这项工作是由美国国立卫生研究院的/ NHGRI基因组学者/教师转型奖（K22 HG002908）给AMD与系统生物学研究所和卢森堡大学之间的战略伙伴关系的支持。请求菌株或质粒，应直接向阿蜜杜德利（aimee.dudley @ gmail.com）。

1株的构建与鉴定<ol><li>构建杂合二倍体菌株交配单倍体菌株，并选择二倍体2或用一种天然存在的杂合菌株被用于在交叉。该菌株不应该心怀绿色荧光蛋白。 </li><li>确认该菌株不能生长在YPD 2 + 200微克/毫升G418。 </li><li>确认该菌株能够形成孢子。最好的是低效率的产孢杂交兼容，但需要孢低频率的增加流式细胞仪分选时间。 <ol><li>设置了该菌株的O / N培养物在5ml YPD和搅拌下在30℃下生长</li><li>洗0.5ml所的O / N培养的三次用PBS（pH值7.4）。 </li><li>沉淀经洗涤细胞，除去在2ml孢子形成介质7上清，重悬。 </li><li>搅动细胞在室温和日常检查使用相衬光学显微镜用40倍物镜四分体形成。 </li> &lt;/ OL&gt; </li><li>由四分少数（〜20）常规清扫2,3估计十字架的孢子生存能力。在不同的应变背景之间的杂交孢子生存能力广泛和不可预知变化。然而，孢子活力的先验期望可用于评估一个具体实验的劳动和成本密集型的步骤，保存和基因分型的菌株之前的效率。因此，该步骤是强烈建议。 </li></ol> 2，转型的二倍体与条形码的质粒的图书馆<ol><li>将pCL2_BC条形码质粒转化利用Gietz和伍兹8所描述的改造方案进行了以下修改杂合二倍体菌株。 <ol><li>后细胞已在30℃下温育的转化混合物20分钟，加入DMSO中的转化混合物体积的8％。加入DMSO已经发现，提高转化效率我Ñ ​​有些紧张的背景9。 </li><li>热休克步骤后，沉淀细胞用短暂离心，滗转化混合物，并在YPD轻轻洗涤细胞。然后，重悬细胞于1ml YPD，让他们恢复在室温3小时，不加搅拌。 </li></ol></li><li>通过镀覆到YPD + G418（200微克/毫升）琼脂平板上选择转化体。为确保质粒条形码的复杂的池，收获了大量的殖民地， 例如 5板，每板〜2,500菌落。 </li><li>一旦菌落可见（2-3天生长在30℃），通过仔细刮他们离开板成液体YPD + G418（200微克/毫升）+ 15％甘油汇集转化。涡旋该细胞悬浮液，转移至冷冻小瓶中，并冷冻在-80℃下使一个单独的冷冻的储存对于每个板。这些冻存作为起始菌株步骤3.1和也可以用于在稍后的日期，以产生更多的四分体，如果需要的话。 </LI&gt; </ol> 3，孢子转化细胞的图书馆获取条形码的四联球菌<ol><li>从2.3按O / N的增长在5毫升的YPD + G418（200微克/毫升）在30℃下搅拌产生的冷冻复苏个股细胞。 </li><li>从将O洗涤细胞/ N培养物3倍的PBS（pH 7.4）中，然后悬浮细胞在孢子形成介质7加200微克/毫升G418。 </li><li>搅拌孢子培养在室温和日常监控产孢的进展，直到新的四分体已经停止形成。 </li><li>一旦文化包含良好的四分体和相对较少的二人组合，从搅拌去除的文化和在室温下至少放置7天。虽然有些十字架可能是服从于板消解和破坏，只要四分体形成（如7.3中所述的评估），额外的孵化（在我们的实验7-10天），对其他的十字架是必不可少的。因此，该步骤是强烈建议。 </li></ol> 4，建立流式细胞仪盖茨的四分体排序在减数分裂表达于p​​CL2_BC质粒SPS2-GFP融合，能够检测和分离从培养物中荧光的孢子形成培养四分体。下面的协议已经制定了一个使用FACSAria II。 <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/51401/51401fig2highres.jpg" width="500" /> 图2流式细胞仪门。一系列的4个连续的门是用来隔离四分。 （A）中所示的门和（B）有助于减少团块的数量。荧光事件在（C） 的选择与门。在（D）的直方图有两个峰。在左边的峰事件是主要四分体（左小图图像），并且在右峰事件通常是四分体用附属芽（右插图图像）。最后一个门应用于此直方图来选择其中大多是四分体（红色条）事件。这个数字已经被修改勒德洛， 等 1。 <ol><li>加载孢子培养到FACS分选器和设置了两个前向散射和侧向散射光闸，以排除碎片，成群的细胞，并且多个单元格的每滴（ 图2A和2B）。 </li><li>应该有荧光的细胞，其使用SPS2-GFP报道基因构建体时，包括二分体和四分体10的群体。