Assessing accuracy and precision for field and laboratory data: a perspective in ecosystem restoration

Unlike most laboratory studies, rigorous quality assurance/quality control (QA/QC) procedures may be lacking in ecosystem restoration (“ecorestoration”) projects, despite legislative mandates in the United States. This is due, in part, to ecorestoration specialists making the false assumption that some types of data (e.g. discrete variables such as species identification and abundance classes) are not subject to evaluations of data quality. Moreover, emergent behavior manifested by complex, adapting, and nonlinear organizations responsible for monitoring the success of ecorestoration projects tend to unconsciously minimize disorder, QA/QC being an activity perceived as creating disorder. We discuss similarities and differences in assessing precision and accuracy for field and laboratory data. Although the concepts for assessing precision and accuracy of ecorestoration field data are conceptually the same as laboratory data, the manner in which these data quality attributes are assessed is different. From a sample analysis perspective, a field crew is comparable to a laboratory instrument that requires regular “recalibration,” with results obtained by experts at the same plot treated as laboratory calibration standards. Unlike laboratory standards and reference materials, the “true” value for many field variables is commonly unknown. In the laboratory, specific QA/QC samples assess error for each aspect of the measurement process, whereas field revisits assess precision and accuracy of the entire data collection process following initial calibration. Rigorous QA/QC data in an ecorestoration project are essential for evaluating the success of a project, and they provide the only objective “legacy” of the dataset for potential legal challenges and future uses.     

Quantification of endogenous steroids in human urine by gas chromatography mass spectrometry using a surrogate analyte approach

Providing “real blank sample” is a problem in determination of endogenous steroids in complex matrices. A new quantification strategy is proposed in the present study, which is based on using isotope-labeled steroids instead of natural steroids for constructing calibration line. This approach is called surrogate analyte and it is shown that its accuracy is better than some of the previously described methods at low concentrations and comparable to standard addition method at medium and high concentration levels. The method was fully validated to satisfy the ICH criteria and it was applied for determination of endogenous steroids in several urine samples.     

Absolute nannofossil abundance estimates: Quantifying the pros and cons of different techniques

A quick and inexpensive method to determine absolute nannofossil abundance in deep sea sediments – the “drop” technique (modified dilution method) – was compared to two other available methods – the filtration and random settling techniques. All techniques rely on the same basic principle, under which a volume of known concentration (bulk sediment weight/mL) is distributed evenly over a known total area (glass slide or filter) to then count particles within a set of (randomly) selected fields of view. The three preparation techniques were also calibrated by spiking the samples with microbeads to approach the “real values” as closely as possible. Significant offsets in abundance estimates between methods mainly reflect bias due to the uneven distribution and/or loss of particles. We show that the drop technique is most consistent and accurate in estimating “real values” and offers similar or better reproducibility than the other techniques. The drop method also allows detection of the same trends with or without calibration with microbeads. The filtration method holds the risk to drastically underestimate absolute abundances, while the settling technique is demanding in terms of time and may suffer from advection processes. The composition of nannofossil assemblages can be reliably determined by any of the three different techniques.     

Identification and classification of cut-flower “Huangyinghua”using DNA marker techniques

rch Laboratory, Nanjing Agricultural University, Nanjing 210095, China) Abstract “Huangyinghua” is a popular cut-flower in China, but it is unclear as to whether “Huangyinghua” is an invasive Solidago canadensis or not. The genetic relationship of a total of 45 samples of “Huangyinghua” with S. canadensis, and S. decurrens were investigated using AFLP technique so as to determine the identity of “Huangyinghua”. Genomic DNA was digested with EcoRI and MseI enzymes and amplified with six E+3 and M+3 primer combinations. AFLP analysis produced 661 endonucleotide discernable bands, of which 639 (96.61%) were polymorphic. Cluster analysis through using UPGMA method indicated that “Huangyinghua” and S. canadensis were clustered into the same group that was different from S. decurrens. Sequence analysis based on the ITS regions showed that their sequences of 5.8S rDNA were the same, and the differences were found only in the ITS1 and ITS2 regions. ITS phylogenetic trees of the tested samples and closely-related species were reconstructed based on our sequence data in combination with those from GenBank. Based on the trees, “Huangyinghua” was found to belong to S. canadensis complex, but not to S. decurrens. Moreover, it was found that there were considerable genetic variation in both “Huangyinghua” and S. canadensis. Therefore, the cut-flower “Huangyinghua” may be invasive, and proper measures should be taken to control the further spread of its propagules.     

