text:
- Research
- Open Access
A statistical model-building perspective to identification of MS/MS spectra with PeptideProphet
- Kelvin Ma1,
- Olga Vitek1, 2 and
- Alexey I Nesvizhskii3Email author
© Ma et al.; licensee BioMed Central Ltd. 2012
- Published: 5 November 2012
PeptideProphet is a post-processing algorithm designed to evaluate the confidence in identifications of MS/MS spectra returned by a database search. In this manuscript we describe the "what and how" of PeptideProphet in a manner aimed at statisticians and life scientists who would like to gain a more in-depth understanding of the underlying statistical modeling. The theory and rationale behind the mixture-modeling approach taken by PeptideProphet is discussed from a statistical model-building perspective followed by a description of how a model can be used to express confidence in the identification of individual peptides or sets of peptides. We also demonstrate how to evaluate the quality of model fit and select an appropriate model from several available alternatives. We illustrate the use of PeptideProphet in association with the Trans-Proteomic Pipeline, a free suite of software used for protein identification.
- False Discovery Rate
- Discriminant Function
- Gumbel Distribution
- Spectrum Identification
- Incorrect Identification
In mass-spectrometry shotgun proteomics, the first phase of analysis is the identification of peptides in complex biological mixtures digested by enzymes such as trypsin. Dependent on the peptides in the biological mixture, an experiment will produce a certain number of spectra (call it N). MS/MS spectra are individually matched to peptides by searching through a database of peptides predicted from the genome of the organism. The way the searches are performed can be constrained using different search parameters, such as the number of tryptic termini (NTT), number of missed cleavages (NMC) or the mass difference of the observed precursor ion mass and the weight of the theoretical peptide (ΔM).
We will discuss PeptideProphet in the context of two database search algorithms: SEQUEST [1] and Tandem with the k-score plugin [2, 3]. SEQUEST attempts to determine a direct correlation between an observed spectrum and sequences of amino acids in a protein sequence database. Typical quantities associated with SEQUEST include: XCorr, ΔCn, SpRank. Typical quantities associated with Tandem with the k-score plugin include: logDot (logarithm of dot product between observed and theoretical spectrum) and ΔDot. PeptideProphet can be used with any database search algorithm that returns a quantitative score.
Given a database search algorithm, every spectrum that is observed will be scored against the peptides in the database. For each spectrum, the highest scoring peptide (depending on the scoring criterion) is typically chosen as the best match. The best match is the potential peptide sequence that generated its corresponding observed spectrum. Thus, we have N spectra that have been matched to a peptide and we will refer to these spectra as identified spectra.
The necessity of PeptideProphet arises because the spectra are subject to noise making it difficult to determine if the peptide that it is matched to is correct. The spectrum itself is generated from a peptide sequence and peaks can be missing or reduced in intensity. Because the spectrum that is being generated is subject to noise the database-based criterion will vary when comparing theoretical spectra to observed spectra. Additionally, when searching the database, the correct peptide sequence may be absent. Because of this noise, how do we determine confidence in an identified spectrum? Traditional standards (such as just accepting all above XCorr > 2.5) does not reflect the quality of the identification. Such a rule may accept too many incorrectly identified spectra. Thus, statistical inference is needed to model the presence of noise.
PeptideProphet [4] is a post-processing and rescoring algorithm for determining confidence in identified spectra found using a database search. PeptideProphet is one of the first methods for the assessment of confidence. It is based on a probability model and an Empirical Bayesian approach to model fitting. It is now not a single model, but a family of models [5].
- 1.
Rescoring: produce a score which reflects the quality of an identified spectrum, while summarizing multiple quantities, such as XCorr and ΔCn or logDot and ΔDot. The rescoring separates incorrectly and correctly identified spectra scores as much as possible.
- 2.
Modeling: produce a probability-based model for the distribution of correctly and incorrectly identified spectra. The model must be then fit to the scores of all identified spectra.
- 3.
Evaluation of the Quality of Fit: determine how well the scores fit the probability-based model.
- 4.Inference
- (a)
Evaluation of confidence in individual identified spectra using the posterior probability.
- (b)
Evaluation of confidence in sets of identified spectra: produce a cutoff on the scores to determine a set of correctly identified spectra while controlling the False-Discovery Rate, defined as the expected proportion of false positives.
- (a)
We will first discuss the basic version of PeptideProphet and then discuss the three extensions.
Human plasma dataset
This dataset uses the first LC-MS/MS replicate file from the Western Consortium of the National Cancer Institute's Mouse Models of Human Cancer [6]. The data was obtained using the Multiple Affinity Removal System and was matched using a semitryptic SEQUEST search against an IPI human protein database allowing a 3 Dalton mass tolerance and 0-1 missed cleavage sites. More details on the spectra can be seen in [7].
Controlled mixture
This dataset uses spectra generated from a linear ion trap Fourier transform instrument that was published in [8]. In particular the spectra from Mixture 3 was used, where 16 known trypsin-digested proteins from different mammals were analyzed. Spectra were also matched using a semitryptic SEQUEST search against a database file with the 16 known proteins concatenated with human influenza proteins allowing a 3 Dalton mass tolerance and 0-2 missed cleavage sites. Matches to human influenza proteins are known to be incorrect. More details on the dataset can be seen in [8].
Statement of the problem from a statistical perspective, and terminology
- 1.
PeptideProphet works with the observed spectra as the experimental unit where we have N observed spectra with N being generally large (in the thousands or more). Since the number of spectra N is typically very large, the identified spectra can be viewed as the underlying population.
- 2.
An observed score is interpreted as a test statistic. In statistics the summarized score S is called a test statistic because it is the function of the observed experimental unit that is being used to answer our hypotheses.
- 3.
PeptideProphet assumes that the test statistic comes from a mixture of two distributions: one from the distribution of correct identifications, and the other from the distribution of the incorrect identifications. The distributions may be characterized by a few parameters (parametric) or many parameters (semi or non-parametric).
- 4.The goal of PeptideProphet is to test two competing hypotheses for each identified spectrum. Let T i be the true status of identified spectrum i where T i = 0 indicates that the identified spectrum was incorrectly identified and where T i = 1 indicates that the identified spectrum was correctly identified. We then wish to compare:
- 5.Inference: confidence is determined for individual spectra or sets of spectra.
If the researcher is interested in a set of spectrum identifications, the False Discovery Rate should be controlled.
We determine the confidence in a set of spectra by controlling the False Discovery Rate. The False Discovery Rate, given a cutoff δ, is the expected proportion of all scores S i >δ that are truly incorrect (the proportion of accepted identified spectra that are false positives). This situation is synonymous to performing N multiple hypothesis tests where using the values in Table 1. P (R > 0) is assumed to be 1 when we perform many tests (N is large). The False Discovery Rate is the expected proportion of incorrectly rejected null hypotheses out of the total rejected hypotheses. For a given cutoff if we were to repeat the experiment an infinite number of times and use the same cutoff each time the expected False Discovery Rate is the average proportion of incorrectly identified and accepted spectra out of the total number of incorrectly identified spectra.
An alternative confidence rate that is rarely used is the False Positive Rate (FPR). The False Positive Rate, given a cutoff δ is the expected proportion of all truly incorrectly identified spectra that are considered to be correctly identified. From the terms in Table 1 it is represented by
Many users prefer the q-value which is the minimum False Discovery Rate required for a score s i to be considered significant. It is represented by , where Γ represents the set of all possible cutoff scores [9]. This confidence measure is used to describe a score s i at a single point but examines the False Discovery Rate of all possible scores. Unlike the False Discovery Rate, the q-value is a monotonic quantity with respect to the score cutoff.
If the researcher is interested in specific spectrum identifications the posterior error probability is most commonly used as it quantifies the confidence of a single identified spectrum.
The posterior error probability represents which we also denote as PEP. In other words using a probability model for S i , we can find the probability of an identified spectrum being incorrect given its test statistic. Note that we can also calculate which is the probability of an identified spectrum being correct given its test statistic. The posterior error probability is also called the local false discovery rate (locfdr) [10, 11].
Alternatively the p-value can be used. If s i is the i th observed score then the p-value represents , or the probability of observing a score equal to or greater than s i assuming that the i th identified spectrum was incorrectly identified. The p-value is similar to the FPR in that the p-value is the probability of observing a score equal to or greater than s i assuming that it is one of the N0 truly null hypotheses.
Table of multiple hypothesis testing quantities
# Not Rejected
# Rejected
# True Nulls
# True Alternatives
For each spectrum, PeptideProphet establishes a score reflecting the quality of an identified spectrum
such that S > 0 for correctly identified spectra and S < 0 for incorrectly identified spectra.
In the basic version of PeptideProphet the β's are estimated empirically from a controlled mixture and are dependent on the precursor ion charge (i.e. a separate discriminant function was trained for 1+, 2+, 3+ precursor ion charges).
PeptideProphet relates observable and unobservable quantities via a joint probability distribution
The last equality is due to the fact that all scores are independent and identically distributed (iid). Due to different discriminant functions being used for each charge, a different sampling distribution and set of parameters are produced for each precursor ion charge (we will refer to this simply as the charge).
Note that the density functions of f T =0, NTT , f T =0, NMC, f T =0, ΔM, f T =1, NTT, f T =1, NMC , and f T =1, ΔMare discrete. It is assumed, conditional on the identified spectrum being incorrect or correct, that the members of (S i , NTT i , NMC i , δM i ) are independent, as shown above.
PeptideProphet estimates parameters of interest in an Empirical Bayesian approach
PeptideProphet is considered an Empirical Bayesian approach because it uses each identified spectrum twice: once to estimate via the Expectation-Maximimzation [14] algorithm the parameters of the sampling distribution (π0, μ, σ, α, β, and γ) and second to estimate the confidence in the correctness of an identified spectrum. The EM-algorithm iterates between two steps, called the E-step and the M-step in order to estimate the value of model parameters. With a large enough set of identified spectra (say 100), the EM-algorithm will always converge [14]. The algorithm starts with initial values of model parameters π0, μ, σ, α, β, and γ.
