这项研究是支持的，部分由美国国立卫生研究院（GM071440 CH和NS051630/MH076090/MH078972的PJ）。

1。基因组DNA碎片适合的全基因组测序平台使用所希望的大小范围内的超声处理的片段的基因组DNA。 （我们通常超声〜300个基点。）验证的粒度分布零散的基因组DNA，1％琼脂糖凝胶电泳（ 图1）。 2。 DNA的制备确定起始DNA金额5-HMC丰富的基因组DNA的基础上。由于5 - HMC水平的显着不同组织类型的不同，启动DNA金额的依赖样品5-HMC水平。请参阅表1的例子。 3。 β-GT催化反应（葡萄糖转移反应） <ol><li>混匀移液混合物，详情载于表2和孵化，在37°C水浴1小时。 </li><li>培养结束后，清理用QIAquick核苷酸去除试剂盒的反应，使用10微克的DNA每列。每列30μl水洗脱结合起来。 </li></ol> 4。生物素化反应（点击化学） <ol><li>添加DBCO-SS-PEG3-生物素共轭的工作溶液（1mM）中洗脱的DNA溶液（从步骤3）至最终浓度为150μM（ 即 ，每30微升DNA溶液的工作溶液的5微升）。 </li><li>混合移液和孵育在37℃水浴2小时。 </li><li>清理用QIAquick核苷酸去除试剂盒的反应。理想的总洗脱体积为100微升。 </li><li>量化使用回收的DNA量微升规模分光光度计（ 如 。的NanoDrop）。 </li></ol> 5。含HMC-DNA捕获<ol><li> 50微升的Dynabeads MyOne链霉亲和素C1 3次洗净，用1ml 1X B＆W缓冲液根据制造商的说明。珠粒分离的磁性的立场。 </li><li>加入等体积的2倍B＆W缓冲恢复的生物素化ðNA（100微升）洗过的小珠。 </li><li>孵育15分钟，在室温下在旋转器上轻轻旋转。 </li><li>带有磁性支架分隔珠粒珠粒和洗3次，用1毫升1X B＆W缓冲。 </li><li>洗脱DNA孵育中的小珠100微升新鲜制备的50mM的DTT为2小时，在室温下在旋转器上轻轻旋转。 </li><li>珠粒分离的磁性的立场。吸取洗脱液和负载到微生物分拆列根据生产指令删除DTT。现在是在溶液中的目标DNA。 </li><li>净化从以前的步骤由Qiagen公司MinElute PCR纯化试剂盒进行纯化，洗脱的DNA在10μlEB缓冲液洗脱的DNA。量化DNA荧光量子比特，或的NanoDrop如果浓度高于20毫微克/ UL。 DNA是下游的全基因组测序文库制备做好准备。 </li></ol> 6。代表性的成果如果质量Øf基因组DNA是高的，典型的β-GT和生物素化反应后恢复产量约60-70％。然而，捕获效率取决于样品5-HMC水平与不同的组织类型的显着变化。通常情况下，脑组织的基因组DNA的捕获效率是〜4-9％，并且在一些极端的情况下，效率可高达12％。对于ES细胞，平均捕获效率是〜2-4％，在对比〜0.5％的神经干细胞。效率最低为癌症细胞的基因组DNA。所有丰富的DNA是准备为下一代标准库准备协议。此外，也可以使用所捕获的DNA作为模板的实时PCR检测到输入DNA相比，富集的一些片段，如果相关的引物是可用的。 <img alt="图1" src="/files/ftp_upload/4441/4441fig1.jpg" /> 图1。经声波处理的人基因组DNA片段在1％琼脂糖凝胶电泳。10μg的人iPS细胞在120μl的1X TE缓冲液中分离出的基因组DNA，使用超声波处理装置（Covaris）超声处理。超声处理后，2微升的超声处理的DNA加载到1％琼脂糖凝胶上，使用100 bp的DNA标记，用来比较的超声处理的DNA片段的大小。 <table border="1"><tbody><tr><td> 元件 </td><td> 量 </td><td> 终浓度 </td></tr><tr><td>水</td><td> _微升</td><td></td></tr><tr><td> 10 Xβ-GT反应缓冲液</td><td> 2微升</td><td> 1 X </td></tr><tr><td>高达10微克基因组DNA的</td><td> _微升</td><td>高达500纳克/微升</td></tr><tr><td> UDP-6-N 3 -葡萄糖（3毫摩） </td><td> 0.67微升</td><td> 100μM </td></tr><tr><td> β-GT（40μM） </td><td> 1微升</td><td> 2μM </td></tr><tr><td>总成交量</td><td> 20微升</TD&gt; <td></td></tr></tbody></table> i）对于组织基因组DNA（5-HMC含量&gt; 0.1％） <table border="1"><tbody><tr><td> 元件 </td><td> 量 </td><td> 终浓度 </td></tr><tr><td>水</td><td> _微升</td><td></td></tr><tr><td> 10 Xβ-GT反应缓冲液</td><td> 10微升</td><td> 1 X </td></tr><tr><td>高达20微克基因组DNA的</td><td> _微升</td><td>高达500纳克/微升</td></tr><tr><td> UDP-6-N3-葡萄糖（3毫摩） </td><td> 1.33微升</td><td> 100μM </td></tr><tr><td> β-GT（40μM） </td><td> 2微升</td><td> 2μM </td></tr><tr><td>总成交量</td><td> 40微升</td><td></td></tr></tbody></table> ii）对于干细胞的基因组DNA（中位数为5-HMC含量约为0.05％） <table border="1"><tbody><tr><td>元件</td><td>量</td><td>终浓度</td></tr><tr><td>水</td><td> _微升</td><td></td></tr><tr><td> 10 Xβ-GT反应缓冲液</td><td> 10微升</td><td> 1 X </td></tr><tr><td>高达50μg的基因组DNA的</td><td> _微升</td><td>高达500纳克/微升</td></tr><tr><td> UDP-6-N3-葡萄糖（3毫摩） </td><td> 3.33微升</td><td> 100μM </td></tr><tr><td> β-GT（40μM） </td><td> 5微升</td><td> 2μM </td></tr><tr><td>总成交量</td><td> 100微升</td><td></td></tr></tbody></table> iii）对于癌症细胞基因组DNA（5-HMC低含量约0.01％） 表1作为输入的DNA的量的和使用的样本的标记反应通过选择性化学标记方法与各种5-HMC水平。 <table border="1"><tbody><tr><td> 样品 </td><td> 5 HMC水平的 </strong&gt; </td><td> 开始的DNA（微克） </td><td> 恢复后的标签（输入珠）（微克） </td><td> 回收率 </td><td> 下拉DNA（纳克） </td><td> 下拉产量 </td></tr><tr><td>成年小鼠小脑</td><td> 0.4％ </td><td> 10 </td><td> 7.5 </td><td> 75％ </td><td> 236 </td><td> 3.1％ </td></tr><tr><td>产后第7天小鼠小脑</td><td> 0.1％ </td><td> 11 </td><td> 9 </td><td> 82％ </td><td> 140 </td><td> 1.6％ </td></tr><tr><td>小鼠胚胎干细胞E14 </td><td> 0.05％ </td><td> 60 </td><td> 42 </td><td> 70％ </td><td> 350 </td><td> 0.8％ </td></tr></tbody></table> 表2中。从小鼠脑组织和ES细胞的代表性的结果。

