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KFA SA – polskie przedsiębiorstwo
 1. KFA Wielkopolska – polski zespół futbolu amerykańskiego
 KFA () – Stowarzyszenie Przyjaźni Koreańskiej

redpajama_set_name: RedPajamaWikipedia


We�ve been flooded with questions about the exit of Tom Pelphrey (Jonathan Randall) and Stephanie Gatschet (Tammy Winslow Randall) this week so, just to clarify for everyone: They are departing from the cast. This was first announced and reported here in September after several months of speculation. Originally the pair were supposed to exit by the end of December or early January. However, thanks to some convincing from the writers and producers, they agreed to stay a little longer to finish the storyline without a rush.
Both of the wonderful young actors decided to leave of their own accord with the desire to move on to other projects. Their final air dates have not been officially given by the network as a means of safeguarding the dramatic effect of their exiting. While the character of Tammy did die this week, we can expect to see Gatschet reappear in upcoming episodes in the dreams and thoughts of her mother and other loves ones. Pelphrey is expected to have his final air dates in early February. They completely filming their final scenes late last year and Pelphrey has already moved on to film projects.
While we are sad to see the characters go, particularly in such a tragic manner, they have certainly given us some of the best moments in the soap�s recent history and will be sorely missed. We wish them all the best and will not forget the wonderful contribution they made.


The South Australian Standards and Guidelines for Breeding and Trading Companion Animals are made under the provisions of the Animal Welfare Act 1985 . The purpose of this document is to:... The South Australian Code of Practice for the Care and Management of Animals in the Pet Trade provides guidelines for businesses that sell companion animals for profit (eg pet stores).
Travelling with your pet Over two-thirds of Australian households own pets, making Australia a country with one of the highest rates of pet ownership in the world i . Considered by many as integral family members ii , pets regularly travel in the family car. how to find a word in pdf The Driving Companion for class 'C' vehicles The information contained within this publication has been prepared to help you become better informed about the provisions of the driver licensing system.
The South Australian Code of Practice for the Care and Management of Animals in the Pet Trade provides guidelines for businesses that sell companion animals for profit (eg pet stores).
Advanced Search. Advanced search can be used to cut down the number of products listed based on your product requirements.


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Q: How to remove API activated by default in Google Cloud Platform projects? I have a Google Cloud Platform account with multiple projects.
Every time I create a new project, I have the following API activated by default.

*

*What are these APIs ?

*Are they optional ?

*Is it safe to disable them ?

*Is it possible to prevent Google Cloud Platform to add them by default when I create a new project ?

A: *

*These APIs are for very different purposes, you can find what are for here.

*In the previous link, it's mentioned that they can be disabled. Check how to disable them here.

*The documentation doesn't mention any risk or side effect of doing so.

*It doesn't look possible, as there is no mention of this in the public documentation.

redpajama_set_name: RedPajamaStackExchange


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Central Park or Reyes Park is a green, cozy, safe and quiet amid the routine and congestion of the city of Bluefields. This site has all the amenities for visitors to enjoy a relaxing afternoon in the shade of leafy trees, or a refreshing drink at one of the kiosks nearby.
This site is well known for the monument it has in its main entrance, which represents the six ethnic groups in the Caribbean Region of the country. The sculpture is surrounded by flowering trees and behind it, there is a basketball court in good condition, which is often used for sports or municipal activities.
Behind the stadium are located some swings for the amusement of children and an ancient relic steamroller, which was first found in the city steamroller. This device is also used by infants as a playground where they usually make their imagination in an open space.
Besides recreation areas for adults and youth, the park has two kiosks or booths that sell soft drinks and snacks that people get after physical activity, or while enjoying good company. Around the park there are large platforms that allow visitors to tour the area on skates, bicycle or just walking. In every sidewalk you can find wooden or concrete benches shaded where you can relax while reading a book or chatting with friends. You can also do night hikes since there is enough lighting in the area.
In the corners of the park's large trees provide more shade and in its center is a gazebo. This is a small building with a domed roof and thick columns, also known as "temples of music" as they originally were considered spaces for concerts. Nowadays this is a place where visitors can relax and children play. Note that this construction has English influence in their finishes.
Another attraction of Kings Park is a painting that represents the theme of gender, masculinity, cultural, ethnic diversity, environment and teenage pregnancy. The paintings displayed on the wall were made by children and young people between 10 and 18 years.
The park is located right in the central area of the city, and is one of its main reference sites. It's just a few blocks away from the pier, so you can walk there at any time.


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Home Health & Medicine
A DNA-ring pill might diagnose any cancer fast and accurately
Stanford scientists proved that it's possible use DNA minirings that code the production of a specific protein that can't be found in healthy cells to diagnose any form of cancer. The mini-rings were injected in the bloodstream of mice and allowed for accurate diagnosis up to two days from the injection. In authors envision using the same solution to diagnose any cancer in humans, while also relaying how large is the extent of the tumour. Moreover, they hope they can achieve this with an orally administered pill, instead of an intravenous injection, thus making it the least invasive form of cancer diagnosis. The only pain you'll experience is when doctors will prick a needle in your finger to get a drop of blood. Take that, biopsy!
in Health & Medicine, News
Stanford scientists proved that it's possible use DNA minirings that code the production of a specific protein that can't be found in healthy cells to diagnose any form of cancer. The mini-rings were injected in the bloodstream of mice and allowed for accurate diagnosis up to two days from the injection. In authors envision using the same solution to diagnose any cancer in humans, while also relaying how large is the extent of the tumour. Moreover, they hope they can achieve this with an orally administered pill, instead of an intravenous injection, thus making it the least invasive form of cancer diagnosis. The only pain you'll experience is when doctors will prick a needle in your finger to get a drop of blood. Take that, biopsy!
Looking for cancer in all the right places
Researchers inserted tiny DNA minicircles into the genes of mice, which caused cancer cells to emit a substance that could be detected from a blood sample. Credit; Jim Strommer
Cancer biomarkers – substances that flag the presence of a particular kind of tumour – aren't new in medicine. We've heard about quite effective efforts where prostate cancer, for instance, was detected judging from the high blood levels of a prostate-specific antigen. Similar biomarkers were identified for ovarian and colorectal cancer. Each of these biomarkers, however, is unique to its cancerous form which means you'd need to take a separate test for each cancer. Also, some of these proteins surface in a healthy cells as well leading to false positive diagnosis.
Sanjiv "Sam" Gambhir, professor and chair of radiology and director of the Canary Center at Stanford for Cancer Early Detection, believes the progress his team made with DNA mini-circles will solve most of the challenges faced by cancer biomarker detection techniques.
The Stanford researchers' DNA minicircle is a tiny, artificial, single-stranded DNA ring about 4,000 nucleotides in circumference. Its function is to code for the production of a protein called secreted embryonic alkaline phosphatase. SEAP is naturally produced in human embryos as they form and develop, but it's not present in adults. The minicircles, which contained a single gene coding for SEAP, were mixed with a chemical agent to facilitate their uptake by both healthy and cancerous cells alike.
The real ingenious part was adding a genetic promoter in front of the gene that codes SEAP production. In genetics, promoters are DNA sequences that instead of making proteins are involved in regulating them. Like a switch, promoters will tell the gene its linked to when and if it needs to activate its function, or how many proteins to make. This particular promoter normally regulates a gene called survivin that, in adults, is only "on" in cancer cells. This way, the SEAP proteins were made only in organisms were cancer is present. With this in mind, it's only a matter of a taking a blood sample and looking for any signs of SEAP proteins.
In experiments, scientists injected cells from human melanoma cell lines into immune-compromised laboratory mice. Because their immune system was jacked, antibodies didn't attack the cancer cells and were free to develop into tumours throughout the body – the lugs were particularly affected. Other mice of the same strain were given otherwise-identical injections that didn't contain the cancer cells; they didn't develop tumors. According to the Stanford press release:
About two weeks later, the animals' blood was checked to make sure their baseline levels of SEAP were zero. Then the investigators injected the minicircles intravenously into the animals' tail veins and measured SEAP levels in the mice's blood one, three, seven, 11 and 14 days later. Within 48 hours, SEAP was present in the blood of mice with tumors, but not in that of the tumor-free animals. That signal began declining in strength as early as 72 hours post-injection, fading to insignificance within the next two weeks or so. Its maximum strength varied with the total tumor volume in a mouse's lungs, suggesting that the test may be sensitive not only to the presence of cancer but also to its extent.
DNA mini-circles boost several advantages over other similar methods, like defanged viruses or specially engineered bacteria. The mini-circles do not replicate, nor do they elicit an immune response.
"We want to translate this strategy into humans, so we've set it up in a way that's most likely to be effective, safe and convenient," said Gambhir, who is also the Virginia and D.K. Ludwig Professor for Clinical Investigation in Cancer Research. Although the minicircles were injected intravenously to the mice in this study, it should eventually prove possible to deliver them orally via a pill, he said. "We haven't got it down to a pill yet, but the oral delivery part of this is likely a solvable problem — only a few years off, not five or 10 years off."
Findings appeared in the journal Proceedings of the National Academy of Sciences.
Tags: cancerdna