In the E-step, given the estimated values of the model parameters, the probability of each score being correct (or incorrect) is calculated. Given a single observed score s i and its correctness status T i , usage of Bayes Theorem yields which corresponds to the ratio of the Gamma density scaled by π0 over the sum of the Gamma and Normal densities scaled by π0 and 1 - π0 at score s i .
Evaluation of the quality of fit of PeptideProphet
Deviations of the assumptions, or a low number of identified spectra can lead to an inadequate or unstable model fit and incorrect conclusions. This can be diagnosed by visual inspection, and also by the bootstrap. We recommend using visual inspection over goodness of fit tests as tests do not explore the specific fitting issues that may influence subsequent inference of the identified spectra. In fact goodness of fit tests simply attempt to summarize the goodness of fit into one summary statistic whereas we are typically interested in the fit at certain locations of the mixture distribution. There are several visual attributes of the mixture distribution that researchers should be aware of and some remedies for them.
Do the empirical scores follow the fitted curves well? Particular attention needs to be paid to the tails of the distributions, especially the right tail of the distribution of scores of incorrect identifications (red) and the left tail of the distribution of scores of correct identifications (blue). This is often of most interest to researchers as the identified spectra in these regions are considered to be borderline correct or incorrect. In the case of Figure 2b the curves fit the histogram well but in Figure 2a there are many mismatches in the bars and the fitted curves. The culprit of these mismatches is likely due to the small number of spectra. The right portion of the Normal distribution is fit with approximately only 30 spectra. If the data is comprised of a large number of spectra but is deviating from the fitted curves, robust procedures can also be considered and will be discussed later.
Do the curves highly overlap? Although high overlap does not necessarily indicate a poor fit it will lead to smaller sets of confidently identified spectra. Overlaps that occur in situations of highly constrained searches can be remedied with techniques in later sections. Overlap in the case of a small number of spectra (Figure 2a) may be remedied by artificially adding observations using decoys which will also be subsequently demonstrated.
An issue that is not commonly addressed however is the number of identified spectra available to fit the mixture model. The number of identified spectra required to fit a reliable model depends highly on the separation and the form of the observed scores. A statistical approach to examine the stability of the fitted model can be done via the bootstrap.
Bootstrapping can be performed by sampling with replacement B samples (spectra) where each is of size N from the original dataset. At least 100 to 500 bootstrapped samples are recommended. For each bootstrapped sample b, we can refit the PeptideProphet model to receive bootstrapped estimates of , , , , , and . The bias, variance, and mean squared error (MSE) of the procedure used to estimate a parameter can be found using the bootstrapped estimates. In the case of μ, the bootstrap bias estimate is . Large biases imply that the estimation procedure is systematically over or underestimating the true value of a parameter. Note that as B increases the bias does not move towards 0. The bootstrap variance estimate is defined as . Smaller variability is desired. The bias and variability of an estimation procedure is often summarized using the mean squared error, which is .
Estimating the confidence of spectrum identifications
Estimating the confidence of a set of spectrum identifications
In order to determine the correctness of the spectrum identifications, a decision rule is defined where any spectrum identification with a score above δ is concluded to be correct. In many experiments we are interested in the statistical properties of the list of spectrum identifications with scores above δ.
The False Positive Rate for a cutoff t can also be estimated using the area under the fitted frequency curve of the distribution of scores for incorrect identifications as seen in Figure 5. Mathematically this is equivalent to the p-value, or since each incorrect score follows the same distribution. Note that the False Positive Rate ignores the distribution of scores for correct identifications.
The estimation of the q-value at a specific point ρ requires the estimation of the False Discovery Rate at every point s i from i = 1, 2, ..., N. The q-value for a point ρ is the minimum False Discovery Rate among all points s i such that s i ≤ ρ. The estimated False Discovery Rate can be found using the model-based estimates or by interpreting each posterior error probability as a local false discovery rate. The q-value is often useful if a monotonically increasing error rate is desired for decreasing cutoff values. For example, in the case of Figure 5 suppose the experimenter was only interested in scores around 4. Using model-based estimates, the estimated False Discovery Rate with a cutoff at 4 is 0.01503874 but the estimate of the False Discovery Rate with a cutoff at 3.8 is 0.01489971 suggesting that the error rate is lower for a lower cutoff value. To avoid this issue, the q-value can be used as it finds the minimum False Discovery Rate at each cutoff value. The q-value at 4 is 0.01489939 (found using increments of 0.01 searching all FDR values from -4 to 4).
Estimating the confidence of an individual spectrum identification
We now discuss the estimation of the posterior error probability and the p-value. These measures are properties of a single spectrum and are synonymous to performing a single hypothesis test. In Figure 5 the posterior error probability and p-value only apply to spectra at a single point.
According to Bayes Theorem the posterior probability of T i = 0 (our hypotheses of interest) given its test statistic is . Following the Empirical Bayesian step where parameters are estimated we have that . Because the posterior error probability is equivalent to the local false discovery rate we also have that .
The p-value is estimated as which is the right tail-end of the Gamma density past s i .
The posterior error probability may be preferred over the p-value because it also yields an estimate for the probability of an identified spectrum to being correct (1 - PEP). The advantage of the p-value is that it only requires the use of the distribution of scores for incorrect identifications as it ignores the distribution of scores for correct identifications. Notice that in Figure 5 the p-value at a score of 1 is a low value of 0.004 but that the Posterior Error Probability at 1 is a much higher value at 0.156.
PeptideProphet can use a decoy database to estimate the parameters of the distributions of scores for incorrect identifications
When there is significant overlap between the two density functions or a low number of identified spectra it is difficult for the EM-algorithm to estimate π0 and the parameters of the Gamma and Normal distributions. In this case PeptideProphet employs the Target-Decoy approach to better estimate the Gamma distribution. We first describe the two forms of Target-Decoy: the concatenated strategy and the separate strategy [16, 17]. The objective of both strategies is to introduce decoys in order to estimate the error rate since decoys are known to be incorrectly identified spectra. Reversed sequences (decoy sequences) are commonly generated by taking the target database and reversing each target sequence. Alternative methods are to use randomized sequences where amino acid sequences are generated using a pre-specified probability distribution [16].
In the concatenated Target-Decoy strategy each spectrum is searched in a single database that is composed of both target and decoy sequences. This involves competition between the best correct peptide sequence, the best incorrect forward peptide sequence, and the best (incorrect) decoy peptide sequence. Hits where the best incorrect decoy peptide sequence is found to be the match are used to estimate the FDR.
In the separate Target-Decoy strategy each spectrum is searched once in the forward database and searched again independently in the decoy database. The distribution of scores from the peptides identified via the decoy database is used to estimate the form of the distribution of incorrectly identified spectra. This approach ignores competition between forward and decoy sequences.
PeptideProphet can use a decoy database for semiparametric estimation of the probability distribution
The quality of fit of the Gamma and Normal distributions may rely on how the database is searched (constrained versus unconstrained search) or the search algorithm that is used [12]. As is the case in many statistical modelings, there is no guarantee that the scores of the identified spectra necessarily follow the Gamma and Normal distributions. Previously, decoys were used to estimate parameters of pre-specified distributions. Now we will use decoys for data-dependent estimation of the distributions themselves.
An example of this approach can be seen in Figure 7. The parametric fit of the distribution of scores for correct identifications clearly deviates from the Normal curve as the mode of the correct hits is shifted to the right. The semiparametric approach produces a curve that more robustly fits the left-skewed distribution of scores for correct identifications.
To avoid overfitting, this approach should only be used in the cases of strong deviations between the fitted distributions and the observed scores, such as the parametric fit (dashed-lines) in Figure 7. Overfitting typically occurs in experiments with a small number of spectra, such as in Figure 2a. Overfitting can be checked via bootstrapping by seeing if bootstrapped samples do not reflect the same need for a semiparametric fit at certain score values. This can be done via quantile to quantile plots or by checking mean squared errors. If users anticipate good separation, parametric PeptideProphet is often sufficient for practical purposes.
PeptideProphet can be extended to dynamically estimate the coefficients of the discriminant function from the data
Overlap in the distributions of scores of correct and incorrect identifications can be due to a suboptimal scoring function, which does not discriminate well between the properties of correct and incorrect identifications. This often occurs in cases of constrained searches where the database that is searched is much smaller than the unconstrained search space that was used to find the coefficients in the fixed discriminant function. For additional information on constrained versus unconstrained searches, see [5]. A solution to this is to adapt the discriminant function to each experiment or search approach which can improve the separation between the distribution of scores for incorrect and correct identifications [13].
The improvement of the adaptive discriminant function over the fixed discriminant function for the Controlled Mixture dataset in a constrained search space is displayed in Figure 9. Only tryptic peptides with a narrow mass tolerance were searched.
This approach is also useful for incorporating lower ranked peptide matches (i.e. for a given spectrum, instead of only considering the best peptide sequence match, use the new discriminant function to also rescore peptide sequence matches that ranked close to the best peptide sequence match). Every time a new discriminant function is estimated (when the I are averaged) a new summarized score is calculated for the top 5 (can be changed of course) Peptide Matches for every spectrum. The highest scoring peptide-spectrum-match is used in the training of the next discriminant function.
