我们要感谢 Helty Adisetiyo, 博士和 Shangxin 杨博士的发展的协议。对国际妇幼青少年艾滋病临床试验组 (IMPAACT) 的总体支助是由国家卫生研究院 (NIH) 国家过敏和传染病研究所 (NIAID) 根据奖励数字提供的。UM1AI068632 (IMPAACT), UM1AI068616 (IMPAACT SDMC) 和 UM1AI106716 (IMPAACT LC), 与来自尤尼斯·肯尼迪-国家儿童健康和人类发展研究所 (发育研究院) 和国家心理健康研究所 (镍氢) 的共同资助。内容完全是作者的责任, 不一定代表 NIH 的官方观点。

对于所有的协议步骤, 必须佩戴适当的个人防护设备 (PPE), 并需要采取严格的防污染措施。观察从预放大工作区到放大后工作区域的工作流程, 以尽量减少样品的污染。所有使用的用品是无菌的, 没有 RNase, DNase, DNA 和热原。所有吸管提示都被过滤。提供了协议步骤的流程图 (图 1)。
1. 样品裂解
注: 样品裂解和核酸提取是在洁净室环境中使用 DNA/RNA 萃取试剂盒进行的, 在这两个工程和程序控制都到位, 以尽量减少向样品中引入环境细菌。
<ol><li>
工作区准备
	<ol>
<li>清洁生物安全柜 (BSC) 工作区与适当的表面清洁剂, 以消除任何核酸污染。</li>
		<li>打开温度控制的 vortexer, 并将其设置为37摄氏度。</li>
	</ol>
</li>
	<li>
胍硫氰酸盐裂解缓冲液的制备 (见材料表)
	<ol>
<li>检查沉淀的溶解缓冲。在37摄氏度的温度下重新溶解沉淀。</li>
		<li>为每个样品准备600µL 的裂解缓冲液和6µL β巯基乙醇 (β i)。考虑每个样本额外的20% 卷。</li>
	</ol>
</li>
	<li>
样品准备
	<ol>
<li>如果整个牛奶都结冰了, 就在冰上解冻。整除5毫升的全脂牛奶成15毫升或5毫升不育管在平衡计分卡, 并保持在冰上。</li>
		<li>旋转5毫升牛奶整除10分钟在 5000 x g 在4°c 到颗粒细胞。</li>
		<li>去除脂肪层, 现在的顶层在管, 用塑料铲或大口径吸管尖。</li>
		<li>不干扰颗粒, 除去所有上清, 除100µL。</li>
		<li>用 resuspending 在1毫升无菌磷酸盐缓冲盐水 (PBS) 中清洗颗粒。</li>
		<li>通过将1毫升不育 PBS 添加到5毫升管中, 准备1负控制。</li>
		<li>将悬浮液转移至清洁的1.5 毫升离心管, 并在离心中旋转1分钟 (室温 (RT) 5000 x g)。</li>
		<li>使用1000µL 无菌过滤吸管提示丢弃整个上清/脂肪层。</li>
		<li>如果不提取同一天, 通过将细胞颗粒放入乙醇/干冰泥浆中, 并立即将其转移到-80 摄氏度冷藏库中, 将其冻结。</li>
	</ol>
</li>
	<li>
样品中断和均匀化 (珠敲打)
	<ol>
<li>加入600µL 的裂解缓冲液, 其中含有β i 的颗粒, 并将悬浮液转移到珠管上。</li>
		<li>每个抽取批次12包含10个样本, 1 个负控制 (在步骤3.7 中编写), 1 个正控制 (在下一步中准备)。<ol>
<li>用裂解缓冲剂和20µL 的细菌模拟样品制备1阳性对照 (模拟社区使用的浓度, 一旦提取, 大约 0.2 ng/µL 的 DNA)。</li>
		</ol>
</li>
		<li>漩涡所有珠管大力十五年代。</li>
		<li>在温度控制的 vortexer 的热样品在37°c 为10分钟, 与震动在 700-800 转每分钟。</li>
		<li>将管道加载到自动的示例干扰器适配器集中。</li>
		<li>珠打1分钟在30赫兹。</li>
		<li>拆卸适配器集, 并手动将适配器集与从左机械手到仪器的右侧机械臂的样本板一起切换。</li>
		<li>珠打1分钟, 在30赫兹。</li>
		<li>离心管在 17200 x g, 3 分钟在25摄氏度。</li>
		<li>用200µL 吸管取出所有上清液, 小心不要在样品的顶端或试样底部的珠子残留物上得到任何脂肪层, 并应用于均质体柱。</li>
		<li>离心机均质柱最大速度为 17200 x 克, 3 分钟25摄氏度。</li>
