Parallel tagged amplicon sequencing of transcriptome-based genetic markers for Triturus newts with the Ion Torrent next-generation sequencing platform

Next-generation sequencing is a fast and cost-effective way to obtain sequence data for nonmodel organisms for many markers and for many individuals. We describe a protocol through which we obtain orthologous markers for the crested newts (Amphibia: Salamandridae: Triturus), suitable for analysis of interspecific hybridization. We use transcriptome data of a single Triturus species and design 96 primer pairs that amplify c. 180 bp fragments positioned in 3-prime untranslated regions. Next, these markers are tested with uniplex PCR for a set of species spanning the taxonomical width of the genus Triturus. The 52 markers that consistently show a single band of expected length at gel electrophoreses for all tested crested newt species are then amplified in five multiplex PCRs (with a plexity of ten or eleven) for 132 individual newts: a set of 84 representing the seven (candidate) species and a set of 48 from a presumed hybrid population. After pooling multiplexes per individual, unique tags are ligated to link amplicons to individuals. Subsequently, individuals are pooled equimolar and sequenced on the Ion Torrent next-generation sequencing platform. A bioinformatics pipeline identifies the alleles and recodes these to a genotypic format. Next, we test the utility of our markers. baps allocates the 84 crested newt individuals representing (candidate) species to their expected (candidate) species, confirming the markers are suitable for species delineation. newhybrids, a hybrid index and hiest confirm the 48 individuals from the presumed hybrid population to be genetically admixed, illustrating the potential of the markers to identify interspecific hybridization. We expect the set of markers we designed to provide a high resolving power for analysis of hybridization in Triturus

Balanced lethal systems: an evolutionary mystery

NWO023.016.006NaturalisAnimal science

Efficient screening for ‘genetic pollution’ in an anthropogenic crested newt hybrid zone

Genetic admixture between endangered native and non-native invasive species poses a complex conservation problem. Decision makers often need to quickly screen large numbers of individuals and distinguish natives from morphologically similar invading species and their genetically admixed offspring. We describe a protocol using the fast and economical Kompetitive Allele Specific PCR (KASP) technology for genotyping on a large scale. We apply this protocol to a case study of hybridization between a native and an invasive crested newt species. Using previously published data, we designed a panel of ten nuclear and one mitochondrial diagnostic SNP markers. We observed only minor differences between KASP and next-generation sequencing data previously produced with the Ion Torrent platform. We briefly discuss practical considerations for tackling the insidious conservation problem of genetic admixture between native and invasive species. The KASP genotyping protocol facilitates policy decision making for the crested newt case and is generally applicable to invasive hybridization with endangered taxa

Impact of EMG-triggered neuromuscular stimulation of the wrist and finger extensors of the paretic hand after stroke: a systematic review of the literature

Objective: To assess whether EMG-triggered neuromuscular electrical stimulation (EMG-NMES) applied to the extensor muscles of the forearm improves hand function after stroke. Design: Systematic review of randomized controlled trials. Methods: A computer-aided literature search up to June 2006 identified articles comparing EMG-NMES of the upper extremity with usual care. Methodological quality was rated on the Physiotherapy Evidence Database scale (PEDro), and the Hedges' g model was used to calculate the summary effect sizes (SES) using fixed or random models depending on heterogeneity. Results: Eight studies, selected out of 192 hits and presenting 157 patients, were included in quantitative and qualitative analyses. The methodological quality ranged from 2 to 6 points. The meta-analysis revealed non-significant effect sizes in favour of EMG-NMES for reaction time, sustained contraction, dexterity measured with the Box and Block manipulation test, synergism measured with the Fugl-Meyer Motor Assessment Scale and manual dexterity measured with the Action Research Arm test. Conclusion: No statistically significant differences in effects were found between EMG-NMES and usual care. Most studies had poor methodological quality, low statistical power and insufficient treatment contrast between experimental and control groups. In addition, all studies except two investigated the effects of EMG-NMES in the chronic phase after stroke, whereas the literature suggests that an early start, within the time window in which functional outcome of the upper limb is not fully defined, is more appropriate. © SAGE Publications 2008