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<p style="margin-left:40px">This Review focuses on commercially available technologies from<strong> Roche/454, Illumina/Solexa, Life/APG and Helicos BioSciences, the Polonator instrument </strong>and the near-term technology of<strong> Pacific Biosciences, </strong></p>
<p style="margin-left:40px">Here, I present a technical review of<strong> template preparation, sequencing and imaging, genome alignment and assembly</strong>, and current NGS platform performance to provide guidance on <em>how these technologies work and how they may be applied to important biological questions. </em></p>
<p style="margin-left:40px"><img alt="" src="/ckfinder/userfiles/images/Picture2(1).png" style="height:13px; width:200px" /></p>
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<p > </p> <p> </p> <h3 style="margin-left: 80px;"><strong>Clonally amplified templates.</strong> most imaging systems have<br />
not been designed to detect single fluorescent events, so<br />
amplified templates are required. The two most common<br />
methods are <strong>emulsion PCR (emPCR)9 and solid-phase<br />
amplification</strong></h3> <h3 style="margin-left: 80px;"><strong><img alt="" src="/ckfinder/userfiles/images/Picture3(1).png" style="height:97px; width:400px" /><u>emulsion PCR (emPCR)</u></strong></h3> <p style="margin-left:80px"><strong><u><img alt="" src="/ckfinder/userfiles/images/Picture4(1).png" style="height:235px; width:400px" />solid-phase amplification </u></strong></p> <p style="margin-left:80px"> </p> <p style="margin-left:80px"> </p> <p style="margin-left:80px"> </p> <h3 style="margin-left: 40px;"><strong>Single-molecule templates.</strong> Although <strong>clonally amplified</strong><br />methods offer certain advantages over bacterial cloning,<br />some of the protocols are cumbersome to implement<br />and<strong> require a large amount of genomic DNA material<br />(3–20 μg)</strong>.</h3> <p style="margin-left:40px"><strong>The preparation of single-molecule templates is more straightforward </strong>and requires less starting material (<1 μg). more importantly, these methods <strong>do not require PCR,</strong><em> which creates mutations in clonally amplified templates</em> that masquerade as sequence variants. AT-rich and GC-rich target sequences may also show amplification bias in product yield, <em>which results in their underrepresentation in genome alignments and assemblies.</em><br /> </p> <p style="margin-left:40px">Two different methods are used to attach the template to a solid support : </p> <p style="margin-left:40px"><img alt="" src="/ckfinder/userfiles/images/Picture5.png" style="height:212px; width:200px" /> <img alt="" src="/ckfinder/userfiles/images/Picture6.png" style="height:180px; width:300px" /></p> <p style="margin-left:40px"> </p> <p style="margin-left:40px"> </p> <p style="margin-left:40px"> </p> <p style="margin-left:40px"> </p>
<p style="margin-left: 80px;40px"><strong><img alt="" src="/ckfinder/userfiles/images/Picture3(1).png" style="height:97px width:400px" /><u>emulsion PCR (emPCR)</u></strong></p>
<p style="margin-left: 80px;40px"><strong><u><img alt="" src="/ckfinder/userfiles/images/Picture4(1).png" style="height:235px width:400px" />solid-phase amplification</u></strong></p>
<p style="margin-left: 80px;40px"> </p>
<p style="margin-left:40px"> </p>
