Name: tick microbiome characterization by next-generation 16s rrna amplicon sequencing
Uploaded: 2018-08-25T14:45:00.000+00:00
Duration: 7 min 21 s
Description: Watch this Scientific Journal Video about Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing at JoVE.com

这项工作得到国家科学基金会赠款的支持, 犍为县 (DEB #1427772, 1745411, 1750037)。

1. 蜱收集和表面灭菌
<ol><li>通过在蜱相关的栖息地上拖动1米2白色布来收集刻度, 去除附着在寄主物种上的蜱, 或在实验室15、16中饲养蜱。使用细钳操纵蜱和储存在-80 摄氏度。</li>
	<li>将蜱在各自的 PCR 管和去除表面污染物由涡流连续十五年代与 500 ul 过氧化氢 (H2O2), 70% 乙醇和 ddH2O。</li>
	<li>将刻度放在新的 PCR 管中, 让它们风干。</li>
	<li>在这个管子里, 机械地扰乱蜱组织通过用灰浆和杵粉碎蜱 (在本研究中使用), 使用小珠子在一个珠子搅拌器, 或削减蜱分离与手术刀。</li>
</ol>2. DNA 纯化
<ol><li>按照商业上可用的 dna 提取试剂盒中提供的指示, 从单个刻度中纯化 DNA。洗脱为 100 ul 的最后容量。 注: 有关步骤 2.2-2.8 的概述, 请参阅图 1 。替代萃取方法包括苯酚-氯仿萃取17,18, 乙醇沉淀19, 或萃取与螯合物质20,21。</li>
	<li>使用荧光计或分光光度计确认成功的 DNA 纯化。对于荧光法, 在 190 ul 双链 dna 检测中使用 10 ul 的 dna 模板。预期产量为 0.1-1.0 ng/ul22。对于分光光度法, 在核酸定量中使用 1 ul 的 DNA 模板。预期的 A260/280 比率约为 1.823。</li>
	<li>如果不立即进行放大, 将样品存储在摄氏-20 摄氏度。</li>
</ol>3. 16S rRNA 基因放大
<ol><li>建立扩增子 PCR 在 27.5 ul 反应包含: 5 ul 每底漆在1微米;12.5 ul 商业上可利用的 PCR 组合;和 5 ul 的 DNA 从单个蜱提取的 5 ng/ul 浓度。 注: 使用以下引物放大 16S rRNA 基因13的高变 V3-V4 区域: 5 '-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3 ', 5 '-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3 '</li>
	<li>将管移动到 thermocycler 程序中: 初始变性在95°c 为3分钟;25周期 1) 95 °c 为三十年代, 2) 57 °c 为三十年代, 3) 72 °c 为三十年代;最后延伸在72°c 为5分钟;最后举行10摄氏度。</li>
	<li>pcr 完成后, 通过在1.5% 琼脂糖凝胶上加载 4-6 ul/样品, 可视化 pcr 产品。寻找一个 460 bp 的波段来确认放大。 注: 如果样品浓度过低 (< 10), 扩增子 PCR 产品在凝胶上可能不可见。底漆二聚体存在是常见的低浓度样品。如果存在其他非目标带, 则调整退火温度或减少循环次数。</li>
	<li>如果不立即进行净化, 将样品贮存在摄氏4摄氏度。</li>
</ol>4. 16S 扩增子净化
<ol><li>使用新的 pcr 管为每个样品, 结合 pcr 产品与 ddH2O 获得 60 ul 总数。对于低 DNA 浓度的样品, 进行三扩增子 PCR, 以减少扩增偏倚, 并汇集样品集中。</li>
	<li>在使用前, 将顺磁珠放在室温下, 使其涡流良好。</li>
	<li>添加 48 ul 的顺磁珠到 60 ul 的样品和孵化5分钟. 把管子放在磁力架上5分钟, 直到溶液变得清晰。移除上清。 注: 此珠浓度的目标是去除底漆脂肪酸 (< 60 bp)。如果需要, 调整珠浓度, 以消除其他非特定的带状。</li>
