这项工作的经费部分是由 NSF 植物基因组研究 (IOS-1127155) 的赠款提供的。

注意: 图 1 显示了数组计算全息协议的五步骤。它们是: 1) 制备植物材料;2) 分离高质量的 DNA 样本;3) DNA 样品的标记和纯化;4) 对整个基因组阵列进行杂交、冲洗和扫描;和 5) 计算全息数据分析。 m 蒺藜 整个基因组平铺阵列包含总共971041种独特的低聚体探针, 其目标是基因组中超过5万基因或基因模型 (参见 材料表 )。独特的探针间隔约每150基对 (bp) 在 exonic 地区和 261 bp 在子地区的 m 蒺藜 基因组. 
 1. 植物材料的制备 <ol> <li> 划破野生类型 (重量; m 蒺藜 cv Jemalong A17) 和 FN6191 突变种子, 浓缩硫酸为8分钟 (参见 材料表 )。将硫酸移至废液容器中. </li>
	<li> 用蒸压去离子水冲洗种子三次. </li>
	<li> 表面消毒20% 漂液的种子10分钟 </li>
	<li> 用蒸压去离子水冲洗种子三次. </li>
	<li> 将种子储存在4和 #176; C 为3天。在春后, 在土壤中将种子在 16 h/8 h 光/暗循环和150和 #181 的生长腔中迁移和发芽一周; 电子/m 2 /秒的光照强度. </li>
	<li> 将幼苗移植到1加仑的花盆中, 在一个温室中生长 16 h/8 h 光/暗循环, 150 和 #181; 电子/m 2 /s 光强度为三周至四星期。收集来自植物的幼叶样本进行 DNA 分离. </li>
</ol> 2。高质量 DNA 样品的分离 <ol> <li> 从一株植物中提取1克幼叶组织, 并立即将其冷冻在液氮中. </li>
	<li> 在液氮中用砂浆和杵将冷冻叶组织研磨成细粉. </li>
	<li> 使用 dna 隔离套件分离基因组 dna (请参阅 材料表 ). </li>
	<li> 重50和 #181 纯化的 DNA 样品; 高压蒸双去离子 H 2 O (ddH 2 o) 或 1x TE 缓冲器 (10 毫米三盐酸, 1 毫米 EDTA, pH 7.0). </li>
	<li> 用分光光度计测量 DNA 浓度 (请参阅 材料表 ). </li>
	<li> 评估 260 nm/280 nm 和 260 nm/230 纳米 DNA 样本的比值. 
	注: 高质量的 DNA 样品应具有 260 nm/280 纳米比和 #8805; 1.8 和 260 nm/230 nm 比值和 #8805; 2.0. </li>
	<li> 通过在1% 琼脂糖凝胶中运行, 进一步评估 DNA 样本. 
	注: 高质量的 DNA 样品应具有较高的分子量, 不应被 RNA 污染。理想的情况下, DNA 样本的最终浓度应该是 150 ng/和 #181; L. </li>
</ol> 3。DNA 标记和纯化 <ol> <li> 稀释1和 #181; g 基因组 DNA 样品与 ddH 2 O 到最后的容量20和 #181; l 在500和 #181; l 离心管。
	<li> 添加5和 #181; L 随机入门 (请参阅 材料表 ) 到管道. </li>
	<li> 涡流管短暂, 并把它放入一个 thermocycler 孵化和变性 DNA 在98和 #176; C 为 10 min. </li>
	<li> 把管子从 thermocycler 中取出, 立即放在冰水上, 冷却5分钟. </li>
	<li> 准备两个标记混合, 如下所示 表 1 . </li>
	<li> 添加25和 #181; 标记混合1和 2, 分别引用 dna 和取样 dna 管. </li>
	<li> 将标签混合和 DNA 混合移三倍, 然后简单地旋转试管. </li>
	<li> 将管子放入 thermocycler, 在37和 #176 孵育; c 为2小时, 然后在65和 #176; c 为20分钟, 使外克莱诺酶钝化 (请参阅 材料表 ). </li>
	<li> 添加430和 #181; 1x 和 #160 的 L; 每根管子的缓冲器. </li>
	<li> 混合内容并简单地旋转管子. </li>