		<li>将350µL 的洗脱液转移到2毫升制造商的离心管中, 用于纯化 DNA 和 RNA 的自动仪器 (不要使用任何其他管)。注意不要转移任何残留的脂肪层。</li>
		<li>如果流经体积小于350µL, 添加裂解缓冲与β-我到最后的体积350µL。</li>
		<li>在使用自动 DNA 纯化仪进行核酸隔离之前, 将样品留在 RT 上长达2小时, 或在-80 摄氏度处冻结。</li>
	</ol>
</li>
	<li>
清洁工作区
	<ol>
<li>清洁所有表面使用非酶净化溶液, 离开10分钟, 然后喷雾与70% 乙醇和擦拭表面。</li>
	</ol>
</li>
</ol>2. 分离 DNA/RNA
<ol><li>
工作区准备
	<ol>
<li>打开散热块, 将温度设置为70摄氏度。</li>
		<li>打开温度控制的 vortexer, 将温度设置为37摄氏度。</li>
		<li>预热洗脱缓冲 (EB) 包含10毫米三氯, pH 8.5, 在50毫升管到70°c。</li>
		<li>加热350µL 冷冻样品裂解物2毫升管在37°c, 直到完全解冻没有任何沉淀 (约10分钟)。</li>
	</ol>
</li>
	<li>
DNA 纯化
	<ol>
<li>漩涡和离心机2毫升管与样品简要地 (3000 x g 十年代)。</li>
		<li>根据制造商的说明, 在自动 DNA/RNA 净化仪器的加载图后, 将2毫升管插入到振动筛中。</li>
		<li>根据样品数量, 获取转子适配器并将其设置在托盘上。</li>
		<li>根据样品的标识 (ID) 对每个转子适配器进行标签。</li>
		<li>切下盖子, 平滑各个旋转柱的边缘, 用于 DNA 和 RNA。</li>
		<li>插入没有收集管的 DNA 旋转柱进入转子适配器。丢弃收集管。</li>
		<li>标签1.5 毫升收集管, 并插入到转子适配器。</li>
		<li>根据自动 DNA/RNA 纯化仪的加载图, 将转子适配器设置为离心机。</li>
		<li>插入制造商的1000µL 过滤器-提示到尖端机架。</li>
		<li>根据样品数量 (根据特定机器协议指令), 在2毫升制造商的离心管中添加 RNase 水。</li>
		<li>将管插入管插槽 "A" 的离心管插槽。</li>
		<li>丢弃试剂瓶中剩下的试剂, 并填充最小体积10毫升。</li>
		<li>将试剂瓶插入试剂瓶架中 (EB 瓶除外)。</li>
		<li>从50毫升管中加入热 EB 到试剂瓶位置6。</li>
		<li>检查1.5 毫升管放在转子适配器紧密。</li>
		<li>关闭仪器的盖子并选择: "RNA" → "提取套件" → "动物, 组织和细胞" → "套件的名称350µL 部分自定义 DNA" →编辑洗脱量为100µL (默认) 或50µL 低生物量样品→ "开始"。</li>
		<li>完成后, 从离心机上取出转子接头, 放在托盘上。</li>
		<li>从转子适配器位置3丢弃 DNA 自旋柱。</li>
		<li>不要丢弃转子适配器, RNA 在位置2。</li>
		<li>去除1.5 毫升收集管含有洗脱 DNA 在位置 3, 并存储在−20°c。</li>
		<li>收集从振动筛和存储在−20°c 样品, 如果任何样品留在, 否则丢弃。</li>
		<li>继续与协议 "试剂盒的名称350µL 部分 B rna" 进一步纯化 RNA。</li>
		<li>RNA 纯化将由大约350µL 流经, 在转子适配器的中间位置。</li>
	</ol>
</li>
	<li>
RNA 纯化
	<ol>
<li>插入 RNA 旋转柱没有它的收集管和盖子到转子适配器。</li>
		<li>标签新的1.5 毫升收集管和插入到转子适配器, 如手册中所示。</li>
		<li>在自动 DNA/RNA 纯化仪的加载图后, 将转子适配器设置为离心机。</li>
		<li>关闭仪器的盖子, 并选择: "RNA" → "制造商的套件" → "动物, 组织和细胞" → "标准部分 B RNA" → "开始"。</li>
		<li>完成后, 从离心机上取出转子接头, 放在托盘上。</li>