	<li>使用磁性机架上的管, 添加 500 ul 新准备的80% 乙醇。在将乙醇加入所有管子后立即将液体吸出。不要去掉珠子。重复乙醇添加和删除一次。</li>
	<li>空气干燥的样品, 以消除过剩的乙醇通过留管打开在磁架上5分钟, 或直到小裂纹是可见的珠子。</li>
	<li>添加 20 ul 的 TE 缓冲器, 并在室温下将样品从磁铁上孵化出来, 5-10 分钟。</li>
	<li>把管子放回磁铁上。一旦珠和液体分离, 转移上清到一个新鲜的管, 以获得清洁的 PCR 产品。</li>
	<li>选通过在1.5% 凝胶上加载 4-6 ul/样品, 可视化产品以确认成功的扩增子纯化。460 bp 波段应该是可见的, 不应该有底漆二聚体。</li>
	<li>如果不立即进行指数 PCR, 存储样品4°c。</li>
</ol>5. 样品条形码和纯化
<ol><li>通过从商业上可用的库索引工具包中选择正向或反向底漆, 或同时为每个样本分配唯一的底漆组合。 注: 独特的标签样本允许在测序后的差异。套件通常提供足够的引物, 以序列96-384 样品。</li>
	<li>将双引物 (或指数) 附在 25 ul 反应中, 其中包括每个底漆的 2.5 ul (N7xx 和 S5xx), 12.5 ul 的商用 pcr 主混合, 5 ul ddH2O, 和 2.5 ul 清洁扩增子产品。</li>
	<li>将管子移动到 thermocycler 程序中: 最初变性在95°c 为3分钟;8-14 周期 1) 95 °c 为三十年代, 2) 55 °c 为三十年代, 3) 72 °c 为三十年代;最后延伸在72°c 为5分钟;最后举行10摄氏度。</li>
	<li>pcr 完成后, 通过在1.5% 琼脂糖凝胶上加载 4-6 ul/样品, 可视化 pcr 产品。寻找一个 550 bp 的波段来确认放大。 注: 使用可视化结果告知指数 PCR 循环条件。降低循环计数以减少非特定绑定或增加循环计数以获取每个示例的可见带区。</li>
	<li>重复步骤4中列出的清理过程。为了避免在清理过程中稀释, 在重复执行指数 PCR, 并在这里汇集产品。</li>
	<li>如果不立即进行图书馆量化和规范化, 将样品储存在摄氏4摄氏度。</li>
</ol>6. 图书馆的量化和规范化
<ol><li>使用荧光计或分光光度计估计步骤5.5 中每种纯化的条形码产品的浓度 (见步骤 2.2)。稀释在 TE 缓冲器中的样品, 以获得大约下午1点的浓度。 注: 为实现测序平台推荐的样品加载浓度, 需要进行库量化。图书馆量化是通过 qPCR 实现的, 但在 qPCR 之前估计样本浓度会节省试剂和时间。建议在下午1点集中提高图书馆量化的准确性, 因为这是量化工具包24中提供的 qPCR 标准的平均集中。</li>
	<li>执行 qPCR 在 10 ul 反应包含 6.0 ul qPCR 大师混合 (从图书馆定量套件), 2.0 ul 的 ddH2O, 和 2.0 ul 的样本或标准。每三个样品和标准三份, 以提高准确性和精确度。以三或更多稀释级别 (e.、0.1 pm、下午1点和下午10点) 运行每个样本, 以确定起始浓度, 以确保准确量化。 注: 有关所提供的底漆和标准的详细信息将根据所使用的量化试剂盒而有所不同, 并可在产品技术数据表中获得。请参阅本研究中使用的量化试剂盒的材料表。</li>
	<li>将 qPCR 板或管移动到一个实时的 PCR 仪器程序:95 °c 的初始变性为5分钟;35周期 1) 95 °c 为三十年代和 2) 60 °c 四十五年代;并且离解步 1) 95 °c 为十五年代, 2) 60 °c 为三十年代和 3) 95 °c 为十五年代。</li>
	<li>计算每个样本的平均起始浓度, 使用从 qPCR 结果中获得的量化值和所使用的样本稀释水平。 注: 例如, 平均浓度为下午2点的样品稀释1:100,000 产生初始浓度为 200 nM。如果给定样本的稀释水平不属于标准曲线的范围, 则量化结果可能不准确;在这种情况下, 调整稀释水平和重新执行 qPCR。</li>
