From NGS assembly challenges to instability of fungal mitochondrial genomes: A case study in genome complexity.

The presence of repetitive or non-unique DNA persisting over sizable regions of a eukaryotic genome can hinder the genome's successful de novo assembly from short reads: ambiguities in assigning genome locations to the non-unique subsequences can result in premature termination of contigs and thus overfragmented assemblies. Fungal mitochondrial (mtDNA) genomes are compact (typically less than 100 kb), yet often contain short non-unique sequences that can be shown to impede their successful de novo assembly in silico. Such repeats can also confuse processes in the cell in vivo. A well-studied example is ectopic (out-of-register, illegitimate) recombination associated with repeat pairs, which can lead to deletion of functionally important genes that are located between the repeats. Repeats that remain conserved over micro- or macroevolutionary timescales despite such risks may indicate functionally or structurally (e.g., for replication) important regions. This principle could form the basis of a mining strategy for accelerating discovery of function in genome sequences. We present here our screening of a sample of 11 fully sequenced fungal mitochondrial genomes by observing where exact k-mer repeats occurred several times; initial analyses motivated us to focus on 17-mers occurring more than three times. Based on the diverse repeats we observe, we propose that such screening may serve as an efficient expedient for gaining a rapid but representative first insight into the repeat landscapes of sparsely characterized mitochondrial chromosomes. Our matching of the flagged repeats to previously reported regions of interest supports the idea that systems of persisting, non-trivial repeats in genomes can often highlight features meriting further attention.

Insulin resistance and beat-to-beat cardiovascular dynamics: a constant relationship across different body mass index and blood pressure categories.

CONTEXT: Epidemiological studies have shown a progressive increase in insulin resistance (IR) accompanying body weight gain and blood pressure (BP) increase. This has led to the consideration that hemodynamic effects of IR might depend on its relationship with body mass index (BMI) and BP. OBJECTIVE: The aim of our study was to determine whether IR is associated with changes in hemodynamic indices of cardiovascular function across different categories of BMI (normal weight, overweight, and obese), and BP levels (normal, high normal, and hypertension). DESIGN, SETTING AND PARTICIPANTS: This was a cross-sectional study conducted in a population sample of nondiabetic individuals (n = 731). MEASURES: Insulin resistance was evaluated with the homeostasis model assessment of insulin resistance (HOMA) and subjects were classified into quartiles according to HOMA index values. Synchronized beat-to-beat recordings of stroke volume (impedance cardiography) and R-R interval, along with repeated auscultatory BP measurements were performed. Derived hemodynamic parameters were computed and averaged. RESULTS: Analysis of covariance adjusting for confounders showed significant differences for most hemodynamic parameters among different quartiles of HOMA index both in the general population and within each BMI and BP category. Overall, increasing values of HOMA index were associated with significantly higher BP; and reduced R-R interval, stroke index, cardiac index, pre-ejection period and left ventricular ejection time (P < .01) across different categories of BMI and BP. CONCLUSIONS: These findings suggest that even small increases in HOMA index (not necessarily in the range to define IR) may induce significant changes on indices of cardiovascular function even in normal-weight and normotensive subjects, emphasizing the importance of IR at an early stage of the cardiovascular risk continuum.

The complex task of choosing a de novo assembly: lessons from fungal genomes.

Selecting the values of parameters used by de novo genomic assembly programs, or choosing an optimal de novo assembly from several runs obtained with different parameters or programs, are tasks that can require complex decision-making. A key parameter that must be supplied to typical next generation sequencing (NGS) assemblers is the k-mer length, i.e., the word size that determines which de Bruijn graph the program should map out and use. The topic of assembly selection criteria was recently revisited in the Assemblathon 2 study (Bradnam et al., 2013). Although no clear message was delivered with regard to optimal k-mer lengths, it was shown with examples that it is sometimes important to decide if one is most interested in optimizing the sequences of protein-coding genes (the gene space) or in optimizing the whole genome sequence including the intergenic DNA, as what is best for one criterion may not be best for the other. In the present study, our aim was to better understand how the assembly of unicellular fungi (which are typically intermediate in size and complexity between prokaryotes and metazoan eukaryotes) can change as one varies the k-mer values over a wide range. We used two different de novo assembly programs (SOAPdenovo2 and ABySS), and simple assembly metrics that also focused on success in assembling the gene space and repetitive elements. A recent increase in Illumina read length to around 150 bp allowed us to attempt de novo assemblies with a larger range of k-mers, up to 127 bp. We applied these methods to Illumina paired-end sequencing read sets of fungal strains of Paracoccidioides brasiliensis and other species. By visualizing the results in simple plots, we were able to track the effect of changing k-mer size and assembly program, and to demonstrate how such plots can readily reveal discontinuities or other unexpected characteristics that assembly programs can present in practice, especially when they are used in a traditional molecular microbiology laboratory with a 'genomics corner'. Here we propose and apply a component of a first pass validation methodology for benchmarking and understanding fungal genome de novo assembly processes.