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没有利益冲突的声明。

5 - 羟甲基胞嘧啶（5-HMC）是最近确定的表观遗传修饰在某些哺乳动物的细胞类型中的大量存在。这里介绍的方法是确定基因组中广泛分布的5-HMC。我们使用T4噬菌体的β-葡糖基转移酶，转移含有叠氮基团上的羟基基团的5-HMC一个精心设计的葡萄糖部分。叠氮基团，可以通过化学方法用生物素修饰，用于检测，亲和富集，和5-HMC-含在哺乳动物基因组的DNA片段测序。该协议具有优势超过5 HMC抗体为基础...

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 产品编号 </td><td> 评论 </td></tr><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td> 5M氯化钠（NaCl） </td><td> Promega公司</td><td> V4221 </td><td></td></tr><tr><td> 0.5M pH值8.0乙二胺四乙酸（EDTA） </td><td> Promega公司</td><td> V4231 </td><td></td></tr><tr><td> ，1M Trizma碱（三）pH7.5的</td><td> Invitrogen公司</td><td> 15567-027） </td><td></td></tr><tr><td> 1M HEPES，pH7.4的</td><td> Invitrogen公司</td><td> 15630 </td><td></td></tr><tr><td>氯化镁（MgCl 2）中1M </td><td> Ambion公司</td><td> AM9530G </td><td></td></tr><tr><td>二甲亚砜（DMSO） </td><td>西格玛</td><td> D8418 </td><td></td></tr><tr><td>吐温20 </td><td>费舍尔生物</td><td> BP337-100 </td><td></td></tr><tr><td> DBCO-SS-PEG3-生物素共轭</td><td>点击化学工具</td><td> A112P3 </td><td></td></tr><tr><td> 1,4 - 二硫苏糖醇（DTT），超超纯</td><td> Invitrogen公司</td><td> 15508-013 </td><td></td></tr><tr><td> QIAQUICK核苷酸去除试剂盒</td><td> Qiagen公司</td><td> 28304 </td><td></td></tr><tr><td>微型生物分拆列</td><td> Bio-Rad公司</td><td> 732-6222 </td><td></td></tr><tr><td>磁珠MyOne </td><td> Invitrogen公司</td><td> 650-01 </td><td></td></tr><tr><td>链霉亲和素C1 </td><td></td><td></td><td></td></tr><tr><td> Qiagen公司MinElute PCR Purification Kit纯化</td><td> Qiagen公司</td><td> 28004 </td><td></td></tr><tr><td>超纯琼脂糖</td><td> Invitrogen公司</td><td> 16500500 </td><td></td></tr><tr><td> UDP-6-N 3-葡萄糖</td><td>主动母题</td><td> 55013 </td><td></td></tr><tr><td></td><td></td><td></td><td> 酶 </td></tr><tr><td> β-葡糖基转移酶（β-GT） </td><td>新英格兰生物实验室</td><td> M0357 </td><td></td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>超声波处理设备</td><td> Covaris </td><td></td><td></td></tr><tr><td>台式离心机</td><td></td><td></td><td></td></tr><tr><td>水浴</td><td> Fisher Scientific则</td><td></td><td></td></tr><tr><td>凝胶运行设备</td><td> Bio-Rad公司</td><td></td><td></td></tr><tr><td> NanoDrop1000 </td><td> Thermo Scientific的</td><td></td><td></td></tr><tr><td> Labquake管摇床</td><td> Barnstead </td><td></td><td></td></tr><tr><td> Labquake管摇床</td><td> Thermolyne </td><td></td><td></td></tr><tr><td>磁分离架</td><td> Promega公司</td><td> Z5342 </td><td></td></tr><tr><td>量子比特2.0荧光</td><td> Invitrogen公司</td><td></td&gt; <td></td></tr><tr><td></td><td></td><td></td><td> 试剂设置 10所述β-GT的反应缓冲液（500毫摩尔HEPES PH值7.9，250mM的MgCl 2的 ）2所述绑定和洗涤（B＆W）缓冲液（10mM的Tris pH值7.5，1mM EDTA的溶液，2M氯化钠，0.02％吐温20） 。 </td></tr></tbody></table>