	<li>根据步骤7.5 中计算的平均浓度, 将每个纯化的条形码样品稀释到 TE 缓冲器中的 4 nM。 注: 例如, 对于平均样品浓度为200毫微米, 稀释样品1:50 在 TE 缓冲器达到 4 nM 浓度。精确的样品浓度将根据所使用的测序系统和试剂盒而异。有关推荐浓度, 请参阅顺序系统用户指南。</li>
	<li>通过将所有单个库的相等卷 (通常为 5-10 ul) 添加到单个管道中, 创建组合库。 注: 由于所有样品的浓度均应为 4 nM, 添加等量的体积可达到汇集库中所有样品的同等浓度。</li>
	<li>对组合库重复步骤 6.2-6.4, 以确认 4 nM 浓度。</li>
	<li>计算组合库浓度, 稀释或重新构成最终的, 汇集库使用三缓冲器, 以达到 4 nM。</li>
	<li>如果不立即进行样品装载, 存储样品在-20 摄氏度。在量化后不久执行顺序运行, 以最大限度地减少存储期间 DNA 浓度的损失或变化。</li>
</ol>7. 图书馆变性和稀释, 以及顺序运行 (当天执行)
<ol><li>变性4毫微米联合图书馆从步骤6.9 与氢氧化钠。 注意: 这些最终的图书馆准备步骤因每个测序系统而异。有关更详细和更新的协议, 请参阅序列系统用户指南。新用户可能需要接受对测序平台使用的培训, 或者可能将他们的库发送到一个核心排序设施。</li>
	<li>使用序列试剂盒 (材料表) 中提供的缓冲区稀释变性库, 使其达到所需的负载浓度。 注: 最佳负载浓度因测序系统而异, 通常范围从 1-250 pM25不等。</li>
	<li>变性和稀释顺序控制。 注: 添加顺序控制将纠正低多样性库产生的排序问题。变性的顺序控制使用氢氧化钠和稀释与库相同的浓度。</li>
	<li>将库和排序控制结合起来。 注意: 顺序控制与组合库的比例取决于所使用的库多样性和排序系统, 但通常为 1:126。</li>
	<li>将组合库和排序控制混合物加载到排序流单元。</li>
</ol>8. 扩增子序列分析
<ol><li>通过检查与所使用的排序系统对应的云计算环境的运行指标, 评估运行的总体成功。 注意: 运行指标 (如顺序读取数) 将根据使用的顺序平台和试剂盒而异。表 1列出了常用测序系统的目标值。</li>
	<li>下载原始测序数据和所需的生物信息学开源软件, 定量洞察到微生物生态学 (QIIME)27或 Mothur28。 注意: QIIME 和 Mothur 都是开源的, 可以免费下载。关于使用这些程序的详细说明可以在线找到, 并且对于第一次用户来说是足够详细的。下面的步骤提供了在 QIIME 中进行的生物信息学管道的广泛概述。不需要熟悉 python, 但会促进以下脚本的实现</li>
	<li>使用 QIIME 中的 "validate_mapping_file" 脚本创建和验证映射文件。 注意: 映射文件包含执行数据分析所需的所有元数据, 包括示例 ID、扩增子底漆序列和示例说明。验证步骤检查是否以正确的格式输入了所有必需的数据。</li>
	<li>解和过滤器序列使用 "split_libraries" 脚本 (图 1)。 注意: 此脚本根据索引底漆组合和映射文件中的示例 id 输入, 为示例分配条形码读取。它还执行几个质量过滤步骤, 以控制排序错误的基础上, 用户定义的削减最低质量分数, 序列长度和最终修剪。</li>
	<li>使用 "pick_open_references_otus" 脚本将操作分类学单元 (OTUs) 分配给序列。 注意: 在此步骤中, 序列根据标识的阈值 (通常为 97%) 聚集在引用序列集合上。与引用序列集合不匹配的读取将彼此聚集在一起。此外, 还提供了 OTU 和闭合引用的领料选项, 但 QIIME 开发人员建议打开参考领料。</li>
	<li>使用 "alpha_rarefaction" 或 "normalize_table" 脚本规范化 OTU 表。 注意: 这一步纠正了列总和的变化, 或每个样本的总序列读数, 这是现代测序技术的结果。规范化可以使用传统的稀疏, 或通过其他方法 (如累计和缩放) 来执行。</li>
	<li>使用 "core_diversity_analysis" 脚本一次执行几种 alpha 多样性、β多样性和分类成分多样性分析。 注意: 或者, 这些分析可以单独使用单独的脚本运行 (例如, "alpha_diversity")。</li>
</ol>

