RIPCAL: a tool for alignment-based analysis of repeat-induced point mutations in fungal genomic sequences

Background Repeat-induced point mutation (RIP) is a fungal-specific genome defence mechanism that alters the sequences of repetitive DNA, thereby inactivating coding genes. Repeated DNA sequences align between mating and meiosis and both sequences undergo C:G to T:A transitions. In most fungi these transitions preferentially affect CpA di-nucleotides thus altering the frequency of certain di-nucleotides in the affected sequences. The majority of previously published in silico analyses were limited to the comparison of ratios of pre- and post-RIP di-nucleotides in putatively RIP-affected sequences – so-called RIP indices. The analysis of RIP is significantly more informative when comparing sequence alignments of repeated sequences. There is, however, a dearth of bioinformatics tools available to the fungal research community for alignment-based RIP analysis of repeat families. Results We present RIPCAL http://www.sourceforge.net/projects/ripcal, a software tool for the automated analysis of RIP in fungal genomic DNA repeats, which performs both RIP index and alignment-based analyses. We demonstrate the ability of RIPCAL to detect RIP within known RIP-affected sequences of Neurospora crassa and other fungi. We also predict and delineate the presence of RIP in the genome of Stagonospora nodorum – a Dothideomycete pathogen of wheat. We show that RIP has affected different members of the S. nodorum rDNA tandem repeat to different extents depending on their genomic contexts. Conclusion The RIPCAL alignment-based method has considerable advantages over RIP indices for the analysis of whole genomes. We demonstrate its application to the recently published genome assembly of S. nodorum

Genome sequencing and comparative genomics of the broad host-range pathogen Rhizoctonia solani AG8

Rhizoctonia solani is a soil-borne basidiomycete fungus with a necrotrophic lifestyle which is classified into fourteen reproductively incompatible anastomosis groups (AGs). One of these, AG8, is a devastating pathogen causing bare patch of cereals, brassicas and legumes. R. solani is a multinucleate heterokaryon containing significant heterozygosity within a single cell. This complexity posed significant challenges for the assembly of its genome. We present a high quality genome assembly of R. solani AG8 and a manually curated set of 13,964 genes supported by RNA-seq. The AG8 genome assembly used novel methods to produce a haploid representation of its heterokaryotic state. The whole-genomes of AG8, the rice pathogen AG1-IA and the potato pathogen AG3 were observed to be syntenic and co-linear. Genes and functions putatively relevant to pathogenicity were highlighted by comparing AG8 to known pathogenicity genes, orthology databases spanning 197 phytopathogenic taxa and AG1-IA.We also observed SNP-level “hypermutation” of CpG dinucleotides to TpG between AG8 nuclei, with similarities to repeat-induced point mutation (RIP). Interestingly, gene-coding regions were widely affected along with repetitive DNA, which has not been previously observed for RIP in mononuclear fungi of the Pezizomycotina. The rate of heterozygous SNP mutations within this single isolate of AG8 was observed to be higher than SNP mutation rates observed across populations of most fungal species compared. Comparative analyses were combined to predict biological processes relevant to AG8 and 308 proteins with effector-like characteristics, forming a valuable resource for further study of this pathosystem. Predicted effector-like proteins had elevated levels of non-synonymous point mutations relative to synonymous mutations (dN/dS), suggesting that they may be under diversifying selection pressures. In addition, the distant relationship to sequenced necrotrophs of the Ascomycota suggests the R. solani genome sequence may prove to be a useful resource in future comparative analysis of plant pathogens

Equine transcriptome quantification using human GeneChip arrays can be improved using genomic DNA hybridisation and probe selection.

Affymetrix GeneChip arrays are a powerful tool for transcriptome profiling and have been applied to a wide range of species. A genomic DNA (gDNA)-based probe selection method has been developed which broadens the range of species to which GeneChips may be successfully applied. This study demonstrated that gDNA-based probe selection on the Affymetrix U133+2 GeneChip array can be used to study the equine transcriptome which, to date, has received only limited attention. More than 29,000 transcripts can be detected in equine brain and liver and in primary cultures of equine articular chondrocytes. Gene ontology analysis of differentially expressed genes revealed the presence of expected categories within each tissue. The level of gene expression could also be correlated with the phenotypes and specialised functions of each tissue. The results demonstrated that probe selection on a human chip can be successfully used to study the equine transcriptome

Equine transcriptome quantification using human GeneChip arrays can be improved using genomic DNA hybridisation and probe selection.

Affymetrix GeneChip arrays are a powerful tool for transcriptome profiling and have been applied to a wide range of species. A genomic DNA (gDNA)-based probe selection method has been developed which broadens the range of species to which GeneChips may be successfully applied. This study demonstrated that gDNA-based probe selection on the Affymetrix U133+2 GeneChip array can be used to study the equine transcriptome which, to date, has received only limited attention. More than 29,000 transcripts can be detected in equine brain and liver and in primary cultures of equine articular chondrocytes. Gene ontology analysis of differentially expressed genes revealed the presence of expected categories within each tissue. The level of gene expression could also be correlated with the phenotypes and specialised functions of each tissue. The results demonstrated that probe selection on a human chip can be successfully used to study the equine transcriptome

A crossover randomised controlled trial of oral mandibular advancement devices for obstructive sleep apnoea-hypopnoea (TOMADO)

Rationale Mandibular advancement devices (MADs) are used to treat obstructive sleep apnoea-hypopnoea syndrome (OSAHS) but evidence is lacking regarding their clinical and cost-effectiveness in less severe disease. Objectives To compare clinical- and cost-effectiveness of a range of MADs against no treatment in mild to moderate OSAHS. Measurements and methods This open-label, randomised, controlled, crossover trial was undertaken at a UK sleep centre. Adults with Apnoea-Hypopnoea Index (AHI) 5–˂30/h and Epworth Sleepiness Scale (ESS) score ≥9 underwent 6 weeks of treatment with three non-adjustable MADs: self-moulded (SleepPro 1; SP1); semi-bespoke (SleepPro 2; SP2); fully-bespoke MAD (bMAD); and 4 weeks no treatment. Primary outcome was AHI scored by a polysomnographer blinded to treatment. Secondary outcomes included ESS, quality of life, resource use and cost. Main results 90 patients were randomised and 83 were analysed. All devices reduced AHI compared with no treatment by 26% (95% CI 11% to 38%, p=0.001) for SP1, 33% (95% CI 24% to 41%) for SP2 and 36% (95% CI 24% to 45%, p˂0.001) for bMAD. ESS was 1.51 (95% CI 0.73 to 2.29, p˂0.001, SP1) to 2.37 (95% CI 1.53 to 3.22, p˂0.001, bMAD) lower than no treatment (p˂0.001 for all). Compliance was lower for SP1, which was the least preferred treatment at trial exit. All devices were cost-effective compared with no treatment at a £20 000/quality-adjusted life year (QALY) threshold. SP2 was the most cost-effective up to £39 800/QALY. Conclusions Non-adjustable MADs achieve clinically important improvements in mild to moderate OSAHS and are cost-effective. Of those trialled, the semi-bespoke MAD is an appropriate first choice.</p

Meta-analysis of randomised controlled trials of oral mandibular advancement devices and continuous positive airway pressure for obstructive sleep apnoea-hypopnoea

Obstructive sleep apnoea-hypopnoea (OSAH) causes excessive daytime sleepiness, impairs quality-of-life, and increases cardiovascular disease and road traffic accident risks. Continuous positive airway pressure (CPAP) treatment and mandibular advancement devices (MAD) have been shown to be effective in individual trials but their effectiveness particularly relative to disease severity is unclear