selective capture of 5-hydroxymethylcytosine from genomic dna

5 - 甲基胞嘧啶（5-MC）构成约2-8％，总在人类基因组DNA胞嘧啶和影响广泛的生物学功能，包括基因表达，维持基因组的完整性，亲本印记，X染色体失活，调节发育，衰老和癌症。最近，氧化的5-MC，5 -羟甲基胞嘧啶（5-HMC），存在下，在哺乳动物细胞中被发现，特别是在胚胎干（ES）细胞和神经元细胞2-4。 TET家族铁（II）/α-酮戊二酸依赖性双加氧酶2，3 5-MC催化氧化所产生的5-HMC。 5-HMC提出参与胚胎干细胞（MES），正常的造血和恶性肿瘤，和受精卵开发2，5-10维护。为了更好地理解5-HMC的功能，可靠的和直接测序系统是必不可少的。传统的亚硫酸氢钠测序可以不区分5-HMC从5-MC 11 </sup&gt;。为了解开生物5 - HMC，我们已经开发出一种高效，高选择性的化学标记方法和捕获5-HMC，利用噬菌体酶，葡萄糖部分，增加了一个5-HMC特别是12。 在这里，我们描述一个简单的两步骤程序选择性化学标记的5-HMC。在第一标记步骤中，基因组DNA中的5-HMC被标记为与6 - 叠氮-葡萄糖催化β-GT，葡糖基转移酶从T4噬菌体，在从一种方式，将6 - 叠氮-葡萄糖以5-HMC改性的辅助因子，:UDP-6-N3-Glc的（6-N3UDPG）。在第二步骤中，生物素化，二硫键的生物素连接器附着的叠氮基，通过点击化学。这两个步骤是非常具体的，高效的，从而完成标签，无论5-HMC丰富的基因组区域和极低的背景。生物素化的5 - HMC后，5-HMC-含DNA片段，然后选择性地捕获使用抗生蛋白链菌素珠粒的密度无关的方式。所得的5-HMC-富集的DNA片段可用于下游分析，包括下一代测序。 我们的选择性标记和捕获协议赋予高灵敏度，适用于任何变量/ 5-HMC不同丰度的基因组DNA的来源。虽然此协议的主要目的，是其下游的应用程序（ 即 ，下一代测序绘制出了在基因组的5-HMC分布），它是兼容的单分子实时SMRT（DNA）测序，这是能够提供单碱基分辨率的测序5 - HMC。

Watch this Scientific Journal Video about Selective Capture of 5-hydroxymethylcytosine from Genomic DNA at JoVE.com

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

描述的是一个两步骤的标记过程中使用的β-葡糖基转移酶（β-GT）来传输的叠氮化物5-HMC-葡萄糖，然后通过点击化学生物素链接器为容易和密度独立富集转移。这种高效的和具体的标记方法使富集5 - HMC具有极低的背景和高通量表观映射的通过新一代测序。

从基因组DNA选择性捕获的5  - 羟甲基

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

selective-capture-of-5-hydroxymethylcytosine-from-genomic-dna

Research

JoVE Journal

Biology

11.9K 次观看.  Emory University School of Medicine. 描述的是一个两步骤的标记过程中使用的β-葡糖基转移酶（β-GT）来传输的叠氮化物5-HMC-葡萄糖，然后通过点击化学生物素链接器为容易和密度独立富集转移。这种高效的和具体的标记方法使富集5 - HMC具有极低的背景和高通量表观映射的通过新一代测序。

视频: Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

Isolation of Genomic DNA from Mouse Tails

从鼠尾基因组DNA提取

科研

生物学

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

大型插入环境基因组文库生产

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

超高分子量基因组DNA的提取土壤和沉积物

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.

We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.

引物延伸捕获：从严重退化的DNA来源有针对性的顺序检索

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

改造使用含RNA寡核苷酸基因组DNA的RNA / DNA杂交一代

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.

This protocol is a cost effective alternative for efficient parallel clarification and plasmid DNA purification from E. coli cultures. The AcroPrep Advance process starts with an optimized lysate clarification filter plate followed by purification on a high binding capacity DNA binding filter plate.

从原核文化精简纯化的质粒DNA

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

磁共振成像基因 - 公共资源为研究基因组DNA序列内模式

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Competitive Genomic Screens of Barcoded Yeast Libraries

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

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

条形码酵母图书馆竞争力的基因屏幕

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

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

超高分子量DNA提取微生物席

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

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

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

罕见的基因变异检测从集资使用斯普林特测序

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