The Quantification of Representative Sequences pipeline for amplicon sequencing: case study on within-population ITS1 sequence variation in a microparasite infecting Daphnia.

Next generation sequencing (NGS) platforms are replacing traditional molecular biology protocols like cloning and Sanger sequencing. However, accuracy of NGS platforms has rarely been measured when quantifying relative frequencies of genotypes or taxa within populations. Here we developed a new bioinformatic pipeline (QRS) that pools similar sequence variants and estimates their frequencies in NGS data sets from populations or communities. We tested whether the estimated frequency of representative sequences, generated by 454 amplicon sequencing, differs significantly from that obtained by Sanger sequencing of cloned PCR products. This was performed by analysing sequence variation of the highly variable first internal transcribed spacer (ITS1) of the ichthyosporean Caullerya mesnili, a microparasite of cladocerans of the genus Daphnia. This analysis also serves as a case example of the usage of this pipeline to study within-population variation. Additionally, a public Illumina data set was used to validate the pipeline on community-level data. Overall, there was a good correspondence in absolute frequencies of C. mesnili ITS1 sequences obtained from Sanger and 454 platforms. Furthermore, analyses of molecular variance (amova) revealed that population structure of C. mesnili differs across lakes and years independently of the sequencing platform. Our results support not only the usefulness of amplicon sequencing data for studies of within-population structure but also the successful application of the QRS pipeline on Illumina-generated data. The QRS pipeline is freely available together with its documentation under GNU Public Licence version 3 at http://code.google.com/p/quantification-representative-sequences.

Genetics of anti-EHV antibody responses in a horse population.

Individual variation in immune responses to herpesviruses was observed in various species. Here, associations between polymorphic molecular markers and life-long anti-EHV-1/4 antibody immune responses were analyzed in a model EHV-infected population of the Old Kladruber horses. Two-dimensional analysis including overall mean titers and titer dynamics expressed by differences between spring and autumn titers allowed identification of low-responders. 50 randomly selected microsatellites and nine single nucleotide polymorphisms in nine immunity-related candidate genes were genotyped. Due to differences (p<0.001) in antibody titers between two color varieties of Old Kladruber horses, separate association studies were performed in the two sub-populations by using the Fisher's exact test. In black horses, the interleukin 4 receptor and MxA protein coding genes, and the microsatellite TKY325 were associated with the responder status. In the grey population, the microsatellite TKY343 showed significant association with anti-EHV antibody responsiveness after Bonferroni corrections.

Spinal cord injury and anxiety: a comprehensive review.

Although there have been past literature reviews which have addressed the psychological adjustment, consequences, and impact/reaction to spinal cord injury, as well as reviews of depression after spinal cord injury, there appears to be an absence of reviews which have focused primarily on the relationship between spinal cord injury and anxiety. The purpose of this paper is to present a comprehensive review of the relatively recent (the past 23-31 years depending on the database utilized) scientific literature as it pertains to anxiety reactions in spinal cord injured individuals. Specifically, this paper provides reviews of the prevalence/presence of anxiety reactions, as well as the correlates of anxiety, in the spinal cord injured population. Furthermore, this paper reviews the relatively few articles which have addressed the treatment of such symptomatology in spinal cord injured individuals. Methodological concerns and limitations of the existing literature and directions for future research are also provided.

Utilization of a comprehensive sensory stimulation program with a comatose tetraplegic patient.

To date, there has been a relative absence of studies which have examined the utility and effectiveness of sensory stimulation procedures in comatose, spinal-cord-injured patients. This report describes the interdisciplinary utilization (within an acute care setting) of a comprehensive sensory stimulation programme with a 21-year-old, comatose, tetraplegic male patient. To promote behavioural arousal, multiple sensory stimulation sessions were conducted with the patient daily. Within each session (and across sensory modalities), the effectiveness of sensory stimulation procedures was evaluated via use of A-B-A within-subject designs. Results indicated that the patient exhibited significantly greater behavioural arousal during sensory stimulation (B1), as compared to pre-(A1) and post-(A2) treatment baselines. There was also a non-significant trend of increased behavioural arousal during the post- as compared to the pre-treatment baselines (i.e. carry-over effect). By termination of this programme, the patient exhibited an overall increased level of arousal/behavioural responsiveness (i.e. decreased coma) as compared to his level at the initiation of this programme. These findings are discussed in light of factors that may affect the effectiveness and implementation of such sensory stimulation programmes with comatose, tetraplegic patients in acute care settings.