作者没有什么可透露的。

16S rRNA 的下一代测序已成为微生物鉴定的标准方法, 并使研究载体微生物如何影响病原体的传播。这里概述的协议详细介绍了利用此方法来调查莱姆病的一个载体pacificus的微生物群落组装。然而, 它可以很容易地用于研究其他蜱种或节肢动物的载体物种。
事实上, 微生物分析的 16S rRNA 测序法已广泛应用于研究微生物, 包括蚊子, psyllids, 和采蝇蝇29,30,31。其他可用于微生物鉴定的方法包括显微镜、培养和组织学染色。如果目标是识别和描述一种新的微生物 (显微学), 评估抗菌药物 (培养基) 的效果, 或者在载体内定位特定和已知的微生物 (组织学染色), 这些方法可能比测序更合适。然而, 这些方法具有低特异性、检测能力和可扩展能力, 因此不太适合在不同的生态和实验条件下识别载体内的微生物群落或表征载体微生物群条件。反之, 16S rRNA 基因的高通量测序能够识别出低丰度和非可培养细菌, 提供高分辨率和检测的综合参考数据库, 并可以提供高复制取决于覆盖需求。
虽然 16S rRNA 测序现在已广泛用于微生物鉴定, 但这种技术并非没有限制。微生物污染主要是对测序结果的解释模糊, 并混淆了生物意义32。此外, 由于使用了普遍的细菌底漆和敏感性低起始浓度是固有的 16S rRNA 测序, 微生物污染是常见的32。污染源包括 PCR 试剂、DNA 提取试剂盒、实验室表面以及研究员33、34、35的皮肤和衣物。微生物污染物的影响可以通过在无菌实验室环境中工作、使用阴性控制和技术复制, 并记录所有使用36的试剂盒和药剂来减少。
除了微生物污染外, 低质量的测序输出也会严重阻碍微生物测序数据的可用性。数据质量可以由许多运行指标来评估, 其中包括读取通过筛选器的次数、高于 Q 分数的读数百分比 (在基数调用中预测错误概率的度量) 和簇密度。这些指标的值将根据运行的样本数、排序系统、试剂盒和所使用的百分比排序控制的不同而有所不同, 但基于运行条件的最佳值可以联机使用。特别是在测序前, 流单元的给定平面上的簇密度、库簇数是优化数据质量和产量的关键参数。过度和低聚类可以减少数据输出, 并导致样本因覆盖率不足而被排除在分析之外。簇密度差往往反映了图书馆量化的不准确;因此, 应注意进行适当的 DNA 清理、个体样本量化和整个图书馆量化。
假设数据质量和产量达到了较高的水平, 用于克服大型数据集统计挑战的数据分析中的不同方法构成了这种方法的另一个局限性。例如, 存在多种方法来解决示例之间读取计数的变化。稀疏, 其中包括次抽样读取, 以达到相同的测序深度, 在所有样本, 经常使用, 但最近受到批评的浪费大量的数据和主观选择最低测序深度37 ,38,39。累积总和缩放 (CSS), 它保持所有序列读取, 但根据给定百分比内的累计计数来衡量它们, 作为一种替代技术开发。然而, 由于它的相对新颖性和在存在/缺席分析之前对规范化的确认效用, CSS 没有被广泛采用。
标准数据分析程序也缺乏处理顺序读在消极控制和区分这些从低丰盈读。如前所述, 建议从 DNA 提取步骤产生的阴性控制, 以帮助区分真正的向量菌群居民从微生物污染物的分析。然而, 缺乏识别和过滤可疑污染物的标准和统计严谨的方法。一种常见的方法是从所有样本中移除负控制中的 OTUs。然而, 这种方法可能过于保守, 因为许多这些微生物可能来源于实际样品, 而不是试剂盒40。另一种常见的技术包括将 < 1% 中的微生物分组为 "稀有" 类别, 假设微生物污染物在实际样品8、41中是罕见的。然而, 这种方法可能会去除存在于低丰度的真正的载体微生物, 特别是当微生物群由一 endosymbiont 控制, 如i. pacificus和i. holocyclus7、42中所见.在这些情况下, 检测稀有微生物需要更深层次的测序, 开发出抑制扩增子 PCR42中 endosymbiont 放大的引物, 或在序列分析过程中计算去除 endosymbiont7. 在处理稀有和污染 OTUs 的各种方法中进行选择, 在微生物数据分析中产生了主观性和偏见的机会, 这可能限制了在研究中比较结果的能力。
参考数据库的选择, 是将分类学和系统系统信息分配给序列阅读所必需的, 为发散和主观性提供了另一个机会。虽然相关序列的任务从可变基因区域读取到已确定的父基因组本身具有挑战性, 但可靠的参考数据库对于精确的系统发育分配至关重要。出现了多个数据库来应对这一挑战, 如席尔瓦43、Greengenes44、RDP45和 NCBI46。席尔瓦是十六年代的分类法中最大的, 但席尔瓦, 以及 RDP, 只提供分类信息到属的水平。Greengenes 提供了物种级信息, 并包含在宏基因组分析包中, 如 QIIME, 但四年来没有更新。NCBI 虽然不是分类信息的主要来源, 但从用户提交的序列中提供每日更新的分类, 但 uncurated。虽然数据库中的选择通常取决于用户对解决方案、覆盖率和货币的需求, 但它对系统的分配和下游分析47、48有重大影响。因此, 调查人员必须就使用哪个参考数据库达成共识, 以避免分析中出现额外的偏见。
对于这些数据处理挑战的标准方法可能会出现, 因为 16S rRNA 测序成为越来越普遍的技术。继续降低排序成本和时间将进一步推广这种方法。这种技术的使用量的增加将有助于对不同条件下的向量微生物的组成和生态学进行更深入的研究。由于对向量菌群生物学的认识尚处于起步阶段, 这些类型的描述性研究是在尝试利用微生物作为媒介控制手段之前的重要第一步。然而, 要真正了解微生物在病原体传播中的作用, 微生物鉴定必须与这些微生物的功能作用相结合。RNA 测序和 transcriptomic 方法, 包括映射和量化基因表达, 使推断成微生物的功能作用, 但需要深测序和完全组装的目标物种的基因组。为了规避这些挑战, 最近开发了用于预测 16S rRNA 测序数据功能成分的计算工具, 但目前还没有得到广泛采用。这种工具的发展, 以及生态学理论, 将微生物身份与载体内的功能作用联系起来, 将增加 16S rRNA 测序数据在载体微生物研究中的效用。