		<li>从位置3丢弃 RNA 自旋列。</li>
		<li>去除1.5 毫升收集管包含30µL 洗脱 RNA 在位置 3, 并且存放在−80°c。</li>
		<li>取出试剂瓶。</li>
		<li>通过适当的危险废物通道处理转子适配器的内容。</li>
	</ol>
</li>
	<li>
清洁工作站
	<ol>
<li>喷雾所有的自动 DNA/RNA 净化仪器的配件, 如试剂架, 托盘, 和任何其他表面使用非酶净化解决方案, 离开10分钟, 并冲洗与去离子 (DI) 水, 然后让他们干。</li>
		<li>喷雾自动仪器只有制造商批准的非酶净化解决方案, 擦拭内部的离心机连同所有的表面使用, 离开10分钟, 然后擦拭70% 乙醇。不要使用其他类型的净化解决方案, 因为他们可以损坏仪器。</li>
	</ol>
</li>
</ol>3. 靶向 16S PCR 设置
注: 16S PCR 的设置是在清洁室内指定的预放大工作区进行的。试剂和样品的制备, 然后加载到一个液体处理程序, 以执行 PCR 为每个样本三 (30 样品, 其中包括真实样品和提取阳性和阴性对照, 加上 2 PCR 水控制, 总共96组合的样本和控件)。一旦 PCR 反应组装和密封, 样品板被转移到一个热循环仪位于后放大区域的自行车。
<ol><li>
工作区准备
	<ol>
<li>清理 PCR 工作站。喷雾所有表面使用 RNase, DNase, DNA decontaminant, 其次是 DI 水两次, 最后70% 乙醇。</li>
		<li>使用精确的样本列表准备 16S pcr 工作表 (参见目标 16S pcr 工作表), 并为每个示例9分配不同的条形码引物。打印出工作表和板块图 (见板 1)。</li>
	</ol>
</li>
	<li>
PCR 主混合制剂 注: 16S 底漆选择是基于感兴趣区域的特定研究, 并可通过研究或样本类型改变。因此, 在订购底漆之前, 重要的是要确认 16S rRNA 的哪个区域最好能放大特定研究中感兴趣的微生物。本协议的编写是为了放大 16S V4 区域。如果选择不同的引物/区域, 则热循环中的退火温度可能需要调整。<ol>
<li>在 PCR 工作站工作, 准备一切。</li>
		<li>从-20 摄氏度取出 50-100 µL 的 DNA 样本, 以及所有需要的试剂, 然后在冰上解冻。漩涡和简要地旋转下来。</li>
		<li>引物预稀释至5µM 的工作浓度, 最小体积为20µL。</li>
		<li>根据工作表上的计算, 在特定的5毫升管中准备 PCR 主混合, 只用正向底漆。</li>
	</ol>
</li>
	<li>
为自动 PCR 装置准备机器人液体处理程序
	<ol>
<li>对于样品, 拿出一个32井仪器的样品适配器, 并根据制造商的指示, 按照96井板图载入 50-100 µL 的 DNA 样本。<ol>
<li>对于每一个 pcr 板, 设置2阴性控制通过放置30µL PCR 水在一个干净的样品管。</li>
			<li>将所有样品放置在32井仪器的样品适配器上, 并将瓶盖锁定在打开位置。</li>
		</ol>
</li>
		<li>对于反向引物9<ol>
<li>删除每个反向底漆的上限与特定的条码 # 的一个在一次 (更换手套之间, 以避免交叉污染)。</li>
			<li>在仪器的试剂适配器上放置最多32个底漆。</li>
		</ol>
</li>
		<li>拿出一个96良好的 PCR 板, 并标签它的研究名称, 样本数, 日期和技术员的缩写。</li>
		<li>装载机器人液体处理程序<ol>
<li>将试剂适配器放置在 B1 位置。为了避免边缘效应, 小心地将适配器的一端放在手柄一侧, 然后慢慢地将另一边缘向下。一定要把适配器的所有角落都推上。</li>
			<li>将样品适配器放置在 C1 位置。一定要把适配器的所有角落都推上。</li>
			<li>涡流5毫升主混合, 打开帽, 并将其放置在仪器的主混合和试剂块位置 A。</li>
			<li>将 pcr 盘放在96井仪器的适配器上, 用于控制半掠过的 pcr 板。</li>
			<li>启动运行并另存为新文件。</li>
			<li>按照提示和复选标记每个提示: 一个, 提示可用, 两个, 废物箱可用, 三, 开始。</li>