Multiple Laboratory Comparison of the Doubly Labeled Water Technique*

A double-blind study was conducted to determine between-laboratory variability in the doubly labeled water method for measurement of total energy expenditure in humans, and to compare the accuracy and precision of three widely-used procedures for calculating rates of carbon dioxide production from the original isotope data. Eighteen laboratories from five countries participated in the study. All laboratories were provided with five water standards containing varying amounts of ²H and ¹⁸O, and in addition 11 laboratories were provided with urine and dose specimens from one (six laboratories) or two (five laboratories) healthy elderly subjects of normal height and weight undergoing a calorimetric validation of the doubly labeled water method. The data from the five water standards were analyzed to predict between-laboratory variability in the doubly labeled water technique in all laboratories. In addition, data from the subjects were analyzed using the “slope-intercept”, “2-point” and “modified” methods of calculation. The results confirm that the doubly labeled water method can be an accurate technique for the measurement of energy expenditure in adult human subjects in some laboratories. However, there was substantial between-laboratory variability in the results and some laboratories returned physiologically impossible results. There was no significant effect of calculation procedure on the accuracy of the technique in this limited comparison, although the slope-intercept procedure appeared to be more susceptible to analytical error than the other procedures. The isotope standards analyzed by participants in this study will be made available to other investigators on request.     

A kinetically controlled,isothermal method for the detection of single nucleotide mismatches

We describe an isothermal, enzyme-free method to detect single nucleotide differences between oligonucleotides of close homology. The approach exploits kinetic differences in toe-hold-mediated, nucleic acid strand-displacement reactions to detect single nucleotide polymorphisms (SNPs) with essentially “digital” precision. The theoretical underpinning, experimental analyses, predictability, and accuracy of this new method are reported. We demonstrate detection of biologically relevant SNPs and single nucleotide differences in the let-7 family of microRNAs. The method is adaptable to microarray formats, as demonstrated with on-chip detection of SNP variants involved in susceptibility to the therapeutic agents abacavir, Herceptin, and simvastatin.     

Evaluating somatic tumor mutation detection without matched normal samples

Background

Observations of recurrent somatic mutations in tumors have led to identification and definition of signaling and other pathways that are important for cancer progression and therapeutic targeting. As tumor cells contain both an individual’s inherited genetic variants and somatic mutations, challenges arise in distinguishing these events in massively parallel sequencing datasets. Typically, both a tumor sample and a “normal” sample from the same individual are sequenced and compared; variants observed only in the tumor are considered to be somatic mutations. However, this approach requires two samples for each individual.

Results

We evaluate a method of detecting somatic mutations in tumor samples for which only a subset of normal samples are available. We describe tuning of the method for detection of mutations in tumors, filtering to remove inherited variants, and comparison of detected mutations to several matched tumor/normal analysis methods. Filtering steps include the use of population variation datasets to remove inherited variants as well a subset of normal samples to remove technical artifacts. We then directly compare mutation detection with tumor-only and tumor-normal approaches using the same sets of samples. Comparisons are performed using an internal targeted gene sequencing dataset (n = 3380) as well as whole exome sequencing data from The Cancer Genome Atlas project (n = 250). Tumor-only mutation detection shows similar recall (43–60%) but lesser precision (20–21%) to current matched tumor/normal approaches (recall 43–73%, precision 30–82%) when compared to a “gold-standard” tumor/normal approach. The inclusion of a small pool of normal samples improves precision, although many variants are still uniquely detected in the tumor-only analysis.

Conclusions

A detailed method for somatic mutation detection without matched normal samples enables study of larger numbers of tumor samples, as well as tumor samples for which a matched normal is not available. As sensitivity/recall is similar to tumor/normal mutation detection but precision is lower, tumor-only detection is more appropriate for classification of samples based on known mutations. Although matched tumor-normal analysis is preferred due to higher precision, we demonstrate that mutation detection without matched normal samples is possible for certain applications.

