TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A genome is assembled by connecting large scaffolds via a collection of thousands of mate-paired ends of various sizes (3-20 kbp). Terminal ends of long DNA fragments can be obtained by sequencing large insert clones by Sanger sequencing, a labour-intensive and expensive approach. Here we describe a high-throughput protocol to tag 11,520 BAC plasmids randomly with barcoded oligonucleotides at the end of a random hexamer by PCR.This method assisted the kiwifruit (<em>Actinidia chinensis</em>) genome assembly in a similar way as mate paired end datasets.  Although not fully sequenced, each randomly tagged BAC clone provides a collection of short sequence tags mapped to a genome across one or multiple scaffolds under three different scenarios: validate a single scaffold, join two scaffolds, or group several scaffolds by proximity only. Our method provides a simple way of cataloging BAC libraries to assist genome assembly projects.</p>

[BEFORE_START]
<p>NOTES</p>
<ol>
<li>When <strong>pipetting dense solutions</strong>, like the PCR master mix, prime the pipette tips by drawing and expelling the liquid slowly, one time. Multichannel pipettes (and robotic stations) allow you to select the speed of this operation. Also, if the required volume is 47.5 µL, set the pipette to 46.7 µL, and don’t dip the tips too far into the solution. The amount of carry over solution outside the tip will compensate for the “missing” µL in the settings.</li>
<li>This method was developed in 2011/2012, and since then the library preparation and sequencing technologies have changed rapidly. You can select any approach as long as the amplicon ends are recovered during the library preparation. Do not use a tagmentation based protocol to prepare the libraries. And we recommend to obtain a pair end read data set to increase the read mapping rate.</li>
<li>We are aware that a small amount of <em>Escherichia coli</em> genomic DNA co-purifies with the BAC plasmid and is tagged and amplified along with the target BAC DNA. The <em>E. coli</em> DNA can be digested with enzyme blends such as <a href="https://www.lucigen.com/Plasmid-Safe-and-trade-ATP-Dependent-DNase/" target="_blank" rel="noopener noreferrer">PlasmidSafe</a> but given the number of BAC plasmid preps needed, it becomes too expensive. An option is to remove the contaminant by <a href="https://en.wikipedia.org/wiki/Suppression_subtractive_hybridization" target="_blank" rel="noopener noreferrer">genomic subtractive hybridization</a>. A subtracter is prepared with purified <em>E. coli</em> DNA which is sheared, end repaired and biotinylated at the 3'-end with terminal deoxynucleotidyl transferase. The subtracter is hybridized with the plate pool amplicons and the double stranded <em>E. coli</em> molecules (subtracter+random tagged <em>E. coli</em> DNA) are removed with streptavidin magnetic beads. After 3 rounds of subtraction, the remaining amplicon sample should be fairly clean of host DNA and ready for library preparation.</li>
</ol>
<p> </p>
<p>Consumables</p>
<ul>
<li>ABgene 96 well polypropylene storage microplate, conical bottom (cat# AB1058)</li>
<li>2 mL deep well plate (NUNC cat# 278752)</li>
<li>96-well Filter plate (NUNC cat# 278011)</li>
<li>Conical polypropylene sterile tubes, 15 mL</li>
<li>Disposable sterile reservoirs</li>
<li>Breathable tape (rayon acrylate NUNC cat# 241205)</li>
<li>Silicone mat that fits the 2 mL deep well plate or a piece of silicone rubber sheet (1.5 mm thickness)</li>
<li>Clear packaging tape (48 mm wide) for temporary plate sealing</li>
<li>Cutter with retractable blade</li>
<li>Microcentrifugues, 1.5 and 2 mL, sterile, and racks</li>
<li>PCR plates (Thermo-Fast AB-0600)</li>
<li>Aluminium tape for PCR reactions and long term storage (NUNC cat# 232698, or similar)</li>
<li>Paper towels, low lint</li>
<li>Rectangular plastic box</li>
<li>Waste containers for biological material and isopropanol</li>
</ul>
<p> </p>
<p>Solutions</p>
<ul>
<li><a href="http://cshprotocols.cshlp.org/content/2006/1/pdb.rec8141.full?text_only=true" target="_blank" rel="noopener noreferrer">LB medium</a></li>
<li>12.5 mg/mL tetracycline hydrochloride, dissolved in absolute ethanol</li>
<li>RNase A 100 μg/μL (Qiagen cat# 19101)</li>
<li>Resuspension buffer (25 mM Tris-HCl pH 8, 10 mM EDTA, sterile). Add RNase A to a final concentration of 50 μg/mL before resuspending the bacterial cells</li>
