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Microsatellite markers have played a major role in ecological, evolutionary and conservation research during the past 20 years. However, technical constrains related to the use of capillary electrophoresis and a recent technological revolution that has impacted other marker types have brought to question the continued use of microsatellites for certain applications. We present a study for improving microsatellite genotyping in ecology using high-throughput sequencing ( HTS). This approach entails selection of short markers suitable for HTS, sequencing PCR-amplified microsatellites on an Illumina platform and bioinformatic treatment of the sequence data to obtain multilocus genotypes. It takes advantage of the fact that HTS gives direct access to microsatellite sequences, allowing unambiguous allele identification and enabling automation of the genotyping process through bioinformatics. In addition, the massive parallel sequencing abilities expand the information content of single experimental runs far beyond capillary electrophoresis. We illustrated the method by genotyping brown bear samples amplified with a multiplex PCR of 13 new microsatellite markers and a sex marker. HTS of microsatellites provided accurate individual identification and parentage assignment and resulted in a significant improvement of genotyping success (84%) of faecal degraded DNA and costs reduction compared to capillary electrophoresis. The HTS approach holds vast potential for improving success, accuracy, efficiency and standardization of microsatellite genotyping in ecological and conservation applications, especially those that rely on profiling of low-quantity/quality DNA and on the construction of genetic databases. We discuss and give perspectives for the implementation of the method in the light of the challenges encountered in wildlife studies. [ABSTRACT FROM AUTHOR]

Ecological understanding of the role of consumer-resource interactions in natural food webs is limited by the difficulty of accurately and efficiently determining the complex variety of food types animals have eaten in the field. We developed a method based on DNA metabarcoding multiplexing and next-generation sequencing to uncover different taxonomic groups of organisms from complex diet samples. We validated this approach on 91 faeces of a large omnivorous mammal, the brown bear, using DNA metabarcoding markers targeting the plant, vertebrate and invertebrate components of the diet. We included internal controls in the experiments and performed PCR replication for accuracy validation in postsequencing data analysis. Using our multiplexing strategy, we significantly simplified the experimental procedure and accurately and concurrently identified different prey DNA corresponding to the targeted taxonomic groups, with ≥60% of taxa of all diet components identified to genus/species level. The systematic application of internal controls and replication was a useful and simple way to evaluate the performance of our experimental procedure, standardize the selection of sequence filtering parameters for each marker data and validate the accuracy of the results. Our general approach can be adapted to the analysis of dietary samples of various predator species in different ecosystems, for a number of conservation and ecological applications entailing large-scale population level diet assessment through cost-effective screening of multiple DNA metabarcodes, and the detection of fine dietary variation among samples or individuals and of rare food items. [ABSTRACT FROM AUTHOR]

Ecosystems across the globe are threatened by climate change and human activities. New rapid survey approaches for monitoring biodiversity would greatly advance assessment and understanding of these threats. Taking advantage of next-generation DNA sequencing, we tested an approach we call metabarcoding: high-throughput and simultaneous taxa identification based on a very short (usually <100 base pairs) but informative DNA fragment. Short DNA fragments allow the use of degraded DNA from environmental samples. All analyses included amplification using plant-specific versatile primers, sequencing and estimation of taxonomic diversity. We tested in three steps whether degraded DNA from dead material in soil has the potential of efficiently assessing biodiversity in different biomes. First, soil DNA from eight boreal plant communities located in two different vegetation types (meadow and heath) was amplified. Plant diversity detected from boreal soil was highly consistent with plant taxonomic and growth form diversity estimated from conventional above-ground surveys. Second, we assessed DNA persistence using samples from formerly cultivated soils in temperate environments. We found that the number of crop DNA sequences retrieved strongly varied with years since last cultivation, and crop sequences were absent from nearby, uncultivated plots. Third, we assessed the universal applicability of DNA metabarcoding using soil samples from tropical environments: a large proportion of species and families from the study site were efficiently recovered. The results open unprecedented opportunities for large-scale DNA-based biodiversity studies across a range of taxonomic groups using standardized metabarcoding approaches. [ABSTRACT FROM AUTHOR]

M. Fèvre-Montange, A. Vasiljevic, D. Frappaz, J. Champier, A. Szathmari, M.-H. Aubriot Lorton, F. Chapon, A. Coulon, I. Quintin Roué, M.-B. Delisle, D. Figarella-Branger, A. Laquerrière, C. Miquel, J.-F. Michiels, M. Péoch, M. Polivka, F. Fauchon and A. Jouvet (2012) Neuropathology and Applied Neurobiology 38, 87-94 Utility of Ki67 immunostaining in the grading of pineal parenchymal tumours: a multicentre study Aims: Pineal parenchymal tumours (PPTs) are rare neoplasms that are divided into pineocytoma (PC), pineoblastoma (PB) and PPT of intermediate differentiation (PPTID). Factors affecting the survival of patients with PPTs are morphological subtype and histological grading according to mitotic index and neurofilament immunostaining. Grading criteria to distinguish PPTIDs are difficult to define, particularly when using small specimens. The Ki67 labelling index (LI) might be helpful in distinguishing between grade II and III PPTIDs. Our study was performed to assess the predictive value of the Ki67 LI in a large cooperative series of PPTs and to evaluate whether inclusion of this data would improve and refine the World Health Organization classification. Methods: A retrospective analysis of 33 PPTs was performed. The histological features of the tumours were reviewed and Ki67 LI scoring was evaluated by immunohistochemistry. Data were correlated with the patients' survival. Results: The mean Ki67 LI was significantly different for tumour grades (0 in PC, 5.2 ± 0.4 in PPTID grade II, 11.2 ± 2.0 in PPTID grade III, 36.4 ± 6.2 in PB; P < 0.0001). However, there was no statistically significant difference in either overall or disease-free survival evaluated by the Kaplan-Meier method for patients with different grade tumours or Ki67 LI, possibly due to the different clinical management of patients in different centres. Conclusions: The Ki67 LI may be a useful additional tool for grading PPTs, more particularly in small tumour samples. [ABSTRACT FROM AUTHOR]

The article reports on childhood gangliogliomas with ependymal differentiation which constitute 10% of paediatric central nervous system tumours and are considered to be the most frequent chronic epilepsy-associated tumours. Three such cases are also discussed in this article and all cases presented numerous granular eosinophilic bodies and perivascular lymphocytic cuffing.

In noninvasive studies, the intersample variance in DNA quality and quantity is large, and produces multilocus genotypes of highly variable quality. Here we propose a standardized method for testing the reliability of the genotyping procedure when using the multiple-tube approach. The quality indexes generated will allow reliable comparisons among samples, loci, studies, and field and/or laboratory protocols. These indexes represent a powerful tool for the quality management of noninvasive studies. [ABSTRACT FROM AUTHOR]

We developed seven microsatellite loci in the common frog, Rana temporaria. There were between 2 and 27 alleles per locus and the expected heterozygosities ranged from 0.28 to 0.96 in a sample of frogs collected in the French Alps. Adding these seven markers to the 15 previously available microsatellite loci for this species should facilitate studies of genetic structure of Rana temporaria populations at a fine geographical scale. [ABSTRACT FROM AUTHOR]