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<p><img alt="" src="/ckfinder/userfiles/images/Picture1(1).png" style="height:220px; width:400px" /></p>
<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 how<br /em>how these technologies work and how they may be applied<br />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>
<p style="margin-left: 40px;80px"> Sequencing technologies include a number of methods<br />that are grouped broadly as <strong>template preparation,<br />sequencing and imaging, and data analysis.</strong></p>
<ul> <li> <p style="margin-left><em>There are two methods used in preparing templates for NGS reactions</em>: 40px;"<strong>clonally amplified templates</strong> originating from single DNA molecules, and<strong> single DNA molecule templates</strong> </p> </li></ul>
<p style="margin-left: 40px;"> </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 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> <ul> <li style="margin-left: 80px;"><span style="font-size:16px"><strong>Sequencing methods</strong> are classified as <strong>cyclic reversible termination (CRT), single-nucleotide addition (SNA) and real-time sequencing.</strong></span></li></ul> <p style="margin-left: 80px;"><span style="font-size:16px"><strong></strong></span></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:40px"> </p> <p style="margin-left:40px"> </p> <p style="margin-left:40px"> </p> <p style="margin-left:40px"> </p>
