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微生物测序指南-原核转录组

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organic substance transport amino acid transport ion transport amino acid transmembrane transp... bioluminescence membrane cellular_component transporter activity anion transmembrane transporte... organic acid:sodium symporter ... organic acid transmembrane tra... organic anion transmembrane tr... carboxylic acid transmembrane ... GTPase activity transmembrane transporter acti... solute:sodium symporter activi... amino acid transmembrane trans... inorganic cation transmembrane... neutral L−amino acid secondary ... neutral amino acid transmembran... alanine:sodium symporter activi...
GO:0044765 single−organism tran... 0.00519 73 / 454
GO:0015849 organic acid transpo... 0.04420 14 / 49
GO:0006811 ion transport NA 37 / 217
பைடு நூலகம்
GO:0006820 anion transport 0.00511 19 / 64
GO:0044699 single−organism proc... NA 177 / 1603
GO:0006810 transport 6.84e−09 122 / 710
GO:0071702 organic substance tr... NA 38 / 217
GO:0071705 nitrogen compound tr... 0.01239 16 / 52
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−4
log2(fold change)
−1 1
4
GO:0008150 biological_process NA 277 / 2632
GO:0051179 localization 3.93e−09 129 / 752
GO:0051234 establishment of loc... 6.84e−09 122 / 712
The Most Enriched GO Terms (Vp_15421vsVp_1616) localization * transport * establishment of localization * anion transport * single−organism transport * nitrogen compound transport * organic anion transport * organic acid transport * carboxylic acid transport *
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Vp_15421 vs Vp_1616
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− log10(qval)
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DEGs ( 881 )
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up: 439 down: 442
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GO:0015711 organic anion transp... 0.04420 14 / 49
GO:0046942 carboxylic acid tran... 0.04420 14 / 49
2
Statistics of Pathway Enrichment
Valine, leucine and isoleucine degradation Two−component system

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微生物学研究中的高通量测序技术使用方法

微生物学研究中的高通量测序技术使用方法

微生物学研究中的高通量测序技术使用方法高通量测序技术是一种在微生物学研究中广泛应用的先进技术。

它能够快速、准确地测定微生物样品中的DNA序列，帮助研究人员了解微生物的物种组成、功能以及它们在不同环境中的分布和相互作用。

本文将介绍高通量测序技术的使用方法，包括样品处理、测序仪选择、实验操作等内容。

1. 样品处理在进行高通量测序之前，首先需要对微生物样品进行处理。

这包括DNA的提取、纯化和扩增。

DNA提取方法应根据样品的不同特点进行选择。

常见的方法有酚/氯仿法、磁珠法和商业试剂盒法等。

DNA纯化是为了去除携带有其他杂质的DNA片段，保证测序结果的准确性。

DNA扩增通常使用PCR方法，可以选择通用引物或特异引物进行扩增，以获取感兴趣的DNA片段。

2. 测序仪选择高通量测序技术有多种选择，常见的有Illumina HiSeq、ABI SOLiD 和Ion Torrent等。

选择合适的测序仪需要根据研究目的、实验预算和实验室资源等因素进行综合考虑。

不同的测序仪有不同的优势和适用范围。

比如，Illumina HiSeq具有高通量、高精度和较低的错误率，适用于研究微生物的物种组成和功能；而ABI SOLiD则适用于重测序、基因表达和SNP检测等研究。