     

Rapid and simple method for the determination of tryptophan in cereal grains.

Alkaline extracts of cereal proteins are treated in the prescribed sequence: Glacial acetic acid-ferric chloride and 25.8 n H₂SO₄ solutions. Specified amounts of these solutions and the sample are added in order to obtain the required sulfuric acid concentration in the reaction mixture. The reaction mixture is heated at 60°C for 45 min (cereal proteins) or 110 min (standard tryptophan). The absorbance of the resulting violet solution is read at 545 nm. Results are highly precise and agree quite favorably with those of the modified Spies and Chambers method using Pronase hydrolyzates. As little as 6–7 μg of tryptophan in cereal proteins may be determined reliably in samples with negligible blank corrections.The factors affecting color development and the accuracy and precision of the method are discussed.     

Taking the stress out of blood collection: comparison of field blood‐sampling techniques for analysis of baseline corticosterone

Many ecological studies use stress hormones to assess the condition, health or disturbance levels of wild organisms. Common blood sampling protocols for this research involve trapping individuals and taking blood within three minutes to obtain a “baseline” for analysis of stress hormones (“conventional method”). In some situations it may be difficult to get an accurate measure of baseline values; therefore, alternative sampling techniques may be preferable. We compared corticosterone levels in samples taken via a newly developed, minimally invasive blood sampling technique with corticosterone levels in blood taken via the conventional method. We collected samples from incubating adult common terns Sterna hirundo via blood sucking bugs (Heteroptera, Triatominae) contained in “dummy eggs” (“bug method”) and compared measured corticosterone concentrations to concentrations in blood taken from the same birds using the conventional method. We found no significant differences in mean or variance of baseline corticosterone levels between samples collected via the different methods. This suggests that the bug method offers a viable alternative for hormone sampling.     

On the origin of the genetic code and the stability of the translation apparatus

An “error theory” is developed which can be applied to determine the stability of a macromolecular translation machinery which reproduces itself. It is shown that the overall effects of a multitude of possible error versions of macromolecules can be treated statistically, and that such a statistical approach is of considerable usefulness in the theoretical treatment of complex macromolecular systems. The theory is developed within the context of a detailed treatment of the “frozen accident” hypothesis for the origin of the genetic code. A model is described which permits some thermodynamic characterization of the components involved in the code nucleation. The model also proves useful in resolving a stability “paradox” described by Orgel, which relates to the translation stability in present-day organisms and mechanisms of ageing. It indicates that any experimentally found decrease in translation accuracy with age is probably not due to an inherent instability in the translation apparatus. Relevant experiments are suggested.     

一种测定蛹虫草中虫草酸含量的酶标仪微量反应方法

研究和建立一种基于酶标仪-96孔板高通量测定虫草酸含量的检测方法,并对该方法进行性能评价。以酶标仪为检测仪器,在96孔板内按照设定反应条件微量加入样品和试剂进行显色反应,利用酶标仪测定吸光度值并计算虫草酸含量。通过检测精密度、重复性、回收率,并与分光光度计法进行比较,综合评价该方法的准确度、精确度。结果表明,测定数据具有较高的精密度(样品CM1的RSD值0.829%;样品CM2的RSD值1.772%)、重复性(标准样品B40的RSD值2.061%;样品CM2的RSD值1.599%)、回收率(平均回收率99.24%,RSD值3.666%),测定结果与分光光度法检测结果无显著差异(P>0.05)。结果表明,酶标仪微量法测定准确、重复性好,并可大大减少样品和试剂的用量,该方法方便、快捷、高效,可以替代分光光度法用于虫草酸含量的测定。     

The scanning behavior of juncos: A game-theoretical approach

Flocking birds frequently look up or “scan” while they are feeding on the ground. High scanning rates increase the probability that the birds in a flock will see an approaching predator in time to avoid predation; however, high scanning rates also decrease the feeding rates of the scanning individuals. Since the scanning rate that maximizes the survival probability of one individual depends on how frequently other birds in the same flock are scanning, the optimal scanning behavior must be modeled as a game. We develop a realistic model of scanning behavior and use it to find two game theoretical solutions—the “co-operative” or Pareto optimum and the “selfish” or Nash optimum. The observed scanning rates do not differ significantly from the co-operative optimal scanning rate. We argue that in a game where players meet again and again such apparent co-operation may be an evolutionarily stable strategy.     

