We are thankful to Samuel Shepard, Peter Bazeley, and John David Bell for the administration of the Genomic MRI web pages. This work was supported by National Science Foundation Career award "Investigation of intron cellular roles" [grant number MCB-0643542].

All the used programs in the paper have been written using perl, and all the web pages have been created using PHP.
1. Starting Point:
Open the home page of the online Genomic MRI package at http://mco321125.meduohio.edu/~jbechtel/gmri/ . The web resource also provides instructions/explanations on the programs in the "Help (How-to/README)" link, while all published materials on Genomic MRI and similar algorithms are listed in the "Links to relevant resources" link.
2. Preparation and Uploading of Input Sequence(s).
Create a file with FASTA-formatted sequence(s) to start a GMRI analysis session. Each nucleotide sequence in this format should be preceded with a single line starting with the "&gt;" character that represents an identifier, followed on the same line by a short description of this sequence. Nucleotide sequences for GMRI analysis also permits characters like R, Y, N, X, etc. Hwever, non-A, T, C, G characters will not be processed by the program and will be skipped. Sequences in which repetitive elements have been "masked" (replaced by "N"s) can be used as input. Note that sequence characters are case insensitive.
<ol>
	<li>Begin a GMRI session by clicking on the "Start or Resume" button on the Genomic MRI home page. This takes the user to a page where nucleotide sequences can be uploaded. </li>
	<li>Copy-and-paste your FASTA-formatted sequences or upload a file containing the sequences from your local computer using the "choose file" button.</li>
	<li>Click on the "start new session with this file" button. A confirmation message should appear above the input window stating that "Your sequence has been successfully uploaded" and you should also get an alphanumeric "GMRI identifier" [the site calls it a "session label"] for your session (e.g. b16yMj), which can be used to retrieve and continue a session for up to two weeks after first use. </li>
</ol>
NOTE: Henceforth the input sequences are referred to as "userfile".
3. Get an Oligonucleotide Frequency Distribution of the Input Sequences (optional).
Click on the "SRI Analyzer" tab (top row) in order to get a distribution of oligonucleotide frequencies for the entire set of input sequences. The acronym SRI stands for short-range inhomogeneity. At this juncture, the user may specify the highest length of oligonucleotides (from 2 up to 9 nucleotides, default 6 nts) for which frequencies will be calculated. This selection is made by clicking on the desired option within the "Maximum oligomer size" list box. Then press the "Analyze File" button to initiate computation. A rough representation of the input sequence composition will immediately appear as a short table in the middle of this web page and downloadable as "userfile.comp.tbl". This table represents only the most and the least abundant oligonucleotides within the input sequences.
The entire frequency table for all possible oligonucleotides is generated as a file named "userfile.comp", which can be obtained via the "Download composition file" link. 
NOTE: SRI analyzer counts the entire set of all overlapping oligonucleotides.
4. Generate Random Sequences with the Same Oligonucleotide Composition As in the Input Sequences (optional).
(Completion of step 3 of the protocol is required for this task).
<ol>
	<li>Click on the "SRI Generator" tab (top row) to open up a new web page that creates random sequences. Choose the number of samples of random sequences to be generated using the list box on this web page. Each of these sample files will contain random sequences of the same number and length as the input sequences in "userfile". Moreover, if an input sequence contains non-A, T, C, or G characters, the random sequence will have "N"s at exactly the same positions as in the input sequence. </li>
	<li>Choose the longest length of oligonucleotides for which frequencies will be approximated in the random sequences. This can be chosen by checking the radio button for the desired oligomer level (e.g. "4-mers" for four-base oligonucleotides) in the table at the center of the screen. It is to be noted here that random sequences will consist of not only the approximate frequencies at the chosen oligomer level, but also the corresponding frequencies of shorter oligomer levels, as in the input sequences. Small fluctuations in the oligonucleotide frequencies of input and random sequences are possible due to the Markov Model procedure applied for the generation of random sequences.</li>
	<li>Start the program by clicking the "Generate File" button. If the input sequences are large it could take a couple of minutes to generate random sequences. Thus, a user should wait until blue "Download" links appear at the bottom of this page. The random sets are placed in files with names such as "userfile.randX_Y" where X is the number of the random set and Y is the chosen oligomer level (e.g. "userfile_rand2_4").</li>
</ol>
5. Analysis of Mid-Range Inhomogeneity (MRI) of Input and Random Sequences.
<ol>
	<li>Click on the "MRI Analyzer" tab (top row), which opens up a new web page that analyzes the mid-range inhomogeneity of the nucleotide composition of sequences.</li>
	<li>Select a sequence to be analyzed from the "File to analyze" list box (a choice between the input sequence and generated sets of random sequences can be made here).</li>
	<li>Choose the content type of MRI to be analyzed via the provided list box. (Seven content options are available: G+C; G+A; G+T; A; G; C; or T.)</li>
	<li>Choose the length of the window for which content-rich and content-poor sequences will be examined via the "Window size" list box (default is 50 nucleotides; the valid range is from 30 to 1000).</li>
	<li>Choose the upper threshold and lower threshold for content-rich and content-poor regions, respectively. These thresholds can be defined by the exact number of particular nucleotides in the current window (using the by number option in the list box) or by percentage of these nucleotides in the window (using the by percentage option)</li>
	<li>After all five choices have been made (for example: Sequence = "userfile"; Content = GC; Window size = 50; Upper threshold = 35; Lower threshold = 15), invoke the program by pressing the Analyze File button. The program scans through all sequences from the selected input consecutively. At each step it obtains a segment of the current sequence with length equal to the specified window size and computes whether the number or percentage of nucleotides of the chosen content is above the upper threshold or below the lower threshold. If the window does not match either criteria, the next overlapping window (shifted by one nucleotide) is selected for the same analysis. When a window is found where the sequence meets one of the threshold requirements for content-rich or -poor composition, the program saves the sequence of this window in the output file and generates a spike on the graphical output. After this, the program jumps to the next non-overlapping adjacent window and resumes the scanning process until the end of the sequence is reached.</li>