		</ol>
</li>
		<li>运行完成后, 通过检查计算机中是否有错误消息来验证没有错误。</li>
		<li>用12或8条带帽密封板, 漩涡大力, 并向下旋转1分钟使用96井板微调, 并把它放在冰或冰箱。</li>
		<li>除去含有反向底漆和样品的管子。</li>
		<li>将96井板带到后放大室, 并根据热循环条件表将该板加载到热循环仪并循环 (请参见表1)。</li>
	</ol>
</li>
</ol>4. 采用基于胶带的凝胶电泳平台进行靶向16S 后 PCR 质量控制
注: PCR 后质量控制 (QC) 和所有后续步骤都在实验室指定的后放大区域进行。dna 被分析在一个自动化的 dna/RNA 片段分析仪。
<ol><li>
工作区准备
	<ol>
<li>通过喷洒 RNase、DNase、DNA decontaminant 的所有表面, 然后用二次水, 最后70% 乙醇, 清洁工作站。</li>
		<li>收集所有需要的用品和设备。</li>
	</ol>
</li>
	<li>
制备试剂
	<ol>
<li>将样品缓冲器和梯子放在温度控制的 vortexer 中, 在摄氏25摄氏度, 最小为30分钟。</li>
	</ol>
</li>
	<li>当试剂正在升温时, 通过将每个样品的 3 pcr 复制汇集到一根管子中, 制备 pcr 样品。</li>
	<li>在仪器上装载样品。</li>
	<li>简要地涡旋和自旋向下管与汇集 PCR 产品。</li>
	<li>将仪器的96井板放在 RT 上的机架上并贴上标签。</li>
	<li>简要涡旋和向下旋转样本缓冲区和梯子。</li>
	<li>将3µL 样本缓冲区添加到96井板的每个井 (至少需要53µL/丝网磁带)。</li>
	<li>运行偶数样本;如果有奇数的样本, 将4µL 的样本缓冲区添加到空井中。</li>
	<li>在梯子上加1µL 梯子。</li>
	<li>按照地图, 将1µL 的聚合 PCR 产品添加到其指定的井中。</li>
	<li>铝箔密封盖板</li>
	<li>用仪器的 vortexer 和适配器在2000转1分钟内涡流板。</li>
	<li>如果有气泡再次旋转, 向下旋转以将样品放置在管子的底部。</li>
	<li>启动软件。</li>
	<li>加载屏幕磁带和加载提示到仪器。</li>
	<li>将样品装入带96井适配器的碎片分析仪中。</li>
	<li>设置与控制器软件的运行。</li>
	<li>一定要选择连编号井。</li>
	<li>编写示例 id。</li>
	<li>检查屏幕磁带的所有油井是否以灰色突出显示。</li>
	<li>请确保分析器识别所有的吸管提示。</li>
	<li>选择 "开始" 并指定一个文件名以保存结果 (研究名称、示例编号和日期)。</li>
	<li>有关详细信息, 请参阅制造商的说明。</li>
	<li>运行完成后, 丢弃笔尖、屏幕胶带和管子。</li>
	<li>分析数据为16S 峰值 nM (315-450 bp 为 V4)。此峰值将根据所选引物的不同而有所不同。</li>
</ol>5. 图书馆的计算、汇集、清理和 QC
<ol><li>在确定了所有示例的扩增子大小和浓度之后, 请将库池存储起来, 以实现池库的最终所需卷和 nM (请参见示例计算)。</li>
	<li>根据制造商的协议 (请参阅材料表), 清理并集中使用基于硅膜的 PCR 产品纯化试剂盒的池库。<ol>
<li>洗脱的 DNA 的最终体积为50µL。</li>
	</ol>
</li>
	<li>根据制造商的协议, 使用双链 DNA 高灵敏度套件对荧光计, 立即测量清理后的库的浓度。<ol>
<li>稀释2µL 的汇集和被清洗的图书馆与198µL 稀释缓冲器加上染料 (1:100 稀释)。</li>
		<li>从荧光器件中记录测量值, 并根据稀释因子进行转换。</li>
	</ol>
</li>
	<li>使用集中阅读 (ng/µL) 的汇集库, 计算图书馆浓度在 nM。使用1µL 未稀释的图书馆测量 microvolume 分光光度计上的 OD260/280。 注: 值 1.8-2.0 表示库的纯度足够。</li>
	<li>将最终库存储在-20 摄氏度, 直到为下一代排序做好准备。</li>
</ol>