	<li> 将管道中的溶液转换为2毫升收集管的净化柱 (请参阅 材料表 ). </li>
	<li> 离心纯化柱在 1.4万 x g 为10分钟. </li>
	<li> 放弃传递解决方案. </li>
	<li> 在每列中添加480和 #181; L 1x 和 #160; TE 缓冲区. </li>
	<li> 在 1.4万 x g 上再次离心该列10分钟. </li>
	<li> 丢弃 pass-through 解决方案. </li>
	<li> 将残留在柱底部的标记 DNA 样本转移到新的离心管上. </li>
	<li> 使用吸管测量标记的 DNA 样本的体积, 并将最后的体积带到80和 #181; L 带有 1x TE 缓冲区. </li>
	<li> 使用分光光度计测量标记的 DNA 样本的浓度 (请参见 材料表 ). </li>
	<li> 混合等量的标记样本 dna 和参考 dna, 并将最终的体积带到160和 #181; L 与 #160; 1x TE 缓冲区. </li>
	<li> 添加256和 #181; l 2x 杂交缓冲, 50 和 #181; 人类 Cot-1 DNA 和50和 #181; l 10 和 #215; aCGH 阻塞代理 (请参阅 材料表 ), 并将它们移为三次. </li>
	<li> 旋转导管并将其放入 thermocycler, 在98和 #176 中孵育; C 为 10 min. </li>
	<li> 在37和 #176 孵育试管; C 为20分钟. </li>
</ol> 4。基因组阵列的杂交、洗涤和扫描 <ol> <li> 将杂交烤箱预热到67和 #176; 在杂交开始前至少4小时. </li>
	<li> 将垫片滑动到杂交室的底部. </li>
	<li> 从步骤3.23 中加载490和 #181; L 的杂交溶液到垫片滑动. </li>
	<li> 用一条 苜蓿蒺藜 基因芯片芯片来覆盖衬垫滑动, 形成杂交室. </li>
	<li> 投入杂交室盖子和紧固用夹子的房间. </li>
	<li> 将组装的杂交室放入杂交烤箱, 孵育67和 #176; C 为 40-48 h. </li>
	<li> 准备三滑动洗碗碟: 两个与250毫升洗涤缓冲区, 我保持在室温下, 一个与洗涤缓冲器 II 保持在37和 #176; C. </li>
	<li> 准备两个滑动洗涤罐: 一个带有70毫升乙腈, 一个带有70毫升稳定和干燥溶液; 在室温下保持通风罩 (请参阅 材料表 ). </li>
	<li> 将杂交室从烤箱中取出, 取出箱夹和盖子. </li>
	<li> 将微阵列芯片和垫片滑动到带洗涤缓冲器的滑动洗涤盘中, 并将芯片与盖板分开. </li>
	<li> 将微阵列芯片移动到第二个带洗涤缓冲的滑动洗涤盘中, 然后在室温下用搅拌板轻轻地将溶液循环到5分钟. </li>
	<li> 将微阵列芯片转移到第三个滑动洗涤盘上, 用5ml 洗涤缓冲器 II, 在37和 #176 的磁性搅拌板上轻轻地用搅拌棒冲洗; C 为 1 min. </li>
	<li> 将微阵列芯片转移到一个通风柜中, 并将其洗净三十年代. </li>
	<li> 将微阵列芯片转移到带有稳定和干燥溶液的滑动洗涤罐中, 并将其洗净三十年代. </li>
	<li> 慢慢取出芯片, 晾干1分钟. </li>
	<li> 使用扫描仪扫描微阵列芯片 (请参阅 2 和 #181; m 分辨率下的材料表 )。设置参数以保存多个图像和整个幻灯片扫描。将通道1和2的增益设置为 100%, 并取消自动增益. </li>
</ol> 5。计算全息数据分析 <ol> <li> 提取 Cy3 和 Cy5 荧光使用扫描软件对阵列上每个探测器的强度 (请参见 材料表 ). </li>
	<li> 使用软件的分段分析来检测样本 dna 和参考 dna 之间的拷贝数变化 (cnv)。使用段的阈值意味着日志 2 采样/参考比和 #177; 2.5 标准偏差用于确定 cnv. </li>
	<li> 使用信号映射软件 (请参阅 材料表 ) 在整个基因组中检查日志的分布情况 2 样本/参考比率. </li>
</ol>

