“Two Are Better Than One”: Case Studies of Brief Effective Therapy

Conversions of cytosine and its modified derivatives upper row during BS and oxBS middle row as well as their appearance after sequencing lower row. Black straight arrows indicate the intended conversion reaction; red dashed arrows indicate possible conversion errors. The PCR uses gene specific primers to amplify a specific target region ligated to the hairpin linker. After incubation, the amplified product needs to be purified to remove PCR residues, such as nucleotides, salt and primers which would interfere with downstream processes.

Amplicon preparation for sequencing is finalised by subjecting the purified product to a second PCR Table 3 and Figure 2. In this amplification, primers are not gene specific, but bind to the adapter part introduced during the first PCR. The second primer pair provides the adapter sequence which facilitates the binding to the sequencing platform and in addition carries a sample specific ID. The program aligns the sequencing reads against a given reference sequence and determines the methylation state for each cytosine.

To exploit all the information contained in the hairpin amplicon, four individual analysis steps have to be performed:. CpG methylation is analysed by providing a genomic reference sequence without for each locus, consisting of the unconverted DNA sequence from top and bottom strand with the converted C replaced by T hairpin linker sequence in between Supplement S.

Cytosines of the hairpin linker will be analysed independently and therefore have to be replaced by Ts. BiQHT provides several filter options to dispose unwanted sequencing reads. However, filtering might be optimised for individual amplicons by including additional parameters such as conversion rate, alignment score or fraction of unrecognized sites.

For an unbiased detection of nonCpG methylation, all CpGs in the reference file and also in the sequencing read file must be replaced by NpNs. Usually, the filter conditions from the CpG methylation analysis can be applied. The unmodified cytosines in the hairpin linker allow the determination of cytosine conversion, unbiased by nonCpG methylation.

To extract the information, sequencing reads are aligned to the genomic sequence of the hairpin linker Supplement S. The variable loop sequence creates UMIs which cannot be described by one reference sequence alone.

Franklin Miller, Inc.

This function can also be used to determine the state of 5mC and 5hmC within the hairpin linker. The Hairpinanalyzer is a python based script which accepts the output of BiQHT, restores the ds information and generates the following output: A map of methylation pattern in form of a portable network graphic png.

A text file for each sample containing the CpG methylation information of each read and in addition, position specific nonCpG methylation, conversion rates and SNPs. Note that the results of BS and oxBS are stored as individual files and the level of 5hmC must be calculated by comparing both outputs. The most simple calculation is the subtraction of the mean methylation level of oxBS from BS results. Additionally, to also gain the distribution of 5hmC, this calculation has to be done for fully methylated CpGs, hemimethylated CpG on the top- and hemimethylated CpGs on the bottom strand, respectively.

The script of the Hairpinanalyzer is available on GitHub https: The conversion scheme in Figure 4 suggests that 5hmC levels are simply determined by subtracting mean methylation levels of BS reactions from those of oxBS reactions. We observe that this may lead to inaccurate estimates due to random sampling of different cells and the omission of conversion errors.

By this, we can accurately estimate 5hmC levels. Based on the information provided, H O TA considers the ds information and conversion rates to estimate accurate 5mC and 5hmC level as well as their ds distribution.

In addition, it predicts the efficiencies of Dnmts and Tets. Furthermore, based on the ds information, H O TA provides a more accurate discrimination of maintenance and de novo methylation compared to single strand based models. All tools come with detailed instruction for easy use.

  • Upcoming Events.
  • .
  • US Army, NBC DECONTAMINATION, FM 3-5, Survival Medical Manual, Army Field Manual.

In addition, we included a test data set to the supplement information, which includes raw data from MiSeq sequencing, BiQHT and Hairpinanalyzer output as well as the input files and the results of the H O TA analysis. This section is followed by a brief summary of use cases on mouse primordial germ cells and human monocytes, respectively. A full description for the additional data can be found in supplement sections S. Following the method outlined in Figure 2 we sequenced PCR products on an Illumina MiSeq platform obtaining a mean read coverage of per locus. Each column represents one CpG position and each row one unique sequence read, which corresponds to the region specific pattern of one chromosome.

Hairpin Methylation Pattern Maps. Each column represents one CpG dyad, each row one sequenced chromosome. The very left column gives the mean methylation pattern over all analysed CpGs. Mean methylation level of BS upper panel and oxBS middle panel samples as well as the predicted 5hmC amount and distribution lower panel. Based on the rates, individual conversion erros for BS and oxBS were calculated. A scheme of all possible conversions and conversion errors are given in Figure 4. However, for simplicity, we here predicted the mean levels over all CpGs across one amplicon.

Fasting vs. Eating Less: What's the Difference? (Science of Fasting)

The ds information demonstrates that 5hmC in most cases occurs in an asymmetric pattern paired either with C Figure 6 , lower diagram, light green or 5mC Figure 6 , lower diagram, dark green. Only the minority of CpGs contain 5hmC in a symmetrical state Figure 6 , lower diagram, yellow. In addition to the 5hmC distribution, H O TA calculates the enzyme efficiencies for Dnmts maintenance and de novo methylation and Tets hydroxylation for each time point. Our analysis shows that the efficiencies differ clearly between the distinct regions.

In general, we can observe a loss in maintenance and de novo methylation activity together with an increase in hydroxylation activity. At this time point, PGCs are known to undergo a rapid replication dependent demethylation, probably supported by Tet mediated oxidation In previous work we identified several deferentially methylated regions DMRs derived from such active demethylation and showed that the loss of 5mC is likely to be caused by Tet mediated oxidation We indeed detect a region specific presence and dynamic change of 5hmC during this time course Supplement Section 4.

