FamPipe: An Automatic Analysis Pipeline for Analyzing Sequencing Data in Families for Disease Studies.

In disease studies, family-based designs have become an attractive approach to analyzing next-generation sequencing (NGS) data for the identification of rare mutations enriched in families. Substantial research effort has been devoted to developing pipelines for automating sequence alignment, variant calling, and annotation. However, fewer pipelines have been designed specifically for disease studies. Most of the current analysis pipelines for family-based disease studies using NGS data focus on a specific function, such as identifying variants with Mendelian inheritance or identifying shared chromosomal regions among affected family members. Consequently, some other useful family-based analysis tools, such as imputation, linkage, and association tools, have yet to be integrated and automated. We developed FamPipe, a comprehensive analysis pipeline, which includes several family-specific analysis modules, including the identification of shared chromosomal regions among affected family members, prioritizing variants assuming a disease model, imputation of untyped variants, and linkage and association tests. We used simulation studies to compare properties of some modules implemented in FamPipe, and based on the results, we provided suggestions for the selection of modules to achieve an optimal analysis strategy. The pipeline is under the GNU GPL License and can be downloaded for free at http://fampipe.sourceforge.net.

SeqSIMLA2: simulating correlated quantitative traits accounting for shared environmental effects in user-specified pedigree structure.

Simulation tools that simulate sequence data in unrelated cases and controls or in families with quantitative traits or disease status are important for genetic studies. The simulation tools can be used to evaluate the statistical power for detecting the causal variants when planning a genetic epidemiology study, or to evaluate the statistical properties for new methods. We previously developed SeqSIMLA version 1 (SeqSIMLA1), which simulates family or case-control data with a disease or quantitative trait model. SeqSIMLA1, and several other tools that simulate quantitative traits, do not specifically model the shared environmental effects among relatives on a trait. However, shared environmental effects are commonly observed for some traits in families, such as body mass index. SeqSIMLA1 simulates a fixed three-generation family structure. However, it would be ideal to simulate prespecified pedigree structures for studies involving large pedigrees. Thus, we extended SeqSIMLA1 to create SeqSIMLA2, which can simulate correlated traits and considers the shared environmental effects. SeqSIMLA2 can also simulate prespecified large pedigree structures. There are no restrictions on the number of individuals that can be simulated in a pedigree. We used a blood pressure example to demonstrate that SeqSIMLA2 can simulate realistic correlation structures between the systolic and diastolic blood pressure among relatives. We also showed that SeqSIMLA2 can simulate large pedigrees with large chromosomal regions in a reasonable time frame.

Photoacoustic and absorption spectroscopy imaging analysis of human blood.

Photoacoustic and absorption spectroscopy imaging are safe and non-invasive molecular quantification techniques, which do not utilize ionizing radiation and allow for repeated probing of samples without them being contaminated or damaged. Here we assessed the potential of these techniques for measuring biochemical parameters. We investigated the statistical association between 31 time and frequency domain features derived from photoacoustic and absorption spectroscopy signals and 19 biochemical blood parameters. We found that photoacoustic and absorption spectroscopy imaging features are significantly correlated with 14 and 17 individual biochemical parameters, respectively. Moreover, some of the biochemical blood parameters can be accurately predicted based on photoacoustic and absorption spectroscopy imaging features by polynomial regression. In particular, the levels of uric acid and albumin can be accurately explained by a combination of photoacoustic and absorption spectroscopy imaging features (adjusted R-squared > 0.75), while creatinine levels can be accurately explained by the features of the photoacoustic system (adjusted R-squared > 0.80). We identified a number of imaging features that inform on the biochemical blood parameters and can be potentially useful in clinical diagnosis. We also demonstrated that linear and non-linear combinations of photoacoustic and absorption spectroscopy imaging features can accurately predict some of the biochemical blood parameters. These results demonstrate that photoacoustic and absorption spectroscopy imaging systems show promise for future applications in clinical practice.

A combined association test for rare variants using family and case-control data.

Statistical association tests for rare variants can be classified as the burden approach and the sequence kernel association test (SKAT) approach. The burden and SKAT approaches, originally developed for case-control analysis, have also been extended to family-based tests. In the presence of both case-control and family data for a study, joint analysis for the combined data set can increase the statistical power. We extended the Combined Association in the Presence of Linkage (CAPL) test, using both case-control and family data for testing common variants, to rare variant association analysis. The burden and SKAT algorithms were applied to the CAPL test. We used simulations to verify that the CAPL tests incorporating the burden and SKAT algorithms have correct type I error rates. Power studies suggested that both tests have adequate power to identify rare variants associated with the disease. We applied the tests to the Genetic Analysis Workshop 19 data set using the combined family and case-control data for hypertension. The analysis identified several candidate genes for hypertension.

Family-based association test using both common and rare variants and accounting for directions of effects for sequencing data.

