Accurate determination of DNA yield from individual mosquitoes for population genomic applications

Abstract  Accurate estimates of DNA quantity are likely to become increasingly important for successful genomic screening of insect populations via recently developed, highly multiplexed genotyping assays and high-throughput sequencing methods. Here we show that genomic DNA extractions from single Anopheles gambiae Giles using a standard commercial kit-based methodology yield extracts with concentrations below the linear range of spectrophotometric absorbance at 260 nm. Concentrations determined by spectrophotometry were not reproducible, and are therefore neither accurate nor reliable. However, DNA quantification using a fluorescent nucleic acid stain (PicoGreen^®) gave highly reproducible concentration estimates, and indicated that, on average, single mosquitoes yielded approximately 300 ng of DNA. Such a total yield is currently insufficient for many high-throughput genome screening applications, necessitating whole genome amplification of all or most individuals in a population prior to genotyping.     

Genetic markers validate using the natural phenotypic characteristics of shed feathers to identify individual northern goshawks Accipiter gentilis

The recognition of individual animals is essential for many types of ecological research, as it enables estimates of demographic parameters such as population size, survival and reproductive rates. A popular method of visually identifying individuals uses natural variations in spot, stripe or scar markings. Although several studies have assessed the accuracy of these methods in mammals, crustaceans and fish, there have been few attempts to determine whether phenotypic characteristics are accurate when used for birds. Furthermore, even less is known about whether shed or moulted body parts can be reliably used to visually identify individuals. Here we assessed the accuracy of using phenotypic characteristics to identify avian individuals using a double‐marking experiment, whereby nine microsatellite genetic markers and natural markings on shed feathers were used to independently identify northern goshawks Accipiter gentilis. Phenotypic and genetic identification of individuals was consistent in 94.4% (51/54) comparisons. Our results suggest that the phenotypic characteristics of shed feathers can be reliably used as a non‐invasive and relatively inexpensive technique to monitor populations of an elusive species, the northern goshawk, without having to physically re‐capture or re‐sight individuals. We posit that using natural markings on shed feathers will also be a reliable method of identifying individuals in avian species with similar phenotypic characteristics, such as other Accipiter species.     

Inference of individual ploidy level using codominant markers

A significant portion of plant species are polyploids, with ploidy levels sometimes varying among individuals and/or populations. Current techniques to determine the individual ploidy, e.g., flow cytometry, chromosome counting or genotyping‐by‐sequencing, are often cumbersome. Based on the genotypic probabilities for polysomic inheritance under double‐reduction, we developed a model to estimate allele frequency and infer the ploidy status of individuals from the allelic phenotypes of codominant genetic markers. The allele frequencies are estimated by an expectation‐maximization algorithm in the presence of null alleles, false alleles, negative amplifications and self‐fertilization, and the posterior probabilities are used to assign individuals into different levels of ploidy. The accuracy of this method under different conditions is evaluated. Our methods are freely available in a new software package, ploidyinfer , for use by other researchers which can be downloaded from http://github.com/huangkang1987/ploidyinfer .     

Measurement of dendritic mRNA transport using ribosomal markers

mRNA is transported to the dendritic regions by forming RNA granules, an aggregate of mRNA, ribosomal proteins, rRNA, and RNA-binding proteins such as Staufen. In this study, the dendritic transport of RNA granules was measured using the individual antibodies to ribosome-specific markers such as ribosomal L4 or S6 protein, and Y10B, a monoclonal antibody specific to rRNA. All the markers showed significant immunoreactivity in the dendritic regions of the hippocampal neurons. In addition, a GFP-tagged Staufen, a marker protein of the RNA granules, was colocalized with the Y10B and S6 signals in the dendrites. The S6 signals were also colocalized with the Y10B signals in the dendrites. Consistent with previous studies, the depolarization induced by KCl stimulation increased the ribosomal level, revealed by the S6 or Y10B immunostaining in the distal dendrites. These results demonstrate the utility of ribosomal markers for detecting the RNA granules or mRNA transport in dendrites.     

Imaging individual mRNA molecules using multiple singly labeled probes

We describe a method for imaging individual mRNA molecules in fixed cells by probing each mRNA species with 48 or more short, singly labeled oligonucleotide probes. This makes each mRNA molecule visible as a computationally identifiable fluorescent spot by fluorescence microscopy. We demonstrate simultaneous detection of three mRNA species in single cells and mRNA detection in yeast, nematodes, fruit fly wing discs, and mammalian cell lines and neurons.     

The determination of growth rates of individual colonies in agarose using high-resolution automated image analysis

This paper describes the evaluation of a colony formation assay using automated image analysis, which permits the tracking of growth at the individual colony level, such that a growth rate can be estimated for each colony followed. In principle, this will permit quantitative characterization of cellular heterogeneity in growth rate and cellular heterogeneity in response to proliferation-modifying agents. In addition, we have demonstrated the possibility of using correlative microscopy to relate growth rate to other parameters, using metabolic viability as an example. This should be useful for determining cellular characteristics associated with proliferative behavior and response to proliferation-modifying agents.     

Age determination of Antarctic krill using fluorescence and image analysis of size

Summary The ages of 252 mature female krill, collected from Prydz Bay in January 1985, were determined using length frequency analysis and the fluorescent age pigment (FAP) technique. Results of both methods suggest 6 years classes for adult krill. Correspondence between the ages determined by the two techniques is generally within one year. The animals were also analyzed by a computerized image analysis system, which recorded a large suite of size and shape parameters. The accuracy of discriminant functions constructed to relate the image analysis parameters to age approached 90% for the ages defined by length frequency, and 52% for physiological age.     