门的绿色荧光蛋白与金管会包括这些荧光事件（ 图2C）。 </li><li>根据不同的菌株背景上它可能是必要的，包括一个额外的栅极以去除团块，包括四分体和其它细胞。如果这是必要的，检查荧光事件的FSC直方图，并指定一个门，它包括较低的FSC（ 图2D，门事件是红色的）事件。 </li><li>检查T的效率<html>他门控机制通过排序〜1500四分体在显微镜载玻片上，并用相衬显微镜在400倍放大计数四分体的比例，以非四分体。四分体的百分比可通过计算只〜200排序事件精确估计，但是排序〜1500减少的时间量使用了搜寻在细胞上的显微镜载玻片上。或者，四分体以非四分之比可以在步骤4.5中进行评估。 </li><li>定期检查该分选器是由分拣25四分体在显微镜载玻片上，并用相衬显微镜在400倍放大检查包含在液滴中的细胞正确地报告事件的数量。应在熔滴25四分体。重复3至4倍，以评估流式细胞仪的方差。 </li></ol> 5，排序四联球菌到一个琼脂平板p_upload/51401/51401fig3highres.jpg“宽度=”500“/&gt; 图3：放置在琼脂平板上的FACS分拣机。为了四分整理成一小滴的酶解酶液放置在一个标准（圆）琼脂平板上，一个标准的100毫米（矩形）的中间96孔板是第一放置在细胞分选阶段和分类器被编程以沉积四分体到一个特定的孔（在该情况下D5）。然后，圆形琼脂平板放置在96孔板的顶部，并对准，使得酶解酶的压降为中心在规定的井。 <ol><li>温暖的YPD + G418（200微克/毫升）板块，至少1.5小时在37℃培养箱放置靠近流式细胞分选仪所需的号码。 </li><li>分拣协议的一个关键特征是能够准确定位FACS分选至四分体琼脂平板上，它包含的酶解酶液池的特定位置的能力。要做到这一点，放置一个“里程碑”的96孔板上吨他的自动分拣机的细胞沉积单元（ACDU）。制备一种布局，将25四分体分类到一个96 - 孔格式中的一个很好。选择一个井附近的板的中间， 如井D5。 图3显示了一个培养皿和96孔板上样到细胞分选器。排序每板25个四分体得到良好分离的菌落。 </li><li>把一叠10琼脂板上从37℃培养箱中培养到分类器。 </li><li>取1琼脂平板和吸管将25μl的酶解酶溶液（1毫克/毫升0.7M的山梨糖醇）上靠近板的中心的琼脂。然后，放置在界标96孔板的板，从步骤5.2， 例如井D5对准酶解酶液滴在目标井。 </li><li>启动排序和沉积25四分体插入琼脂平板的酶解酶液滴的中间。然后，删除并覆盖板。 </li><li>重复步骤5.4和5.5，直到25四分体已经被分类到堆栈中的每个板酶解酶液滴。 </li&gt; <li>返回一个包含四分体 - 板的堆叠到37℃培养箱中培养。 </li><li>重复步骤5.3至5.7，直到所有的板块有四分。 </li></ol> 6。孢子分离<ol><li>孵育各堆含四分-琼脂平板上，在37℃下进行1.5小时。 </li><li>从孵化器中取出板，加上每板15-25无菌的毫米的玻璃珠，摇板作为一个堆栈（或两个堆栈5板）3-5分钟。最好的孢子分离是通过硬性移动板从一侧到另一侧，或从前向后实现。不移动的板，使得所述珠“漩涡”围绕板的外边缘。完成后留在板的珠子。 </li><li>将板垛珠正视在30℃培养箱中培养。检索来自37℃培养箱中培养下一个堆​​栈板，并重复步骤6.2。 </li><li>孵育板O / N为30°C。 </li></ol> 7，收获殖民地<ol><li>后一个O / N培养，在30℃，小菌落应该形成于板。小心地从培养箱中取出板，而不会干扰玻璃珠。 </li><li>通过仔细地除去玻璃珠，并迅速翻转板在一个深的容器。该容器应足够深，以赶珠而不让他们反弹向上，撞上琼脂平板。轻敲板的底部，同时它被反转可以帮助驱逐被粘到板珠。这些珠子可以通过彻底的清洗用肥皂被重用，以及漂洗用去离子水通过高压灭菌消毒。 </li><li>计数菌落在每个板的数量和使用的菌落数来估计孢子分离和回收的效率。例如，每板25个四分体为具有接近100％的存活率的横可能会产生一个总的每板100个菌落。 </li><li>从板块有足够数量的菌落（&gt; 65为高生存能力十字架），传递细胞从每个结肠NY到96孔板中含有YPD液体+ G418的一个单独的井。此外，请注意其中包含井菌落从相同的琼脂平板上;这些信息将被用于帮助四分体分配软件。 </li><li>使用96孔平板液体种子培养的基因型和保存菌种冷冻甘油股票。在RAD标签测序和序列分析细节可以在Ludlow 等[1]发现。 </li></ol>