近几十年来, 媒介传播疾病的死灰复燃和传播对全球人类和野生动物的健康构成严重威胁。对大多数这些疾病缺乏有效的疫苗, 控制努力受到媒介和媒介宿主相互作用的复杂生物学特性的阻碍。了解在病原体传播媒介中微生物相互作用的作用, 可以为规避这些挑战的新策略的发展提供帮助。特别是, 与载体相关的微生物 commensals、共生和病原体之间的相互作用, 称为微生物, 可能对病原体传播有重要的影响。现在压倒性的证据支持这一断言, 举例说明向量菌群与疟疾、Zika 病毒和莱姆病1、2、3等疾病的能力之间的联系。然而, 将这些发现转化为疾病控制策略需要对向量微生物的结构、功能和起源有更详细的了解。在不同的生态学和实验条件下, 载体微生物群落的识别和表征是这一领域前进的重要途径。
通过利用西部黑腿蜱、 Ixodes pacificus、莱姆病病原体螺旋体螺旋体的载体, 在这里提供了一种鉴定病原体微生物的方法。虽然蜱比任何其他节肢动物更有人类病原体的种类, 但对蜱微生物4的生物学和群落生态学的了解相对较少。很明显, 蜱港有多种病毒, 细菌, 真菌和原生动物, 包括 commensals, endosymbionts 和瞬态微生物居民5,4。先前的工作表明, Ixodes微生物与地理, 物种, 性别, 生命阶段, 和血膳食来源6,7,8的强烈变化。然而, 这种变化所依据的机制仍不得而知, 并对这些微生物群落的起源和组装进行更详细的调查。蜱可以通过垂直传播获得微生物, 与寄主接触, 通过气孔、嘴巴和肛门孔9吸收环境。了解蜱菌群最初形成和发展的因素, 特别是垂直和环境传播的相对贡献, 对于了解蜱的自然形态和变化非常重要。微生物多样性以及这些群落在病原体传播过程中的相互作用, 并可能应用于疾病或媒介控制。
强大的分子技术, 如下一代测序, 现在已经存在, 以确定微生物群落, 并可用于表征向量微生物在不同的环境或实验条件。在这些高通量测序方法出现之前, 微生物的鉴定主要依靠显微学和文化。显微术是一种快速、简便的技术, 其识别微生物的形态学方法固有的主观性和粗糙性, 且灵敏度低, 检测10。基于培养基的方法广泛用于微生物鉴定, 可用于测定微生物对药物治疗的敏感性11。然而, 这种方法也有低灵敏度, 因为它已经估计, 2% 的环境微生物可以培养在实验室设置12。组织学染色方法也被用来检测和本地化的特定微生物的载体, 使调查的不同分类群分布在蜱, 并研究假说的微生物相互作用。然而, 在选择合适的污渍之前, 必须事先了解微生物身份, 使这种方法不适合于微生物的鉴定和鉴定。此外, 组织学染色是一个高度时间密集, 费力的过程, 并没有很好的规模大样本大小。传统的分子方法, 如桑格测序同样有限的敏感性和检测不同的微生物群落。
下一代测序允许从大量样品中快速识别微生物。标准标记基因和参考数据库的存在进一步促进了分类分辨率的提高, 通常是属或物种水平。小亚基核糖体 rna 经常被用于达到这个目标, 16S rRNA 是最普遍的由于存在保守和可变区域在基因之内, 允许创造普遍引物以唯一的 amplicons 为每个细菌种类13,14。本报告详细介绍了通过 16S rRNA 下一代测序在蜱微生物群中识别分类的程序。特别是, 本议定书强调了为测序准备样品所涉及的步骤。提供了关于排序和生物信息学步骤的更广义的详细信息, 因为目前有各种测序平台和分析程序, 每个都有广泛的现有文档。这种下一代测序方法的总体可行性是通过将其应用于一个关键疾病载体内微生物群落组装的研究中得到证明的。

<table><tbody><tr><td>Item</td><td>Name of Material/Equipment</td><td>Company</td><td>Catalog #</td></tr><tr><td>1</td><td>DNeasy Blood &amp;amp; Tissue Kit</td><td>Qiagen</td><td>69504</td></tr><tr><td>2</td><td>Qubit 4 Fluorometer</td><td>ThermoFisher Scientific</td><td>Q3326</td></tr><tr><td>3</td><td>NanoDrop 8000 Spectrophotometer</td><td>ThermoFisher Scientific</td><td>ND-8000-GL</td></tr><tr><td>4</td><td>2x KAPA HiFi HotStart ReadyMix</td><td>Kapa Biosystems</td><td>KK2501</td></tr><tr><td>5</td><td>AMPure XP beads</td><td>Agen Court</td><td>A63880&amp;nbsp;</td></tr><tr><td>6</td><td>Magnetic Rack</td><td>ThermoFisher Scientific</td><td>MR02</td></tr><tr><td>6</td><td>TE buffer</td><td>Teknova</td><td>T0223</td></tr><tr><td>7</td><td>Nextera Index Kit</td><td>Illumina</td><td>FC-121-1011</td></tr><tr><td>8</td><td>KAPA Library Quantification Kit</td><td>Roche</td><td>KK4824</td></tr><tr><td>9</td><td>MiSeq System</td><td>Illumina</td><td>SY-410-1003</td></tr><tr><td>10</td><td>MiSeq Reagent Kit v3&amp;nbsp;</td><td>Illumina</td><td>MS-102-3001</td></tr><tr><td>11</td><td>10 mM Tris-HCl with 0.1% Tween 20</td><td>Teknova</td><td>T7724</td></tr></tbody></table>

tick microbiome characterization by next-generation 16s rrna amplicon sequencing

近几十年来, 媒介传播的疾病以惊人的速度重新出现和扩大, 在全世界造成相当大的发病率和死亡率。大多数这些疾病缺乏有效和广泛使用的疫苗, 这就有必要制定新的疾病缓解战略。为此, 一个有希望的疾病控制途径是针对媒介微生物群, 即居住在载体中的微生物群落。载体菌群在病原体动力学中起着举足轻重的作用, 微生物群的操控导致了少数媒介传播疾病的媒介丰度或病原体传播。然而, 将这些发现转化为疾病控制的应用需要对媒介微生物生态学的透彻理解, 这在历史上受此领域技术不足的限制。下一代测序方法的出现使不同微生物群落的快速、高度平行测序得以实现。以高度保守的 16S rRNA 基因为靶, 促进了在不同生态环境和实验条件下, 在载体内的微生物的刻画。这项技术包括扩增 16S rRNA 基因, 通过 PCR 样品条形码, 将样品加载到流细胞进行测序, 以及生物信息学方法将序列数据与系统进化信息相匹配。对于大量复制, 通常可以通过这种方法实现物种或属类级别的识别, 从而规避传统培养、显微学或组织学染色中低检测、分辨和输出的挑战。技术。因此, 该方法非常适合于不同条件下的载体微生物的表征, 但目前尚不能提供微生物功能、载体内位置或抗生素治疗反应的信息。总体而言, 16S 的下一代测序技术是更好地了解媒介微生物在疾病动力学中的身份和作用的有力手段。