<li>0.2 N NaOH prepared in sterile deionized water and stored in a plastic bottle</li>
<li>10% SDS, sterile</li>
<li>Lysis solution (Use sterile deionized water to prepare 0.2 M NaOH, 0.1% SDS. This solution is freshly made from stocks described above)</li>
<li>Neutralizing solution (3 M potassium acetate, 2 M acetic acid, pH 5.6). To prepare this solution dissolve 295.5 g potassium acetate (MW 98.5 g/mol) in ~400 mL sterile deionized water. Adjust the volume to 600 mL and store in a sterile 1 L glass bottle. Add 115 mL glacial acetic acid and 285 mL sterile deionized water. Do not autoclave and do not adjust the pH, which should be around 5.6. Split the solutions in 2 or 3 sterile glass bottles and store at 4°C</li>
<li>Isopropanol</li>
<li>70% ethanol, in sterile deionized water</li>
<li>Plasmid resuspension buffer (28 mM Tris-HCl pH 8, 1 mM EDTA, 0.6 mM cresol red)</li>
<li>Deionized water, sterile</li>
<li>AccuPrime Taq DNA polymerase High Fidelity 5 U/µL and 10X AccuPrime High Fidelity Buffer I and II (Invitrogen 12346094)</li>
<li>Mineral oil (Sigma-Aldrich cat # M5904-500ML)</li>
<li>2% xylene cyanole FF solution prepared in sterile deionized water</li>
<li>Platinum <em>Pfx</em> DNA polymerase 2.5 U/µL (Invitrogen 11708039)</li>
<li>1.2 M Trehalose, dissolved in sterile deionized water and filtered through 0.2 or 0.4 µm</li>
<li>10X <em>Pfx</em> reaction buffer homemade: 200 mM Tris-HCl, 100 mM KCl, 100 mM (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, pH 8, sterile. <strong><em>Note</em></strong><em>: I have not tried the 10X buffer and enhancer solutions supplied with the Platinum Pfx DNA polymerase (Invitrogen 11708039). It should work, but do a preliminary test</em></li>
<li>50 mM MgSO<sub>4</sub> (use the stock provided with either DNA polymerase)</li>
<li>2 mM dNTP (dilution prepared in deionized sterile water from 100 mM stocks)</li>
<li>2% agarose gels in <a href="https://en.wikipedia.org/wiki/TAE_buffer" target="_blank" rel="noopener noreferrer">1X TAE</a> buffer and 1X TAE running buffer</li>
<li>1 kb+ DNA ladder (Invitrogen) or similar</li>
<li>SybrSafe (Invitrogen)</li>
<li>3 M sodium acetate pH 5.2. To prepare this solution dissolve 24.6 g sodium acetate anhydrous (MW 82.03 g/mol) in ~ 80 mL deionized water. Adjust the pH to 5.2 with glacial acetic acid. Adjust the volume to 100 mL, split into two glass bottles, and autoclave</li>
<li>TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5), sterile</li>
</ul>
<p> </p>
<p>Equipment and kits</p>
<p> </p>
<ul>
<li>Cell culture incubator</li>
<li>Bench top centrifuge with plate rotor (for example, Eppendorf SKU 5810000084 Rotor A-4-62, with 4 MTP buckets; Cat. # 5810711002)</li>
<li>Bench top microcentrifuge for 1.5 mL tubes</li>
<li>
<div><label>Vacuum manifold (for example </label>Supleco PlatePrep 96-well Vacuum Manifold <a href="https://www.sigmaaldrich.com/catalog/product/supelco/575650u?lang=en&amp;region=NZ" target="_blank" rel="noopener noreferrer"><label>SKU:</label>575650-U</a>)</div>
</li>
<li>Vortex with plate adaptor</li>
<li>Qubit 2.0 or above and High Sensitivity dsDNA kit</li>
<li>Horizontal gel electrophoresis unit with power supply</li>
<li>Image capture equipment (for example, GelDoc, LSA3000, etc)</li>
<li>Multichannel pipettes (1-10 µL, 5-200 µL, 50-1000 µL) or liquid handling robotic station with appropriate multichannel tools and tips, for example Biomek series, Hamilton, Integra’s Viaflo, etc.</li>
<li>PCR machine</li>
<li>Timer</li>
</ul>

[GUIDELINES]
<p>A BAC library from kiwifruit (<em>Actinidia chinensis</em>) was constructed by Bio S&amp;T, Québec, Canada. The nuclear genomic DNA was partially digested with <em>Hind</em> III, cloned in the plant transformable binary vector pCLD04541, maintained in <em>Escherichia coli</em> DHB10 and arrayed in 180 382-well plates. The BAC library represents ~ 6.7 times the haploid genome; the average insert size is 71.3 kbp (<em>n</em> = 309), and 0.6% of the library contain organellar DNA clones.</p>
<p>The process of oligonucleotide design will be reported in a separate protocol. But here is an overview:</p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/ybyeupe.jpg" data-src="https://s3.amazonaws.com/pr-journal/ybyeupe.jpg" data-ofn="BACRB oligo design.jpg" /></p>

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