<ol>
	<li>Ludlow, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+tetrad+analysis.">High-throughput tetrad analysis.</a> Nat Methods. 10, 671-675 (2013).</li><li>Rose, M., Winston, F., Hieter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Yeast Genetics A Laboratory Course Manual. , (1990).</li><li>Morin, A., Moores, A. W., Sacher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+Saccharomyces+cerevisiae+asci.">Dissection of Saccharomyces cerevisiae asci.</a> J Vis Exp. 27, (2009).</li><li>Winge, O., Laustsen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+two+types+of+spore+germination,+and+on+genetic+segregations+in+Saccharomyces+demonstrated+through+single+spore+cultures.">On two types of spore germination, and on genetic segregations in Saccharomyces demonstrated through single spore cultures.</a> C.R. Trav. Lab. Carlsberg Ser. Physiol. 24, 263-315 (1937).</li><li>Coluccio, A., Neiman, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interspore+bridges:+a+new+feature+of+the+Saccharomyces+cerevisiae+spore+wall.">Interspore bridges: a new feature of the Saccharomyces cerevisiae spore wall.</a> Microbiology. 150, 3189-3196 (2004).</li><li>Baird, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+SNP+discovery+and+genetic+mapping+using+sequenced+RAD+markers.">Rapid SNP discovery and genetic mapping using sequenced RAD markers.</a> PLoS One. 3, (2008).</li><li>Tong, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+genetic+analysis+with+ordered+arrays+of+yeast+deletion+mutants.">Systematic genetic analysis with ordered arrays of yeast deletion mutants.</a> Science. 294, 2364-2368 (2001).</li><li>Gietz, R. D., Woods, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+yeast+by+lithium+acetate/single-stranded+carrier+DNA/polyethylene+glycol+method.">Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.</a> Methods Enzymol. 350, 87-96 (2002).</li><li>Hill, J., Donald, K. A., Griffiths, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DMSO-enhanced+whole+cell+yeast+transformation.">DMSO-enhanced whole cell yeast transformation.</a> Nucleic Acids Res. 19, 5791-57 (1991).</li><li>Gerke, J. P., Chen, C. T., Cohen, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+isolates+of+Saccharomyces+cerevisiae+display+complex+genetic+variation+in+sporulation+efficiency.">Natural isolates of Saccharomyces cerevisiae display complex genetic variation in sporulation efficiency.</a> Genetics. 174, 985-997 (2006).</li><li>Bloom, J. S., Ehrenreich, I. M., Loo, W. T., Lite, T. L., Kruglyak, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finding+the+sources+of+missing+heritability+in+a+yeast+cross.">Finding the sources of missing heritability in a yeast cross.</a> Nature. 494, 234-237 (2013).</li><li>Fogel, S., Mortimer, R., Lusnak, K., Tavares, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiotic+gene+conversion:+a+signal+of+the+basic+recombination+event+in+yeast.">Meiotic gene conversion: a signal of the basic recombination event in yeast.</a> Cold Spring Harb Symp Quant Biol. 43 Pt 2, 1325-1341 (1979).</li></ol>