Trace element status of hemodialyzed patients

The trace elements Ba, Bi, Cd, Co, Cs, Cu, Hg, La, Mn, Mo, Pb, Rb, Sb, Sn, Sr, Tl, and Zn were determined by inductively coupled plasma mass spectrometry in plasma samples of 68 hemodialysis patients. The same elements (with exception of La and Mn) were also determined in whole blood after mineralization with high-purity nitric acid/hydrogen peroxide in a closed-pressurized microwave system. The accuracy and precision was checked by analyzing two Seronorm “whole blood” reference materials. All samples were contaminated with barium (heparinized tubes) and the plasma samples with tin (collection tubes). The concentrations for Bi, Hg, Pb, Rb, Sb, and Sr in whole blood were within the literature ranges for healthy adults. All of the concentrations for Co, and some of the concentrations for Cd, Cs, Tl, and Zn were higher than the high limits of the normal ranges. Approximately 14% of the Cu concentrations were lower than the low limit of the normal range. The Mo and Sn concentrations are difficult to evaluate, because the normal ranges appears to be unreliable. All concentrations for Cd, Co, Mo, Pb, Sn, and Sr and some of the concentrations for Cu (15%) and Mn (75%) in the plasma samples were higher than the high limits of the normal ranges. The concentrations for Rb tended to be lower than the normal range. To establish unequivocally the causes for elevated and reduced concentrations of trace elements in whole blood and plasma of dialysis patients, all fluids in the dialysis process must be investigated.     

Determination of serum uric acid using high-performance liquid chromatography (HPLC)/isotope dilution mass spectrometry (ID-MS) as a candidate reference method

Uric acid is an important diagnostic marker of catabolism of the purine nucleosides, and accurate measurements of serum uric acid are necessary for proper diagnosis of gout or renal disease appearance. A candidate reference method involving isotope dilution coupled with liquid chromatography/mass spectrometry (LC/MS) has been described. An isotopically labeled internal standard, [1,3-(15)N(2)] uric acid, was added to serum, followed by equilibration and protein removal clean up to prepare samples for liquid chromatography/mass spectrometry electrospray ionization (LC/MS-ESI) analyses. (M-H)(-) ions at m/z 167 and 169 for uric acid and its labeled internal standard were monitored for LC/MS. The accuracy of the measurement was evaluated by a comparison of results of this candidate reference method on lyophilized human serum reference materials for uric acid (Standard Reference Materials SRM909b) with the certified values determined by gas chromatography/mass spectrometry reference methods and by a recovery study for the added uric acid. The method performed well against the established reference method of ion-exchange followed by derivatization isotope dilution (ID) gas chromatography mass spectrometry (ID-GC/MS). The results of this method for uric acid agreed well with the certified values and were within 0.10%. The amounts of uric acid recovered and added were in good agreement for the three concentrations. This method was applied to determine uric acid in samples of frozen serum pools. Excellent precision was obtained with within-set CVs of 0.08-0.18% and between-set CVs of 0.02-0.07% for LC/MS analyses. Liquid chromatography/tandem mass spectrometry electrospray ionization (LC/MS/MS-ESI) analysis was also performed. The LC/MS and LC/MS/MS results were in very good agreement (within 0.14%). This LC/MS method, which demonstrates good accuracy and precision, and is in the speed of analysis without the need for a derivatization stage, qualifies as a candidate reference method. This method can be used as an alternative reference method to provide an accuracy base to which the routine methods can be compared.     

Application and evaluation of limited mass monitoring with a gas chromatograph mass spectrometer computer system

The technique of scanning a preselected set of ions employing a combined gas chromatography mass spectrometer computer system has been investigated to ascertain the advantages and disadvantages of such a procedure. This technique allows one to determine gas chromatographic retention data with with a high degree of precision and accuracy, in rapid temperature programming operation, due to shortening of the mas spectral scanning interval. Signal-to-noise ratio in ion abundance recordings can be enhanced by increasing the dwell time for as many as 100 ions without lenghtening the scanning interval. The utility of such an approach was demonstrated by analysis of complex mixtures isolated form human urine and cerebrospinal fluid.     

