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[DESCRIPTION]
<p>This protocol is for the JetSeqTM DNA Library Preparation Kit. The kit is designed to generate high-quality next generation sequencing (NGS) libraries suitable for sequencing on Illumina MiSeqTM, NextSeqTM or HiSeqTM instruments.</p>

[GUIDELINES]
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<p><strong>1. KIT CONTENTS</strong></p>
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<p>Box1:</p>
<p><strong><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/ftscz36.png" data-src="https://s3.amazonaws.com/pr-journal/ftscz36.png" data-ofn="Screen Shot 2016-11-03 at 2.20.23 PM.png" /></strong></p>
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<div class="column">Box 2: JetSeq DNA Library Preparation Index Set (1-16, 20µl each)</div>
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<p><strong>2. DESCRIPTION</strong></p>
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<p>The success of next-generation sequencing is dependent upon the precise and accurate processing of the input DNA. This requires high-quality library preparation of sheared DNA using a coordinated series of standard molecular biology reactions whilst maintaining high yields during the intermediate puri cation steps.</p>
<p> </p>
<p>The JetSeq<sup>TM</sup> DNA Library Preparation Kit is designed to generate high-quality next generation sequencing (NGS) libraries suitable for sequencing on Illumina MiSeq<sup>TM</sup>, NextSeq<sup>TM</sup> or HiSeq<sup>TM</sup> instruments. The kit contains all of the enzymes and buffers necessary for end-repair, A-tailing, ligation and amplification in convenient master mix formulations as well as 16 barcoded adapters that can be used for single or multiplex reads.</p>
<p> </p>
<ul>
<li>Low input: 0.01-3 μg fragmented DNA </li>
<li>Increased speed: sequencing ready library in under 3 hours</li>
<li>Improved confidence: simpler protocol improves reproducibility</li>
<li>Improved quality: maximum coverage from all sample types</li>
<li>Maximum convenience: all-in-one kit</li>
</ul>
<p> </p>
<p> </p>
<p>By combining end-repair and A-tailing in one unique step, the JetSeq<sup>TM</sup> DNA Library Preparation Kit is able to reduce total NGS library preparation time and minimize the variability caused by additional handling, as well as the risk of contamination.</p>
<p>Please read this manual carefully to familiarize yourself with the JetSeq<sup>TM</sup> DNA Library Preparation protocol before starting (also available on <a href="http://www.bioline. com/jetseq" target="_blank">www.bioline. com/jetseq</a>).</p>
<p> </p>
<p><strong>3. STORAGE</strong></p>
<p>When stored under the recommended conditions and handled correctly, full activity of reagents is retained until the expiry date indicated on the outer box label.</p>
<p>The kit components should be stored at -20 °C. It is recommended that the user avoid repeated freeze-thaw cycles.</p>
<p> </p>
<p><strong>4. SAFETY</strong> <strong>INFORMATION</strong></p>
<p>When working with chemicals, always wear suitable personal protective equipment, including lab coat, gloves and safety glasses.</p>
<p>For detailed information, please consult the material safety data sheets available on our website at www.bioline.com.</p>
<p> </p>
<p><strong>5.</strong> <strong>PRODUCT SPECIFICATIONS</strong></p>
<p>The JetSeq<sup>TM</sup> DNA Library Preparation Kit is designed for Illumina® library construction work ows for a wide range of NGS applications, including: targeted sequencing (capture), whole genome sequencing, de novo sequencing, whole exome sequencing and ChIP sequencing.</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/e7cc3kn.png" width="650" height="915" data-src="https://s3.amazonaws.com/pr-journal/e7bc3kn.png" data-ofn="Screen Shot 2016-09-25 at 10.52.59 AM.png" /> </p>
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<p><strong>Fig. 1 Workflow for JetSeq<sup>TM</sup> DNA Library Preparation Kit</strong></p>
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<div class="page" title="Page 5"><strong>6. EQUIPMENT AND REAGENTS TO BE SUPPLIED BY THE USER</strong>