作者什么都没有透露。

遗传学研究的许多领域，从复杂性状的映射11到相关DNA重组12机制的研究，需要极其大量的后代和高量的DNA测序。那嫁给高通量DNA测序的能力传统方法的电力技术有可能使这在以前是顽固性的研究。尽管一些高通量酵母遗传学方法已经有效地具体问题得到了应用，每个都有功亏一篑传统的四分体分析的广泛适用性的​​限制。通过采用高通量的方法相结合的四分体解剖和基因分型酵母杂交的后代最佳解决了这些挑战。在复用RAD标签测序相结合，提供了最好的一种廉价的方式来收集信息，基因型为每个子代菌株，同时保持四分体的关系由独特的四分体条形码手段。0;所有目前使用的高通量方法，最佳最密切概括了手动解剖酵母交所提供的信息。 最好的有两个主要特点。首先是使用流式细胞仪的迅速隔离四分远离unsporulated细胞培​​养（协议第5-6步）。隔离数以千计的荧光事件（四分体）的能力，为最佳的吞吐量其最大的收益。这种自动化提供了手动清扫十字架与产孢低效率的更大的优势，因为需要直观地识别罕见事件的时间增加。第二个关键特征是使用一种高度复杂的分子条形码（协议步骤2）被发送到一个四分体的每个姊妹孢子。因为这些条形码可以用来计算重建已随机分布在琼脂平板上，需要手动阵列单元中的有序栅格缓解孢子之间的四分体的关系。最后，因为分子条形码和孢子形成特异的GFP报告基因是物理连接的（在相同的质粒），只是保留了分子条形码四分体是通过FACS分选选中。 有两个方面的考虑支配的时间的文化应该在产孢培养基中培养的长度。首先，由于特定的孢子记者（SPS2-GFP）在减数分裂早期表达，绿色荧光蛋白本身并不能从尚未完成减数分裂（ 即二分体）细胞区分完整的4孢子四分体。而另外的流式细胞仪门可能会降低荧光，不完整的四分体的数量，以减少这些不必要的事件最简单的方法是简单地监视孢子培养（步骤3.3），一旦新的四分体已经停止继续形成。在我们的实验中，二价基排序的频率小于1％。其次，在室温中度过产孢培养基中，不加搅拌一些十字架额外的时间（步骤3.4）SIGnificantly提高了四分体由玻璃珠电镀过程中的分离（（协议第6步），事实上，这个步骤是用于一些杂交的破坏绝对必要的。 像所有的高通量方法，最佳比相应的传统方法“喧闹”。在适度调低最佳的效率比传统的四分体解剖，可能会导致从几个因素的组合。一个因素是，否则活孢子的损失增加，这可能是由于该过程的机械应力扩散或增加孢子死亡期间导致粘附到玻璃珠。减数分裂过程中的第二个因素可能是简单的质粒丢失。第三个因素可能是一种有效的减少从混合基因型的菌落产生可用的殖民地，在我们的飞行员殖民地的估计〜5％，跨越1。它很可能是这些殖民地代表妹妹孢子分离的故障。因为快速氟escence排序和孢子分离允许四分的巨大数字的集合，最好的是装备精良，以克服在减少这种规模效益。而最佳的未来的迭代可以提高效率接近于人工清扫，这或许是更可能是DNA测序能力目前指数的轨迹将迅速弥补可用应变损失的这个水平。无论如何，进行四分体分析到最佳可应用于规模的能力，使研究，目前不可行通过手动的方法。