<ol>
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E., Finkel, S. E., Tavare, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+expression+arrays+for+copy+number+detection:+an+example+from+E.+coli.">Using expression arrays for copy number detection: an example from E. coli.</a> BMC Bioinformatics. 8, 203 (2007).</li><li>Werner, J. D., 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=Quantitative+trait+locus+mapping+and+DNA+array+hybridization+identify+an+FLM+deletion+as+a+cause+for+natural+flowering-time+variation.">Quantitative trait locus mapping and DNA array hybridization identify an FLM deletion as a cause for natural flowering-time variation.</a> Proc Natl Acad Sci U S A. 102 (7), 2460-2465 (2005).</li><li>Xi, J., Chen, Y., Nakashima, J., Wang, S. M., Chen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medicago+truncatula+esn1+defines+a+genetic+locus+involved+in+nodule+senescence+and+symbiotic+nitrogen+fixation.">Medicago truncatula esn1 defines a genetic locus involved in nodule senescence and symbiotic nitrogen fixation.</a> Mol Plant Microbe Interact. 26 (8), 893-902 (2013).</li><li>Schnabel, E., Journet, E. P., de Carvalho-Niebel, F., Duc, G., Frugoli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Medicago+truncatula+SUNN+gene+encodes+a+CLV1-like+leucine-rich+repeat+receptor+kinase+that+regulates+nodule+number+and+root+length.">The Medicago truncatula SUNN gene encodes a CLV1-like leucine-rich repeat receptor kinase that regulates nodule number and root length.</a> Plant Mol Biol. 58 (6), 809-822 (2005).</li><li>Horvath, B., 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=Loss+of+the+nodule-specific+cysteine+rich+peptide,+NCR169,+abolishes+symbiotic+nitrogen+fixation+in+the+Medicago+truncatula+dnf7+mutant.">Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant.</a> Proc Natl Acad Sci U S A. 112 (49), 15232-15237 (2015).</li><li>Kim, M., 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=An+antimicrobial+peptide+essential+for+bacterial+survival+in+the+nitrogen-fixing+symbiosis.">An antimicrobial peptide essential for bacterial survival in the nitrogen-fixing symbiosis.</a> Proc Natl Acad Sci U S A. 112 (49), 15238-15243 (2015).</li><li>. Medicago truncatula Mutant Database Available from: <a target="_blank" href="https://medicago-mutant.noble.org/mutant/FNB.php">https://medicago-mutant.noble.org/mutant/FNB.php</a> (2017)</li><li>Burton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNP+genotyping+with+the+next+generation+of+CGH+microarray.">SNP genotyping with the next generation of CGH microarray.</a> MLO Med Lab Obs. 45 (7), (2013).</li></ol>

作者声明没有竞争的金融利益。

我们已经开发了一个基于阵列的计算全息平台, 用于检测和表征快中子轰击 (FNB) 诱发突变体的m 蒺藜cv Jemalong A17。为了证明阵列全息计算方法在检测基因突变中的应用, 我们对突变 FN6191 进行了 aCGH 分析, 并与野生型植物形成了高结瘤表型, 并与S. 根瘤菌 Sm1021进行了接种。对于分段分析, 如果段中的探测器的日志2比率的平均值高于上限阈值或低于该给定数组比较的下限阈值, 则?...