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<p>The following additional items are required:<br /> PCR equipment: Thermal cycler.<br /> Equipment for the determination of DNA concentration such as Nanodrop<sup>TM</sup>, Qubit<sup>TM</sup>, TapestationTM, Bioanalyzer or equivalent.<br /> Equipment for the determination of DNA size distribution such as Tapestation<sup>TM</sup>, Bioanalyzer or equivalent.<br /> Equipment for the puri cation and size selection of DNA fragments such as AMPure<sup>TM</sup>, Dynabeads<sup>TM</sup>, SPRI<sup>TM</sup> beads or other equivalent column-based systems.</p>
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<div class="page" title="Page 5"><strong>7. IMPORTANT NOTES</strong></div>
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<div class="page" title="Page 5"><strong>7.1. DNA preparation and quality control</strong>
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<p>The most important prerequisite for any NGS library preparation is high-quality DNA. Sample handling and DNA isolation procedures are therefore critical<br /> to the success of the experiment. Residual traces of proteins, salts or other contaminants will degrade the DNA or decrease the ef ciency of the enzymatic activities necessary for optimal library preparation.</p>
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<p><strong>7.1.1. Recommended genomic DNA preparation method</strong></p>
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<p>Depending on the sample, we recommend one of the following extraction kits:</p>
<ul>
<li>ISOLATE II Genomic DNA Kit (BIO-52066) for the preparation of genomic DNA from fresh tissues and cells.</li>
<li>ISOLATE II FFPE RNA/DNA Kit (BIO-52087) for the preparation of genomic DNA from FFPE tissue samples.</li>
<li>ISOLATE II Plant DNA Kit (BIO-52069) for isolation of genomic DNA from plants.</li>
</ul>
<p>For more DNA extraction kits, please refer to our ISOLATE II selection tool (www.bioline.com/isolate).</p>
<p> </p>
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<p><strong>7.1.2. Recommendations for DNA fragmentation</strong></p>
<p>DNA can be fragmented using one of the following methods:</p>
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<li>Mechanical fragmentation (acoustics, sonication, nebulization).</li>
<li>Enzymatic fragmentation.</li>
</ul>
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<p>To ensure complete fragmentation of the DNA that is needed for library preparation, only use the recommended parameters given in the manufacturer’s instructions. Check the fragmented DNA to ensure a correct size distribution is obtained.</p>
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<p><strong>8. PROTOCOL</strong></p>
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<p><strong>8.1. End-repair</strong></p>
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<p>Remove the “Step 1” reagents (green cap) and the nuclease free water (blue cap) from storage (-20 °C) and allow them to thaw on ice.</p>
<p>1. Prepare reaction mix on ice using the volumes shown below and mix by pipetting up and down.</p>
<p><strong>Table 1. End-repair reaction mix</strong></p>
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<div class="column"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/e7ec3kn.png" width="506" height="158" data-src="https://s3.amazonaws.com/pr-journal/e7dc3kn.png" data-ofn="Screen Shot 2016-09-25 at 10.58.33 AM.png" /></div>
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<p>2. Incubate for 30 min at 20 °C then 30 min at 72 °C.</p>
<p>3. Transfer the reaction tube on ice (4 °C).</p>
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<p><strong>8.2. Adaptor ligation</strong></p>
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<p>Remove the “Step 2” reagents (yellow cap) from storage (-20 °C) and allow them to thaw on ice.</p>
<p>1. Using the end-repair reaction from section 7.1 assemble the following reagents on ice. Mix by pipetting up and down.</p>
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<p><strong>Table 1. Adaptor ligation reaction mix</strong></p>
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<p><strong><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/e7gc3kn.png" width="641" height="264" data-src="https://s3.amazonaws.com/pr-journal/e7fc3kn.png" data-ofn="Screen Shot 2016-09-25 at 11.01.31 AM.png" /></strong></p>
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<p><strong>Table 2. Recommended adaptor volumes for varying starting amounts of DNA</strong></p>