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SAVI: a statistical algorithm for variant frequency identification
Vladimir Trifonov1,2,3,
Laura Pasqualucci4,5,
Enrico Tiacci6,
Brunangelo Falini6 &
Raul Rabadan1,2,3
Many problems in biomedical research can be posed as a comparison between related samples (healthy vs. disease, subtypes of the same disease, longitudinal data representing the progression of a disease, etc). In the cases in which the distinction has a genetic or epigenetic basis, next-generation sequencing technologies have become a major tool for obtaining the difference between the samples. A commonly occurring application is the identification of somatic mutations occurring in tumor tissue samples driving a single cell to expand clonally. In this case, the progression of the disease can be traced through the trajectory of the frequency of the oncogenic alleles. Thus obtaining precise estimates of the frequency of abnormal alleles at various stages of the disease is paramount to understanding the processes driving it. Although the procedure is conceptually simple, technical difficulties arise due to inhomogeneous samples, existence of competing subclonal populations, and systematic and non-systematic errors introduced by the sequencing technologies.
We present a method, Statistical Algorithm for Variant Frequency Identification (SAVI), to estimate the frequency of alleles in a set of samples. The method employs Bayesian analysis and uses an iterative procedure to derive empirical priors. The approach allows for the comparison of allele frequencies across several samples, e.g. normal/tumor pairs and more complex experimental designs comparing multiple samples in tumor progression, as well as analyzing sequencing data from RNA sequencing experiments.
Analyzing sequencing data through estimating allele frequencies using empirical Bayes methods is a powerful complement to the ever-increasing throughput of the sequencing technologies.
High-throughput sequencing (HTS) has become one of the most important tools in the arsenal of biomedical researchers in their quest for understanding the foundations of diseases with a strong genetic component like cancer [1]. Sequencing technologies have significantly increased the amount of data produced by a single run of an instrument with their modern high-throughput versions producing more than 1Tb in a single run. Thus, in the case of the human genome, the scale of the sequencing experiments has been pushed into the realm of whole-exome [2, 3], whole transcriptome [4, 5], and even whole-genome studies [6, 7]. The sheer volume of this data combined with the inherently random aspect of the sequencing process conspire to introduce uncertainties in the output of the experiments. Furthermore, the intricate chemistry of the novel sequencing instruments often results in systematic errors, which can make artifacts indistinguishable from actual genetic lesions. Although detecting such sources of non-random errors is an important part of analyzing the data produced by sequencing experiments, the purpose of this paper is to develop a framework for the correction of the random component in the noise introduced by the sequencers. Furthermore, our goal is not to establish the biochemical sources of those errors, but to develop a statistical approach applicable in diverse scenarios in which the assumption about the random nature of the source of errors holds.
A major application of HTS is to compare related samples, e.g. healthy vs. disease or various stages in the progression of a disease, based on their genetic makeup. Many such examples are provided in cancer research, where identifying the genetic or epigenetic lesions that contribute to the oncongenic process is an important step towards better diagnosis, evaluation of prognosis, and treatment of the disease. Thus HTS has become a powerful unbiased approach to delineating the landscape of genetic alterations in different tumor types [8, 9]. An often-applied technique involves identifying aberrant somatic alleles fixed in a population of cancer cells, which are absent from a control sample. The wisdom behind this technique is based soundly on the observation that the drivers of oncogenic processes are fixed by the clonal expansion [10]. From a more general perspective, and in addition to making a digital (binary) present/absent call, one could strive for identifying the frequency of alleles in a given population of cells. The importance of this analysis to cancer research is immense since low frequency alleles can have a major contribution to the disease in later stages [11], e.g. by conferring resistance to treatment. Furthermore, detecting low frequency alleles at an early stage of the disease, and even before the disease has manifested itself, can be crucial for its prognosis.
The comparison of two temporally ordered samples with respect to the frequency of an allele could indicate either a gain or a loss of that allele. Both of these alternatives could be the cause (driver) or a consequence (passenger) of the biological process driving the phenotype. For example, a point mutation could inactivate the normal function of a tumor suppressor gene and lead to the formation of cells with a cancer phenotype (Figure 1, left). Alternatively, the abnormal phenotype could be the result of a loss of heterozygosity (Figure 1, right). Although, distinguishing driver from passenger alterations is an important part of the functional analysis of clonally expanding populations of cells, detecting such events regardless of their direction and relevance to the phenotype is the first step towards such analysis.
High-throughput sequencing data provides information about the frequency of an allele in a population of cells. The numbers in parentheses are the 5% confidence interval. Depending on parameters, discretization might distort our perception of the data (left). Alleles can be gained by point mutation (left) or lost by LOH, e.g. copy-neutral LOH (right), in the evolution of the population.
Besides the clinical importance of diagnosing and treating cancer, having a detailed picture of the alleles present at different stages of the evolution of a population of cells is an important step towards understanding the intricate biological processes underlying its existence. Several approaches have been used to identify the genetic alterations differentiating a set of related samples. Some methods [12, 4] involve ad hoc thresholds on the number of reads reporting a variant allele, their quality and/or the total number of reads covering the position of the variant allele to produce a binary present/absent call for that variant. A comparison between different samples is then based on differences in those calls (see Figure 1). Arbitrary thresholds on the number of reads reporting a variant introduce biases due to the uneven distribution of depths along the genome. Such methods can miss variants which have somewhat lower quality but high depth or lower depth, but high quality. Furthermore, decisions based on a discretized view of the data depend strongly on the level of discretization and other parameters of the discretization process. This could be a problem if the samples being compared are not perfectly homogeneous, e.g. by contamination or the natural presence of tumor cells in the control sample. Other methods [13, 14] assume allele distributions particular to a homogenous population of diploid genomes. Similar to our approach, the algorithm SNVMix [15] uses a Bayesian framework and is able to identify variants in samples with different ploidy and tumor cellularity. The algorithm employs an expectation-maximization for constructing a prior.
In this paper we present the statistical Algorithm for Variant frequency Identification (SAVI) developed in the course of analyzing data from sequencing experiments of cancer samples from nine Hairy Cell Leukemia (HCL) patients and the corresponding paired normal samples. The initial findings are published in [2]. The algorithm is based on constructing Bayesian posteriors distributions on allele frequencies and employs an iterative procedure for constructing an empirical prior from a given dataset. Having posterior distributions lets us obtain a high credibility interval for the frequency of a particular allele as well as estimates on its expected and most likely value. In contrast to other applications of Bayesian analysis to the problem of frequency estimation, our prior is not fixed to belong to a predetermined set that might not represent an accurate description of the data. Our desire was to develop a method which is more empirically grounded and governed by the data itself as much as possible. With the advent of large datasets the significance of Bayesian methods using priors based on empirical observations has increased [16, 17]. Thus our goal to tailor the prior to the data was naturally steered towards such considerations. Empirical Bayesian methods have been applied before in the context of gene expression in RNA sequencing experiments [18] and estimates of positive selection in tumors [19]. Although, as outlined above, the techniques developed in this manuscript are quite general, we focus here on two important aspects of its application: detection and genotyping of variant alleles in a population of cells and comparing allele frequencies across different samples.
The methods described in this paper were developed to analyze the sequencing data from a study on HCL - a lymphoid malignancy in which bone marrow, spleen and liver are infiltrated by leukemic B cells showing abundant cytoplasm with characteristic "hairy" projections. In the study, the exome of samples from peripheral blood leukemic hairy cells and paired normal mononuclear cells from 9 HCL patients was sequenced with Illumina GAIIx and HiSeq2000. Genetic material from the exome of those samples was obtained by enrichment with SureSelect. The mate-pair reads produced by the sequencer were aligned to the hg18/NCBI 36.1 human reference using MAQ. On average around 27M of the 28M exonic positions reported in the NCBI 36.3 CCDS database were covered by reads with an average haploid depth of 55. After filtering, described next, on average 16K exonic positions were determined to contain a variant. Of those, 2,500 were novel (not recorded in dbSNP 130) and 1,900 were in addition non-synonymous. Finally, across all samples a total of 81 of the novel non-synonymous variants were predicted to be somatic. These variants were compared against a list of 121 tested variants 67 of which were confirmed to be somatic. Fifty-six of the confirmed somatic variants were predicted and only 1 of the tested, but not confirmed variants, was predicted.
To analyze the variants produced by the sequencer we applied the Bayesian framework outlined in the Methods section. As discussed there, for every potential variant allele we obtained the number of reads confirming the variant, the average Phred score of the nucleotides containing the variant, and the total number of reads covering its locus. Assuming a binomial likelihood of the variant depth given a particular allele frequency, total depth, and a prior distribution on those frequencies, we obtained a posterior distribution for the allele frequency for every tentative allele using Bayes theorem. In the following discussion, the priors and posteriors have precision 1% unless explicitly stated otherwise.
