Digital fragment analysis of short tandem repeats by high‐throughput amplicon sequencing

High‐throughput sequencing has been proposed as a method to genotype microsatellites and overcome the four main technical drawbacks of capillary electrophoresis: amplification artifacts, imprecise sizing, length homoplasy, and limited multiplex capability. The objective of this project was to test a high‐throughput amplicon sequencing approach to fragment analysis of short tandem repeats and characterize its advantages and disadvantages against traditional capillary electrophoresis. We amplified and sequenced 12 muskrat microsatellite loci from 180 muskrat specimens and analyzed the sequencing data for precision of allele calling, propensity for amplification or sequencing artifacts, and for evidence of length homoplasy. Of the 294 total alleles, we detected by sequencing, only 164 alleles would have been detected by capillary electrophoresis as the remaining 130 alleles (44%) would have been hidden by length homoplasy. The ability to detect a greater number of unique alleles resulted in the ability to resolve greater population genetic structure. The primary advantages of fragment analysis by sequencing are the ability to precisely size fragments, resolve length homoplasy, multiplex many individuals and many loci into a single high‐throughput run, and compare data across projects and across laboratories (present and future) with minimal technical calibration. A significant disadvantage of fragment analysis by sequencing is that the method is only practical and cost‐effective when performed on batches of several hundred samples with multiple loci. Future work is needed to optimize throughput while minimizing costs and to update existing microsatellite allele calling and analysis programs to accommodate sequence‐aware microsatellite data

Data from: Digital fragment analysis of short tandem repeats by high-throughput amplicon sequencing

High-throughput sequencing has been proposed as a method to genotype microsatellites and overcome the four main technical drawbacks of capillary electrophoresis: amplification artifacts, imprecise sizing, length homoplasy, and limited multiplex capability. The objective of this project was to test a high-throughput amplicon sequencing approach to fragment analysis of short tandem repeats and characterize its advantages and disadvantages against traditional capillary electrophoresis. We amplified and sequenced 12 muskrat microsatellite loci from 180 muskrat specimens and analyzed the sequencing data for precision of allele calling, propensity for amplification or sequencing artifacts, and for evidence of length homoplasy. Of the 294 total alleles, we detected by sequencing, only 164 alleles would have been detected by capillary electrophoresis as the remaining 130 alleles (44%) would have been hidden by length homoplasy. The ability to detect a greater number of unique alleles resulted in the ability to resolve greater population genetic structure. The primary advantages of fragment analysis by sequencing are the ability to precisely size fragments, resolve length homoplasy, multiplex many individuals and many loci into a single high-throughput run, and compare data across projects and across laboratories (present and future) with minimal technical calibration. A significant disadvantage of fragment analysis by sequencing is that the method is only practical and cost-effective when performed on batches of several hundred samples with multiple loci. Future work is needed to optimize throughput while minimizing costs and to update existing microsatellite allele calling and analysis programs to accommodate sequence-aware microsatellite data

digital Fragment Analysis Histograms

Histograms (in text files) of each microsatellite locus from Ondatra zibethicus (muskrat). These histograms were created with a custom Python script from Illumina MiSeq paired-end sequencing reads that were merged, quality filtered, and de-replicated. A file to map the sample to the specimen ID (and its sampling population) is in the ReadMe file

digital Fragment Analysis Final Genotypes

Final microsatellite genotypes for each muskrat specimen, including muskrat ID, location, allele (0,1), and eleven micorsatellite loci. A text file that maps alleles to their sequence is in the ReadMe file

Zinc status affects neurotransmitter activity in the paraventricular nucleus of rats

Alterations in neurochemical activity in the paraventricular nucleus (PVN) of the hypothalamus may account for decreased intake of zinc-deficient diets. Male Sprague-Dawley rats were fed zinc-deficient (ZD) or zinc-adequate (ZA) diet for 14 d before samples of extracellular fluid in the PVN were collected by microdialysis or push-pull perfusion. A third set of rats was pair-fed (PF) an amount of ZA diet equal to the intake of ZD rats. Samples were collected over a 2-h period spanning the transition from light to dark. All rats then consumed the zinc adequate diet ad libitum for 3 d before a second set of samples was collected. The increase in extracellular norepineprhrine (NE) during h 1 of the dark period to 147 +/- 13% of baseline (P < 0.05) was apparent only in ZA rats at d 14. After the 3-d repletion period, the increase in NE at dark onset occurred in all three groups. An increase in extracellular neuropeptide Y (NPY) at dark onset to 174 +/- 32% of baseline in rats fed ZA (P < 0.01) was measured in all three groups at both d 14 and 17. Basal NPY concentrations were significantly elevated in PF rats on d 14 (7.45 +/- 2.01 vs. 0.58 +/- 0.23 pmol/L, P = 0.01) and returned to ZA levels by d 17. The activities of the NE and NPY systems in the PVN were altered in rats fed a zinc-deficient diet; however, it is unclear whether the disruption in the NE and NPY neural systems in the PVN results in the altered feeding behavior accompanying zinc deficiency

Aerobic Exercise Intervention, CognitivePerformance, and Brain Structure : results from the Physical Influences on Brain in Aging (PHIBRA) Study

Studies have shown that aerobic exercise has the potential to improve cognition and reduce brain atrophy in older adults. However, the literature is equivocal with regards to the specificity or generality of these effects. To this end, we report results on cognitive function and brain structure from a 6-month training intervention with 60 sedentary adults (64–78 years) randomized to either aerobic training or stretching and toning control training. Cognitive functions were assessed with a neuropsychological test battery in which cognitive constructs were measured using several different tests. Freesurfer was used to estimate cortical thickness in frontal regions and hippocampus volume. Results showed that aerobic exercisers, compared to controls, exhibited a broad, rather than specific, improvement in cognition as indexed by a higher “Cognitive score,” a composite including episodic memory, processing speed, updating, and executive function tasks (p = 0.01). There were no group differences in cortical thickness, but additional analyses revealed that aerobic fitness at baseline was specifically related to larger thickness in dorsolateral prefrontal cortex (dlPFC), and hippocampus volume was positively associated with increased aerobic fitness over time. Moreover, “Cognitive score” was related to dlPFC thickness at baseline, but changes in “Cognitive score” and dlPFC thickness were associated over time in the aerobic group only. However, aerobic fitness did not predict dlPFC change, despite the improvement in “Cognitive score” in aerobic exercisers. Our interpretation of these observations is that potential exercise-induced changes in thickness are slow, and may be undetectable within 6-months, in contrast to change in hippocampus volume which in fact was predicted by the change in aerobic fitness. To conclude, our results add to a growing literature suggesting that aerobic exercise has a broad influence on cognitive functioning, which may aid in explaining why studies focusing on a narrower range of functions have sometimes reported mixed results