<ol>
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S., 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=Topographical+continuity+of+bacterial+populations+in+the+healthy+human+respiratory+tract.">Topographical continuity of bacterial populations in the healthy human respiratory tract.</a> Am J Respir Crit Care Med. 184 (8), 957-963 (2011).</li><li>Bittinger, K., 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=Improved+characterization+of+medically+relevant+fungi+in+the+human+respiratory+tract+using+next-generation+sequencing.">Improved characterization of medically relevant fungi in the human respiratory tract using next-generation sequencing.</a> Genome Biol. 15 (10), 487 (2014).</li><li>Jervis-Bardy, J., 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=Deriving+accurate+microbiota+profiles+from+human+samples+with+low+bacterial+content+through+post-sequencing+processing+of+Illumina+MiSeq+data.">Deriving accurate microbiota profiles from human samples with low bacterial content through post-sequencing processing of Illumina MiSeq data.</a> Microbiome. 3, 19 (2015).</li><li>Knights, 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=Bayesian+community-wide+culture-independent+microbial+source+tracking.">Bayesian community-wide culture-independent microbial source tracking.</a> Nat Methods. 8 (9), 761-763 (2011).</li><li>Lazarevic, V., Gaia, N., Girard, M., Schrenzel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decontamination+of+16S+rRNA+gene+amplicon+sequence+datasets+based+on+bacterial+load+assessment+by+qPCR.">Decontamination of 16S rRNA gene amplicon sequence datasets based on bacterial load assessment by qPCR.</a> BMC Microbiol. 16, 73 (2016).</li><li>Kurilshikov, A., Wijmenga, C., Fu, J., Zhernakova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+Genetics+and+Gut+Microbiome:+Challenges+and+Perspectives.">Host Genetics and Gut Microbiome: Challenges and Perspectives.</a> Trends Immunol. 38 (9), 633-647 (2017).</li><li>. Mock microbial communities Available from: <a target="_blank" href="https://www.atcc.org/en/Products/Microbiome_Standards.aspx?utm_id=t170601524l1">https://www.atcc.org/en/Products/Microbiome_Standards.aspx?utm_id=t170601524l1</a> (2017)</li></ol>

