <li id="fn:71"><p>McKernan KJ, Peckham HE, Costa GL, McLaughlin SF, Fu Y, Tsung EF, et al. (September 2009). "Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding". Genome Research. 19 (9): 1527–1541. doi:10.1101/gr.091868.109. PMC 2752135. PMID 19546169. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752135" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752135</a> <a href="#fnref:71" class="footnote-back-ref">↩</a></p></li> <li id="fn:72"><p>"Fundamentals of 2 Base Encoding and Color Space". Appliedbiosystems.cnpg.com. Retrieved 2012-08-05. <a href="http://appliedbiosystems.cnpg.com/Video/flatFiles/699/index.aspx" target="_blank">http://appliedbiosystems.cnpg.com/Video/flatFiles/699/index.aspx</a> <a href="#fnref:72" class="footnote-back-ref">↩</a></p></li> <li id="fn:73"><p>Drmanac R, Sparks AB, Callow MJ, Halpern AL, Burns NL, Kermani BG, et al. (January 2010). "Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays". Science. 327 (5961): 78–81. Bibcode:2010Sci...327...78D. doi:10.1126/science.1181498. PMID 19892942. S2CID 17309571. <a href="https://doi.org/10.1126%2Fscience.1181498" target="_blank">https://doi.org/10.1126%2Fscience.1181498</a> <a href="#fnref:73" class="footnote-back-ref">↩</a></p></li> <li id="fn:74"><p>Shendure J, Porreca GJ, Reppas NB, Lin X, McCutcheon JP, Rosenbaum AM, et al. (September 2005). "Accurate multiplex polony sequencing of an evolved bacterial genome". Science. 309 (5741): 1728–1732. Bibcode:2005Sci...309.1728S. doi:10.1126/science.1117389. PMID 16081699. S2CID 11405973. <a href="https://doi.org/10.1126%2Fscience.1117389" target="_blank">https://doi.org/10.1126%2Fscience.1117389</a> <a href="#fnref:74" class="footnote-back-ref">↩</a></p></li> <li id="fn:75"><p>Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, et al. (June 2013). "Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data". Nature Methods. 10 (6): 563–569. doi:10.1038/nmeth.2474. PMID 23644548. S2CID 205421576. <a href="/wiki/Doi_(identifier)" target="_blank">/wiki/Doi_(identifier)</a> <a href="#fnref:75" class="footnote-back-ref">↩</a></p></li> <li id="fn:76"><p>Monica Heger (March 5, 2013). "PacBio Users Report Progress in Long Reads for Plant Genome Assembly, Tricky Regions of Human Genome". <a href="http://www.genomeweb.com/sequencing/pacbio-users-report-progress-long-reads-plant-genome-assembly-tricky-regions-hum" target="_blank">http://www.genomeweb.com/sequencing/pacbio-users-report-progress-long-reads-plant-genome-assembly-tricky-regions-hum</a> <a href="#fnref:76" class="footnote-back-ref">↩</a></p></li> <li id="fn:77"><p>"PacBio Launches Higher-Throughput, Lower-Cost Single-Molecule Sequencing System". October 2015. <a href="https://www.genomeweb.com/business-news/pacbio-launches-higher-throughput-lower-cost-single-molecule-sequencing-system" target="_blank">https://www.genomeweb.com/business-news/pacbio-launches-higher-throughput-lower-cost-single-molecule-sequencing-system</a> <a href="#fnref:77" class="footnote-back-ref">↩</a></p></li> <li id="fn:78"><p>"PacBio Announces Sequel Sequencing System - Bio-IT World". www.bio-itworld.com. Archived from the original on 2015-10-02. <a href="https://web.archive.org/web/20151002033528/http://www.bio-itworld.com/2015/9/30/pacbio-announces-sequel-sequencing-system.aspx" target="_blank">https://web.archive.org/web/20151002033528/http://www.bio-itworld.com/2015/9/30/pacbio-announces-sequel-sequencing-system.aspx</a> <a href="#fnref:78" class="footnote-back-ref">↩</a></p></li> </ol>