Implementation of the PeptideProphet in the Trans-Proteomic Pipeline
The Trans-Proteomic Pipieline (TPP) is an open source program developed at the Institute for Systems Biology designed for complete proteomic analysis starting from spectrum identification to protein identification and quantification and can be downloaded from http://sourceforge.net/projects/sashimi/[18]. In this section we assume that search results have already been converted to pepXML files, which is the standard input for PeptideProphet. A discussion of this can be found at (http://tools.proteomecenter.org/wiki/index.php?title=TPP_Tutorial).
We present an example using the Human Plasma dataset where the spectra are searched through Tandem with the k-score plugin with TPP version 4.4. PeptideProphet automatically models all precursor ion charges and outputs the probability of correct identification. A mixture model using the Normal for the distribution of correct scores and a Gumbel distribution for the distribution of incorrect scores.
- 1.
False Discovery Rate: estimates of the False Discovery Rate can be obtained three ways. In the upper-right hand corner of Figure 12 estimated False Discovery Rates under the "Error" column is given for 1 - PEP values under the "Min Prob" column. In other words, "Min Prob" represents the minimum posterior probability of being correct in order to conclude that an identified spectrum is correct. For example, a "Min Prob" of 0.95 implies that only identified spectra with PEP's lesser than 0.05 are considered correct or that (1 - PEP) must be greater than 0.95 to be considered correct.A second approach is to use the estimated model parameters in Figure 13 to estimate the False Discovery Rate for identified spectra of charge 2. The estimate for (1 - π0) is 0.04 which yields an estimate of π0 as 0.96. The Normal's (Gaussian) estimated mean μ is 2.6 with an estimated standard deviation σ of 1.90. The Gumbel's estimated μ G parameter is -1.16 with an estimated β parameter as 0.76. Alternatively, the expected value (mean) of the Gumbel is -1.16 with a standard deviation of 0.98. If the experimenter is not interested in NTT, NMC, and ΔM, for a cutoff score t, the estimated FDR can then be estimated by where P(S >t|T = 0) is found using the Normal distribution and P(S >t|T = 1) is found using the Gumbel distribution. Suppose the experimenter wanted to restrict the FDR calculation to identified spectra with only 0 missed cleavages. According to the output in the distribution of correct scores, a randomly selected correctly identified spectra has a 0.926 probability of having 0 missed cleavages and a 0.074 probability of having 1 to 2 missed cleavages. For the distribution of incorrect scores, probabilities are 0.404 and 0.596 for 0 and 1 missed cleavages respectively. The estimated FDR would then be
The calculation takes into account that among the correctly identified spectra, it is estimated that a majority of the identified spectra have 0 missed cleavages.
A third approach of estimating the False Discovery Rate is to download all posterior probabilities, convert them to posterior error probabilities (local false discovery rates) by taking the complement, define a cutoff point t and then to calculate .
- 2.
False Positive Rate or p-value: using the Gumbel's estimated parameters, the false positive rate can be found by looking at the tail area.
- 3.
q-value: the q-value at a specific point δ can be calculated by estimating the False Discovery Rate at the score value of every identified spectra and then by finding the minimum False Discovery Rate among all scores .
- 4.
Posterior Error Probability and Local False Discovery Rate: these are most easily found by finding the complement of the values in the first column of Figure 11 or by looking at the complement of "prob" at the bottom center of Figure 12. Note that these probabilities automatically incorporate NTT, NMC, and ΔM. If the experimenter was interested in the posterior error probability of a score independent of NTT, NMC, and ΔM, this can still be calculated using the estimated model parameters.
All inference for semisupervised and semiparametric PeptideProphet cases are identical. Inference would be identical for the adaptive version of PeptideProphet but it is not implemented in TPP at this time but is available from the authors upon request.
Following the execution of PeptideProphet the next step in analysis is often the identification of proteins present in the sample. In this different analysis, the experimental unit changes from being a spectrum to a peptide. TPP can be used to run ProteinProphet, a computational algorithm that can utilize PeptideProphet's estimated probabilities to determine the probability for the presence of proteins in two steps [19]. In the first step the posterior probability of a peptide being correctly identified from PeptideProphet is decreased for peptides that are the only peptide linked to a protein and increased for peptides that are linked to proteins explained by many peptides. In the second step the probability of a protein being in the sample is calculated as the probability that at least one of its associated peptides were identified in the sample. This is if is the adjusted probability of a peptide being in the sample where i is indexed from 1 to the number of peptides linked to the protein in question.
PeptideProphet is available for use on the Trans-Proteomic Pipeline with many other database search tools (X!Tandem, MASCOT, OMSSA, Phenyx, ProbID, InsPecT, MyriMatch). The statistical approach of PeptideProphet is generalizable to any database search algorithm that returns a quantitative score for each identified spectrum.
Although we used the Gamma and Normal distributions to model the components of the PeptideProphet model, there are no limitation to the choice of parametric distribution for describing the distributions of scores for incorrect and correct identifications in PeptideProphet. The Gumbel distribution, with parameters μ and β is another common distribution used for the distribution of scores of incorrect identifications. A generalization of the Gumbel distribution is the Extreme Value Distribution. Additional information, such as the NTT, may be incorporated into the summarized score by using a different machine learning approach instead of a discriminant function. Quantities like the NTT were left out of the summarized discriminant score due to its discrete nature. For example, the logistic regression function would allow discrete and continuous covariates to be transformed into a single summarized score while separating identified spectra with T = 0 from identified spectra with T = 1.
The Target-Decoy approach used in this manuscript is an approach that pioneered the use of decoys for the estimation of the False Discovery Rate and its results are often compared to other techniques [16]. For the estimation of the False Discovery Rate PeptideProphet and Target-Decoy methods in our experience produce similar results especially when the semisupervised version of PeptideProphet is used as its search approach is similar to the concatenated version of Target-Decoy. In fact, PeptideProphet can be considered as an extension of the concatenated version of Target-Prophet because of its additional modeling objectives. PeptideProphet simply has distributional assumptions and can be used to estimate confidence of individual spectrum identifications or sets of spectrum identifications (local and global FDR estimates) whereas target-decoy is limited to sets (global FDR estimate only). Also, if there is heavy overlap Target-Decoy will outperform basic PeptideProphet but Semisupervised PeptideProphet and Target-Decoy should be similar.
An alternative approach which relaxes the parametric assumptions is the variable component approach which uses an unknown mixture of Gaussians to represent the incorrect and correct distributions of scores [12]. The correct distribution is represented by a mixture distribution of k0 normal distributions (that may have different means and variances) and the incorrect distribution is represented by a separate mixture distribution of k1 normal distributions. Parameters k0 and k1 are unknown. Each score s i is a member of either the overall correct or incorrect distributions, but are then further assigned as a member to one of the sub-components of the mixture representing the correct or incorrect distribution. Gibbs sampling is used to estimate the forms of the sub-components (which also suggests the complexity of this approach). Although the variable component and kernel methods perform similarly there are minor computational and modeling issues to consider [12]. The advantages to the variable component method are that: (1) The model is still parametric, which may help reduce the chance of overfitting, (2) Kernel estimation may over fit, especially if the bandwidth is too low, and (3) It does not completely rely on decoys for the negative whereas kernel density estimation uses decoys only for estimating the negative distribution. The advantages of the kernel approach are that: (1) The variable component method is much more computationally intensive and more complicated (and thus the Kernel Estimation is less intensive), (2) The variable component method requires the specification of priors, and (3) Kernel estimation is very well known and commonly used.
The authors would like to thank Hyungwon Choi for providing R-code for the PeptideProphet model fits. The work was supported in part by the NSF CAREER award DBI-1054826 to OV, and by NIH grants R01-GM-094231 and R01-CA-126239 to AN.
This article has been published as part of BMC Bioinformatics Volume 13 Supplement 16, 2012: Statistical mass spectrometry-based proteomics. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcbioinformatics/supplements/13/S16.
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- Storey J: The positive false discovery rate: a Bayesian interpretation and the q-value. Annals of Statistics. 2003, 31 (6): 2013-2035. 10.1214/aos/1074290335.View ArticleGoogle Scholar
- Elias J, Gygi S: Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nature Methods. 2007, 4 (3): 207-214. 10.1038/nmeth1019.View ArticlePubMedGoogle Scholar
- Käll L, Storey J, MacCoss M, Noble W: Assigning significance to peptides identified by tandem mass spectrometry using decoy databases. Journal of Proteome Research. 2008, 7: 29-34. 10.1021/pr700600n.View ArticlePubMedGoogle Scholar
- Deutsch E, Mendoza L, Shteynberg D, Farrah T, Lam H, Tasman N, Sun Z, Nilsson E, Pratt B, Prazen B, Eng JK, Martin DB, Nesvizhskii AI, Aebersold R: A guided tour of the Trans Proteomic Pipeline. Proteomics. 2010, 10: 1150-1159. 10.1002/pmic.200900375.PubMed CentralView ArticlePubMedGoogle Scholar
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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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retro baby waving at all zimmermaniacs
here is the little girl modelling her yoke sweater. i think she looks pretty pleased with it waving at all zimmermaniacs from alaska to australia. it is still a little big but will fit by winter. it is very retro i think. she looks a little bit like a girl from a time long gone. the wool is from my lys in copenhagen and their own brand. some of you have asked but i didn't find it so relevant to write about it since it is only here they sell it. but it is pretty much the same as indiecita or drops alpaca. oh and some have asked me for the address of my lys's in copenhagen. there are strikkeboden and uldstedet in fiolstræde. strikkeboden sometimes have rarities that i have read about on international blogs and they sometimes have very good offers. and there is sommerfuglen (the butterfly) in vandkunsten that maybe has the greatest variety. they also have different books.