To construct a prior empirically we apply an iterative procedure based on the observation that a prior appropriate to a given set of observations should predict the empirically observed distribution of that data as a certain marginal distribution. The iterative procedure derived from this observation has as a fixed point a prior satisfying it. The details of the procedure are given in the Methods section. Figure 2 (left) shows the result of iterations 2, 5, and 10 of the procedure starting with a prior uniform over all frequencies. As can be seen, the priors obtained from the procedure get consecutively better at picking up the salient features of the allele frequency distribution particular to a diploid genome compared to a haploid reference - most positions are homozygous for the reference, a number of positions are heterozygous, and that number is proportional to the homozygous variant positions. Figure 2 (right) contains the Kullback-Leibler divergence between every two consecutive priors produced by the procedure. As can be seen in this plot, in our case the procedure is converging to a fixed point. Analyzing the convergence properties of this procedure in more detail is certainly of high mathematical interest but beyond the scope of this paper.
Iterative procedure for constructing an empirical prior. Features particular to a diploid genome compared to a haploid reference become pronounced (left). The procedure is converging to a fixed point (right).
Figure 3 (left) shows the aggregation of the posterior distributions by adding the corresponding posterior probabilities across all exonic locations. The figure contains two plots: one in which the prior was assumed to be uniform (in blue) and one (in red) which was obtained empirically. At the scale of the figure, the aggregated posterior obtained from the empirical prior exhibits a shift towards frequency 0 (non-variant alleles) when compared to the aggregated posterior obtained from the uniform prior. Considering that a posterior obtained from the uniform prior peaks at the observed frequency of the allele, we can say that the empirical prior shifts the posteriors of alleles with low frequency towards the 0 frequency since the empirical prior gives more weight to that frequency. The fact that the 0 frequency gets more weight in the empirical prior as compared to the uniform is expected, since most alleles are homozygous and equal to the reference. Thus the information accumulated from all locations is leveraged in a particular locus as an additional corrective factor. This ability of the empirical Bayesian framework to borrow information from all experiments in the decision for a particular experiment and to shrink the observations towards a common mean is considered one of its strongest theoretical and practical properties [17, 20].
Aggregated posteriors before filtering (left) and after filtering (center). Filtering at 2% frequency derived from locations present in dbSNP removes potential sequencing artifacts (right).
Detection of variants
Given the posterior distribution for the frequency of an allele we obtained the posterior probability that the allele has a non-zero frequency. Our decision was to consider as present variants whose posterior probability of having a non-zero frequency was at least 1-10-6 (see the Methods section for the choice of confidence cutoffs). Figure 3 (center) shows in red the aggregated posterior obtained from variants determined to be present with the criterion that the hypothesis "frequency at least 1%" is credible at the 1-10-6 level and using the empirical prior. The plot also contains (in blue) the aggregated posterior corresponding to the uniform prior. A comparison between the two posteriors shows again the shrinking towards a common mean, this time towards the frequency 50% for heterozygous variants and towards 100% for homozygous variant alleles. Also shown here are the aggregated posteriors for variants present in both the tumor and normal samples (in black) and novel variants not documented in the NCBI dbSNP130 database (in green).
Next we analyzed the performance of the 1% criterion on positions reported in the dbSNP database. Figure 4 (left) shows the aggregated posterior of the dbSNP locations for which an allele has been determined present with that criterion. In this figure such locations have been split in two disjoint datasets - those present in both the tumor and normal samples and those present in only one of the samples. Our intuition is that that the variants confirmed in both samples are more likely to be genuine germline polymorphisms. An inspection of the figure shows that the posterior distribution of variants at known locations, which are detected in only one sample exhibits, a peak at low frequencies, confirming our intuition that those variants are likely to be sequencing artifacts. To remove such artifacts we considered the option of raising the cutoff frequency in the filtering criterion. More precisely, our strategy was to obtain for every potential variant the highest frequency f for which the hypothesis "frequency at least f" is credible a posteriori with confidence at least 1-10-6 - i.e. we obtained a one-sided credible interval for that level. Next, for every possible cutoff frequency we obtained the contingency table for the test corresponding to the cutoff on that frequency vs. the known polymorphic locations present with frequency at least 1% with high posterior confidence in both samples. We computed the mutual information corresponding to that contingency table and chose the frequency cutoff which maximizes it. In our case a frequency cutoff of 2% was chosen. Figure 4 (right) shows the result of this analysis and Figure 4 (center) shows the ROC curve for the frequency cutoff parameter, where the chosen cutoff frequency is marked in red. Finally, we revisited the definition of presence of an allele and used a frequency cutoff of 2% at posterior credibility level 1-10-6. The result of this filtering is shown in Figure 3 (right). For comparison, in that plot we have also included the result of filtering with frequency cutoff 1%, which was shown in red in Figure 3 (center). From the plot it is evident that the cutoff at 2% removes a small peak and some of the bulk at low frequencies of the posterior distribution, which we hypothesize are due to sequencing artifacts. A possible extension iterates the process with the newly established cut-off until the aggregated posteriors stop changing substantially as measured, for example, by the Kullback-Leibler divergence.
Establishing frequency cutoff using known SNPs. SNPs present in both samples are more likely to be genuine (right). ROC curve for a varying allele frequency cutoff (center). Obtaining a frequency cutoff by maximizing mutual information (right).
Detection of somatic variants
To detect somatic variants we extend the method of detecting variants by obtaining a posterior probability for each possible difference of frequencies. Figure 5 (left) shows the aggregated posterior of the allele frequency differences for locations not included in the NCBI dbSNP 130 database and restricted further to variant alleles resulting in a non-synonymous change in amino acid. Those criteria were chosen because the goal of the sequencing experiments was to detect nucleotides changes relevant to the oncogenic process. The figure has a spike at difference 0%, which is expected since the majority of mutant alleles are germline variants present in both samples at equal allele frequency. The figure also contains smaller features at ±50% and ±100% due to potential candidates for somatic mutations.
Aggregate posterior distribution of allele frequency differences for novel, non-synonymous variants before filtering (left) and after filtering for a high-confidence difference of at least 10% (right).
Similar to variant detection, the identification of somatic alleles is based on choosing a suitable cutoff on frequency difference. In the variant detection context our search for suitable frequency cutoff was guided by the frequencies of variants at known (as recorded in NCBI dbSNP 130) locations observed in both tumor and normal samples. To establish a variant difference cutoff we leveraged the validation results of 121 potential somatic alleles 67 of which were confirmed to be genuine somatic mutations present only in the tumor samples.
The list of potential somatic alleles was chosen in the following way. First, starting from a uniform prior on frequencies 0%, 50%, and 100%, with the iterative procedure outlined in the Methods section we constructed an empirical prior for those frequencies. Next, amongst non-synonymous variants whose locations do not appear in dbSNP we selected those with posterior confidence of having zero frequency in tumor at most 10-6 and a posterior confidence of having non-zero frequency in normal at most 10-6. Finally, we restricted our attention to the variants having an observed frequency at least 25% in the tumor and observed frequency at most 5% in the normal. Those 121 variants were subjected to validation by a direct Sanger sequencing, and 67 were confirmed to be present in the tumor, but absent from the normal samples.
Figure 6 (left) shows the aggregated posterior distribution of frequency differences for the validated variants. The variants, which were not confirmed, exhibited a bimodal posterior distribution of frequency differences. This feature was a consequence of the bimodal posterior distribution of the tumor frequency distribution for those variants due to the particular data (quality, variant and total depth) for those variants. Informally, the data for those variants was not sufficient to distinguish a posteriori between the variant being absent or present. In retrospect and having the validation results in which such variants were not confirmed in the tumor, the ability of the posterior to mark them as less reliable was remarkable.
Establishing frequency cutoff using validated variants. Confirmed variants have higher frequency difference (left). ROC curve for a varying frequency difference cutoff (center). Obtaining a difference frequency cutoff by maximizing mutual information (right).
To select a frequency difference able to distinguish between the confirmed variants from those which were not confirmed in the validation experiment, we performed a mutual information analysis, similar to the one we used to obtain a variant frequency cutoff in the context of detection of variants. More precisely, for every possible frequency difference cutoff we compared the variants obtained at this cutoff with the result of the validation experiment and chose the cutoff for which the mutual information of the corresponding contingency table was maximized. In this way, a cutoff of 10% on frequency difference was chosen. Figure 6 (center, right) contains the details of this analysis and Figure 6 (left, in green) contains the aggregated posterior of the validated variants, showing an increase in frequency of at least 10% with high posterior confidence. As designed, this cutoff was able to distinguish the confirmed from the non-confirmed variants quite well, so we went back and filtered the novel non-synonymous variants using this criterion. In addition, we filtered for variants for which a decrease of at least 10% in allele frequency was observed. Our hypothesis was that such variants might be due to a loss of heterozygosity, which was confirmed subsequently. The aggregated posterior distribution of the frequency differences of the filtered variants is given in Figure 5 (right).