Techniques for Universal Evaluation of Astringency of Green Tea Infusion by the Use of a Taste Sensor System

A practical method for universal evaluation of the astringency of green tea infusion by a taste sensor system was established. The use of EGCg aqueous solution as a standard enabled analysis with high accuracy and reproducibility. The sensor output was converted into taste-intensity on the basis of Weber’s and Weber-Fechner laws, which was named the “EIT _ast ” value (“EIT” and “ast” are abbreviations for “Estimated Intensity of Taste” and “astringency” respectively). It was clarified that green tea infusion is to be classified into eight grades on the EIT _ast scale. Furthermore, the high correlation of the EIT _ast value with the human gustatory sense and the high stability of the taste sensor were proved.     

重组生物制品十二烷基硫酸钠含量检测方法的建立

目的：用亚甲基蓝-分光光度法检测重组生物制品中十二烷基硫酸钠（SDS）的含量。方法：将送检样品按比例稀释后与亚甲基蓝混合,加入氯仿进行萃取,然后用紫外分光光度计测定各样品的吸光度D值。结果：SDS浓度为0～0.01mg/mL时,SDS与D651nm值呈线性关系。结论：在一定的SDS浓度范围内可用亚甲基蓝-分光光度法检测重组生物制品中SDS的含量。     

Spatial and temporal genetic structure of a river‐resident Atlantic salmon (Salmo salar) after millennia of isolation

The river‐resident Salmo salar (“småblank”) has been isolated from other Atlantic salmon populations for 9,500 years in upper River Namsen, Norway. This is the only European Atlantic salmon population accomplishing its entire life cycle in a river. Hydropower development during the last six decades has introduced movement barriers and changed more than 50% of the river habitat to lentic conditions. Based on microsatellites and SNPs, genetic variation within småblank was only about 50% of that in the anadromous Atlantic salmon within the same river. The genetic differentiation (F_ST) between småblank and the anadromous population was 0.24. This is similar to the differentiation between anadromous Atlantic salmon in Europe and North America. Microsatellite analyses identified three genetic subpopulations within småblank, each with an effective population size N_e of a few hundred individuals. There was no evidence of reduced heterozygosity and allelic richness in contemporary samples (2005–2008) compared with historical samples (1955–56 and 1978–79). However, there was a reduction in genetic differentiation between sampling localities over time. SNP data supported the differentiation of småblank into subpopulations and revealed downstream asymmetric gene flow between subpopulations. In spite of this, genetic variation was not higher in the lower than in the upper areas. The meta‐population structure of småblank probably maintains genetic variation better than one panmictic population would do, as long as gene flow among subpopulations is maintained. Småblank is a unique endemic island population of Atlantic salmon. It is in a precarious situation due to a variety of anthropogenic impacts on its restricted habitat area. Thus, maintaining population size and avoiding further habitat fragmentation are important.     

RESPONSE TO SELECTION IN PARTIALLY SELF-FERTILIZING POPULATIONS. I. SELECTION ON A SINGLE TRAIT

Self-fertilization is a common form of reproduction in plants and it has important implications for quantitative trait evolution. Here, I present a model of selection on quantitative traits that can accommodate any level of self-fertilization. The “structured linear model” (SLM) predicts the evolution of the mean phenotype as a function of three distinct quantities: the mean additive genetic value, the directional dominance, and the mean inbreeding coefficient. Stochastic simulations of truncation selection demonstrate the accuracy of the SLM in predicting changes in the mean and variance of a quantitative trait over the full range of selfing rates. They also illustrate how complex interactions between selection and mating system determine the population distribution of inbreeding coefficients and also the amount of linkage disequilibrium. Changes in the genetic variance due to linkage disequilibria, which are commonly referred to as the “Bulmer effect,” are greatly magnified by selfing. This complicates the relationship between selfing rate and response to selection. Like the random mating theory, the parameters of the SLM can be estimated from phenotypic data.