subset: commoncrawl


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Every week, Texas on the Potomac presents your favorite postings of the previous week. Today, we offer an encore of the top 10 postings for the week ending September 3.
1. Is Rick Perry the new George W. Bush? In many ways, yes – In many ways, no. Perry Watch takes a look at both the similarities and the differences between the two. Lauren French reports>>>
2. Texas Rep. Ron Paul, who's vying for the Republican presidential nomination, certainly sticks to his principles, Richard Dunham writes in TexMessage, after Paul told CNN's Anderson Cooper that the Federal Emergency Management Agency should be abolished. TK's TexMessage also includes more Texas tips and events. Richard Dunham reports>>>
3. Last week's top 10 included Ron Paul's lead in another presidential poll, his lack of media attention and a set of creative Rick Perry campaign slogans. Ingrid H. Storaas reports>>>
4. "Kinky for Perry" is what The Daily Beast titled an article Kinky Friedman wrote on Rick Perry. This led some to say that Kinky Friedman was endorsing Rick Perry. In fact, he says he's not. Kathleen McKinley reports>>>
5. President Obama's absence from a Veterans of Foreign Wars convention in San Antonio insulted National Commander Richard Eubank. The White House responds to the critique stating that President Obama and his administration's deep respect and appreciation for the natio/ servicemen and veterans are clearly seen in their policies and actions. Gary Martin reports>>>
6. The results of Texas on the Potomac`s reader poll "Which Republican presidential candidate do you prefer?" is final. From last week's preliminary results, Ron Paul has gone up from 79 to 83 percent of the total votes, while Perry`s final result is 6 percent compared to last week's 9 percent. See the results>>>
7. Jon Huntsman thinks his own entourage, consisting of his wife Mary Kaye Huntsman, is just as impressive as Rick Perry`s large group of Texas Rangers. Puneet Kollipara reports>>>
8. The top 10 from two weeks ago includes the similarities and differences between former President George W. Bush and Texas Gov. Rick Perry, things about Rick Perry that could worry some conservatives, and Perry questioning the science of global warming. Richard Dunham reports>>>
9. The Texas Tribune staff guide you through Rick Perry`s November 2010 book "Fed Up!" in their guide "'Fed Up!' for dummies." Richard Dunham reports>>>
10. "Governor Goodhair" keeps stealing jobs in California, Lloyd Doggett talks about his No. 1 rival, and President Obama is shaking his pants after the latest poll results. Check out last week`s Political Cartoons>>>