Current family-based association tests for sequencing data were mainly developed for identifying rare variants associated with a complex disease. As the disease can be influenced by the joint effects of common and rare variants, common variants with modest effects may not be identified by the methods focusing on rare variants. Moreover, variants can have risk, neutral, or protective effects. Association tests that can effectively select groups of common and rare variants that are likely to be causal and consider the directions of effects have become important. We developed the Ordered Subset - Variable Threshold - Pedigree Disequilibrium Test (OVPDT), a combination of three algorithms, for association analysis in family sequencing data. The ordered subset algorithm is used to select a subset of common variants based on their relative risks, calculated using only parental mating types. The variable threshold algorithm is used to search for an optimal allele frequency threshold such that rare variants below the threshold are more likely to be causal. The PDT statistics from both rare and common variants selected by the two algorithms are combined as the OVPDT statistic. A permutation procedure is used in OVPDT to calculate the p-value. We used simulations to demonstrate that OVPDT has the correct type I error rates under different scenarios and compared the power of OVPDT with two other family-based association tests. The results suggested that OVPDT can have more power than the other tests if both common and rare variants have effects on the disease in a region.

Incidence and Levels of Fusarium moniliforme , Fusarium proliferatum and Fumonisins in Corn and Corn-Based Foods and Feeds1.

Fusarium moniliforme Sheldon occurs worldwide on corn intended for human and animal consumption. A closely related species Fusarium proliferatum also occurs frequently on corn. Yellow dent corn, white dent corn, white and yellow popcorn and sweetcorn may be contaminated. Both organisms are capable of producing a group of toxins known as fumonisins, of which Fumonisin B1 (FB1), Fumonisin B2 (FB2) and Fumonisin B3 (FB3) are most common. Fumonisins have been found in corn and corn-based foods worldwide. Fumonisins may be found in sound whole kernel corn at levels at or below 1.0 µg/g. By contrast animal disease problems begin to occur at fumonisin levels above 5.0 to 10.0 µg/g. Corn-based food products that have the most frequent and highest fumonisin levels, besides whole kernels, are corn meal and corn grits. In the United States, corn meal has been found contaminated with Fumonisin B1 at levels from 0.5 to 2.05 µg/g, and grits from 0.14 to 0.27 µg/g. Corn flakes, corn pops, corn chips and tortilla chips have been found negative for fumonisins. Popcorn, sweetcorn and hominy corn have been found contaminated with sporadic, low levels (0.01 to 0.08 µg/g) of fumonisins. The effects of processing on fumonisins in corn are still largely unknown. Heating may cause a loss of fumonisins in corn, but it may be a loss of detectability rather than degradation.

Simplified Method for Microscale Production and Quantification of Aflatoxin in Broth 1.

A simplified method for aflatoxin production studies is described. The mold was cultured in 4-dram (15 ml) vials containing 5 ml of yeast extract sucrose broth, and aflatoxin levels were determined by direct spotting of the broth on thin layer chromatography (TLC) plates and quantitating by spectrodensitometry. Equivalent levels of aflatoxins were produced in vials as compared to flasks. When compared to conventional TLC after solvent extraction, direct spotting was rapid, economical and statistically equivalent. Heating broth cultures (121°C, 15 s) before TLC improved the release of aflatoxin from mycelial mats. Aflatoxins were unstable in YES broth during 3 months of frozen storage.

Inhibition of Production of Cyclopiazonic Acid and Ochratoxin A by the Fungicide Iprodione ‡.

Aspergillus flavus NRRL 1290 and Aspergillus ochraceus NRRL 3174 were grown on a glucose-salts medium and yeast extract-sucrose broth containing the fungicide iprodione at concentrations of 0, 1,3,5, 10, 15, and 20 µg of active ingredient per ml of growth medium. Cultures were analyzed for cyclopiazonic acid, ochratoxin A, and mycelium production after 4,7, 10, 14, and 21 days of incubation at 25°C. Increasing concentrations of iprodione in the growth media resulted in greater reduction of cyclopiazonic acid, ochratoxin A, and mycelium production at the end of each incubation period. More than 50% reduction of cyclopiazonic acid, ochratoxin A, and mycelium production was observed when iprodione was added to growth media at a concentration of 5 µg/ml of medium. Higher concentrations of iprodione (10 to 20 µg/ml of growth medium) inhibited the production of cyclopiazonic acid and mycelium by A. flavus NRRL 1290 almost completely, but not the production of ochratoxin A and mycelium by A. ochraceus NRRL 3174.

Toxicity and Sorbate Sensitivity of Molds Isolated from Surplus Commodity Cheeses 1.

A total of 263 mold isolates were obtained from moldy surplus cheese released from government storage for distribution in the surplus commodity food distribution program in 1984. All of the molds belonged to the genus Penicillium , and consisted of four species, P. roqueforti (176), P. cyclopium (46), P. viridicatum (32) and P. crustosum (9). About 10% of the isolates were capable of producing known mycotoxins on laboratory media. The mycotoxins detected were patulin, penicillic acid and ochratoxin. Patulin was detected most often followed by penicillic acid and ochratoxin. When tested in chicken embryos, 10.1% of the isolates were toxic (causing 50% mortality or more) when grown on cheese, and 29.7% of the isolates were toxic when grown on rice. There was no correlation between having the ability to produce known mycotoxins and toxicity to chicken embryos. None of the isolates when grown on cheese contained any mutagenic activity in the Salmonella mutagenesis (Ames) test. The percentage of isolates showing a high or medium degree of resistance to sorbate were 77, 45, 3.6 and 0 at sorbate concentrations of 0.30, 0.45, 0.60 and 0.90%, respectively. There was no apparent relationship between sorbate resistance and toxigenic properties of the molds.