The eukaryotic genome, its reads, and the unfinished assembly.

In recent years, readily affordable short read sequences provided by next-generation sequencing (NGS) have become longer and more accurate. This has led to a jump in interest in the utility of NGS-only approaches for exploring eukaryotic genomes. The concept of a static, 'finished' genome assembly, which still appears to be a faraway goal for many eukaryotes, is yielding to new paradigms. We here motivate an object-view concept where the raw reads are the main, fixed object, and assemblies with their annotations take a role of dynamically changing and modifiable views of that object.

Kinetic analysis of gene expression during mycelium to yeast transition and yeast to mycelium germination in Paracoccidioides brasiliensis.

INTRODUCTION: Paracoccidioidomycosis is an endemic systemic mycosis caused by Paracoccidioides brasiliensis, a thermally dimorphic fungus that in tissues and cultures at 37°C grows as a yeast while at lower temperatures (less than 24°C) it becomes a mold; however the genes that rule these processes and their expression are poorly understood. OBJECTIVE: This research focused on the kinetic expression of certain genes in P. brasiliensis throughout the dimorphic process, one that involves the transition from the mycelium to yeast forms and the germination from the yeast to mycelium form. MATERIALS AND METHODS: A real-time quantitative polymerase chain reaction (RT-qPCR) was optimized to measure the expression of ten genes connected with diverse cellular functions including cell synthesis and wall structure, oxidative stress response, heat shock response, metabolism, proteins' processing, solute transport across the cell membrane and signal transduction pathways at different time points during the mycelia to yeast transition, as well as in the yeast to mycelia germination processes. RESULTS: Genes involved in cell synthesis and wall structure, metabolism and signal transduction were differentially expressed and highly up-regulated during the yeast to mycelia germination process; on the other hand, genes involved in heat shock response, cell synthesis and wall structure were highly up-regulated during the mycelia to yeast transition process. The remaining genes were differentially regulated during both processes. CONCLUSION: In this work the up-regulation of certain genes involved in the morphological changes occurring in P. brasiliensis yeast and mycelia forms were confirmed, indicating that these biological processes play an important role during the host-pathogen interactions, as well as in the fungus adaptation to environmental conditions.

Kinetic analysis of gene expression during mycelium to yeast transition and yeast to mycelium germination in Paracoccidioides brasiliensis / Análisis de la cinética de expresión de genes durante la transición de micelio a levadura y la germinación levadura a micelio en Paracoccidioides brasiliensis

Introduction: Paracoccidioidomycosis is an endemic systemic mycosis caused by Paracoccidioides brasiliensis, a thermally dimorphic fungus that in tissues and cultures at 37°C grows as a yeast while at lower temperatures (less than 24°C) it becomes a mold; however the genes that rule these processes and their expression are poorly understood. Objective: This research focused on the kinetic expression of certain genes in P. brasiliensis throughout the dimorphic process, one that involves the transition from the mycelium to yeast forms and the germination from the yeast to mycelium form. Materials and methods: A real-time quantitative polymerase chain reaction (RT-qPCR) was optimized to measure the expression of ten genes connected with diverse cellular functions including cell synthesis and wall structure, oxidative stress response, heat shock response, metabolism, proteins’ processing, solute transport across the cell membrane and signal transduction pathways at different time points during the mycelia to yeast transition, as well as in the yeast to mycelia germination processes. Results: Genes involved in cell synthesis and wall structure, metabolism and signal transduction were differentially expressed and highly up-regulated during the yeast to mycelia germination process; on the other hand, genes involved in heat shock response, cell synthesis and wall structure were highly up-regulated during the mycelia to yeast transition process. The remaining genes were differentially regulated during both processes. Conclusion: In this work the up-regulation of certain genes involved in the morphological changes occurring in P. brasiliensis yeast and mycelia forms were confirmed, indicating that these biological processes play an important role during the host-pathogen interactions, as well as in the fungus adaptation to environmental conditions.