豆类 (豆科) 是第三大家族的开花植物, 与许多经济上重要的物种, 如大豆 (甘氨酸 max) 和苜蓿 (苜蓿)。豆科植物可以与固氮土壤细菌相互作用, 通常称为根瘤菌来发展根结节, 将大气氮减少到氨供寄主植物使用。因此, 种植豆类作物需要很少的氮肥投入, 从而有助于可持续农业。豆类作物生产的叶子和种子的高蛋白含量, 作为优良的饲料和谷物作物。然而, 种植豆科植物通常有复杂的基因组结构, 使功能研究的基因发挥关键作用在豆类特定的过程繁琐。苜蓿蒺藜已被广泛采用为豆科植物的模型, 主要是因为 (1) 它有一个相对较小的单倍体基因组大小 (~ 550) 的二倍体基因组;(2) 植物可稳定转化为基因功能研究;和 (3) 它与紫花苜蓿 (m), 牧草皇后, 和许多其他经济重要的农作物的转化研究密切相关。最近, m 蒺藜cv Jemalong A17 的基因组序列已被释放12。对基因组的诠释显示, 基因组中有超过5万预测基因或基因模型。要确定m 蒺藜基因组中大多数基因的功能是一项具有挑战性的任务。为了促进基因的功能研究, 在 15万 M1线范围内, 利用快中子轰击 (FNB) 诱变, 在m. 蒺藜cv Jemalong A173中生成了一组突变体的综合集合.,4。快中子, 高能电离诱变, 已被用于在许多植物物种中生成突变体, 包括拟南芥5,6, 大米 (水稻品种)7, 西红柿 (茄汁番茄), 黄豆 (甘氨酸大豆;G. max)8,9, 大麦 (大麦大麦), 和莲花日本人10。从 FNB 诱变衍生出的很大一部分突变是由于 DNA 的缺失, 其范围从几个碱基对到巨型碱基对9,11。许多表型相关基因已被成功识别并具有特征4、12、13、14、15、16、17,18,19. 以前, FNB 突变体的潜在基因的分子克隆依赖于地图的方法, 这是时间消耗和限制分子水平的突变体数量。最近, 一些免费的方法, 包括 transcript-based 方法, 基因组平铺阵列的比较基因组杂交 (计算全息) 的 DNA 拷贝数变异检测, 和整个基因组测序, 已被用来促进不同生物体 (包括动植物) 中缺失突变体的特征20,21,22,2324,25 26,27,28,29,30,31。
为了便于在m. 蒺藜中对 FNB 突变体的定性, 开发并验证了基于基因组的整体比较基因组杂交 (计算全息) 平台。正如在动物系统中报告的那样, 基于阵列的全息计算平台允许在m. 蒺藜FNB 突变体的整个基因组水平检测拷贝数变体 (cnv)。此外, 病变可以通过 PCR 确认和删除边界可以确定的排序。总的来说, 阵列全息计算平台是一个有效的工具, 以识别病变的m 蒺藜FNB 突变体。本文对m 蒺藜FNB 突变体的阵列全息程序和删除边界的 PCR 特征进行了说明。
以下协议提供了实验步骤和有关试剂、设备和分析工具的信息, 这些研究人员有兴趣执行整个基因组阵列的比较基因组杂交 (计算全息) 拷贝数分析植物的变异。例如,苜蓿蒺藜FN6191 突变体用于识别与突变表型相关的缺失区域和候选基因。m 蒺藜FN6191 突变体, 最初是从快中子轰击引发的删除突变的集合中分离出来的32 (参见材料表), 在接种后用土壤展示了一个超级结瘤表型细菌, Sihorhizobium 根瘤菌Sm1021, 与野生型植物形成对比。