	<li>After completion of the program, a link to the output file (with name "userfile_GC_50_35..15" for the example above) appears and a graphical representation of the results is displayed in the middle of the web page (see Figure 1). On this graphical display all input sequences from the userfile are concatenated into a single string and presented as a horizontal black line on the X axis, with length in kilobases (kb) shown below. All content-rich regions along input sequences are marked as blue "upward" spikes, and content-poor regions as red "downward" spikes. The total numbers of content-rich and content-poor windows are shown in parenthses in the legend at the bottom of this figure (32 and 19, respectively). The figure serves to illustrate the relative abundance and the arrangement of MRI regions. Meanwhile specific details are presented in the output file (see Figure 3). In this file, all nucleotide sequence segments that match content-rich or -poor criteria and their coordinates are available to a user as a list according to their consecutive positions along the input file.</li>
	<li>After completion of MRI analysis for the chosen sequence a user can start a new process in the same web page by making changes to parameters and/or input files. For example, in order to examine the previously generated random sample #1 with the same MRI parameters, the user only needs to change the File to analyzeoption and select the "userfile_rand1_4" file, and then press the Analyze File button again. A new file and graphical display will replace the old one. The results and figures of ALL examinations under each "session lable" (GMRI identifier) will be saved and be available for two weeks from the last activity. In order to save the results/figures permanently, the user should select the "Download Files" tab (top row) and download the entire session or individual files, as needed.</li>
	<li>With this MRI Analyzer web page a user can study
	<ul>
		<li>(G+C)-rich and (A+T)-rich regions</li>
		<li>Purine (A+G)-rich and Pyrimidine (C+T)-rich regions</li>
		<li>Keto (G+T)-rich and aMino (A+C)-rich regions</li>
		<li>A-rich and A-poor regions</li>
		<li>G-rich and G-poor regions</li>
		<li>T-rich and T-poor regions</li>
		<li>C-rich and C-poor regions</li>
	</ul>
	</li>
	<li>The latest release of Genomic MRI has a new option for studying regions rich with Purine(R)/Pyrimidine(Y) alternation patterns that might form Z-DNA conformations. Currently, this option is available from the link "Z-DNA" and it works on the same basis as other aforementioned MRI regions. A user should select upper and lower thresholds for the number of (RY+YR) overlapping dinucleotides in the scanning window. The program produces a similar graphical output and a file of DNA segments enriched and depleted by alternating purines and pyrimidines. The putative Z-DNA regions must be highly enriched by alternating R/Y bases (see review F&amp;F 2011).</li>
</ol>
6. Additional Programs Within the Genomic MRI Package (optional).
The Genomic MRI resource also has two advanced options for generation of very specific random sequences. They are available through the "MRI Generator" and "CDS Generator" tabs in the top row.
<ol>
	<li>MRI generator creates randomized sequences with the same oligonucleotide composition as the input file (similar to SRI generator). However, in addition, randomized sequences mimic a particular MRI pattern specified by the user. Within this web page a user should specify from a list box a particular MRI pattern to be mimicked. The list box contains all patterns that have been examined in this session by MRI analyzer (e.g. "userfile_GC_50_35..15"). A random sequence generated with this option will have the same oligonucleotide composition as the selected input file and also the same GC-rich and -poor patterns as seen in "userfile_GC_50_35..15".</li>
	<li>CDS generator is used for randomization of protein coding sequences. It preserves the same amino acid sequence as the one coded by the user-specified input. In addition the program retains the same codon and di-codon biases as specified in the user-chosen input table. The online version of the CDS generator also accepts a protein sequence as an input. All other options for the program are offered only via stand-alone Perl scripts available for download from the main Genomic MRI web page.</li>
</ol>
7. Representative Results
This protocol allows a user to study compositional inhomogeneity of nucleotide sequences. Importantly, it also supports the generation of a variety of randomized sequences with an oligonucleotide composition approximating that of the input sequences. Usually, genomic sequences of complex eukaryotes are not homogeneous in composition, but rather represent a complex mosaic of sequence segments enriched by particular nucleotides (for example, purine-rich, (G+T)-rich, (A+T)-rich, etc.). These patterns at mid-range scale (30-1000 bp) are visualized by the graphical output of MRI analyzer that shows selected content-rich segments as upper blue spikes and content-poor segments as lower red spikes (see Figures 1 and 2). Typically, the number of any content-rich and content-poor regions in a natural sequence (Figure 1) is on the order of times higher than the number of the same types of regions in corresponding randomized sequences (Figure 2) having the same oligonucleotide composition. These sequence segments with mid-range inhomogeneity in nucleotide composition may be of interest to the user. They are available from the Genomic MRI output files for further investigation.
<img src="/files/ftp_upload/2663/2663fig1.jpg" alt="Figure 1" /> 
Figure 1. An example of the MRI analyzer graphical output from step 5.7. The results have been obtained on a sample of 44 human introns. Blue bars represent positions of GC-rich regions along these introns. Red bars represent GC-poor (or AT-rich) MRI regions. The y-axis contains upper and lower thresholds for the given content type.
<img src="/files/ftp_upload/2663/2663fig2.jpg" alt="Figure 2" /> 
Figure 2.MRI analyzer output for the random sequence "userfile.rand1_4". The graphical representation of MRI within a randomly generated sequence using the SRI generator program.
<img src="/files/ftp_upload/2663/2663fig3.jpg" alt="Figure 3" /> 
Figure 3.An example of the beginning of a textual output file from MRI analyzer. All content-rich and content-poor sequences detected by the program are presented in the last (fourth) column. Their relative positions, measured in the number of windows, are shown in the first column. The second and third columns are indicators for content-rich and content-poor regions, respectively.