3. 实验操作高通量测序实验的操作流程主要包括文库构建、测序和数据分析三个步骤。

文库构建是将DNA样品通过特定方法转化为可以用于测序的DNA文库。

常见的文库构建方法包括PCR文库构建、接头连接法和DNA片段捕获法等。

在文库构建过程中，需要注意对文库的质量进行检测和评估，确保测序结果的准确性。

测序过程中，需要根据不同的测序仪和试剂盒选择适当的实验条件。

测序仪的操作通常由供应商提供的说明书进行指导。

需要注意的是要合理安排样品的测序顺序，避免交叉污染和测序顺序对结果产生影响。

数据分析是高通量测序研究的最后一步，对测序得到的数据进行生物信息学分析。

常见的数据分析内容包括序列去除、序列拼接、序列比对和物种注释等。

原核转录组测序

原核转录组测序

原核转录下转录出来的所有mRNA。由于原核生物mRNA没有polyA
尾结构，需要去除rRNA的特点，诺禾致源针对不同的研究物种采取有效的方法去除rRNA，保证数据量。
技术参数样品要求 rRNA去除 类型 测序策略 数据量类型 项目周期
图1 高通量筛选转录因子过表达结构的流程图
［案例二］ 基于副溶血性弧菌基因的表达比较分析感染 期间其调控网络及对环境的应答反应
副溶血性弧菌（Vibrio Parahemolyticus），为革兰氏阴性杆 菌，进食含有该菌的食物可致食物中毒，主要来源于鱼、虾、 蟹、贝类和海藻等海产品。该研究采用RNA-seq对体内和体 外培养的副溶血性弧菌进行分析，主要针对密度感应系统依赖 的T3SS1和T6SS2调控机制研究其致病机理。
［案例一］ 结核分枝杆菌转录因子过表达调控网络的调 控特征
结核分枝杆菌（Mycobacterium tuberculosis）是导致人类 感染结核病的病原菌，全球每年有15亿人被感染，并且有 130万人死于该病。结核分枝杆菌中有近200个转录因子 （TFs）和DNA结合蛋白参与环境变化的应答反应。该研究 借助RNA-seq技术对转录因子过表达进行分析，揭示结核分 枝杆菌中近200个转录因子控制的基因调控网络在其应对宿主 环境变化中的作用。
原核转录组测序 RNA样品总量: ≥3 μg； RNA样品浓度: ≥65 ng/μL
Ribo-Zero rRNA Removal Ki：1 Gb clean data； 高深度：2 Gb clean data 45天
案例解析
[2] Jonathan L, Xiaohui Z, Anjali M, et al. Comparative RNA-Seq based dissection of the regulatory networks and environmental stimuli underlying Vibrio parahaemolyticus gene expression during infection [J]. Nucleic Acids Research, 2014, 42(19):12212-23
转录组测序原理.pptx

转录组测序原理.pptx


激发碱基荧光并收集荧光信号
去除阻断基团和荧光基团
Cycle 2-n:
重复前面的步骤
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ห้องสมุดไป่ตู้基片段杂交
OH
diol
P7 P5
Flow Cell接头
diol diol
模板杂交
diol diol
延长
diol diol
变性
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Cluster station
• 剩下的复制链其一端“固定”在芯片上，
Throughput : up to 25 Gb per day
Output 26-35 Gb 75-100 Gb 150-200 Gb
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基于SBS测序技术
3’-
…-5’
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GTATTTTCGGCACAG
A
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A
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C TG
Cycle 1:按顺序加入反应试剂
合成第一个碱基
清除未反应的碱基和试剂
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鉴定基因可变剪接
exon1
common reads
exon2
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鉴定融合基因
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Paired Reads distribution
Reads cluster
主要内容• 样品检测 • 制备 • Cluster Station • Illumina Sequencing • 生物信息分析
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新一代测序技术
真核转录组测序

真核转录组测序

图2 显著富集的KEGG通路图1 A 新合成异源多倍体小麦中12种差异表达基因 B 非加性表达miRNA与亲本显性表达miRNA的 等级聚类分析和两者的关联图3 菟丝子与拟南芥、番茄转移RNA和非转移RNA的表达和富集分析. mRNA and small RNA transcriptomes reveal insights into dynamic homoeolog regulation of allopolyploid heterosis in nascent hexaploid wheat [J]. The Plant Cell, 2014: tpc. 114.124388.. Effects of Tris(1,3-dichloro-2-propyl) Phosphate (TDCPP) in Tetrahymena Thermophila: [3] Kim G, LeBlanc M L, et al. Genomic-scale exchange of mRNA between a parasitic plant and its hosts [J]. Science, 2014, 345图1 人类着床前胚胎在发育过程中的基因表达聚类分析案例二 利用单细胞RNA-seq手段追踪人类和小鼠胚胎的遗传发育本研究采用单细胞RNA-seq技术对人类和小鼠胚胎从卵母细胞到桑椹胚阶段的转录组动态进行了全面的分析。

通过权重基因共表达网络分析（WGCNA），发现通过少量共表达基因功能性模块就可以简明地描绘每个发育阶段。

小鼠和人类植入前胚胎比较分析结果表明，人类和小鼠之间发育特异性和时序上存在差异，大多数的人类阶段特异性模块（9 个）在物种间具有保守性。

此外，鉴别出人类和小鼠网络中一些保守的模块成员：hub选基因可能参与调控哺乳动物着床期以前的胚胎发育过程。

图2 人类胚胎基因共表达模块[1] Yan L, Yang M, Guo H, et al. Single-cell RNA-Seq profling of human preimplantation embryos and embryonic stem cells [J]. Nature structural & molecular biology, 2013.[2] Xue Z, Huang K, Cai C, et al. Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing [J]. Nature, 2013, 500(7464): 593-597.图1 栽培番茄和野生番茄构建系统进化树案例二 比较转录组揭示野生种苎麻向栽培种苎麻进化机制诺禾致源携手中国农业科学院麻类研究所，利用比较转录组解析栽培种苎麻和野生种苎麻在基因序列水平上的自然变异，找到13个受到正向选择的基因，这些差异可能与植物的抗病或者抗逆境有关，推测生物和非生物胁迫在苎麻的驯化过程中可能起着非常重要的作用；另外，两个受正向选择的基因与苎麻的纤维产量相关，主要是人工选择导致的。