作者没有什么可透露的。

16S rRNA 的目标下一代测序是一种广泛使用的、快速的微生物特征18技术。但是, 许多因素, 包括批处理效果、环境污染、样本交叉污染、灵敏度和重现性都会对实验结果产生负面影响, 并混淆其解释7,19,20. 为了最好地促进强健的16S 分析, 微生物组工作流必须包括良好的实验设计、使用适当的控制、工作流步骤的?...

殖民人类的微生物群落被认为对人类健康和疾病影响新陈代谢、免疫发展、对疾病的易感性以及对疫苗接种和药物治疗的反应至关重要1, 2. 为了解微生物群对人类健康的影响而作出的努力目前强调识别与定义的解剖隔间 (即、皮肤、肠道、口腔、等) 相关的微生物, 以及内部的本地化站点。这些舱室3,4。这些调查工作的基础是迅速出现并增加了下一代测序技术的可获得性, 为样品的微生物遗传量 (微生物) 分析提供了一个大规模的并行平台。对于许多生理样本, 相关的菌群既复杂又丰富 (即, 大便), 但对于一些样本, 微生物群是由低生物量 (即, 人乳, 下呼吸道) 所代表的, 其中敏感度、实验性文物和可能的污染成为主要问题。微生物研究和适当实验设计的共同挑战是多个评论文章的主题5,6,7,8。
本文介绍了一种基于 rRNA 16S V4 区域9的目标测序的鲁棒实验管道, 以表征人乳的微生物群。微生物菌群分析的人奶不仅是复杂的先天低生物量的10, 但另外由高级别的人类 DNA 背景11,12,13,14和PCR 抑制剂的潜在结转15,16在提取的核酸。该协议依赖于商业上可用的提取工具包和半自动平台, 可帮助最大限度地减少样本准备批次之间的可变性。它包含了一个定义良好的细菌模拟社区, 它与样品一起处理, 作为质量控制来验证协议中的每一步, 并提供一个独立的管道健壮性度量。虽然所描述的协议是特定于人乳样本, 它很容易适应其他样本类型, 包括大便, 直肠, 阴道, 皮肤, 晕和口服拭子10,17, 并可以作为一个起点希望进行微生物分析的研究人员。