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This is what I think we're seeing:
The president has backed away from a military strike in Syria. But he can't acknowledge this or act as if it is true. He is acting and talking as if he's coolly, analytically, even warily contemplating the Russian proposal and the Syrian response. The proposal, he must know, is absurd. Bashar Assad isn't going to give up all his hidden weapons in wartime, in the middle of a conflict so bitter and severe that his forces this morning reportedly bombed parts of Damascus, the city in which he lives. In such conditions his weapons could not be fully accounted for, packed up, transported or relinquished, even if he wanted to. But it will take time—weeks, months—for the absurdity to become obvious. And it is time the president wants. Because with time, with a series of statements, negotiations, ultimatums, promises and proposals, the Syria crisis can pass. It can dissipate into the air, like gas.
The president will keep the possibility of force on the table, but really he's lunging for a lifeline he was lucky to be thrown.
Why is he backing off? Because he knows he doesn't have the American people and isn't going to get them. The polls, embarrassingly, show the more people hear the less they support it. The president's problem with his own base was probably startling to him, and sobering. He knows he was going to lose Congress, not only the House but very possibly—likely, I'd say—the Senate. The momentum was all against him. And he never solved—it was not solvable—his own Goldilocks problem: A strike too small is an embarrassment, a strike too big could topple the Assad regime and leave Obama responsible for a complete and cutthroat civil war involving terrorists, foreign operatives, nihilists, jihadists, underemployed young men, and some really nice, smart people. Obama didn't want to own that, or the fires that could engulf the region once Syria went up.
His plan was never good. The choices were never good. In any case he was going to lose either in terms of domestic prestige, the foreign result or both. Likely both.
He got himself into it and now Vladimir Putin, who opposes U.S. policy in Syria and repeatedly opposed a strike, is getting him out. This would be coldly satisfying for Putin and no doubt personally galling for Obama—another reason he can't look as if he's lunging.
A serious foreign-policy intellectual said recently that Putin's problem is that he's a Russian leader in search of a Nixon, a U.S. president he can really negotiate with, a stone player who can talk grand strategy and the needs of his nation, someone with whom he can thrash it through and work it out. Instead he has Obama, a self-besotted charismatic who can't tell the difference between showbiz and strategy, and who enjoys unburdening himself of moral insights to his peers.
But Putin has no reason to want a Syrian conflagration. He is perhaps amused to have a stray comment by John Kerry be the basis for a resolution of the crisis. The hidden rebuke: It means that when Putin met with Obama at the G-20 last week Obama, due to his lack of competence, got nothing. But a stray comment by the Secretary of State? Sure, why not rub Obama's face in it.
All this, if it is roughly correct, is going to make the president's speech tonight quite remarkable. It will be a White House address in which a president argues for an endeavor he is abandoning. It will be a president appealing for public support for an action he intends not to take.
We've never had a presidential speech like that!
So what will he say? Some guesses.
He will not really be trying to "convince the public." He will be trying to move the needle a little, which will comfort those who want to say he retains a matchless ability to move the masses. It will make him feel better. And it will send the world the message: Hey, this isn't a complete disaster. The U.S. president still has some juice, and that juice can still allow him to surprise you, so watch it.
He will attempt to be morally compelling and rhetorically memorable. He will probably, like Susan Rice yesterday, attempt to paint a graphic portrait of what chemical weapons do—the children in their shrouds, the suffering parents, what such deaths look like and are. This is not meaningless: the world must be reminded what weapons of mass destruction are, and what the indifference of the world foretells.
He will claim the moral high ground. He will temporarily reserve the use of force and welcome recent diplomatic efforts. He will suggest it was his threat of force that forced a possible diplomatic solution. His people will be all over the airwaves saying it was his deft leadership and steely-eyed threat to use force that allowed for a diplomatic break.
The real purpose of the speech will be to lay the predicate for a retrospective judgment of journalists and, later, historians. He was the president who warned the world and almost went—but didn't go—to war to make a point that needed making.
Before or after the speech there will be some quiet leaking to the press that yes, frankly, the president, with so many difficult domestic issues facing him and Congress in the fall, wanted, sympathetically, to let lawmakers off the hook. They never wanted to vote on this.
Once that was true, they didn't. But now, having seen the polls and heard from their constituents, a lot of them are raring to go, especially Republicans. It is Democrats who were caught in the crosshairs between an antiwar base and a suddenly hawkish president. But again, a Democratic White House can't admit it put its people in a fix like that.
In any case it's good for America that we've dodged either bad outcome: Congress votes no and the president moves anyway, or Congress votes no and he doesn't. Both possibilities contained dangers for future presidents.
The president will assert that as a lover of peace he welcomes the Russian move and reports of the positive Syrian reaction, that he will closely monitor the situation, set deadlines. He will speak of how he understands the American people, after the past 12 years, after previous and painful mistakes by their leaders, would feel so reluctant for any military engagement. He not only understands this reluctance, he shares it. He knows he was elected, in part, because he would not think of war as the first, or even second or third, option. But he has a higher responsibility now, and it is to attempt to warn the world of the moral disaster of the use of weapons of mass destruction. If we don't move in the firmest opposition our children will face a darker future.
The speech will end. Polls will be taken. Maybe a mild uptick, maybe a flatline. Probably more or less the latter—people have made up their mind. They sense the crisis has passed or is passing. They're not keen for more presidential rhetoric.
* * *
Then get ready for the spin job of all spin jobs. It's already begun: the White House is beginning to repeat that a diplomatic solution only came because the president threatened force. That is going to be followed by something that will grate on Republicans, conservatives, and foreign-policy journalists and professionals. But many Democrats will find it sweet, and some in the political press will go for it, if for no other reason than it's a new story line.
It is that Syria was not a self-made mess, an example of historic incompetence. It was Obama's Cuban Missile Crisis—high-stakes, eyeball-to-eyeball, with weapons of mass destruction and an implacable foe. The steady waiting it out, the inner anguish, the idea that crosses the Telex that seems to soften the situation. A cool, calibrated, chancy decision to go with the idea, to make a measured diplomatic concession. In the end it got us through the crisis.
Really, they're going to say this. And only in part because this White House is full of people who know nothing—really nothing—about history. They've only seen movies.
The only question is who plays Bobby. Get ready for a leak war between Kerry's staff and Hillary Clinton's
An important thing. The president will be tempted, in his embarrassment, to show a certain dry and contemplative distance from Putin. The Obama White House should go lightly here: Putin could always, in his pique, decide to make things worse, not better. It would be good for Obama to show graciousness and appreciation. Yes, this will leave Putin looking and feeling good. But that's not the worst thing that ever happened. And Putin has played this pretty well.


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All of the above cameras have clean HDMI out, which makes use with a Ninja (Prores HQ @220 mbps) a very big advantage over the internal codecs. I will not go into the details of my comparative tests other than to say that I'm only interested in optimum video image quality under normal circumstances...so noise at very high ISO is irrelevant for my selection criteria. I normally shoot at 400 ISO and rarely above 1000 ISO.
To get to the point...for my requirements the Nikon D5200 (with and without the Ninja) produced the best video image in my topside tests. Slightly better than the D800; significantly better than the hacked GH2 and GH3 (which really isn't that different to the GH2's video quality...a bit disappointing, actually).
In terms of DR, color purity, color gradation, color banding (lack of it), moire and aliasing, and...particularly NOISE, the D5200 was significantly better than the GH2/GH3, and somewhat better than the D800. The new Toshiba sensor in the D5200 with the way the processor handles the 24 mp image results in what is to my eyes a very beautiful, neutral and very clean video image. Two other important features of the D5200 (for me, anyway) is decent autofocus (AF-F) during video which is almost as good as Panasonics; and that the HDMI also carries audio...important for topside work with the Ninja. Unlike the D800 it is also possible to have very accurate auto exposure (AE-S) during Live-view video acquisition with the D5200 (and still control shutter preference and ISO).
Of course each camera has its feature advantages (the 50/60p of the GH3 is excellent, whereas the 50/60i of the D5200 is retarded). For still photographers the RAWs out of the D5200 are way better than anything out of the GH3.
If you are wondering why there are no Canon DSLRs on my short list...three reasons: 1) Canon have decided not to allow clean HDMI out for cynical marketing reasons (the HDMI out will soon be miraculously "unlocked" in the Canon 5D3 by a simple firmware update...no doubt in response to the success of the D800); 2) Canons (including the 5D3) have relatively soft video; and 3) I dislike the "Pink Disease" color cast of current Canon lenses. I had a 5D2 with several L lenses for a year...but went back to Nikon and Zeiss glass.
Please regard this as just my personal opinion and a heads up about the cheap and humble D5200. I'm happy to answer questions...but I have neither the time nor desire to post examples to "prove" anything. There's pleanty out there already:
GH2 vs GH3
"If you're looking for a big leap in video performance over the GH2, the GH3 doesn't appear to be it."
D5200 vs GH2
This has a couple of frame grabs that clearly shows the noise difference...which is much more noticeable when comparing the video material.
I hope someone makes a good housing for the D5200 asap...or it's another Subal conversion job for me.
Edited by HDVdiver, 14 February 2013 - 04:41 AM.


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For more information about themes, see Use or create themes in PowerPoint.
PowerPoint provides a variety of design themes, including coordinating color schemes, backgrounds, font styles, and placement of placeholders. Using pre-designed themes makes it easy for you to change the overall look of your presentation quickly.
By default, PowerPoint applies the plain, Office theme to new blank presentations. However, you can easily change the look and feel of your presentation by applying a different theme.
1. By default, the default theme is applied to your presentation
2. In the Themes gallery, you can change to the Civic theme
3. The Civic theme is now applied to your presentation
To apply a different theme to your presentation, do the following:
On the Design tab, in the Themes group, click the document theme that you want to apply.
To preview how the current slide looks with a particular theme applied, rest your pointer on the thumbnail of that theme.
To view more themes, on the Design tab, in the Themes group, click More .
Note: Unless you specify otherwise, PowerPoint applies themes to the entire presentation. To apply more than one theme to a single presentation see, Apply multiple themes to a presentation.


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Hand-Made & Made to Order (2-3 weeks)
100% Leather Sole
Suede Lining Between Toes for Added Comfort
Embellished with Czech Crystals & Rhinestone Chain
Made in Bali, Shipped from Los Angeles
3 Heel Heights- 1 ½ Inches, 2 ½ Inches, 3 Inches
* Our wedges are offered in multiple heel heights. Please check our size guide to see the different wedge height options.
* If you want to change a heel height, please include the heel size you would like in the order comments section
* Email firstname.lastname@example.com to discuss these changes


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|Pueraria grandiflora Bo Pan & Bing Liu
A new species, Pueraria grandiflora, (Fabaceae, Phaseoleae), is described and illustrated. It resembles Pueraria tuberosa, but differs in lobed leaflets, larger flowers, single or once branched inflorescences, and flowering and fruiting season. This species is only known from dry-hot valleys in Sichuan and Yunnan, and is rare and narrowly distributed.
Key words: Dry-hot valley, Fabaceae, Phaseoleae, tuber
|FIGURE 1. Pueraria grandiflora (from type locality).
A. Habit. B. Flowers. C. Entire leaflets. D. Tubers. E. Tuber opened up. F. Sagittate stipule. G. Lobed leaflets.
Photos by Bo Pan and Bing Liu.
Pueraria grandiflora Bo Pan & Bing Liu, sp. nov.
Etymology: The species epithet refers to the large flowers of this species, the largest within the genus to date.
Bo Pan, Bing Liu, Zhi-Xiang Yu and Yong-Qiong Yang. 2015. Pueraria grandiflora (Fabaceae), A New Species from Southwest China. Phytotaxa. 203(3); 287–291. DOI: 10.11646/phytotaxa.203.3.8