从三个单独的卵子离合器和两个环境暴露期, 0 和2周的土壤中, 对微生物序列进行了总共42个刻度。每个治疗组, 被认为是一个单一的离合器和曝光时间, 包含6-8 复制蜱样本。这些经过处理的蜱提取物加载到下一代排序器上, 产生了12885713配对端读取传递滤波器。此运行中包含的是从抽取步骤中产生的3个负控制项, 总共生成211214个读取 (包括在上一个计数中)。表 2详细说明了进一步运行质量指标以及每个指标的最佳值。稀疏曲线将测序工作与每个样本的 OTUs 数联系起来, 表明测序深度, 或每个样本的唯一序列读数的数量为2129读, 足以充分捕获微生物群落的多样性 (图 2)。稀疏级别将根据样本类型而异, 并且必须分别为每个排序运行确定。在 rarefying 到这个深度之后, 所有样本中都发现了 1714 OTUs, 平均每个样本有 93.3 4.3 OTUs。为了避免由稀疏矩阵引起的下游分析问题, 并去除潜在的污染物, 在≥1% 丰度中至少有一个样品中没有发现的所有 OTUs 都被汇集成一个稀有的一般类别。此外, R 中的 decontam 包用于识别相对于实际样本的负控制中的 OTUs 超过表示, 这些 OTUs 从下游分析中删除。
利用 QIIME 多样性分析工作流进行社区生态学分析, 以表明使用简单生物信息学管道生成的α多样性、β多样性和分类学组成多样性输出的类型。例如, 使用 "core_diversity_analyses" 脚本生成加权和加权 Unifrac 主坐标分析, 并揭示了基于离合器识别的幼虫蜱的空间聚类 (图 3)。Boxplots 的 OTU 计数在不同的环境暴露时间也产生了通过这个剧本, 表明不同的微生物物种丰富的时间 (图 4)。这些 alpha 和 beta 多样性分析是根据映射文件中列出的用户定义的类别执行的, 但也为所有样本生成了关于一般分类信息的数字和统计 (图 5)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58239/58239fig1.jpg"> 图 1:微生物群测序工作流程.微生物群测序工作流的主要步骤显示为库准备和排序分析步骤的调用。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58239/58239fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58239/58239fig2.jpg"> 图 2: 序列计数规范化的稀疏曲线.稀疏曲线, 显示每队列的蜱, 相关观察 OTU 计数的取样努力。由于在每个离合器中都复制了样本, 所以存在误差线, 它们表示一个标准偏差。测序深度应在曲线趋于稳定的点处选取, 以充分捕捉 OTUs 的完全多样性。在这里, 2000 读取的排序深度看起来是适当的。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58239/58239fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58239/58239fig3.jpg"> 图 3:离合器主坐标分析。加权 Unifrac 主坐标分析 (PCoA) 显示不同离合器幼虫蜱与成虫之间微生物组成的变化。每个数据点表示单个刻度。这个数字是通过 QIIME "core_diversity_analyses" 脚本自动生成的。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58239/58239fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58239/58239fig4.jpg"> 图 4: 阿尔法多样性 boxplots 的暴露时间.Boxplots 描述了环境暴露群体的 OTU 计数, 显示了随着时间的推移微生物多样性的变化。这个数字是通过 QIIME "core_diversity_analyses" 脚本自动生成的。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58239/58239fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58239/58239fig5.jpg"> 图 5: 一、离合器一级微生物鉴定.从离合器中分离出的单个蜱样的微生物组成, 显示在一级。这个数字以及较低分类级别的摘要信息是通过 QIIME "core_diversity_analyses" 脚本自动生成的。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58239/58239fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>度量</td>
			<td colspan="2">定义</td>
			<td>我们的结果 (MiSeq V3)</td>
			<td>MiSeq V3 的优化</td>
			<td>HiSeq 快速模式的优化</td>
		</tr>
<tr>
<td>读取 PF</td>
			<td colspan="2">通过贞操筛选器读取的排序次数。如果在前25个周期中不超过1个基调用的贞操值低于 0.6, 则读取传递筛选器</td>
			<td>12885713</td>
			<td>14-16 元</td>
			<td>6亿</td>
		</tr>
<tr>
<td>错误率</td>
			<td colspan="2">基于与 PhiX 控件对齐的读取, 在周期中错误地调用基对的百分比</td>
			<td>3.28 ±0.10</td>
			<td>* 取决于其他度量值</td>
			<td>* 取决于其他度量值</td>
		</tr>
<tr>
<td>% ≥Q30</td>
			<td colspan="2">基数与 Q 分数≥30的百分比, 表示基调用精度为99.9%</td>
			<td>68.94</td>
			<td>> 70%</td>
			<td>> 80%</td>
		</tr>
<tr>
<td>群集 PF (%)</td>
			<td colspan="2">通过贞操筛选器的群集的百分比。</td>
			<td>85.61 ±0.81</td>
			<td>90%</td>
			<td>90%</td>
		</tr>
<tr>
<td>密度</td>
			<td colspan="2">flowcell 的簇密度--数据质量和输出的关键指标</td>
			<td>552 @ 35 cluster/mm^2</td>
			<td>1200-1400 cluster/mm^2</td>
			<td>850-1000 cluster/mm^2</td>
		</tr>
</tbody></table>表 1:输出的关键性能参数.根据本研究 (材料表) 和25% 序控制加法所使用的测序平台和试剂盒报告的用户数据和目标值。