<table><tbody><tr><td>AllPrep RNA/DNA Mini Kit</td><td>Qiagen</td><td>80204</td><td>DNA/RNA extraction kit</td></tr><tr><td>Eliminase</td><td>Fisher Scientific</td><td>435532</td><td>RNase, DNase, DNA decontaminant</td></tr><tr><td>Thermo Mixer</td><td>Fisher Scientific</td><td></td><td>temperature-controlled vortexer</td></tr><tr><td>Buffer RLT plus</td><td>Qiagen</td><td>1053393</td><td>guanidinium thiocyanate lysis buffer/ Part of Allprep kit</td></tr><tr><td>&amp;szlig;-Mercaptoethanol</td><td>Sigma Aldrich</td><td>63689-25ML-F</td><td>&amp;szlig;-ME is a reducing agent that will irreversibly denature RNases by reducing disulfide bonds</td></tr><tr><td>LME Beads</td><td>MP Biomedicals</td><td>116914050</td><td>bead tube</td></tr><tr><td>QIAgen TissueLyzer</td><td>Qiagen</td><td>85300</td><td>automated sample disruptor adapter set</td></tr><tr><td>QIAshredder column</td><td>Qiagen</td><td>79654</td><td></td></tr><tr><td>QIAgen RB tube</td><td></td><td></td><td>manufacturer&#039;s microcentrifuge tube in kit</td></tr><tr><td>QIAcube and related plasticware</td><td>Qiagen</td><td>9001292</td><td>automated DNA/RNA purification instrument</td></tr><tr><td>DNA exitus plus</td><td>Applichem</td><td>A7089</td><td>non-enzymatic decontamination solution</td></tr><tr><td>EB Buffer</td><td>Qiagen</td><td>19086</td><td>elution buffer</td></tr><tr><td>QIAgility and related plasticware</td><td>Qiagen</td><td>9001532</td><td>robotic liquid handler</td></tr><tr><td>PCR water</td><td>MO BIO</td><td>17000-</td><td></td></tr><tr><td>5PRIME HotMasterMix</td><td>Quantabio</td><td>2200400</td><td></td></tr><tr><td>Barcoded reverse primers</td><td>Eurofin</td><td>No Catalog #&#039;s</td><td>designed and ordered</td></tr><tr><td>96 well PCR plate</td><td>USA scientific</td><td>1402-9708</td><td></td></tr><tr><td>Tapestation 2200 and related plasticware</td><td>Agilent</td><td>G2964AA</td><td>automated DNA/RNA fragment analyzer</td></tr><tr><td>D1000 reagents for Tapestation</td><td>Agilent</td><td>5067-5585</td><td>Sample buffer and ladder are part of this kit</td></tr><tr><td>OneStep PCR Inhibitor Removal Kit</td><td>Zymo Research</td><td>50444470</td><td>PCR inhibitor removal is done per the manufacturer&#039;s instructions.</td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td>DNA clean up kit: silica-membrane-based purification of PCR products</td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Thermo Fisher</td><td>Q32854</td><td>dimethylsulfoxide-based dilution buffer and dye are part of this kit.</td></tr><tr><td>Qubit Fluorometer</td><td>Thermo Fisher</td><td>Q33216</td><td></td></tr><tr><td>NanoDrop</td><td>Thermo Fisher</td><td></td><td>microvolume spectrophotometer</td></tr><tr><td>MiSeq 300 V2 kit</td><td>Illumina</td><td>15033624/15033626</td><td></td></tr><tr><td>MiSeq</td><td>Illumina</td><td>No Catalog #&#039;s</td><td>next generation sequencer</td></tr></tbody></table>

a method for targeted 16s sequencing of human milk samples

随着相对便宜、快速和高吞吐量测序的发展, 微生物群落的研究变得越来越普遍。然而, 与所有这些技术一样, 可重现的结果取决于实验室工作流, 其中包含适当的预防措施和控制。这是特别重要的低生物量样本, 污染细菌 DNA 可以产生误导的结果。本文详细介绍了一种半自动的工作流程, 即使用16S 核糖体 RNA (rRNA) V4 区域的目标测序, 在低到中吞吐量范围内, 识别出人类母乳样本中的微生物。该协议描述了从全牛乳样品的制备方法, 包括: 样品裂解、核酸提取、16S rRNA 基因 V4 区的扩增和质量控制措施的库准备。重要的是, 该议定书和讨论考虑了对低生物量样品的制备和分析具有显著意义的问题, 包括适当的阳性和阴性对照、PCR 抑制剂去除、环境样品污染、试剂或实验源和实验性最佳实践, 旨在确保重现性。虽然所描述的议定书是特定于人乳样本, 但它适用于许多低和高生物量样本类型, 包括收集在拭子上的样本, 冷冻整齐, 或稳定在保存缓冲区。

此处介绍的协议包括重要的质量控制 (QC) 步骤, 以确保所生成的数据符合协议灵敏度、特异性和污染控制的基准。该协议的第一个 QC 步骤是 16S V4 区域的 PCR 放大 (图 2)。通过电泳分析了每种样品的 PCR 产物的一个µL, 以确认其在 315-450 bp (图 2、红色箭头) 的预期尺寸范围内。一些人奶样本产生了较低数量的特定产品 (<strong class="xf...