We have presented an empirical Bayesian framework for estimating allele frequencies in a given sample and for comparing such frequencies across samples. The method constructs an empirical prior through an iterative procedure with a fixed-point distribution predicting the empirically observed distribution on data as a marginal. Given the prior, the method combines the data of a particular position with Binomial likelihood to produce a posterior allele frequency distribution. We use high credibility intervals derived from that distribution to decide between the competing variant present/absent hypotheses and distinguish alleles existing in the sample from variants introduced as artifacts by the sequencing technology. For a particular sample involving the study of a human/cancer genome, the parameters of that decision can be inferred from the variants present at locations polymorphic in the human population. Finally, when confronted with a pair of samples, we obtain a posterior distribution on the allele frequency differences between them and detect the alleles presenting a substantial differential. This differential can constitute the appearance of oncogenic alleles, as well as a loss of alleles, e.g. loss of heterozygosity, as a result of the oncogenic process. In our work we used a set of validated variants to deduce the cutoff on frequency difference and have shown that the chosen cutoff distinguishes variants confirmed to distinguish the samples, e.g. somatic variants, from those which do not, e.g. germline variants. Regarding cutoffs we can make the general comment that although obtaining theoretical guarantees on those cutoffs is important, in experiments in which subsequent independent validation is available the stress is more on the cutoff parameter as setting a priority in the validation and leaving the decision of a cutoff to be guided by the design and the resources of the experiment.
This method was developed in order to analyze the sequencing data from tumor and paired normal samples from HCL patients and was successful in identifying important somatic mutations present in the tumor sample. The goal of the project is to obtain the mutational landscape of that disease and will be reported in a separate publication. We saw the study as an opportunity to explore the larger goal of estimating the frequency of alleles in a population of cells. Our motivation for this was enabled by the ability of the novel high-throughput sequencing technologies to detect alleles at a growing depth, and hence at a decreasing frequency. The importance of this to the future of biomedical research is immense, since it will let us fine-tune to a higher precision our understanding of complex populations of cells. More precisely, it will let us detect low frequency alleles, which might have been or will be selected for a clonal expansion in the evolution of that population. Furthermore, as is often the need in biomedical research, this approach will let us study in finer detail the differences between two samples as captured by their genetic makeup. This is important in contexts in which impurities in the sample might confound a more coarsely discretized view of the data. Our conclusion is that the approach to analyzing sequencing data through estimating allele frequencies using empirical Bayes methods is a powerful complement to the ever-increasing throughput of the sequencing technologies.
Bayesian framework
Consider a random source, which produces bits ("0"s and "1"s) with probability/frequency of "1" equal to f. Assume also a probability e of error in reading the bit. Then the probability of observing a "1" is
1 - e f + e 1 - f = f + e - 2 e f
Assume a prior density p(f) of the frequency f and a prior for the error e uniform on the interval [0, E] for a fixed 0 ≤ E ≤ 1. A random experiment produces a string of m bits with n "1"s. Assuming a binomial likelihood of the data, the posterior density on (f, e) is
P f , e = p f E ⋅ C ( f + e - 2 e f ) n ( 1 - f - e + 2 e f ) m - n
where the constant C ensures that ∫P f , e dfde=1. For the marginal posterior on f we obtain
P f = p ( f ) E ⋅ C ∫ 0 E f + e - 2 e f n 1 - f - e + 2 e f m - n d e = P ( f ) C B n , m ( f , f + E - 2 E f )
B m , n a , b = 1 b - a ∫ a b x n ( 1 - x ) m - n dx
An event/hypothesis H is a set of frequencies. We compute the prior p(H) and posterior P(H) of H, by summing/integrating over the frequencies in H. Following the Bayesian framework, p(H) reflects our prior knowledge about the hypothesis H and P(H) our belief in the hypothesis after we have observed the data/evidence.
If {H 1 , ..., H k } is a disjoint and complete set of hypotheses, then ∑ i P ( H i ) =1 and we assume the hypothesis with the highest posterior to hold. Alternatively, given a confidence threshold α, we can find a set of hypotheses, which are satisfied with confidence at least 1 - α. A particular choice for this set orders the hypotheses in decreasing order of their posterior and then selects the smallest l so that the total posterior weight of the top l hypotheses is at least 1 - α.
A particular example of the above framework is obtained by taking the prior to be concentrated on 0, ½, and 1 with densities p 0 , p 1/2 , and p 1 , i.e.
p f = p 0 ⋅δ f + p 1 / 2 ⋅δ f - 1 / 2 + p 1 ⋅δ f - 1
Consider the hypothesis H fair = {½} and H fake = {0,1}. Interpreting the random experiment as a sequence of m coin flips of which n have come up "Heads", the posterior P(H fair ) reflects our belief that the coin we are observing is fair.
In general, a random experiment can consist of k independent pieces of data/evidence (n 1 , m 1 ), ..., (n k , m k ). A hypothesis H in this setting is a set of sequences of frequencies. For example, for a given frequency f the hypothesis H f = {f = f 1 = ... = f k } consists of all sequences which have all of their components equal to f. Since hypotheses are sets, we can combine them with the usual operations permitted on sets, i.e. union, set, and intersection. Thus, to continue our example, we can form the hypothesis H eq consisting of all sequences with all components being equal, regardless of their common value, as the union ∪ f H f . We define the prior probability p(f 1 , ..., f k ) of a sequence of frequencies (f 1 , ..., f k ) to be the product p(f 1 )...p(f k ) of the priors of its components. Since the pieces of evidence are independent, we obtain the posterior probability P(f 1 , ..., f k ) of the sequence to be the product P(f 1 )...P(f k ) of the posteriors of its components. The prior/posterior of a hypothesis H is formed by summing the priors/posteriors of its elements.
Genotyping a diploid organism
In sequencing data, each position observed by the sequencing instrument acquires an independent piece of evidence regarding the frequency of the allele at that position. Diploidity means that for each position there are three possible zigosity types: both homozygous and equal to the reference genome (two alleles equal to the reference), heterozygous (exactly one allele equal to the reference), or both homozygous and different from the reference genome (two alleles different from the reference). The reference genome in this case refers to the particular genome to which the reads of the sequencing experiment have been aligned. These three possibilities correspond naturally to three possible frequencies 0, ½, and 1 for the random experiment discussed in the general section on the Bayesian framework, where the frequency of a bit being "1" captures the frequency of the non-reference/variant allele.
In a given sample, the data at a particular position consists of the total number m of reads mapping at that position and the number n of those reads confirming the variant allele. We interpret the data (n, m) as the evidence for the zygosity type of the particular position. Following the Bayesian framework, we compute the posteriors P 0 , P ½ , and P 1 for the three possible zygosity types and choose the type with highest posterior to be the type of the position. The upper bound E on the error in reading the nucleotide allele corresponds to the probability of a non-systematic/random sequencing error. Letting Q to be the average of the Phred scores provided by the sequencing instrument for all variant nucleotides mapping at the position, we set E to be 10-Q/10.
Detection of variants in diploid genomes
To detect the presence of a variant at a particular position, we form two hypotheses one for the position being a variant (heterozygous or homozygous) and one for the position being a non-variant (homozygous for the reference), and consider their corresponding posteriors P var and P ref .
Comparing variants across samples from diploid organisms
The discussion so far has focused on the reads obtained from the sequencing of a single sample. In the cancer sequencing experiment discussed in this paper, we have two types of samples for each patient: one from the cancer tissue and one from a normal tissue. The goal is to set the variants obtained from the cancer tissue against the background provided by the normal and obtain the somatic variants which are present in tumor tissue, but do not appear in the normal. Thus, the evidence for the lineage type, i.e. germline vs. somatic, of the variant at a particular position in the genome consists of the two independent pieces of evidence for the zygosity type of that position in the tumor and the normal tissue. Since the data for the zygosity type is simply the total counts, then the evidence for the lineage type is (n t , m t ) and (n n , m n ). Using this data one can assign the posterior probability to the hypothesis that the variant is somatic to be
P s o m = P t , v a r × P n , r e f + P t , r e f × P n , v a r
and then set the non-somatic posterior to P nsom = 1 - P som .
Detection of variants in non-diploid genomes
An objection to the approach outlined so far is that although the cancer genome descends from the diploid human genome, the zigosity of a particular allele can be distorted considerably by the oncogenic process, to the point where the diplodity of that allele is dubious. For example, it is known that wildly varying copy numbers are an important characteristic of many cancer genomes. Furthermore, impure samples containing several types of genomes, e.g. mixture of tumor and normal cells, can produce non-diploid frequencies. To account for this, one has to abandon the notion that alleles come in only three possibly frequencies, and allow for more possibilities in the priors and posteriors. Further applications of sequencing experiments, e.g. sequencing cellular transcriptomes or experiments characterizing metagenomic samples, point to the necessity of a wider vocabulary of allele frequencies.