<ol>
	<li>Bechtel, J. M., Wittenschlaeger, T., Dwyer, T., Song, J., Arunachalam, S., Ramakrishnan, S. K., Shepard, S., Fedorov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+mid-range+inhomogeneity+correlates+with+an+abundance+of+RNA+secondary+structures.">Genomic mid-range inhomogeneity correlates with an abundance of RNA secondary structures.</a> BMC Genomics. 9, 284-284 (2008).</li><li>Prakash, A., Shepard, S., Mileyeva-Biebesheimer, O., He, J., Hart, B., Chen, M., Amarachiniha, S., Bechtel, J., Fedorov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+forces+shaping+human+genomic+sequence+at+mid-range+scales.">Molecular forces shaping human genomic sequence at mid-range scales.</a> BMC Genomics. 10, 513-513 (2009).</li><li>Fedorov, A., Fedorova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3:+An+Intricate+Mosaic+of+Genomic+Patterns+at+Mid-range+Scale.">Chapter 3: An Intricate Mosaic of Genomic Patterns at Mid-range Scale.</a> Advances in Genomic Sequence Analysis and Pattern Discovery. , 65-91 (2010).</li><li>Shepard, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+4:+Binary-abstracted+Markov+models+and+their+application+to+sequence+classificatio.">Chapter 4: Binary-abstracted Markov models and their application to sequence classificatio.</a> The characterization and utilization of middle-range sequence patterns within human genome [dissertation]. , 57-157 (2010).</li></ol>