Introducción. La paracoccidioidomicosis es una micosis sistémica causada por el hongo termodimorfo Paracoccidioides brasiliensis. En tejidos y cultivos a 37°C crece como levadura, mientras que a temperaturas menores de 24°C crece como un moho. Sin embargo, se conoce poco sobre los genes que regulan estos procesos. Objetivo. Se evaluó la cinética de expresión de algunos genes en P. brasiliensis mediante el proceso de dimorfismo incluida la transición del micelio a levadura y de la germinación de levadura a micelio. Materiales y métodos. Se optimizó una PCR cuantitativa en tiempo real (RT-qPCR) para medir la expresión de diez genes relacionados con diversas funciones celulares que incluyeron: síntesis de pared, respuesta al estrés oxidativo, respuesta al choque térmico, metabolismo, procesamiento de proteínas, trasporte de solutos a través de membranas y transducción de señales, todo ello a diferentes tiempos durante la transición de micelio a levadura, así como de la germinación de levadura a micelio. Resultados. Se encontró que los genes relacionados con síntesis de pared, metabolismo y transducción de señales, se expresaban de manera diferencial y con regulación positiva durante la germinaciónlevadura a micelio, mientras que algunos genes relacionados con respuesta a choque térmico y a síntesis de pared estaban sobreexpresados en la transición de micelio a levadura. Los genes restantes se regularon de manera diferencial en ambos procesos. Conclusiones. En este trabajo se confirma la regulación positiva de algunos genes relacionados con los cambios morfológicos de las fases levadura y micelio en P. brasiliensis, procesos biológicos que juegan un papel de importancia durante la interacción huésped-parásito y durante la adaptación del hongo al ambiente, respectivamente.

Evaluación del efecto de cuatro polimorfismos en el gen del receptor adrenérgico beta - 2 en el parto pretérmino / Role of four polymorphisms of the beta - 2 adrenergic receptor gen in patients with preterm delivery

Objetivo: Determinar si los polimorfismos de nucleótido único encontrados en la secuencia del receptor β-2 adrenérgico β2AR) se asocian con el parto pretérmino espontáneo. Método: Se realizó un estudio de casos y controles no pareado en el que se determinaron y compararon las frecuencias alélicas, genotípicas y haplotípicas de cuatro polimorfismos, -47 C/T, -20 C/T, Arg16Gly y Gln27Glu del gen del β2AR, entre 35 mujeres con parto pretérmino (casos) y 105 mujeres con parto a término (controles). El estudio se hizo con pacientes de tres instituciones hospitalarias de la ciudad de Medellín, Colombia. Se aisló el ADN genómico de sangre periférica, se amplificó el segmento del gen mediante la reacción en cadena de la polimerasa (PCR) y se determinaron los polimorfismos por secuenciación directa. Resultados: No se encontraron diferencias significativas de las frecuencias alélicas de los loci polimórficos -47 C/T, -20 C/T, Arg16Gly y Gln27Glu entre el grupo de casos (21.5%, 21.5%, 51.5% y 21.5%, respectivamente) y los controles (21.5%, 21.5%, 57% y 21.5%, respectivamente (p>0.05). Las frecuencias genotípicas también fueron similares entre las pacientes con parto pretérmino y las de parto a término (p>0.05 para todos los genotipos). De los 16 posibles haplotipos, resultantes de la combinación entre los distintos alelos, sólo se presentaron tres en toda la población de estudio (CCGG, TTGC, TTAC). Las frecuencias haplotípicas tampoco tuvieron diferencias significativas entre los grupos comparados (p>0.05). Conclusión: No se observó una asociación entre el parto pretérmino y cualquiera de los polimorfismos o sus haplotipos. Estos resultados no apoyan una participación de los polimorfismos del β2AR en el trabajo de parto pretérmino espontßneo en estas pacientes.

Objective: To determine whether single nucleotide polymorphisms found in the sequence of the β-2 (β2AR) adrenergic receptor are associated with spontaneous preterm delivery. Method: A case-control study in which the allelic, genotype, and haplotype frequencies of four polymorphisms, -47 C/T, -20 C/T, Arg16Gly, and Gln27Glu of the β2AR gene, were determined and compared between 35 women with preterm delivery (cases) and 105 women with term delivery (controls). The study was performed with patients of three hospitals in the city of Medellín, Colombia. The genomic DNA of peripheral blood was isolated, the segment of the gene was amplified by means of the chain reaction of polymerase (PCR), and the polymorphisms were determined by direct sequence method. Results: No significant differences for the allelic frequencies were found for polymorphic loci -47 C/T, -20 C/T, Arg16Gly, and Gln27Glu between the cases group (21.5%, 21.5%, 51.5% and 21.5%, respectively) and controls (21.5%, 21.5%, 57% and 21.5%, respectively; p>0.05). The genotype frequencies were also similar between patients with preterm delivery and those with term delivery (p>0.05 for all the genotypes). Only three of the 16 possible haplotypes (CCGG, TTGC, and TTAC), from the combination between the different alleles, appeared in the participants of this study. The haplotype frequencies were not significantly different between the compared groups either (p>0.05). Conclusion: No association was observed between preterm delivery and any of the polymorphisms or its haplotypes. These results do not support a role of the polymorphisms of the β2AR in spontaneous preterm delivery for these patients.