To simplify our exposition, and since the resolution of current sequencing technologies is rather limited, we decided to consider as a possibility the set {0, 0.01, 0.02, 0.03, ..., 1} of frequencies with resolution 0.01. Under this setting for a given position in the sequenced genome we have a hypothesis for every possible frequency f of the non-reference allele with is corresponding posterior P f obtained from the data (n, m) for that position. Given the posteriors probability of every possible frequency, one can find the most likely and the expected frequency. Furthermore, given a confidence threshold α, we can obtain a credible interval such that the posterior weight of the frequencies outside of this interval is at most α.
The decision whether a variant allele is present is based on selecting a frequency cutoff f and confidence threshold α < 0.5 and then calling present the variants for which the event "frequency at least f" has posterior confidence at least 1 - α. The frequency cutoff f can be chosen to be equal to 1%, i.e. variant is present, if it has non-zero frequency, or selected according to a set of validated variants, for example from a paired SNP array analysis.
Comparing variants across samples from non-diploid genomes
The setting can be extended to the comparison of two samples in the following way. For every two frequencies f 1 and f 2 we can obtain their posteriors P 1 , f 1 and P 2 , f 2 according to each of the samples. Then assuming that the samples are independent we can assign the posterior of the pair P f 1 , f 2 to be P 1 , f 1 × P 2 , f 2 . Next, for every difference Δ we can form the posterior probability P Δ that the difference f 1 - f 2 is equal to Δ, namely
P Δ = ∑ f 1 - f 2 = Δ P f 1 , f 2
Finally, we can obtain the most likely and the expected frequency difference, as well as a credible interval for confidence level 1 - α. One can use those posterior measurements to focus on a subset of the alleles, e.g. somatic alleles, which differentiate the two samples.
Similar to the case of detection of variants, the decision whether a variant allele has different frequency in the two samples is based on selecting a frequency difference cutoff d and confidence threshold α < 0.5. If one is interested only in variants in which the allele frequency has increased, for example somatic point mutations, then one can focus only on those variants for which the event "frequency difference at least d" has posterior confidence at least 1 - α. If in addition the goal is to obtain locations with a loss of allele, e.g. as part of obtaining the loci of a loss of heterozygosity, then the event is "frequency difference at least d in absolute value". The frequency cutoff d can be chosen to be equal to 1% or adjusted to an existing set of validated variant positions.
Construction of priors
The prior distribution is an essential component of the Bayesian framework. Generally speaking, the prior captures our belief in a particular hypothesis before we have observed the data. From a practical point of view, when many repetitions of a random experiment are available our belief should coincide with the frequency of occurrence of the hypothesis.
For the application of this framework to sequencing data of diploid organisms, a prior should capture our knowledge about the relative prevalence of the three zygosity types in the genome of the organism. Ignoring our knowledge in that respect, we can set the prior distribution to be uniform over the three types. Concerning a sequencing experiment focused on the coding part of the human genome, a better-informed prior takes into consideration that this part of the human contains around 107 nucleotides, and that the average number of variants (heterozygous and homozygous and different from the reference) with respect to a reference genome is around 104 [21]. Furthermore, considering that the reference genome is given as haploid, whereas the data is obtained from the diploid cancer/normal samples one can show that the number of heterozygous variants is expected to be twice as many as the homozygous ones. This holds because fixing the order of the two homologues of the human genome, a heterozygous variant can be located on either one, whereas for the homozygous variant there is a single choice. This leads to a prior in which the proportion of the three zygosity types is roughly 103: 2: 1.
An empirical prior can be obtained if we acknowledge that we have two samples from a patient - from tumor and normal tissue. Hence one can estimate the proportion of the three zygosity types from one of the samples and use this estimate to form the prior for the other. In addition to this, an empirical prior can be obtained form previous sequencing experiment using the same sequencing technology. This approach is applicable also to the more general frequency setting.
More precisely, to obtain an empirical prior we use that a sequencing experiment consists of multiple random experiments - one for each location of the genome. Thus an empirical prior based on a particular sequencing experiment should reflect the allele distribution observed in the genome by that experiment. Using that in the Bayesian framework the prior and the likelihood determine the distribution of the data as a certain marginal, the empirical prior derived from a particular experiment should predict the distribution of the data as observed in that experiment. In particular we must have p e f =∫ P e f | D P e ( D ) dD where p e f is the empirical prior, P e f | D the posterior based on that prior, and P e ( D ) is the empirically observed distribution of the data. Thus we establish the following iterative procedure for constructing an empirical prior. Let p 0 be the prior before the experiment. In the case of lack of prior knowledge we can set p 0 to be the uniform distribution. Then on iteration i=0,1,… we set
p i + 1 f =∫ P i f | D P e ( D ) dD= 1 N ∑ j = 1 N P i f | D j
where summation is over all pieces of data and P i f | D j is the posterior probability of frequency f based on prior p i for the j- th piece of data. The empirical prior p e is a stationary point of this process. In practice we terminate the process as soon as there is no substantial change in the prior. For the data obtained from the sequencing the human exome and using frequencies of resolution 1% we used around 10 iterations at which point the Kullback-Leibler divergence between the consecutive priors was 0.0013 bits (see Figure 2).
In the case of the human genome the empirical priors obtained in this way were similar to the theoretical prior outlined above and exhibited the expected modes at 0.5 and 1 (variant alleles), and 0 (reference alleles). Furthermore, the frequency distribution in the vicinity of the 0.5 mode was observed to be fitted well by a beta distribution. This empirical observation is consistent with the classically established prescription for choosing the beta distribution as a conjugate prior to a binomial likelihood. Furthermore, the beta distribution was justified because we observed that the depths follow a negative binomial distribution, and hence the observed allele frequencies are a ratio of such distributions, which is approximated well by a beta distribution. As far as the observed negative binomial distribution of the depths, our conjecture is that this is related to the fact that locally the depth distribution is Poisson, but that the means of these Poissons vary widely due to the heterogeneity in the nucleotide content of the human genome and are long tailed over the whole genome. Hence globally the depth distribution is a convolution of Poisson distributions with a long tailed distribution of the means, which is the nature of the negative binomial distribution. The variance of the beta distribution fitting the distribution of the heterozygous alleles depends on the ability of the sequencing technology to detect the two alleles at a heterozygous locus: the more uncorrelated the observed frequencies of the two alleles the wider the variance.
Choosing confidence cutoffs
Classically, the Bayesian framework prescribes the choice between two competing hypotheses to the one with higher posterior weight. In our case we follow this prescription with the addition that we do not make a decision in the case that the posterior weight of the more likely hypothesis is not high enough. Our decision is justified by the observation that, assuming independent random experiments, maximizing the posterior when deciding on a hypothesis in each will maximize the posterior over all choices because in this case the posterior over all choices is equal to the product of the individual choices. This is not necessarily true if the data is not independent and in this case a more sophisticated analysis is necessary. Furthermore, restricting our attention only to the experiments for which the most likely hypothesis has a high enough posterior guarantees a high enough overall posterior. We answer the question of how high is high enough by noting that if for each experiment the most likely hypothesis has a posterior confidence at least 1 - α and we have N experiments, then the overall posterior is at least ( 1 - α ) N ≈1-Nα. Thus to reach a high enough overall posterior confidence, a reasonable posterior confidence for each experiment can be obtained by taking α proportional to the inverse of the number of experiments in a manner similar to the Bonferroni correction in the frequentist setting.
Since for current estimates the human exome contains around 107 nucleotides, in the case of sequencing experiments related to that part of the human genome, when deciding a posteriori between two competing hypothesis, e.g. present/absent or germline/somatic, we can to take α = 10-6 for each genomic location.
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We would like to thank Joseph Chan, Riccardo dalla Favera, Oliver Elliott, Giulia Fabbri, and Hossein Khiabanian, for helpful discussions, comments, and help in the preparation of this manuscript.
The publication of this article was funded by the Northeast Biodefence Center (U54-AI057158), the National Institute of Health (U54 CA121852-05), and the National Library of Medicine (1R01LM010140-01).
Department of Biomedical Informatics, Columbia University, New York, NY, 10032, USA
Vladimir Trifonov & Raul Rabadan
Department of Systems Biology, Columbia University, New York, NY, 10032, USA
Center for Computational Biology and Bioinformatics, Columbia University, New York, NY, 10032, USA
Institute for Cancer Genetics and Herbert Irving Comprehensive Cancer Center, Columbia University College of Physicians and Surgeons, New York, NY, USA
Laura Pasqualucci
Department of Pathology and Cell Biology, Columbia University, New York, NY, 10032, USA
Institute of Hematology, University of Perugia, Perugia, Italy
Enrico Tiacci & Brunangelo Falini
Vladimir Trifonov
Enrico Tiacci
Brunangelo Falini
Raul Rabadan
Correspondence to Vladimir Trifonov or Raul Rabadan.
VT and RR designed the experiment, developed the method, analyzed the experimental data, and wrote the manuscript. LL designed the experiment and participated in the development of the method. BF and ET conceived the experimental study, designed the experiment, and provided samples for it. All authors read and approved the manuscript.
Trifonov, V., Pasqualucci, L., Tiacci, E. et al. SAVI: a statistical algorithm for variant frequency identification. BMC Syst Biol 7 (Suppl 2), S2 (2013). https://doi.org/10.1186/1752-0509-7-S2-S2
Posterior Distribution
Hairy Cell Leukemia
Sequencing Experiment
Random Experiment