减数分裂基因作图，俗称四分体分析，是中央的酵母遗传学。简单地说，双单倍体品系，每个包含每个染色体的一个副本，交配形成二倍体杂株式两份每个染色体，分别来自父母。氮饥饿二倍体细胞诱导减数分裂（孢子），其中染色体复制，进行重排，并分为四个单倍体细胞（孢子或分离子）与等位基因分别来自父母的新组合。从单一减数分裂产生四个孢子可以通过一个手动过程，称为四分体解剖分离。 这是自原始出版物已经改变相对较少，1937年4传统的四分体解剖2,3，有三个目标。首先，已经历减数分裂（四分体）细胞必须从营养细胞的背景分离。在传统的分析，这是通过一个研究者谁完成的，使用显微镜，在视觉上识别在琼脂平板上四分体和采用了微操作装置可视地识别四分体琼脂平板上，并用显微操作器的四分体移动到所述板的无细胞区域。接着，4个孢子在四分体必须物理分离，以防止interspore交配。四分体中的孢子通过既是子囊，原二倍体细胞的细胞壁的残余，以及一组interspore桥5固定在一起。在传统的四分体分析子囊被酶消化去除，研究员使用显微操纵器，打破interspore桥梁和捉弄除了孢子。最后，个人孢子被排列在该保持孢子之间的关系的网格图案， 也就是说 ，所有的子代在一排四个是相同的原四分体的妹妹孢子。一位经验丰富的酵母研究者可以完成解剖10四分体（一种普遍接受的最低数目的过程每个交叉）中约15分钟。 我们已经开发了一种新的高通量基因分型四分体的方法，我们称之为乙arcodeÉ对 T etrads nabled S equencing（BEST）1。通过使大量的后代是孤立的，基因分型，并保持在一个方式，允许所有四个减数分裂产物的妹妹孢子关系到恢复个体的产生和基因分型的最优扩展了现有方法中的高通量基因。这是通过使用三种主要的策略（ 图1）来完成的。首先是引进一名记者构造，标签已经历减数分裂与GFP细胞，并允许他们通过荧光激活细胞分选（FACS）来分离。第二是一个高度复杂的DNA条形码的形式掺​​入（共15个随机核苷酸的字符串）被发送到所有4个孢子四分体中，并且可以通过重组Progen公司的DNA测序阅读y和从而识别来自同一四分妹妹孢子。三是基因分型的步骤，最好就是与众多的基因分型平台，捕捉这两个一组的基因组标记以及四分体特异的条形码兼容。我们采用了限制性位点相关的DNA标记的测序（RAD-SEQ）6的方法，指示基因组测序，以特定的限制性位点1，由此捕获的子代菌株的基因组中的一个一致的2-3％，以及该质粒携带的条形码。四分体的关系以及从十字架的经验源性基因分型数据恢复允许丢失的信息，包括与低覆盖的序列标记的状态和不能存活（因此不可恢复）个人的基因型完全准确的推断。在这里，我们描述了在最常用的微生物为减数分裂映射，酵母S.方法的应用酵母 ， 但与机体特定意图的次要取代男装，融合到GFP，例如不同的孢子特异性蛋白，该方法应该是很容易移植到其他微生物，包括微生物，其中减数分裂映射显著更加劳动密集的或当前棘手。 <img alt="图1" fo:content-width="4in" src="/files/ftp_upload/51401/51401fig1highres.jpg" width="400" /> 图1的最优方法（A）的pCL2_BC质粒是2微米质粒与孢子形成特异的GFP融合，对G418的抗性，和一个15聚体随机条形码。质粒文库的构建在Ludlow 等[1]中描述。 （ 二）条形码质粒的池改造成从跨二倍体菌株。 （ 三）转化，然后生长在选择和〜10,000转化的菌落汇集和孢子。在减数分裂的四分孢子各继承一个共同PY印有条码的质粒。 （ 四）四联球菌是从unsporulated细胞用流式细胞仪分离和收集琼脂平板上，在那里他们被消化和破坏，使每个孢子形成一个殖民地。 （E）的菌落，然后挑入96孔板中，表型和基因分型。在基因分型质粒条形码读取和用于识别每个四分体的四名成员。这个数字已经被修改勒德洛等[1]。 <a href="https://www.jove.com/files/ftp_upload/51401/51401fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table border="0" cellpadding="0" cellspacing="0" width="726">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">FACSAria II (Special order unit)</td>
			<td style="width:149px;">BD Biosciences</td>
			<td style="width:127px;">-</td>
			<td style="width:155px;">Other cell sorters should work for this procedure. The key factors are: (1) a laser/detection system for the fluorescent reporter being used (488nm laser to excite EGFP in our case), (2) the ability to sort single events, (3) the ability to sort onto microscope slides for assessing the sort quality and (4) the ability to sort onto petri dishes (which may need</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Automated Cell Deposition Unit (for FACSAria II)</td>
			<td>BD Biosciences</td>
			<td colspan="2">643155 (if purchased separately from the FACSAria instrument)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zymolyase 100T</td>
			<td>Seikagaku Biobusiness</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">G418</td>
			<td>A.G. Scientific</td>
			<td></td>
			<td>http://www.agscientific.com/molecular-biology/gene-expression/g-418-sulfate-solid.html</td>
		</tr>
	</tbody>
</table>