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by Mike Topliff
SOCORRO, NM, Oct. 5, 2004 -- Twelve Socorro-area students are participating in the 15th annual New Mexico Adventures in Supercomputing Challenge, called "the Challenge" by those in the know. The students, all from Socorro High School, are Doug Haworth, Megan Pearson, Anthony Zecco, Scott Engler, Nathan Zecco, Bryan Haworth, Omar Soliman, Thor Johnson, Antonio Carrejo, Justin Greene, Emily Walker and Joshua Farmer.
Sponsors are Peggy Mitchusson from Socorro High School and Dr. Lorie Liebrock and Dr. Mike Topliff from New Mexico Tech.
The Challenge is a school-year long program where teams of middle school and high school students from around the state design and implement computer programs that model and simulate interesting real-world problems.
At the October Kickoff Conference at Glorieta Conference Center, students and teachers learn to write computer programs, collect, analyze and model data, write technical reports and develop sophisticated presentations.
Professional scientists and engineers from Los Alamos National Laboratory, Sandia National Laboratories, New Mexico State University, the University of New Mexico, New Mexico Tech, Eastern New Mexico University, and private companies work with students and teacher-sponsors to provide initial and ongoing training in computational methods and scientific modeling. These volunteer professionals evaluate the students' project ideas, steer student teams towards successful completion of their work, and evaluate projects at mid-term and final judging events.
The Challenge year culminates in an expo, showing the work of all the teams who completed the program, and an awards ceremony in April at Los Alamos National Laboratory, where student scholarships and school computing equipment and software are presented by sponsoring organizations.
"As New Mexico and other states consider their workforce needs for the 21st century," says Topliff, "it has become increasingly obvious that the 7,500 plus students who have previously participated in the Challenge program are uniquely prepared for careers in high technologies industries. Challenge participants will benefit as our state colleges and universities and potential future employers are constantly seeking the technical and planning skills that the Challenge participants develop."
For more information, please send inquires to firstname.lastname@example.com or visit the Challenge web site at http://www.challenge.nm.org.


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This all-analog pedal uses bucket-brigade circuitry to create classically lush, liquid textures that you just can't get with digital circuitry. Rate, Level, and Depth controls, as well as knobs for cutting High and Low frequencies allow ultimate tone control. .
Our product catalog varies by country due to manufacturer restrictions. If you change the Ship-To country, some or all of the items in your cart may not ship to the new destination.


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End-of-life notice: American Legal Ethics Library
As of March 1, 2013, the Legal Information Institute is no longer maintaining the information in the American Legal Ethics Library. It is no longer possible for us to maintain it at a level of completeness and accuracy given its staffing needs. It is very possible that we will revive it at a future time. At this point, it is in need of a complete technological renovation and reworking of the "correspondent firm" model which successfully sustained it for many years.
Many people have contributed time and effort to the project over the years, and we would like to thank them. In particular, Roger Cramton and Peter Martin not only conceived ALEL but gave much of their own labor to it. We are also grateful to Brad Wendel for his editorial contributions, to Brian Toohey and all at Jones Day for their efforts, and to all of our correspondents and contributors. Thank you.
We regret any inconvenience.
Some portions of the collection may already be severely out of date, so please be cautious in your use of this material.
Ohio Rules of Professional Conduct
Comment - 7.1
[1] This rule governs all communications about a lawyer's services, including advertising permitted by Rule 7.2. Whatever means are used to make known a lawyer's services, statements about them must be truthful.
[2] Truthful statements that are misleading are also prohibited by this rule. A truthful statement is misleading if it omits a fact necessary to make the lawyer's communication considered as a whole not materially misleading. A truthful statement is also misleading if there is a substantial likelihood that it will lead a reasonable person to formulate a specific conclusion about the lawyer or the lawyer's services for which there is no reasonable factual foundation.
[3] An advertisement that truthfully reports a lawyer's achievements on behalf of clients or former clients may be misleading if presented so as to lead a reasonable person to form an unjustified expectation that the same results could be obtained for other clients in similar matters without reference to the specific factual and legal circumstances of each client's case. Similarly, an unsubstantiated comparison of the lawyer's services or fees with the services or fees of other lawyers may be misleading if presented with such specificity as would lead a reasonable person to conclude that the comparison can be substantiated. The inclusion of an appropriate disclaimer or qualifying language may preclude a finding that a statement is likely to create unjustified expectations or otherwise mislead a prospective client.
[4] Characterization of rates or fees chargeable by the lawyer or law firm such as "cut-rate," "lowest," "giveaway," "below cost," "discount," or "special" is misleading.
[5] See also Rule 8.4(e) for the prohibition against stating or implying an ability to influence improperly a government agency or official or to achieve results by means that violate the Ohio Rules of Professional Conduct or other law.
Comparison to former Ohio Code of Professional Responsibility
Rule 7.1 corresponds to DR 2-101. Rule 7.1 does not contain the prohibitions found in DR 2-101 on client testimonials or self-laudatory claims. However, the rule does retain the DR 2-101 prohibition on unverifiable claims.
In addition, Rule 7.1 contains none of the other directives found in DR 2-101(B), the definition of misleading found in DR 2-101(C) (see comment [2] of Rule 7.1), or the directives found in DR 2-101(D), (E), and (G).
For DR 2-101(F) and DR 2-101(H) see Rule 7.3.
Comparison to ABA Model Rules of Professional Conduct
Rule 7.1 is similar to Model Rule 7.1 except for the inclusion of a prohibition on the use of nonverifiable communications about the lawyer or the lawyer's services.


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HOUSTON - NBC's new show "Revolution" explores a post-electric world. So, what would happen if the real world went dark?
Houston area residents have gotten a taste of the loss of power after natural occurrences, like Hurricane Ike, and when electric companies used rolling blackouts to protect power grids.
It is not just the end-of-the world types exploring alternatives to signing up for power from a grid. Dan Bretch is one of those people. His property has no power lines or cables, no water pipes or connections. He is proud to be "totally off-the-grid."
"We derive all our own power from the sunlight or a generator that runs on biodiesel," said Bretch, the owner of Industrial Country Market.
It wasn't the fear of Armageddon or the complete collapse of the United States, but the simple issue of money that pushed Bretch and his family to cut the cord.
Bretch sampled living off-the-grid first with a cabin on the property before opening a 6,000-square-feet store. The Industrial Country Market is just off Highway 71 near Interstate 10 on the way to Austin.
The store has gadgets, both fun and serious, along with a sampling of just about anything you could find in a roadside store. The walls are filled with all kinds of art from local artists. Bretch has a little work station in the back to continue his experiments and build contraptions that can be found everywhere at ICM.
Bretch's crowning jewel is a room with a huge bank of batteries and large inverter boxes that funnel the energy being collected by solar panels in the yard.
"At this facility you are looking at 8,000 to 10,000 watts coming out of this from the sun right now," Bretch said.
He said the panels can feed up to 100,000 watts of electricity a day. What isn't used is collected in batteries and not only keeps the lights on but even powers a few small air conditioners.
"What we have is the standard power you would have at your house -- 110 volt and 220 volt," Bretch said.
It goes beyond just the power -- composting toilets, plants that filter water for ponds -- everything in his store serves a purpose.
Bretch said the cost for things like solar panels has dropped dramatically, making off-the-grid living more feasible. Panels that were once $800 now hover around $100.
Bretch has found a way to make it even cheaper by bargain hunting. He said most of his supplies are reclaimed or acquired when other stores have either remodeled or gone out of business.
"Get on Internet, look on Craigslist. See what is on eBay," Bretch said.
Many of his bigger ticket items were acquired from people he said prepared for the Y2K disaster that never happened. Those people began trying to get rid ofwhat at the time was very expensive gear at rock-bottom prices.
Little is wasted at ICM. Most of the buildings are made of reclaimed metal or recycled items. Even the water is acquired. Some of the water comes from a drilled well, but most comes from Mother Nature. Rain barrels and gutter systems can be found all over the property. Bretch said even during last year's drought they had no problems living off the water they had already collected.
"Part of the codicil was to do this whole thing without giving anything up. I have my satellite television. I have my microwave oven. I have my shower. I have my air conditioner," Bretch said.
Solar power runs a water heater. A sun stove can even cook a meal if need be.
Bretch and his wife, Michele, are currently building a self-sustaining house on the property.
"It is just like the house you have, except no electric bill," Bretch said.
And no water bill, too. The slopping roof allows the rain water to flow to a single gutter that carries the water to two giant tanks. A water filtration system cleans the water before it enters the house.
Bretch said it is difficult for people living in the city or those with lots of trees to live off-the-grid, but everyone should be able to use collected rainwaterto water their lawns.
"I would simply have a valve that turns off city water to rain water," Bretch said.
Bretch teaches classes for $71 to anyone interested in going solar.
For Bretch, the self-sustaining lifestyle is about being financially secure and keeping his costs down.
"My biggest expense from my Social Security check will be my DirecTV," Bretch said.Photos: Industrial Country Market
Copyright 2012 by Click2Houston.com. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.