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text:
"the fire / that burns the longest": remembering Alan Horvath
By Ingrid Swanberg
Alan Horvath, cover for (Scheduled) Random Sightings by d.a.levy (Kirpan Press)
Though we never met in person, Alan was my good friend.
I have a big cardboard file box filled with our correspondence, which began in early 1999 when Alan contacted the d.a.levy homepage I co-curate with Karl Young, asking for permission to reprint some of the levy paintings in my possession in a forthcoming Kirpan Press publication of levy's work. I had not yet connected with the larger group of people concerned with d.a.levy in Cleveland, and so did not know of Alan, who had been actively publishing poetry there from 1974 until he moved to San Francisco in 1981.
I tended to keep the envelopes. This was not only for the postmarks, as Alan did not usually date his letters, but also because they were creative and playful expressions in themselves. They often came hand-stamped with Chinese characters for such words as 'wisdom,' 'grace,' 'forever', with crossed kirpan daggers, and/or with Native American petro-glyphs from the Columbia River Gorge where Alan liked to go. They also came decorated (sometimes abundantly) with an array of different brightly-colored stickers: an Impressionist painting, a flower, a woman doing yoga, a description of "Peruvian Organic" coffee, "Cleveland Bridges" (the label for the floppy disc included with the Kirpan Press book Surfacing), a football player, American flags, butterflies, the unasked-for 'gift' sticker from the National Parks & Conservation Association soliciting contributions. . . These sticker collages-collations are somewhat in keeping with the Cleveland small press tradition of using everyday materials at hand — from Burger King wrappers to city maps — to make art. The envelopes are done in a way that communicated Alan's having taken time and care and deliberation, qualities he brought to all of his work. It was the same with the boxes that yielded the wonderful Kirpan Press editions. Even the plain boxes were remarkable; so thoroughly were they sealed with clear tape that opening them was a challenge. Again, this was a communication—about valuable contents Alan and Kathy had poured hours into making, about what can happen in the mails, about how things are done right.
Our correspondence was mainly focused on our shared interest in levy. Alan's archival work on and textual editing of levy, which I had the privilege of helping with—at times even collaborating on, is unequaled among levy's collectors and editors. When we first connected, Alan's Random Sightings project of reprinting the body of levy's work —from obscure hand-written manuscripts to often-published (and then often with errors) well-known poems—was just getting underway. He'd first contacted Karl Young about the levy paintings appearing on the levy homepage, and Karl had forwarded his message to me:
beginning:
I printed a couple dozen handmade chapbooks/magazines/etc in cleveland, ohio in the mid-to-late 70s. in fact the mimeo I used was a gift from rjs & was the one used to print levy's ukanhavyrf**kinciti bak. besides reprinting some of levy's work, I printed two collections of rare levy which hadn't been distributed very much in the cleveland area: barking rabbit (t.l. kryss silkscreen cover) and red cat of reason (barking too).
after the 80s, I'm beginning to publish again. I am planning a series of four very small press chapbooks (65 copies/per) of rare levy material. some of the reprints will include "the box lunch travelog of freemont gulch," "5 cleveland prints," "the in group" play and a collection of poems which were printed in small press publications that were never reprinted in citi or any of the collections.
recently I've gained access to the internet at my work & was really surprised to see so many links for d.a. I think this is a good sign.
I believe it was Alan's goal to preserve the breadth and depth of levy's work in accessible and readable editions, reprinting everything he could get his hands on so as to offer the most rounded sense of the man and his work possible. He approached this project with tremendous energy, tenacity and resourcefulness, guided by a love for what he might have called the honest poem. Alan had a keen distrust of literary and political facade. He was interested in poetry as direct communication, free from the interference of what is said about the poem or what is said about the poet. As he wrote in a letter to Russell Salamon (which he copied for me):
. . .the goal is to let levy speak for himself. much of the content in the Random Sightings series has never seen the light of day or has never been reprinted for 45 years.
levy seems to be in fashion again. A lot of people are interested in reading someone else's interpretation of levy instead of f**king reading about what levy had to say for himself. that should be the starting point. ground zero. . . . one thing I know: after excavating all my contacts for missing levy pieces for 40 years, there will never be The Complete Collected Works of d.a.levy. . . . It is the missing pieces that tell me that my job is not finished . . .
(undated, Autumn 2007)
Alan's work in 'letting levy speak for himself' is nothing less than stupendous. He reprinted levy's early lyrical poems that he found in obscure and forgotten little magazines from the 60s, poems that fly in the face of the stereotypes and myth-making that continue to surround levy. He reprinted levy's unpublished work, his early books, his picture-poems, his "destructive writing," his Concrete poetry, his collages (in color!), his hand-written manuscripts, his paintings, prints, drawings, and other artwork. He published levy's major poetry. He gathered and published levy's correspondence. With the collaboration of Kent Taylor, he compiled and published an exhaustive two-volume levy bibliography. He published everything of levy's he could find in lovely hand-assembled editions, with the intention of getting them into the hands of those who ought to hear "what levy had to say for himself." The Kirpan books were generally issued in editions of from 30 to 130 copies; most of the levy editions were done in 65 to 70 copies. Such work is demanding—of energy, time, patience—and it can be thankless. But Alan was not doing it for personal gain or recognition. And he was doing it as levy did (though of course by 1999 the printing methods were different)—largely by hand, largely given away. As he wrote in our initial exchange,
. . . these are not museum books, but meant to be seen by people who appreciate it. you price it too high, the wrong people read it. you make it too slick or too big & you send the wrong message. levy never made any money on his art/poetry. why should anyone else?
I once sent Alan a check for one of the Kirpan Press editions he'd sent me; he returned it to me cut into a snowflake.
Alan's publication of the nine working mss drafts of levy's long poem Cleveland undercovers coincided with my writing at length about that poem in my dissertation (in which levy is a central figure). Perhaps because that particular Kirpan edition was so helpful to me, I have often wondered if that may have been a motivating factor in its timing. That certainly would have fit Alan's generous character. His support throughout the dissertation ordeal was unflagging. During that time he presented me with one of a Kirpan edition of two copies, (Specific) Random Sightings: All the News that Fits d.a.levy, a collection of newspaper clippings about levy from 1965 to 1999.
Once, during the writing of the dissertation, when I'd asked him for information about the Old Viaduct, which looms in Cleveland undercovers, he shared a story (in a letter I cannot now find) about playing touch football with some of his friends on that bridge one afternoon or evening. 'It was a miracle no one fell off,' I think he wrote.
Throughout our collaborative work on levy, perhaps because it ran the risk of becoming obsessive, we were on guard against taking ourselves too seriously and/or making levy into a saint. One time, when I had to disassemble a rare levy pamphlet from my collection in order to photocopy it for Alan, I sent him the "original" binding staples along with the photocopies, as if to say, 'Here are the staples, the ones actually stapled by the great poet himself.' He responded in kind, sending me a large envelope filled with multi-colored paper shredding, labeled "binding from (Completely Covered) Random Sightings." Holy confetti!
He also sent me pages of printed-out listings of levy publications he had found being sold on online venues at exorbitant prices, including my own levy edition that was still in print and available from my press at the original price of $27.50, being offered online for as much as $100.
But if you really want to discover Alan's sense of humor, read his poems. See dirty dishes" and "the secret" (Surfacing). Alan's poetry is direct, spare, personal, ironic, often plain funny, at times stunning. He had the honesty and willingness to confront himself and the pain of life. He wrote in the spirit of the proactive, fearless Sikh soldier-saints whose kirpan daggers are worn as physical instruments of non-violence and defense of the vulnerable and disenfranchised. Symbolically, the kirpan means the power of truth to cut through lies.
The Random Sightings editions were well-received, with positive reviews in American Book Review, Small Press Review, Rattlesnake, and the online Big Bridge. They early on gained Alan the attention of other small press publishers such as Richard Hansen of 24th street irregular press and the well-known avant-garde poet and micropublisher jwcurry. They sold to libraries, bookstores and collectors. But many more were simply given away to those whom Alan thought would appreciate them.
Alan did not, in deference to levy's wishes, copyright the publications: ". . . I have never nor will ever file a copyright status claim to the library of congress for any of d.a.'s work" (February 21, 2001). However, he was impeccable about crediting people's work and had little patience for publishers who omitted attribution (in our first exchange he pointed out my mistaken statement, in Zen Concrete & Etc., that levy's "Last Dynasty Notes" were previously unpublished—actually, these visual poems were included in bill bissett's earlier blewointment press edition, Zen Concrete). Alan was also absolutely conscientious in handling copyrighted work. The annotated credits pages appearing at the end of the Kirpan editions are wonderfully detailed and complete, and many of them make for fascinating reading in their own right. Several of my Kirpan books are signed by the authors on the credits pages. A miniscule, handwritten number on the lower right corner of the credits page gives which number of the edition one has in hand.
Throughout our correspondence, Alan stressed the great importance of James Lowell to levy and to the literary scene in Cleveland, and to the whole country, for that matter. As he wrote in response to my description of the "levy fest" I attended in Cleveland in 2005, a year and ten months after Jim died,
It's too bad they didn't squeeze a bit more of jim lowell into the levy fest. Particularly when discussing the 60's mimeo revolution. Would there have been a Cleveland scene without jim lowell? Would levy have been exposed to as many people if he didn't have an outlet for his work? Would levy have found out about what was going on outside of Cleveland (particularly the Canadian concrete-ists) without Lowell making the effort to stock their publications in the Asphodel? Like the chicken & the egg. They just go together.