Watch this Scientific Journal Video about A Method for Targeted 16S Sequencing of Human Milk Samples at JoVE.com

A Method for Targeted 16S Sequencing of Human Milk Samples

提出了一种半自动化工作流, 用于从人乳和其他低生物量样品类型中进行 16S rRNA 的目标排序。

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 ...

a-method-for-targeted-16s-sequencing-of-human-milk-samples

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

9.7K 次观看.  University of California at Los Angeles. 提出了一种半自动化工作流, 用于从人乳和其他低生物量样品类型中进行 16S rRNA 的目标排序。

视频: A Method for Targeted 16S Sequencing of Human Milk Samples

Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, optimization of their biotechnological synthesis is limited by the relatively low throughput of detection and quantification of monosaccharide and linkages. Conventional techniques of glycan analysis include chromatographic/mass-spectrometric methods with throughput on the order of hundreds of samples per day without automation. We demonstrate here, a genetically encoded bacterial biosensor for the high-throughput, linkage-specific detection and quantification of the fucosylated HMO structures, 2&#8217;-fucosyllactose and 3-fucosyllactose, which we achieved via heterologous expression of fucosidases. As the presence of lactose in milk or in biotechnological processes could lead to false positives, we also demonstrate the reduction of signal from lactose using different strategies. Due to the high throughput of this technique, many reaction conditions or bioreactor parameters could be assayed in parallel in a matter of hours, allowing for the optimization of HMO manufacturing.

We describe here the high-throughput detection and quantification of fucosylated human milk oligosaccharides (HMOs) using a whole-cell biosensor. We also demonstrate here, the adaptation of this platform towards analysis of HMO production strains, focusing on improving the signal to noise ratio.

生物技术环境下的氟黏膜化人乳三糖--用基因编码生物传感器分析

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, ...

科研

生物化学

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

免疫与感染

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 ...

生物学

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

从复杂的动物相关样品测序宏基因组和Metatranscriptomes:净化不纯

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and ...

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 ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

评估细菌素对小鼠肠道微生物的影响的方法

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

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. ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

食品和环境样品中沙门氏菌的准总体基因体分析

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

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 ...

<meta charset="utf8"></head><body>使用母亲自己的乳汁恢复巴氏杀菌的捐赠乳微生物群

Individualized Reconstitution of Human Milk Microbiota: A Feasible Approach in Real-World Settings

Mother&#39;s own milk (MOM) is the most complete nutritional resource for newborns. In cases where mothers are unable to produce sufficient milk or cannot breastfeed, the preferred alternative is pasteurized donor human milk (PDM), which is routinely provided by human milk banks. PDM offers a superior range of nutritional and immunological elements compared to any commercially available formula. However, to ensure biosafety, PDM undergoes pasteurization, a process that inactivates commensal microbiota and reduces certain bioactive compounds. This study presents a protocol designed to restore the microbiota of PDM using MOM as a microbial source, adapting the approach to a real-world clinical setting.
The protocol was implemented in a clinical trial conducted at a maternity hospital and its associated human milk bank, with the aim of providing personalized donor milk to preterm infants whose mothers cannot produce sufficient milk. The methodology involves inoculating PDM with 10% of MOM, followed by incubation at 37 &#176;C for 4 h. Microbiological analysis demonstrated successful bacterial growth in the inoculated milk (IM) post incubation, with the microbiota profile of the reconstituted milk (RM) closely resembling that of MOM, indicating effective microbiota restoration. These results suggest that the reconstitution protocol is feasible for implementation in neonatal care, with the potential to enhance the nutritional and immunological quality of PDM, thereby supporting the health and development of non-breastfed newborns.

<meta charset="utf8"></head><body>母乳微生物群的个体化重构:现实世界中的可行方法

The article describes a protocol for reconstituting pasteurized donor milk microbiota using the mother's own milk microbiota in practical, real-world settings. It demonstrates effective bacterial growth and microbiome modulation, supporting the feasible application of this procedure within the routine care of a maternity hospital and its associated human milk bank.

母乳微生物群的个体化重构:现实世界中的可行方法

Mother&#39;s own milk (MOM) is the most complete nutritional resource for newborns. In cases where mothers are unable to produce sufficient milk or cannot ...