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WE04September 25, 2004
Leaving for the airport in a few hours, so here's the plan. A brief flight to LA and a bit of a layover will be followed by the long trek across the Pacific, Equator, and International Date Line — all in the same flight.
Scheduled arrival time in Sydney: the future. Something like early the 27th, all I know is that I'll completely miss the 26th of September. (Aw, my favorite day of the year, too.)
Plan for Sydney: a day of sleeping. Two days of exploring. And then the whole reason for the trip, WE04. Can't wait to meet everyone, it's going to be an invigorating couple of days.
Afterwards a drive down to Melbourne (yes, I'm aware it's a big spread-out country… but remember where I live) and a few days enjoying the sights. Yes, I'd love to have lunch/coffee/whatever with the local Melbourne crowd. Here's the catch — I'd rather not coordinate it. Leave your contact details in the comments, and have a type A personality take the ball and figure it all out, and I'll be there. Good day to plan for: the 5th.
So that's that. Don't expect much activity on here until mid-October. Take care, and enjoy the turning leaves for me.
Reader Comments
While you're in Sydney, go to Chinatown. It's got *real* Chinese food. You won't be disappointed.
A drive from Sydney to Melbourne… well, if you must drive (seriously… a flight between the two is inexplicably cheap), perhaps some pointers:
-Left side, left side! (best way to keep yourself from unconsciously drifting over to the wrong side is to remember that, at least on a two lane highway, just keep your bum in the centre of the road… if it's near the shoulder, you're on the wrong side)
-Don't drive after dark. Having done so many times, I can say you never know what you might hit. Remember that a kangaroo may seem like a wondrous curiosity in the daylight, but on a highway late at night, it's something of a bouncing deer… and one that hitting will most likely total the car. Also, wombats hit at speed react as you might expect a speedbump hit at speed to do: they're fine, but your car is suddenly catapulted into the stratosphere.
-Photo-cops: I've never had a problem with them in the north of Victoria, but we got a notice in the mail for doing 20km/h over the limit along the the Great Ocean Road (I still to this day have no idea where it was hidden).
Australia is a wonderful place. Enjoy it. I lived in and around Melbourne for the better part of two years, and I miss it every day.
Return Flight Sydney - Melbourne: AUS $120
Hire Car to drive Great Ocean Road: AUS $45 per day.
Time Saved by not driving the boring hours from Sydney to Melbourne: Priceless.
Honestly, hop a flight, pick up your hire car at the airport and on you go, with a saving of at least 8 hours of driving time.
That said, 5th is Tuesday, some of us have to work!
Contact: 0415 730 653 (mobile/cell) if you would like to go for some cheap Thai in Richmond, or a beer at a great little pub.
Good Lord, you're driving! I hope that's not for reasons of financial convenience, because it is improbably cheap to fly between the two now.
If you're driving the Hume Highway route, I found it to be a many-hour stretch of bum-achingly boring terrain. If you want to see some lovely parts of the country, you may wish to consider the Princes' Highway, which goes along the coast and through the Gippsland region of Victoria. The upshot is that it's a somewhat longer drive.
Have a great time at WE04!
Okay, time for one quick comment before I really AM out the door.
The idea is driving to Melbourne, then keeping the car and seeing how far we can get along the Great Ocean Road before we have to turn around and come back.
Welcome to Australia Dave, I wish I had a chance to meet you. I live in Artarmon, a suburb of Sydney. Unfortunately, I couldn't afford to go to WE04 :-(, oh well, maybe another time.
Don't worry about hitting kangaroos or wombats, you won't see any in the city areas, unless you go to the zoo, but there are kangaroo and wombat signs on the country roads to let you know to look out for them in the areas they live.
The great ocean is quite long, but worth taking the trip to see it all.
Oops, forgot to leave my contact details for you. I'll usually be reachable by at least one of these numbers, or e-mail me any time.
Mobile: 0427 202 908
Home phone: (02) 9415 1358
Damn J2EE programming, man! I need to get into design if only for the conferences :-)
I wish I had the money to go to Australia to hear web designers like you.
So….what happens if you're birthday is on the 26th?
Do you magicly not age for a year? Or do you reck the time-space continuum, instantly creating a polarised paralel world where you were never born?
Or not?
Anyway, have fun.
Whatever you do DO NOT miss the Great Ocean Road drive. Look forward to seeing you at the WE04.
And again the drive down from Sydney to Melbourne is really scenic if you go down the Coast. Some beautiful quaint towns, villages, its very rejuvinating.
Looking forward to seeing you at WE04 Dave. I'd tend to confer with everyone else and say "take the plane" and spend more time on the Great Ocean Road - it really is beautiful.
If you're not over-run by adoring fans and need to know where the best watering holes are - St Kilda's a great place to sit outside and have a beer on a warm spring afternoon - contact details are Mob: 0418893116.
Actually that's a good point that Luke makes - the best parts of Melbourne are outside the CBD, so if you haven't organized accommodation yet then think about staying somewhere like St Kilda, Richmond, Carlton or generally one of those lively hubs that are close to the city, well furnished with public transport and more interesting (and probably less expensive) than a city hotel.
I will go out on a limb here and state that Melbourne is absolutely the best place in the world to eat out, so make the most of it! Vietnamese/Turkish in Victoria St, Nepalese/Argentinian/Indonesian/Thai in Bridge Road, Italian/Greek in Lygon St, everything else in St Kilda, you can't go wrong. Have fun.
Don't be put off by these clowns telling you to fly :) Drive! Its the only way to see the place. Take a couple of days and do the coast road. Seriously worth it.
See you later in the week.
It's getting close now. The drive from Sydney to Melb is amazing, for me its Canberra to Melb. It's all about the Twelve Apostles.
Can't wait till Thursday! Anyone staying in the Mercure Hotel Sydney on Broadway?
Yes, like I said earlier. Drive! Thats the only way to see the place and take back some unforgettable memories.
Just got news from Maxine that you have arrive down-under. Enjoy!! ;0) Hope you had a pleasant trip.
I'd be keen for any of the above! Catch me on 0421 616 955 or email cam [at symbol] selfgratification [dot] org.
I'm happy to organise something for Melbourne, so if you're keen to come along, email me at the above address and I'll see what I can do.
Dave: any preference for time of day?
"you'll find that Australians are Canadians' long lost cousins"
Yup. The black sheep of the family.
(Refering to it's former state as inmate island :P)
Welcome to Australia Dave and anybody else who is on the way over here.
I wish I could be at WE04 but cant make it sadly, hopefully next time I'll be there.
Have fun while your here, oh and keep left ;).
Another welcome to Australia message (even if I am in Stockholm at the moment).
Have a great time; you'll find that Australians are Canadians' long lost cousins. Don't forget to go down to Bondi and have lunch. The Icebergs is expensive but well worth it for the view. After lunch I'd recommend doing the cliff-walk from Bondi to Coogee. A few hours well spent.
Why did this happen when I'm overseas? Doh!
Any chance they can hold WE05 in Indianapolis, IN next year :)
You lucky webdesigner ;) I wish I could go to Australia for a nice conference. Right now I´m sitting in Stockholm Sweden, it´s raining and about 10 degrees Celcius. Not even websurfing is fun in this weather :(
Well, you have a great time down under and let us know how it went, I´m sure you will.
Sheesh kebab, thats one heck of a long drive! Well, good luck with the WE04 gig, wish some of the poorer we designers out there could afford to go but hey.
Have a great time.
Okey dokey… I have booked a table at Gurkha's Cafe (360 Bridge Rd, Richmond) for 7pm on Tuesday night (the 5th).
If you want to come along and haven't already been in touch, please email me by about midday Tuesday so I can add you to the booking (my email address is in comment #18).
In case people hadn't heard, the dinner Cam organized above has been superceded by the "Dave 'n' Doug in Melbourne" event. Info at http://webstandardsgroup.org/go/event19.cfm