Simultaneous analysis of lymphocyte markers using triple color markers

The introduction in flow cytometry of the new reagent Streptavidin Duochrome allows the simultaneous three color analysis of biological samples. Streptavidin Duochrome is a phycoerythrin-Texas red fluorochrome complex conjugated to the protein streptavidin. This novel complex exploits the principle fluorescence energy transfer when this reagent is used with FITC, PE and biotin-conjugated monoclonal antibodies; three different emissions of fluorescence are possible simultaneously using an argon laser at 488 nm (FACScan Becton Dickinson). Using Paint-a-gate software which analyses samples and displays the three colors on a screen, antigen distribution on multiple cell populations is obtained. Single, double and triple labeled cells (up to seven combinations) are visualized and quantified quickly and easily. Using a panel of normal donors we have evaluated the following combinations: CD3/CD4/CD8 to visualize T lymphocytes and their subsets; CD3/CD19/CD16 to quantify quickly the T, B, NK populations; CD4/Leu8/Leu18 to analyse the helper subset and CD3/CD8/Leu7. Our purpose is to evaluate the importance of this new kind of analysis to study the heterogeneity of several lymphocytic populations.     

Accurate determination of vegetational change in meadows by successive point quadrat analysis

The point quadrat method can be used for determination of vegetational change in meadows of great species diversity. An appropriate sampling technique is described comprehending an apparatus of high rigidity and a shelter to keep off wind and rain. Sample size related to sampling time expense affects the number of species recorded and methodical error which is empirically determined by repetitive sampling. A setup of fixed points leads to higher accuracy than random sampling. When methodical error is quantitatively known, significant vegetational change can be detected by sampling at successive times.For fluctuation studies of plant populations in meadows the point quadrat technique should be preferred to visual estimates of plant cover because of its higher accuracy.Abbreviations f= frequency - s= standard deviation - Sr= variation coefficient - DWLS= distance weighted least squares     

Genetic analysis of cystic fibrosis using linked DNA markers.

Genetic linkage has been analyzed between cystic fibrosis (CF) and a number of markers on the long arm of chromosome 7, including D7S15, COL1A2, PON, MET, D7S8, and TCRB, using a cohort of 47 Canadian and 13 Danish CF families. The analysis confirms the previous observations that both MET and D7S8 are closely linked to CF. Based on the result from one family, MET appears to be more proximal to the centromere than CF. Our analysis also suggests that genetic heterogeneity may account for the high recombination fraction between CF and D7S8 observed in another family. In addition, a strong linkage disequilibrium has been observed between CF and the two closely flanking markers.     

基于DNA分子标记的花粉流动态分析

花粉介导的基因流是植物有性繁殖世代之间的桥梁, 花粉散布属性是植物繁殖生态学、保护生物学和进化生物学研究关注的焦点。随着DNA分子技术的发展, 花粉流分析所使用的分子标记(尤其是微卫星标记)逐步替代了早期物理标记, 基于最大似然法估计以及新兴的基于贝叶斯推断的父本指派算法的发展, 能有效地估计花粉流散布的方向、距离和强度等重要特征。花粉散布曲线由单一参数向多参数模型发展, 以更好地获得花粉散布特征的拟合效果, 双组分的复合模型利用相互独立的参数空间使得散布曲线在长距离和短距离形状上呈现更大的可塑性。这些革新的技术和方法被成功应用于植物性别表型、隔离种群和杂交物种间花粉流分析, 以探讨进化、生态和保护等多领域的基础理论问题。近年来, 高通量测序技术的发展将进一步加快以分子标记为基础的花粉流动态分析在更广泛的植物类群中运用。     

A reliable method for northern blot analysis using synthetic oligonucleotide probes.

We have developed a method for using short (30-42 base pair) synthetic oligonucleotide DNA probes in Northern blot assays. The method involves labeling the probes to high specific activity, very stringent hybridization and wash conditions, and the presence of several inhibitors of nonspecific binding in the hybridization buffer. We have tested this method with several probes obtained from local and commercial sources. The results with every probe used were high signal-to-noise ratios in an exposure time range of 30 min to 7 days.     

Translation of individual species of vesicular stomatitis viral mRNA.

Vesicular stomatitis virus mRNAs from three of the four bands fractionated by polyacrylamide gel electrophoresis in 99% formamide have been eluted from gels and translated in the Krebs II ascites cell-free system. Band 2 mRNA (0.7 times 10-6 daltons) directed the synthesis of the protein moiety of the glycoprotein (G), and band 3 (0.55 times 10-6 daltons) coded for the nucleocapsid (N) protein. Band 4 mRNA (o.28 times 10-6 daltons) directed the synthesis of the NS and matrix (M) proteins. The authenticity of viral proteins synthesized in vitro was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by analysis of (35-S)metionine-labeled tryptic peptides. These results are consistent with the complexity analysis and coding capacities for the vesicular stomatitis virus mRNA species presented in the accompanying paper.     

Sample size determination for matched-pair equivalence trials using rate ratio

In this article, we compare Wald-type, logarithmic transformation, and Fieller-type statistics for the classical 2-sided equivalence testing of the rate ratio under matched-pair designs with a binary end point. These statistics can be implemented through sample-based, constrained least squares estimation and constrained maximum likelihood (CML) estimation methods. Sample size formulae based on the CML estimation method are developed. We consider formulae that control a prespecified power or confidence width. Our simulation studies show that statistics based on the CML estimation method generally outperform other statistics and methods with respect to actual type I error rate and average width of confidence intervals. Also, the corresponding sample size formulae are valid asymptotically in the sense that the exact power and actual coverage probability for the estimated sample size are generally close to their prespecified values. The methods are illustrated with a real example from a clinical laboratory study.