No conflicts of interest declared.

Regions with inhomogeneous nucleotide composition at mid-range scales (30-1000 nucleotides) are overabundant in the genomes of complex eukaryotes and can be found anywhere (intergenic regions, introns, untranslated regions of exons, repetitive elements). These regions are frequently associated with unusual DNA conformations. For instance, purine-/pyrimidine-rich sequences tend to form DNA triplexes (H-DNA); sequences with alternating purine/pyrimidine bases are associated with Z-DNA conformations; (G+C)-rich regions exhibit structural abnormalities in B-DNA and could be prone to backbone cleavage; (A+T)-rich regions might form an unusual structure - a DNA unwinding element; etc. (reviewed by Fedorov &amp; Fedorova 2010). Some of these mid-range patterns (e.g. (G+T)-rich regions) are scarcely investigated and still await thorough exploration and recognition. The main aim of our Genomic MRI web resource is to help users in the identification of these MRI regions for their further experimental analysis and for exploration of their possible functions. Knowledge of MRI regions could be incorporated into and improve the new generation of gene predictor programs (Shepard 2010) and advance our understanding of genome functions and properties.

<ul>
 <li>Computer with Internet</li>
 <li>Files with nucleotide sequences for examination.</li>
</ul>

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.

Watch this Scientific Journal Video about Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA at JoVE.com

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

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.

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

genomic-mri-public-resource-for-studying-sequence-patterns-within

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Biology

10.0K ビュー.  University of Toledo Health Science Campus. 我々は、ゲノム配列の解析のために公共の計算のウェブサイトを提示する。それは様々な非ランダムな塩基組成とDNA配列のパターンを検出します。このリソースは、また複雑さの多様なレベルでランダム化されたシーケンスを生成します。

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

Isolation of Genomic DNA from Mouse Tails

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

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.

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.

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

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.

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

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.

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

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. The simplicity and power of the yeast aging model can also be explored to study DNA damage and genome maintenance as well as their contributions to diseases during aging. Here, we describe a system to study age-dependent DNA mutations, including base substitutions, frame-shift mutations, gross chromosomal rearrangements, and homologous/homeologous recombination, as well as nuclear DNA repair activity by combining the yeast chronological life span with simple DNA damage and mutation assays. The methods described here should facilitate the identification of genes/pathways that regulate genomic instability and the mechanisms that underlie age-dependent DNA mutations and cancer in mammals.

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Here we describe a set of DNA mutation assays that can be combined with the yeast chronological life span model to study the genes/pathways that regulate or contribute to genomic DNA instability during aging.

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

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 expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

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 ...