The understanding of dynamic changes of DNA methylation during development and disease is a major research area in the field of epigenetics. Such a task can only be realised if DNA modifications can be measured accurately. This is especially challenging for oxidative derivatives of 5mC considering their low abundance and unequal distribution in the genome. Furthermore there is a clear lack of reproducible and easy-to-handle assays for determination of their distribution at single base resolution.

The precise knowledge however would allow to model their presumed influence on epigenetic inheritance and temporal stability. In addition, most chemical assays only allow measuring DNA methylation on one DNA strand, making it impossible to determine the precise rates of symmetric methylation and its consequences.

Log in to view the full text

"Two Are Better Than One": Case Studies of Brief Effective Therapy eBook: Seymour Hoffman: domaine-solitude.com: Kindle Store. Two Are Better Than One: Case Studies of Brief Effective Therapy [Seymour Hoffman] on domaine-solitude.com *FREE* shipping on qualifying offers. This book presents.

Our workflow not only describes the generation of sequencing data, but also bioinformatic tools applicable for data analysis and modelling. In addition, we combine this approach with a double bisulfite chemistry, allowing the discrimination between 5mC and 5hmC in the DNA 32 , We also introduce the novel concept to incorporate 5mC and 5hmC nucleotides into the hairpin linker. This allows us to directly measure their conversion rates following BS and oxBS treatment, respectively.

Typically, conversion rates are determined using spike ins, i. Such oligos are difficult to titrate and frequently perform with a different conversion efficiency. As an integrated part of the analysed DNA region, the ligated hairpin linker improves the sample specific conversion rate detection and at the same time serves as a UMI.

Such ds data provide a new resource for mathematic modelling of proposed DNA methylation maintenance and de novo methylation activities as well as active processes of DNA demethylation.


More importantly, ds information allows a more accurate discrimination of maintenance and de novo methylation compared to singe strand data. We observe that indeed individual loci display individual combinations of enzyme efficiencies and DNA demethylation dynamics. During the ESC culturing, i. Predicted enzyme efficiencies for Dnmts and Tets.

Here, both active and passive demethylation processes are known to take place but the exact involvement of oxidation processes is still debated 47 , Rapid locus specific demethylation can also be found in somatic cells and are likewise thought to be Tet mediated. One such example is the generation of region specific demethylation during monocyte-to-macrophage maturation. Our analysis shows that indeed the active loss of 5mC clearly relies on a strong increase of 5hmC level Supplement Section S.

All three examples show the broad application possibilities for HPoxBS. Moreover, these three examples demonstrate possible variations in design DNA vs cells , molecular performance high or low amount of material and data analysis and modeling. Taken together, we present a step by step protocol of HPoxBS which allows the detection and distribution of both 5mC and 5hmC.

Overall, the outlined procedures can be modified and implemented for a number of biological questions, e. Ultimately, in combination with new detection methods, our pipeline could easily be adjusted to likewise, describe the distribution and the behavior 5fC or 5caC 38—40 , 50 , We thank Jasmine Kirch for the technical assistance concerning next generation sequencing using the Illumina MiSeq system, Mathias Bader for programming the Hairpinanalyzer , Gilles Gasparoni for the Hairpinizer v2. Funding for open access charge: Oxford University Press is a department of the University of Oxford.

It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Chromatin and Epigenetics All Journals search input. Close mobile search navigation Article navigation. Two are better than one: To whom correspondence should be addressed.

  • Life Lessons From Your Cat: Were so vain, we probably think this book is about us.;
  • The Peacemakers (American Family Portrait Book 8).
  • Document Preview.

Abstract The controlled and stepwise oxidation of 5mC to 5hmC, 5fC and 5caC by Tet enzymes is influencing the chemical and biological properties of cytosine. View large Download slide. Concerning conversion quality of the oxBS reactions, we determined the conversion rate of the known C, 5mC and 5hmC positions within the hairpin linker Figure 3.

The conversion rates were calculated by dividing the number of sequenced thymines at given cytosine positions by the total number of obtained reads Table 4 and Table 5. Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a.

Reader for the Orthodox Jewish Psychotherapist: Issues, Case Studies and Contemporary Responsa

Human DNA methylomes at base resolution show widespread epigenomic differences. Cloning and characterization of a family of novel mammalian DNA cytosine-5 methyltransferases. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Np95 interacts with de novo DNA methyltransferases, Dnmt3a and Dnmt3b, and mediates epigenetic silencing of the viral CMV promoter in embryonic stem cells.

Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements. TET1, a member of a novel protein family, is fused to MLL in acute myeloid leukemia containing the t 10; 11 q22; q Recently, the plant, which treats 2. A barminutor, installed in the s as a bar screen and grinder, had rusted and developed holes, allowing rags, wood, rocks and other debris to pass through. The debris caused blockages and clogs in the aerators and also contributed to a sludge buildup in the lagoon. Management at the facility was determined to install a more efficient system and end the chronic problems.

Based on assessment, we decided that two units would do a better job of meeting peak flows and minimizing the probability of an overflow.

The comminutors were placed side by side in two existing channels. In Kitimat, effluent flows by gravity to a wet well and is then pumped by three screw pumps into the headworks, where the two comminutors are located.

BookREVIEW: Two Are Better Than One

All the wastewater solids are finely ground before entering an aerated lagoon. The treated effluent is then discharged into the river. Installation of the units has made a noticeable difference. The Dimminutor offers effective automatic screening and disintegrating of wastewater solids in straight-through channels and wet wells. This powerful unit reduces plastics, wood, rags and other solids to fine bits, improving the reliability of pumps and other downstream equipment.

The unit employs a smooth, continuously rotating design with high torque.