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<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/e7ic3kn.png" width="832" height="327" data-src="https://s3.amazonaws.com/pr-journal/e7hc3kn.png" data-ofn="Screen Shot 2016-09-25 at 11.02.31 AM.png" /></p>
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<p>*Note: Lower starting amounts may need further optimization for optimal ligation.</p>
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<p><strong>Appendix A: Adaptor indexes</strong></p>
<p>The nucleotide sequences for the 16 indexes provided are detailed in the table below.</p>
<p> </p>
<p> </p>
<p>Index Number         Sequence</p>
<p>        1                    AACGTGAT</p>
<p>        2                    AAACATCG</p>
<p>        3                    AGTGGTCA</p>
<p>        4                    ACCACTGT</p>
<p>        5                   GATAGACA</p>
<p>        6                   GTGTTCTA</p>
<p>        7                   TGGAACAA</p>
<p>        8                   TGGTGGTA</p>
<p>        9                   ACATTGGC</p>
<p>       10                  CAGATCTG</p>
<p>       11                  CATCAAGT</p>
<p>       12                  AGTACAAG</p>
<p>       13                  AGATCGCA</p>
<p>       14                 GACTAGTA</p>
<p>       15                 GGTGCGAA</p>
<p>       16                TGAAGAGA</p>
<p> </p>
<p> </p>
<p><strong>Appendix B: Low multiplexing guidelines</strong></p>
<p> </p>
<p>Illumina platform such as MiSeq and HiSeq use a red laser to sequence A/C and a green laser to sequence G/T. To ensure accurate registration of the index read, both a red and green signal must be present at each cycle. It is also important to maintain color balance where possible. <br />If pooling less than eight samples in the final sequencing pool we suggest using the following index combinations</p>
<p><br />Number of samples in pool                Index</p>
<p>--------------------------------------------------------------------------------<br />         1                                        Any index</p>
<p>         2                                          2 &amp; 6<br />         3                                   Option A: 4, 6 &amp; 7<br />                                          Option B: 1, 11 &amp; 16     </p>
<p>          4                             Option A: 2, 6, 10 &amp; 14</p>
<p>                                       Option B: 9, 12, 15 &amp; 16<br />          6                          Option A: 1, 2, 4, 6, 7 &amp; 8    <br />                                    Option B: 2, 8, 9, 12, 15 &amp; 16 <br />          8                                     Option A: 1-8       <br />                                                Option B: 9-16</p>
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<p> </p>
<p><strong>A TECHNICAL SUPPORT AND TROUBLESHOOTING </strong></p>
<p>    For technical assistance or more information on this product, please email us at tech@bioline.com<br /><strong>B ASSOCIATED PRODUCTS</strong></p>
<p> </p>
<p>Product                                      Size                      Cat. #</p>
<p>ISOLATE II Genomic DNA Kit     50 prep                BIO-52066</p>
<p>ISOLATE II FFPE RNA/DNA Kit  50 prep                BIO-52087</p>
<p>ISOLATE II Plant DNA Kit           50 prep                BIO-52069</p>
<p>JetSeq Library Quantification Kit  TBD                  Please enquire</p>
<p><br /><strong>C PRODUCT WARRANTY AND DISCLAIMER </strong></p>
<p>Bioline warrants that its products will conform to the standards stated in its product specification sheets in effect at the time of shipment. Bioline will replace any product that does not conform to the specifications free of charge. This warranty limits Bioline’s liability to only the replacement of the product.</p>
<p><br /><strong>D TRADEMARK AND LICENSING INFORMATION</strong></p>
<p>JetSeq™ was developed jointly by OGT and Bioline. JetSeq™ (Bioline Reagents Ltd), HiSeq™, MiSeq™, NextSeq™ (Illumina Inc.); Qubit® (ThermoFisher Scientific); Dynabeads™ (Dynal Inc.); AMPure™ (Backman Coulter Inc.)<br /><br /><br /><strong>Ordering Information</strong><br />Product                                                 Size            Cat. #</p>
<p>JetSeq DNA Library Preparation Kit    16 Reactions  BIO-68025</p>
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