(November 21, 2005)
Elsewhere he wrote that an important aspect of Lowell's contribution was that he paid up-front for levy's and others' publications to be sold at the Asphodel—thus they could count on immediate payment for their work. This is quite in contrast to the sort of consignment arrangement usually offered to small press publishers by local bookstores, in which one receives payment only after a book has sold, months after, more often than not, and one has to keep track of sales oneself, an arrangement very often more trouble than it's worth.
Alan published three volumes of Jim Lowell's correspondence with Kent Taylor and Tom Kryss, — letters about poetry, literature, books, politics, government corruption, baseball, football, readings, poets, writers, family, gardening, mutual friends, Ohio snow storms and heat waves. . . The facsimile pages of handwritten and typed letters are interspersed with art by Taylor and Kryss, photos of all of them, a portrait of Jim by Hilary Krzywkowski, and a moving photo of Jim's empty chair at his desk after he passed by Cindy Marx. . . In this correspondence we are able to gain a glimpse of the deep camaraderie of the Cleveland 'brotherhood' of poets and artists (one which by no means excludes sisters) that first gathered in the 60s around levy and Lowell and continues. Alan found Lowell's lifelong dedication to literature an inspiration.
even as he was dying,
jim lowell typed
a few pages
for the next
asphodel book shop
catalogue.
Alan also published a collection of levy's letters to various poets and friends—a beautiful edition of multi-colored pages, including letters presented both in facsimile and retyped, details of levy paintings, a flyer for a fund-raising benefit for levy and Lowell, a photo of levy and friends from The Tibetan Stroboscope, and other ephemera from the time. Like all the Kirpan editions, it is the sort of book that had to be assembled by hand. The pages are of different weights, colors and finishes—a facsimile "P.S." page scrawled on a legal pad sheet is reproduced legal size, folded, and set into the spiral binding sideways, preserving its readability, as the handwriting would have been too small reduced.
Alan was as thoroughgoing an editor as I have ever known, from his encouragement of writers to his meticulous care in the presentation of their work. When it came to reprinting problematic levy material, we both tended to prefer to err on the side of inclusion. I consulted Alan when I was about to present a previously unpublished levy poem in a forthcoming issue of Abraxas. The mss I was working from had a number of hand-written indications of lines to be cut (who had indicated the cuts was unclear). He advised me to leave the lines in, and to simply indicate with brackets where the markings occurred. This was typical of his open approach as a textual editor, sharing all the information possible while upholding the highest standards of presentation.
The physical book itself was of central importance to Alan, in the sense of the pleasure taken in the paper, printing, and compilation, but also in the sense of a recognition of production limitations. His books were hand-assembled once enough pages had been printed and when the time was right, and thus there were natural constraints governing a book's editing and design that offered a positive organizing principle now largely lost in the economies of internet publishing.
Alan wrote in a letter of October 21, 2001:
. . . tom has been sending packets of poems. . . they seem to me to be an explosion of ideas which he has to toss to the wind. a lot more personal than the batches he sent me a while ago which I've assembled. forget when I laid that project down (months ago), but I have an impetus to complete it soon. glad I did take a vacation away from it. I needed to whittle down a few of the poems to both sharpen the book as well as thin it out so I could staple the book together. I think I have finally gotten a new perspective on it. as soon as he reviews it one more time, I think I can pump it out with only a minimal amount of effort (easier said than done). after that, the kent taylor book. & after that, more levy.
Many Kirpan books, like the book of levy correspondence or the 2004 edition of the anthology Measured Steps, present pages of many different colors, sometimes dedicating a set of pages of one color to a poet or a particular work, sometimes not. In every case, the effect is one of total readability.
Alan's books are tactile and personal. Each is unique in the usage of paper and artwork. Some have exquisite, bright, translucent, fine-paper fly leaves, as does Alan's A Series of Sharp Points, which gathers a variety of different colored pages in the body of the text, and Alan's and Dave Pishnery's Surfacing, in which white paper is primarily used for the text. The paper stock is very much a part of Alan's book design. For instance, his book Until the Last Light Goes Out presents a long narrative and meditative (even brooding) poem about a solo camping trip. The poem is printed on a rich, heavy, dark grey-green stock. Here are the final lines of the poem:
I stare awake the coals
until the tiny embers have gone black
& then leave whatever is left
as a memory,
because that is the fire
that burns the longest
The cover page is a lighter grey flecked with white. Following it is a color collage by Alan's friend Richard Werner. The end pages, of different colored stock, offer photographs, including one of Joan and her son, Joey, whom we encounter in the poem, and a candid portrait of Alan by Kathy Horvath. The cover displays one of Alan's digital pastel landscape 'paintings' (at least that's what I imagine them to be).
Office Copy, a collection of poems about Alan's years of work for a large corporation, is also printed on grey stock throughout the poetic text, but it is a softer, textured grey of company fog, subterfuge, tedium.
deaf, dumb & blind
those of you
who cannot believe
that poetry exists in
an office environment
have never heard
the howl of a trapped dog
moments before he chews off
his own leg
Alan's 2006 collection, Learning to Say Goodbye, is, aside from the cover page and credits page, printed on starkly bright, white paper throughout, in keeping with the poems' straightforward seriousness and candor. Rereading this book the other day, I came across one poem with a handwritten note to me from Alan in the lower right margins:
Just when I thought that I never wrote a poem for you, I remembered this one—written in 2002 around the time of levy's 60th birthday—as if levy was talking to everybody as well as you.
Alan is referring to a poetry reading, conference and celebration commemorating levy's 60th birthday that I had attended in Cleveland in 2002. Alan's health problems had prevented his attending. Here's the poem:
one final request
now that I'm dead
I want you to forget
don't think about
the day I died
or how I died
it won't help you
every minute
which we shared
is projected
as wonderful white
clouds filling a
brilliant summer sky
but after this moment
it is time for you to move on
Though he makes clear in his note to me his intention that it is levy speaking here, this poem is the last in the book, and follows several others in which Alan directly contemplates his own mortality. I feel that Alan and levy communicate to the reader as brothers in this poem.
I think that, for Alan, part of letting the poet 'speak for himself,' was a feeling that a poem occupies its own moment, stands by itself, and speaks itself. I think this is the reason Alan printed the anthology The Clevelanders, Vol. III, which included work from eighteen contributors, without the authors' names appearing anywhere in the body of the text, but only on the final credits pages (except where a name was included as part of original art). He did something similar in Surfacing, co-authored with his friend Dave Pishnery, listing the poems by both authors in the table of contents, and then presenting them in the body of the book without designation, in an order seemingly suggested by the poems themselves. This approach helps one to appreciate the work solely on its own merits. Just read the poem, undistracted by who wrote it and by your preconceptions about them.
At the end of The Clevelanders, Vol. III, pasted onto the inside back cover, is an original pencil drawing of Alan's, a dense force field of tangled and energetic graphite lines, titled "MAKING A POINT (TOO)." The anthology is an edition of 75 copies, so he must have done 75 individual drawings! The title may be a glancing reference to his book, A Series of Sharp Points (planned as a Mostly Broken Scabs Press publication in 1978 and finally published in 2003). The drawing itself, the original, unique artwork itself, done in pencil point, communicates with a strong immediacy the care, energy, and delight Alan put into making his books. (Since writing the foregoing, I have learned from Kathy Horvath that these "drawings" were actually pencil point "sharpenings" from the time of Alan's work as a designer twenty-three years earlier! —See Kathy's "Kirpan Press Narrative." This new information explains the title, "MAKING A POINT (TOO)," but in no way alters what the inclusion of these "drawings" in the book communicates, except perhaps to add "foresight" and "humor" to the list.) In this vein, and again, in the Cleveland small press tradition of using materials at hand, a real coffee ring appears on the title page of Alan's book of poems Office Copy.
Alan elicited a variety of original individual works for his editions. Some examples are: the original individual cover collages by Richard Werner on the edition of Lowell correspondence to Taylor and Kryss (Sleeping Nude on Top of the Sheets); the silk screen covers by Mike Schaefer (A Series of Sharp Points), Dave Pishnery (Surfacing), and Tom Kryss (Looking for d.a.levy [Random Sightings: The d.a.levy Bibliography, Volume 2] and the Lowell correspondence to Tom, Notes to Cleveland). Kryss also did individual covers for Grace Butcher's book Horses in the Snow (a combined Kirpan Press/Black Rabbit Press edition), using ink and watercolor on a stamped imprint; some rare copies present the artwork on a tipped-in paper inset.
In the recent broadside card series, "Stories from The Flats" (a collaborative Kirpan Press effort with Tom Kryss's Black Rabbit Press), there is a decided focus on these touches of original handwork and art.
The broadsides were done in editions of 75-100 copies. Unique outer envelopes were designed for each illustrated card, some produced on inkjet printers, others illustrated individually, by hand. The envelopes for my broadside, "the pure," have each been pressed with a red lipstick kiss, thematically relating to the final line of my poem (thank you, Kathy!).
Tom Kryss did original artwork on the covers and envelopes of some 20 of The Flats series, working with ink and painted or sponged watercolor on stampings. Many of these have the artwork on paper insets tipped in. One, Michael Basinki's "Ghosts Live in Closed Eyes," has a watercolor-stamped paper inset tipped in on the cover, and the envelope has ink-stamped botanical prints on canvas that has also been tipped in.
A number of The Flats series envelopes appear with the familiar signatory stampings of Chinese characters, Columbia River Gorge petro-glyphs, and crossed kirpans.
The broadsides come to readers with their envelopes sealed. One has to break the seal. One's attention is brought to this moment of opening, this moment of the poem, from which there is no going back.
. . . wonderful white
Return to Index for Alan Horvath Memorial
Return to Light and Dust Anthology