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Boom Goes the Dynamite: 03.15.09Posted by nvr1983 on March 15th, 2009
It's officially here. . .Selection Sunday is upon us. Time for a bunch of whining and complaining by grown men about how their team that went 20-12 deserved to get in or for Billy Packer to rip some mid-major for getting in at 26-7 over an ACC team that went 17-15. Thankfully, the slate today is a little lighter to let us get some work done on our NCAA tournament preview. We'll be covering all 4 games today and we will be doing a separate live blog (RTC Live style, but we won't be inside the Selection Committee room although give it time). Here are the games today in chronological order and a brief synopsis of what is at stake in each game:
- 1 PM: #22 FSU vs. #8 Duke on ESPN, Raycom, and ESPN360.com: This is only for seeding purposes. FSU is probably a solid #5 after knocking off UNC yesterday. A win here might be able to move them up to the last #4 seed. Duke is pretty much locked into a #2 seed. There is no way they are getting a #1 seed and they won't fall to a #3 seed because the two teams above them and three teams below them in the rankings all had worse weeks.
- 1 PM: Tennessee vs. Mississippi State on CBS: This is the biggest game of the day because of its implications on the bubble. I'm guessing 95% of the people who have any rooting interest in this game will be pulling for Tennessee. The Vols are solidly in the field at a #7 seed in most predictions and I can't see them jumping much higher, which would essentially mean they should be ranked, if they beat a good, but not great MSU team. The Bulldogs on the other hand can wreck a bunch of teams NCAA dreams by winning the SEC title.
- 1 PM: Texas-San Antonio vs. Stephen F. Austin on ESPN2 and ESPN360.com: The winner of this game will end up with a 14 or 15 seed. SFA might be an interesting first round opponent since they actually had a decent RPI for a Southland team (#79) and boast a win over #94 North Dakota State. Honestly though, unless you're a fan/grad of one of the schools, you're not going to be watching this over the other two games.
- 3:30 PM: Ohio State vs. #24 Purdue on CBS: Another game that is about seeding. I think Purdue has moved up about as far as it can after destroying Illinois in a game that wasn't as close as the final score indicated. Ohio State has a chance to move up to a 7 if they can win this after knocking off Michigan State yesterday.
12:20 PM: Ok. Some quick questions for you that ESPN.com posed this morning:
- Which teams are the #1 seeds?
- Is UConn better off as a #2 seed?
- Which conference will send the most teams to the NCAA tournament?
- Will the SEC really only put two of its teams into the NCAA field?
- Will Arizona's 24-year bid streak finally end?
- Which teams will be seeded higher than you think?
- Which teams will be seeded lower than you think?
- Which mid-major teams will the big boys hope to avoid in the first round?
Let me know what you think and I'll give you my thoughts in a little bit.
12:45 PM: Why does CBS drag these Selection Committee people onto the show? I know they're trying to hype up the Selection Special at 6 PM, but they add absolutely nothing. They just give generic, PR firm answers. I almost prefer the bickering that ESPN has arguing whether or not a team deserves to be in.


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I found Galena while on a trip to Florida back in April 2003. I had been looking for a sailboat and had pretty much
settled on the Westsail 32 as the one for me.
I had read a great deal about other people's thoughts on what makes a good cruising boat.
I had read that cruising boats are bought by the pound rather than the foot. i.e. The heavier the better. And that full keel boats track best and are unsurpassed at surviving groundings and other contacts with things below the waterline. Cutter rigs provide more sail plan options than standard sloops. Double-enders handle following seas better than the modern sugar-scoop sterns. Minimal equipment on board reduces opportunities for breakdowns. These are the axioms that guided my search. And as misguided as some might think that was, it's what I went with.
The decision to go with the Westsail was based on the above and the fact that it was the most boat I could afford. That decision was made easier after reading about the Satori. She is a Westsail 32 that was caught in "The Perfect Storm." She survived the storm without damage. And then she survived being washed up on a beach after her crew were forced by the Coast Guard to abandon her. Satori's story was contorted in the screenplay of the movie about the storm and she was mishandled by the Coast Guard (actual USCG video here). The real story as told here by her owner convinced me of the qualtiy of the Westsail's design and construction.
To date (2012) I have completed six significant voyages on Galena: The maiden voyage from Florida to Maryland, a four trips down the east coast with three out to the Bahamas, and a trip off shore to the BVI. Currently I'm on my seventh voyage. I'm not sure where this trip will take me, however I'm sure it will be an adventure.
Click on the Titles/Links below to see the logs/photos/charts that go with each of these voyages. Or you can use the voyage list in the box above.
Maiden Voyage, Spring 2003 (Follow this link to read the logs and view the pictures) Jane and I, along with a good friend from work (yes, I used to work), Greg Moyers, sailed Galena home. We sailed from Ft Lauderdale, FL to Beauford, NC and then up the ICW to Kent Island, MD. Greg and I were on a 2-week vacation from work. We therefore broke the first rule of cruising: "Never adhere to a schedule." That led to the violation of cruising rule #2: "Wait for a good weather window." With only beginner's luck on our side we headed off-shore. We paid for those mistakes with a couple of bad days. And out there a bad day can be pretty bad. Now I hope I know better.
First Season of Cruising, Winter '04-'05 (Logs and photos here...) As a first cruise my ICW-Bahamas adventure was just right: Not too long; not too extreme. I felt comfortable on the ICW. I had run the 200-mile section between Beaufort, NC, and Norfolk, VA once already. The rest of the coastline down to Florida was bound to be more of the same. I planned on a couple of overnight off-shore runs but after the maiden voyage in '03 I felt comfortable with that. I had studied the ink off the charts of the Bahamas and was comfortable with our marine preparations.
Second Season of Cruising, Winter '05-'06 (Logs and photos here...) As a second trip, heading off-shore to the British Virgin Islands sounded about right. I planned to run down the ICW, yet again, to Beaufort, NC. Then head off-shore to a point just south of Burmuda. There I would turn south on what is sometimes called "I-65." That would be 065° West longitude. I would follow that line from about 29° North down to the BVI at 18° N. That leg of the trip would consist of about 1400 miles of ocean and would take about 2-weeks. Following that, I'd turn west and cruise the USVI, the southern coast of Puerto Rico, the northern coast of the Dominican Republic and the Bahamas before returning to the US and the Chesapeake Bay. It was a good plan. It sort-of worked out.
Third Season of Cruising, Winter '07-'08 (Logs and photos here...) Last winter I stayed home and gave Galena some much needed TLC. Then I did a single-handed voyage through the Bahamas. I cruised the Exumas, Eleuthera, and the Abaco's. I started by travelling down the ICW with my friend Tom (s/v Evergreen). Then I traveled to Nassau and down the Exumas with s/v Stella Polaris (Tom and Joyce) and s/v Moonlight Serenade (Bill and Sarah). Then, on the return trip north, I visited Eleuthera and a good deal of the Abaco Islands of the northern Bahamas. Jane visited me and sailed from Nassau to George Town, Grand Exuma and then flew back home. The rest of the trip was a very interesting singled-handed voyage.
Fourth Season of Cruising, Winter '08-'09 (Logs and photos here...)
It's been a long summer on the Chesapeake. A lot has happened in my life but I really have not done much to prepare Galena for her fourth trip south. Mostly I've just installed a few more
bells and whistles, hauled and bottom-paint, and did a little bit of overdue maintenance.
This winter I'll do the same thing as last winter (is this getting boring or what?). Because of my recent change in marital status I'll be single-handing again this year. My plan is to finally get down to Key West by running down the Atlantic ICW. After a week or so there I'll head over to Nassau and back down the Exuma Island chain to George Town for the Cruiser's Regatta that starts on March 4th. After that, well, we'll see which way the wind is blowing.
Fifth Season of Cruising, Winter '09-'10 (Logs and photos here...) I moved galena's home port to Boca Chica Naval Air Station, Key West, FL. Mostly this was just the standard run down the ICW to Beaufort, NC. Then a bunch of off-shore hops south along the east coast to Key West. I had planned on cruising south to Central America. But instead just stayed in Boca Chica for the winter.
Sixth Season of Cruising, 2010- ???? (Logs and photos here...) Somewhat ashamed of myself for spending an entire year in Key West I headed off-shore to finally see some of the world. The plan is to head east to the Bahamas, then south through the Windward Passage (between Cuba and Haiti). Then a long 8-day run straight south to Catagena, Columbia. Then I started heading west. The plan was to get through the Panama Canal by mid-March 2011. But I missed that by about three months. Eventually I made it to the South Pacific Islands and spent a lot of time enjoying the part of the world.


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Singer Alicia Keys has shot down ongoing reports she began dating husband Swizz Beatz while he was still married.
The music producer and R&B star Mashonda separated in 2008 just before Beatz, real name Kasseem Dean, started dating Keys.
The "Empire State of Mind" hitmaker has been dogged by rumors she broke up the former couple's four-year union ever since, and now the soul singer has addressed the cheating allegations, insisting she has done nothing wrong.
She tells Jet magazine, "(Mashonda and Beatz) were apart for some time before we ever got together… (but) that doesn't matter to those who take pleasure in trying to knock others down."
And Keys, who exchanged vows with Beatz in 2010, insists she is tired of having to defend herself to the media: "There's no need to fight what's not true."