best: barcode enabled sequencing of tetrads

四分体分析是酵母遗传学一个有价值的工具，但这个过程的繁琐的人工性质已阻碍了其应用在大尺度上。条码启用四分体测序（BEST）1代替隔离，破坏和间距四分体的手动流程。凭借一个孢子形成特异的GFP融合蛋白，其允许四分体的荧光激活细胞分选，直接到琼脂平板上，其中所述子囊被酶消化和孢子被打乱，并随机由玻璃珠电镀排列的最优隔离四分体。单倍体克隆，然后分配妹妹孢子的关系， 即有关哪些孢子起源于同一个四分体，利用基因分型中读取分子条形码。通过去除手工剥离的瓶颈，数百四分甚至上千可被分离以分钟计。在这里，我们提出了以表现最佳的酵母所需的实验步骤的详细说明酿酒酵母 ，首先通过从单倍体减数分裂后代衍生的菌落隔离杂合二倍体菌株。

最好能大大改善从四分孢子可以分离的速度。作为该方法可以提供的吞吐量的一个例子，1研究者进行的FACS分选和孢子分离（步骤5.1至6.3）与149琼脂平板在3小时1。等效收益率（约3,725四分）将需要超过90小时人工清扫。然而，与许多高通量方法，所述方法的当前迭代具有一定的效率损失比手动剥离。这种损失表现为菌落数目减少回收相比基于交叉的孢子生存，由于手工剥离（步骤1.4）来确定所计算的期望。假设孢子损失（讨论）的原因影响孢子随机，估计可制成回收的菌落多少都会四分体，其中1，2，3，或4个孢子已被追回的成员（ 图4）。 图4。在四分体的水平。未能成功地恢复孢子（如集落） 仿真方法的效率可能是由于孢子inviability，孢子附着到玻璃珠，未孢子彼此分离等二项式分布，使用计算四分体的预期分数（至少有一个成功收复孢子）生产的基础上成功地恢复了所有的孢子比例4，3，2，1殖民地。此图可作为一个粗略的指南菌株，这将最终组装成3的数量 - 或4 - 孢子四分体。如每板的增加菌落数，所以没有完全恢复的四分体的数量。 在实验中，如上所述，从与孢子存活率接近100％的横25四分体（如DET通过人工清扫ermined）1涂布在149琼脂平板。在该实验中，每板观察到菌落的最大数目为80，这表明20个孢子未能形成离散的菌落。每个平板菌落的平均数是62。菌落从含65菌落或更多的61板收获和测序。 3700菌株通过测序质量控制，72％可以计算分为3 - 或4 - 孢子四分体。

Watch this Scientific Journal Video about BEST: Barcode Enabled Sequencing of Tetrads at JoVE.com

BEST: Barcode Enabled Sequencing of Tetrads

四分体启用条码序列（BEST）取代分离，破坏和间距四分体的手动流程。的最优隔离四分体通过荧光激活细胞分选到琼脂平板上，通过搅拌与玻璃珠分离出孢子，并确定哪个随机排列菌落，从同一原始四分使用分子条形码而得。