Watch this Scientific Journal Video about Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing at JoVE.com

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

在这里, 我们提出了下一代测序协议 16S rRNA 测序, 使识别和鉴定微生物群落在载体内。该方法包括 DNA 的提取、扩增和条形码样品的 PCR、序列化和生物信息学, 将序列数据与系统进化信息相匹配。

下一代 16S rRNA 扩增子测序的蜱菌群鉴定

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

tick-microbiome-characterization-next-generation-16s-rrna-amplicon

Universidad San Sebastian

Research

JoVE Journal

Biology

12.7K 次观看.  San Francisco State University. 在这里, 我们提出了下一代测序协议 16S rRNA 测序, 使识别和鉴定微生物群落在载体内。该方法包括 DNA 的提取、扩增和条形码样品的 PCR、序列化和生物信息学, 将序列数据与系统进化信息相匹配。

视频: Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

科研

遗传学

A Method for Targeted 16S Sequencing of Human Milk Samples

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as with all these technologies, reproducible results depend on a laboratory workflow that incorporates appropriate precautions and controls. This is particularly important with low-biomass samples where contaminating bacterial DNA can generate misleading results. This article details a semi-automated workflow to identify microbes from human breast milk samples using targeted sequencing of the 16S ribosomal RNA (rRNA) V4 region on a low- to mid-throughput scale. The protocol describes sample preparation from whole milk including: sample lysis, nucleic acid extraction, amplification of the V4 region of the 16S rRNA gene, and library preparation with quality control measures. Importantly, the protocol and discussion consider issues that are salient to the preparation and analysis of low-biomass samples including appropriate positive and negative controls, PCR inhibitor removal, sample contamination by environmental, reagent, or experimental sources, and experimental best practices designed to ensure reproducibility. While the protocol as described is specific to human milk samples, it is adaptable to numerous low- and high-biomass sample types, including samples collected on swabs, frozen neat, or stabilized in a preservation buffer.

A semi-automated workflow is presented for targeted sequencing of 16S rRNA from human milk and other low-biomass sample types.

人乳样品靶向16S 测序方法的研究

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as ...

免疫与感染

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

荧光显微镜筛选和新一代测序：参与细胞器完整性的基因鉴定的有用工具

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

生物学

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 ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

高效的核酸提取和16S rRNA基因序列的细菌群落特征

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. Deviations from what is considered a healthy microbiota composition (dysbiosis) may impair vital functions leading to pathologic conditions. Recent and ongoing research efforts have been directed toward the characterization of associations between microbial composition and human health and disease.
Advances in high-throughput sequencing technologies enable characterization of the gut microbial composition. These methods include 16S rRNA-amplicon sequencing and shotgun sequencing. 16S rRNA-amplicon sequencing is used to profile taxonomical composition, while shotgun sequencing provides additional information about gene predictions and functional annotation. An advantage in using a targeted sequencing method of the 16S rRNA gene variable region is its substantially lower cost compared to shotgun sequencing. Sequence differences in the 16S rRNA gene are used as a microbial fingerprint to identify and quantify different taxa within an individual sample.
Major international efforts have enlisted standards for 16S rRNA-amplicon sequencing. However, several studies report a common source of variation caused by batch effect. To minimize this effect, uniformed protocols for sample collection, processing, and sequencing must be implemented. This protocol proposes the integration of broadly used protocols starting from fecal sample collection to data analyses. This protocol includes a column-free, direct-PCR approach that enables simultaneous handling and DNA extraction of large numbers of fecal samples, along with PCR amplification of the V4 region. In addition, the protocol describes the analysis pipeline and provides a script using the latest version of QIIME (QIIME 2 version 2017.7.0 and DADA2). This step-by-step protocol is aimed to guide those interested in initiating the use of 16S rRNA-amplicon sequencing in a robust, reproductive, easy to use, detailed way.

This manuscript describes a detailed standardized protocol of high-throughput 16S rRNA-amplicon sequencing. The protocol introduces an integrated, uniformed, feasible, and inexpensive protocol starting from fecal sample collection through data analyses. This protocol enables analysis of large numbers of samples with rigorous standards and several controls.