<table>
	<tbody>
		<tr>
			<td>Medicago truncatula genome array, 1 x 1 M</td>
			<td>Agilent</td>
			<td>G4123A</td>
			<td></td>
		</tr>
		<tr>
			<td>Medicago truncatula FN6191 (mutant)</td>
			<td>In house</td>
			<td>FN6191</td>
			<td></td>
		</tr>
		<tr>
			<td>Medicago truncatula Jemalong A17 (reference)</td>
			<td>In house</td>
			<td>A17</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Sigma-Aldrich</td>
			<td>320501</td>
			<td></td>
		</tr>
		<tr>
			<td>DNeasy Plant Mini Kit</td>
			<td>Qiagen</td>
			<td>69104</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>1000D</td>
			<td></td>
		</tr>
		<tr>
			<td>SureTag DNA Labeling Kit</td>
			<td>Agilent</td>
			<td>5190-3400</td>
			<td></td>
		</tr>
		<tr>
			<td>Random primer</td>
			<td>Agilent</td>
			<td>5190-3399</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>271004-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>MJ research</td>
			<td>PTC-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Labnet international Inc</td>
			<td>Spectrafuge 24D</td>
			<td></td>
		</tr>
		<tr>
			<td>Stabilization and Drying Solution</td>
			<td>Agilent</td>
			<td>5185-5979</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo aCGH/ChIP-on-chip Hybridization Kit</td>
			<td>Agilent</td>
			<td>5188-5380</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Chamber gasket slides</td>
			<td>Agilent</td>
			<td>G2505</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Cot-1 DNA</td>
			<td>Agilent</td>
			<td>5190-3393</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo aCGH/ChIP-on-chip Wash Buffer 1 and 2</td>
			<td>Agilent</td>
			<td>5188-5221</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Chamber, stainless</td>
			<td>Agilent</td>
			<td>G2534A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Agilent</td>
			<td>G2545A</td>
			<td></td>
		</tr>
		<tr>
			<td>Purification Columns</td>
			<td>Agilent</td>
			<td>5190-3391</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser scanner</td>
			<td>Roche</td>
			<td>MS200</td>
			<td></td>
		</tr>
		<tr>
			<td>NimbleScan 2.6</td>
			<td>Roche Nimblegen</td>
			<td>5225035001</td>
			<td></td>
		</tr>
		<tr>
			<td>Signal Map 1.9</td>
			<td>Roche Nimblegen</td>
			<td>Signalmap1.9</td>
			<td></td>
		</tr>
	</tbody>
</table>

an array-based comparative genomic hybridization platform for efficient detection of copy number variations in fast neutron-induced medicago truncatula mutants

突变体是基因功能研究的宝贵的遗传资源。为了产生突变体集合, 可利用三种诱变, 包括生物如 T-脱氧核糖核酸或转, 化学物质如甲基磺酸乙酯 (EMS), 或物理如电离辐射。所观察到的突变类型因所使用的诱变而异。对于电离辐射诱发突变体, 突变包括删除, 重复, 或重排。虽然 T-DNA 或 transposon-based 诱变仅限于易受转化的物种, 但化学或物理诱变可应用于种类繁多的物种。然而, 从化学或物理诱变衍生的突变的定性传统上依赖于地图的克隆方法, 这是劳动密集型和耗时。在这里, 我们表明, 一个高密度基因组阵列的比较基因组杂交 (aCGH) 平台可用于有效地检测和表征拷贝数变异 (cnv) 的突变从快中子轰击 (FNB) 突变中获得的苜蓿蒺藜, 豆科植物。整个基因组序列分析显示, 在m 蒺藜中有超过5万基因或基因模型。目前, FNB 诱发突变体的m. 蒺藜是从超过 15万 M1 线, 代表了宝贵的基因资源的功能研究的基因组。这里描述的 aCGH 平台是一个有效的工具, 用于表征 FNB 诱导突变体在m 蒺藜。

图 2显示了在整个基因组中, 突变体与重量信号的归一化对数2比值的分布。对计算全息数据的分析显示, 4 号染色体上大约有 22 kb 的删除, 它包含了整个SUNN基因33和 FN6191 突变体中的其他一些注释基因 (图 2,图 3)。候选删除区域被73连续探针覆盖, 该阵列的平均正常化日志2...