Friday, April 20th 2018, 04:00 PM Sunday, April 22nd 2018, 02:00 PM The New Testament is populated with the questions of many people, but this retreat will look at the implications of questions raised by Jesus. "What do you want me to do for you?" "Are you also going to leave me?" "You are a Teacher of Israel and you do not understand these things?" "What were you discussing along the road?" "Have you caught anything?" These questions (and so many others) are raised by Jesus and need to be answered in every generation. Rev. Simeon Gallagher is Director of the Preaching Ministry Office of the Mid-America Province of the Capuchin Order. He conducts retreats, parish development programs, and religious education workshops. To register, please contact a Retreat Captain or you may call the Retreat/Hospitality Coordinator, Susan Halligan at (504) 267-9604 or 1-866-937-9170.


text:
Effects of diet and exercise on maternal body composition and breast milk components
Melanie Joy Bopp (Creator)
Cheryl Lovelady
Abstract: "Long-chain polyunsaturated fatty acids (LCPUFA) are essential for infant growth and development, and amounts in breast milk depend on maternal diet and body stores. Because exercise increases mobilization and utilization of fatty acids, maternal activity may also influence the amount of LCPUFA in breast milk. In addition, exercise has been shown to improve bone mineral density (BMD) in postmenopausal women. During lactation, bone turnover is elevated to provide calcium for breast milk. This results in decreased BMD. Therefore, the purpose of these studies was to: 1) investigate the effects of diet and exercise on fatty acid concentrations in maternal plasma and breast milk, and 2) determine the effects of a specific resistance and aerobic training program and dietary intake on BMD and bone turnover during lactation. In the first study, LCPUFA in plasma and breast milk were measured at 12 weeks postpartum in exercising and sedentary women. Dietary intake was recorded for three days. A subsample of women participated in exercise and rest sessions to examine the acute effects of exercise on breast milk LCPUFA. In the second study, whole body, lumbar spine (LS) and hip BMD and bone mineral content (BMC), and bone turnover markers were measured before and after a 16-wk intervention in breastfeeding women who either participated in a specific resistance and aerobic training program or were sedentary. Dietary intake and body composition were also assessed in all women. In the first study, there were no differences in essential fatty acid intake between the two groups. Intake of linoleic acid (LA) was 11.05 ± 1.39 and 9.34 ± 0.97 and α-linolenic (LNA) was 0.96 ± 0.12 and 0.82 ± 0.09 grams/day for the sedentary and exercise groups, respectively. No differences were found in LCPUFA in plasma and breast milk between groups. After 30 minutes of exercise, there was a trend for an increase in LA and LNA. In the second study, exercising women lost significantly less LS BMD and LS BMC (p<0.05), and had substantial improvements in body composition. Calcium and protein intake were significant predictors of whole body BMD loss with parity being a significant covariate (p<0.05). Bone turnover markers were also significant predictors of LS BMD and LS BMC, when adjusted for parity and exercise (p<0.01. These results suggest that women consuming adequate amounts of LCPUFA can exercise moderately without decreasing the LCPUFA in their breast milk. In addition, increased calcium and protein intake lessens BMD loss during lactation. In breastfeeding women, moderate resistance and aerobic exercise was effective in improving body composition and minimizing BMD losses."--Abstract from author supplied metadata.
Long-chain polyunsaturated fatty acids (LCPUFA), infant, growth, development, breast milk, exercise, lactation, dietary intake
Breast milk--Health aspects
Breast milk--Composition
Lactation--Physiological aspects
Lactation--Nutritional aspects
Exercise for women--Physiological aspects


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