16S rRNA-扩增子测序法对粪便微生物特性的指导协议

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

16S rRNA Sequencing: A PCR-based Technique to Identify Bacterial Species

Source: Ewa Bukowska-Faniband1, Tilde Andersson1, Rolf Lood1 
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund, Sweden
Planet Earth is a habitat for millions of bacterial species, each of which has specific characteristics. Identification of bacterial species is widely used in microbial ecology to determine biodiversity of environmental samples and medical microbiology to diagnose infected patients. Bacteria can be classified using conventional microbiology methods, such as microscopy, growth on specific media, biochemical and serological tests, and antibiotic sensitivity assays. In recent decades, molecular microbiology methods have revolutionized bacterial identification. A popular method is 16S ribosomal RNA (rRNA) gene sequencing. This method is not only faster and more accurate than conventional methods, but also allows identification of strains that are difficult to grow in laboratory conditions. Furthermore, differentiation of strains at the molecular level enables discrimination between phenotypically identical bacteria (1-4).
16S rRNA joins with a complex of 19 proteins to form a 30S subunit of the bacterial ribosome (5). It is encoded by the 16S rRNA gene, which is present and highly conserved in all bacteria due to its essential function in ribosome assembly; however, it also contains variable regions which may serve as fingerprints for particular species. These features have made the 16S rRNA gene an ideal genetic fragment to be used in identification, comparison, and phylogenetic classification of bacteria (6).
16S rRNA gene sequencing is based on the polymerase chain reaction (PCR) (7-8) followed by DNA sequencing (9). PCR is a molecular biology method used to amplify specific fragments of DNA through a series of cycles that include:
i) Denaturation of a double stranded DNA template 
ii) Annealing of primers (short oligonucleotides) that are complementary to the template 
iii) Extension of primers by the DNA polymerase enzyme, which synthesizes a new DNA strand
A schematic overview of the method is shown in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10510/10510fig1.jpg" /> 
Figure 1: Schematic overview of the PCR reaction. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/10510/10510fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
There are several factors that are important for a successful PCR reaction, one of which is quality of the DNA template. Isolation of chromosomal DNA from bacteria can be performed using standard protocols or commercial kits. Special care should be taken to obtain DNA that is free of contaminants that can inhibit the PCR reaction.
Conserved regions of the 16S rRNA gene permit the design of universal primer pairs (one forward and one reverse) that can bind to and amplify the target region in any bacterial species. The target region can vary in size. While some primer pairs can amplify most of the 16S rRNA gene, others amplify only parts of it. Examples of commonly used primers are shown in Table 1 and their binding sites are depicted in Figure 2.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Primer name</td>
			<td>Sequence (5&#39;&#8594;3&#39;)</td>
			<td>Forward/ reverse</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>8F b)</td>
			<td>AGAGTTTGATCCTGGCTCAG</td>
			<td>forward</td>
			<td>-1</td>
		</tr>
		<tr>
			<td>27F</td>
			<td>AGAGTTTGATCMTGGCTCAG</td>
			<td>forward</td>
			<td>-10</td>
		</tr>
		<tr>
			<td>515F</td>
			<td>GTGCCAGCMGCCGCGGTAA</td>
			<td>forward</td>
			<td>-11</td>
		</tr>
		<tr>
			<td>911R</td>
			<td>GCCCCCGTCAATTCMTTTGA</td>
			<td>reverse</td>
			<td>-12</td>
		</tr>
		<tr>
			<td>1391R</td>
			<td>GACGGGCGGTGTGTRCA</td>
			<td>reverse</td>
			<td>-11</td>
		</tr>
		<tr>
			<td>1492R</td>
			<td>GGTTACCTTGTTACGACTT</td>
			<td>reverse</td>
			<td>-11</td>
		</tr>
	</tbody>
</table>
Table 1: Examples of standard oligonucleotides used in amplification of 16S rRNA genes a). 
a) The expected lengths of the PCR product generated using the different primer combinations can be estimated by calculating the distance between the binding sites for the forward and the reverse primer (see Figure 2), e.g. the size of the PCR product using primer pair 8F-1492R is ~1500 bp, and for primer pair 27F-911R ~900 bp. 
b) also known as fD1
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10510/10510fig2.jpg" /> 
Figure 2: Representative figure of the 16S rRNA sequence and the primer-binding sites. Conserved regions are colored in grey and variable regions are filled with diagonal lines. To allow for the highest resolution, primer 8F and 1492R (name based on location on rRNA sequence) are used to amplify the whole sequence, allowing for the sequencing of several variable regions of the gene. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/10510/10510fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Cycling conditions for PCR (i.e. the temperature and time required for the DNA to be denatured, annealed with primers, and synthesized) are dependent on the type of polymerase that is used and the properties of the primers. It is recommended to follow the manufacturer&#39;s guidelines for a particular polymerase.
After the PCR program is completed, the products are analyzed by agarose gel electrophoresis. A successful PCR yields a single band of expected size. The product must be purified prior to sequencing to remove residual primers, deoxyribonucleotides, polymerase, and buffer which were present in the PCR reaction. The purified DNA fragments are usually sent for sequencing to commercial sequencing services; however, some institutions perform DNA sequencing at their own core facilities.
The DNA sequence is automatically generated from a DNA chromatogram by a computer and must be carefully checked for quality, as manual editing is sometimes needed. Following this step, the gene sequence is compared with sequences deposited in the 16S rRNA database. The regions of similarity are identified, and the most similar sequences are delivered.

16S rRNA Sequencing: Identifying Bacterial Species by PCR

16S rRNA测序:基于PCR的技术，用于识别细菌物种

Source: Ewa Bukowska-Faniband1, Tilde Andersson1, Rolf Lood1
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, ...