Watch this Scientific Journal Video about An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutant

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

该协议提供了实验步骤和有关试剂、设备和分析工具的信息, 这些研究人员有兴趣执行整个基因组阵列的比较基因组杂交 (计算全息) 对拷贝数变化的分析植物.

一种基于阵列的比较基因组杂交平台, 有效检测快中子诱导的苜蓿蒺藜突变体的拷贝数变化

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including ...

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An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

Unit 2

Genomes and Evolution

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7.7K 次观看.  Noble Research Institute. 该协议提供了实验步骤和有关试剂、设备和分析工具的信息, 这些研究人员有兴趣执行整个基因组阵列的比较基因组杂交 (计算全息) 对拷贝数变化的分析植物.

视频: An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. This approach to colorimetric DNA detection was based on the hybridization-mediated growth of gold nanoparticle probes (AuNP probes). The growth size and configuration of the AuNP are dominated by the number of DNA samples hybridized with the probes. Based on the specific size- and shape-dependent optical properties of the nanoparticles, the number of mismatches in a sample DNA fragment to the probes is able to be discriminated. The tests were conducted via droplets containing reagents and DNA samples respectively, and were transported and mixed on the pneumatic platform with the controlled pneumatic suction of the flexible PDMS-based superhydrophobic membrane. Droplets can be delivered simultaneously and precisely on an open-surface on the proposed pneumatic platform that is highly biocompatible with no side effect of DNA samples inside the droplets. Combining the two proposed methods, the multi-nucleotide polymorphism can be detected at sight on the pneumatic droplet manipulation platform; no additional instrument is required. The procedure from installing the droplets on the platform to the final result takes less than 5 min, much less than with existing methods. Moreover, this combined MNP detection approach requires a sample volume of only 10 &#181;l in each operation, which is remarkably less than that of a macro system.

This work presents a simple and visual method to detect multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform. With the proposed method, the entire experiment, including droplet manipulation and detection of multi-nucleotide polymorphisms, can be performed near 23 &#176;C without the aid of advanced instruments.

在气动操纵液滴平台多核苷酸多态性的视觉比色法检测

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. ...

科研

遗传学

Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved (Non-model) Organisms

This workflow allows novice researchers to leverage advanced computational resources such as cloud computing to carry out pairwise comparative transcriptomics. It also serves as a primer for biologists to develop data scientist computational skills, e.g. executing bash commands, visualization and management of large data sets. All command line code and further explanations of each command or step can be found on the wiki (<a href="https://wiki.cyverse.org/wiki/x/dgGtAQ">https://wiki.cyverse.org/wiki/x/dgGtAQ</a>). The Discovery Environment and Atmosphere platforms are connected together through the CyVerse Data Store. As such, once the initial raw sequencing data has been uploaded there is no more need to transfer large data files over an Internet connection, minimizing the amount of time needed to conduct analyses. This protocol is designed to analyze only two experimental treatments or conditions. Differential gene expression analysis is conducted through pairwise comparisons, and will not be suitable to test multiple factors. This workflow is also designed to be manual rather than automated. Each step must be executed and investigated by the user, yielding a better understanding of data and analytical outputs, and therefore better results for the user. Once complete, this protocol will yield de novo assembled transcriptome(s) for underserved (non-model) organisms without the need to map to previously assembled reference genomes (which are usually not available in underserved organism). These de novo transcriptomes are further used in pairwise differential gene expression analysis to investigate genes differing between two experimental conditions. Differentially expressed genes are then functionally annotated to understand the genetic response organisms have to experimental conditions. In total, the data derived from this protocol is used to test hypotheses about biological responses of underserved organisms.

Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved Non-model Organisms

This protocol outlines a comparative de novo transcriptome assembly and annotation workflow for novice bioinformaticians. The workflow is available for free entirely through CyVerse and connected by the Data Store. Command line and graphical user interfaces are used, but all code needed is available to copy and paste.

利用CyVerse资源<em&gt; De Novo</em&gt;不合格（非模型）生物的比较转录组学

This workflow allows novice researchers to leverage advanced computational resources such as cloud computing to carry out pairwise comparative ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

通过多色荧光跨染色体畸变稳定的检测<em&gt;原位</em&gt;杂交（渔业部）和光谱核型分析（SKY）的照射小鼠

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver staining is a widely used method for the detection of SSR markers in a polyacrylamide gel. However, conventional protocols for silver staining are technically demanding and time-consuming. Like many other biological laboratory techniques, silver staining protocols have been steadily optimized to improve detection efficiency. Here, we report a simplified silver staining method that significantly reduces reagent costs and enhances the detection resolution and picture clarity. The new method requires two major steps (impregnation and development) and three reagents (silver nitrate, sodium hydroxide, and formaldehyde), and only 7 min of processing for a non-denaturing polyacrylamide gel. Compared to previously reported protocols, this new method is easier, quicker and uses fewer chemical reagents for SSR detection. Therefore, this simple, low-cost, and effective silver staining protocol will benefit genetic mapping and marker-assisted breeding by a quick generation of SSR marker data.

Here, we report a simple and low-cost silver staining protocol which requires only three reagents and 7 min of processing, and is suitable for fast generation of high-quality SSR data in the genetic analysis.

一种快速的银染色协议, 能够使用非变性聚丙烯酰胺凝胶对 SSR 标记进行简单而有效的检测

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver ...

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the transgene, as multi-copy integrations are often subjected to gene silencing. The Gateway-compatible binary vector based on bacterial artificial chromosomes (pBIBAC-GW), like other pBIBAC derivatives, allows the insertion of single-copy transgenes with high efficiency. As an improvement to the original pBIBAC, a Gateway cassette has been cloned into pBIBAC-GW, so that the sequences of interest can now be easily incorporated into the vector transfer DNA (T-DNA) by Gateway cloning. Commonly, the transformation with pBIBAC-GW results in an efficiency of 0.2&#8211;0.5%, whereby half of the transgenics carry an intact single-copy integration of the T-DNA. The pBIBAC-GW vectors are available with resistance to Glufosinate-ammonium or DsRed fluorescence in seed coats for selection in plants, and with resistance to kanamycin as a selection in bacteria. Here, a series of protocols is presented that guide the reader through the process of generating transgenic plants using pBIBAC-GW: starting from recombining the sequences of interest into the pBIBAC-GW vector of choice, to plant transformation with Agrobacterium, selection of the transgenics, and testing the plants for intactness and copy number of the inserts using DNA blotting. Attention is given to designing a DNA blotting strategy to recognize single- and multi-copy integrations at single and multiple loci.

Using a pBIBAC-GW binary vector makes generating transgenic plants with intact single-copy insertions, an easy process. Here, a series of protocols is presented that guide the reader through the process of generating transgenic Arabidopsis plants, and testing the plants for intactness and copy number of the inserts.

用 BIBAC 二元矢量生成单拷贝插入的转基因植物

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

利用 CRISPR/Cas9 Ribonucleoproteins 快速简便地生成线虫基因组点突变体的管道

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

全挂载原位杂交法可视化蜱内脏微生物群

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

慢病毒载体平台, 用于将表观基因编辑工具高效地传递到人类诱导的多能干细胞衍生疾病模型中

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

一种基于阵列的比较基因组杂交平台, 有效检测快中子诱导的苜蓿蒺藜突变体的拷贝数变化

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