最佳：条码启用四分体测序

Tetrad analysis is a valuable tool for yeast genetics, but the laborious manual nature of the process has hindered its application on large scales. ...

best-barcode-enabled-sequencing-of-tetrads

Research

JoVE Journal

Biology

10.0K 次观看.  Pacific Northwest Diabetes Research Institute. 四分体启用条码序列（BEST）取代分离，破坏和间距四分体的手动流程。的最优隔离四分体通过荧光激活细胞分选到琼脂平板上，通过搅拌与玻璃珠分离出孢子，并确定哪个随机排列菌落，从同一原始四分使用分子条形码而得。 

视频: BEST: Barcode Enabled Sequencing of Tetrads

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

科研

生物学

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

罕见的基因变异检测从集资使用斯普林特测序

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

测绘细菌功能的网络和途径<em&gt;大肠杆菌</em使用合成基因阵列

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

的16S核糖体RNA基因的扩增子的新一代测序

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

有针对性的DNA甲基化分析通过新一代测序

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

3' End Sequencing Library Preparation with A-seq2

Studies in the last decade have revealed a complex and dynamic variety of pre-mRNA cleavage and polyadenylation reactions. mRNAs with long 3&#39; untranslated regions (UTRs) are generated in differentiated cells whereas proliferating cells preferentially express transcripts with short 3&#39;UTRs. We describe the A-seq protocol, now at its second version, which was developed to map polyadenylation sites genome-wide and study the regulation of pre-mRNA 3&#39; end processing. Also this current protocol takes advantage of the polyadenylate (poly(A)) tails that are added during the biogenesis of most mammalian mRNAs to enrich for fully processed mRNAs. A DNA adaptor with deoxyuracil at its fourth position allows the precise processing of mRNA 3&#39; end fragments for sequencing. Not including the cell culture and the overnight ligations, the protocol requires about 8 h hands-on time. Along with it, an easy-to-use software package for the analysis of the derived sequencing data is provided. A-seq2 and the associated analysis software provide an efficient and reliable solution to the mapping of pre-mRNA 3&#39; ends in a wide range of conditions, from 106 or fewer cells.

3&#039; End Sequencing Library Preparation with A-seq2

This protocol describes a method for mapping pre-mRNA 3&#39; end processing sites.

3 A-seq2 的末端测序库的研制

Studies in the last decade have revealed a complex and dynamic variety of pre-mRNA cleavage and polyadenylation reactions. mRNAs with long 3&#39; ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

三维白色脂肪组织结构的全装染色可视化

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

Assessment of the Metabolic Effects of Isocaloric 2:1 Intermittent Fasting in Mice

Intermittent fasting (IF), a dietary intervention involving periodic energy restriction, has been considered to provide numerous benefits and counteract metabolic abnormalities. So far, different types of IF models with varying durations of fasting and feeding periods have been documented. However, interpreting the outcomes is challenging, as many of these models involve multifactorial contributions from both time- and calorie-restriction strategies. For example, the alternate day fasting model, often used as a rodent IF regimen, can result in underfeeding, suggesting that health benefits from this intervention are likely mediated via both caloric restriction and fasting-refeeding cycles. Recently, it has been successfully demonstrated that 2:1 IF, comprising 1 day of fasting followed by 2 days of feeding, can provide protection against diet-induced obesity and metabolic improvements without a reduction in overall caloric intake. Presented here is a protocol of this isocaloric 2:1 IF intervention in mice. Also described is a pair-feeding (PF) protocol required to examine a mouse model with altered eating behaviors, such as hyperphagia. Using the 2:1 IF regimen, it is demonstrated that isocaloric IF leads to reduced body weight gain, improved glucose homeostasis, and elevated energy expenditure. Thus, this regimen may be useful to investigate the health impacts of IF on various disease conditions.

The current article describes a detailed protocol for isocaloric 2:1 intermittent fasting to protect and treat against obesity and impaired glucose metabolism in wild-type and ob/ob mice.

小鼠间间间禁食的代谢效应评估 2：1 间歇禁食

Intermittent fasting (IF), a dietary intervention involving periodic energy restriction, has been considered to provide numerous benefits and counteract ...