Computational methods for transcriptome annotation and quantification using RNA-seq

High-throughput RNA sequencing (RNA-seq) promises a comprehensive picture of the transcriptome, allowing for the complete annotation and quantification of all genes and their isoforms across samples. Realizing this promise requires increasingly complex computational methods. These computational challenges fall into three main categories: (i) read mapping, (ii) transcriptome reconstruction and (iii) expression quantification. Here we explain the major conceptual and practical challenges, and the general classes of solutions for each category. Finally, we highlight the interdependence between these categories and discuss the benefits for different biological applications

Sex estimation in a Turkish population using Purkait’s triangle: a virtual approach by 3-dimensional computed tomography (3D-CT)

Sex estimation is considered one of the first steps in the forensic identification process. Morphological and morphometrical differences between males and females have been used as means for morphoscopic and metric methods on both cranial and postcranial skeletal elements. When dry skeletal elements are not available, virtual data can be used as a substitute. The present research explores 3-dimensional (3D) scans from a Turkish population to test a sex estimation method developed by Purkait (2005). Overall, 296 individuals were used in this study (158 males and 138 females). Purkait’s triangle parameters were measured on computed tomography (CT) scans obtained from both right and left femora of each patient at the Bakirkoy Dr. Sadi Konuk Training Research Hospital (Istanbul, Turkey). Intra- and inter-observer errors were assessed for all variables through technical error of measurements analysis. Bilateral asymmetry and sex differences were evaluated using parametric and non-parametric statistical approaches. Univariate and multivariate discriminant function analyses were then conducted. Observer errors demonstrated an overall agreement within and between experts, as indicated by technical error of measurement (TEM) results. No bilateral asymmetries were reported, and all parameters demonstrated a statistically significant difference between males and females. Fourteen discriminant models were generated by applying single and combined parameters, producing a total correct sex classification ranging from 78.4% to 92.6%. In addition, over 67% of the total sample was accurately classified, with 95% or greater posterior probabilities. Our study demonstrates the feasibility of 3D sex estimation using Purkait’s triangle on a Turkish population, with accuracy rates comparable to those reported in other populations. This is the first attempt to apply this method on virtual data and although further validation and standardisation are recommended for its application on dry bone, this research constitutes a significant contribution to the development of population-specific standards when only virtual data are available

Computational methods for transcriptome annotation and quantification using RNA-seq

High-throughput RNA sequencing (RNA-seq) promises a comprehensive picture of the transcriptome, allowing for the complete annotation and quantification of all genes and their isoforms across samples. Realizing this promise requires increasingly complex computational methods. These computational challenges fall into three main categories: (i) read mapping, (ii) transcriptome reconstruction and (iii) expression quantification. Here we explain the major conceptual and practical challenges, and the general classes of solutions for each category. Finally, we highlight the interdependence between these categories and discuss the benefits for different biological applications

Impact of increasing levels of adaptive statistical iterative reconstruction on image quality in oil-based postmortem CT angiography in coronary arteries.

Postmortem multi-detector computed tomography (PMCT) has become an important part in forensic imaging. Modern reconstruction techniques such as iterative reconstruction (IR) are frequently used in postmortem CT angiography (PMCTA). The image quality of PMCTA depends on the strength of IR. For this purpose, we aimed to investigate the impact of different advanced IR levels on the objective and subjective PMCTA image quality. We retrospectively analyzed the coronary arteries of 27 human cadavers undergoing whole-body postmortem CT angiography between July 2017 and March 2018 in a single center. Iterative reconstructions of the coronary arteries were processed in five different level settings (0%; 30%; 50%; 70%; 100%) by using an adaptive statistical IR method. We evaluated the objective (contrast-to-noise ratio (CNR)) and subjective image quality in several anatomical locations. Our results demonstrate that the increasing levels of an IR technique have relevant impact on the image quality in PMCTA scans in forensic postmortem examinations. Higher levels of IR have led to a significant reduction of image noise and therefore to a significant improvement of objective image quality (+ 70%). However, subjective image quality is inferior at higher levels of IR due to plasticized image appearance. Objective image quality in PMCTA progressively improves with increasing level of IR with the best CNR at the highest IR level. However, subjective image quality is best at low to medium levels of IR. To obtain a "classic" image appearance with optimal image quality, PMCTAs should be reconstructed at medium levels of IR

Vestibular Perception following Acute Unilateral Vestibular Lesions.

Little is known about the vestibulo-perceptual (VP) system, particularly after a unilateral vestibular lesion. We investigated vestibulo-ocular (VO) and VP function in 25 patients with vestibular neuritis (VN) acutely (2 days after onset) and after compensation (recovery phase, 10 weeks). Since the effect of VN on reflex and perceptual function may differ at threshold and supra-threshold acceleration levels, we used two stimulus intensities, acceleration steps of 0.5°/s(2) and velocity steps of 90°/s (acceleration 180°/s(2)). We hypothesised that the vestibular lesion or the compensatory processes could dissociate VO and VP function, particularly if the acute vertiginous sensation interferes with the perceptual tasks. Both in acute and recovery phases, VO and VP thresholds increased, particularly during ipsilesional rotations. In signal detection theory this indicates that signals from the healthy and affected side are still fused, but result in asymmetric thresholds due to a lesion-induced bias. The normal pattern whereby VP thresholds are higher than VO thresholds was preserved, indicating that any 'perceptual noise' added by the vertigo does not disrupt the cognitive decision-making processes inherent to the perceptual task. Overall, the parallel findings in VO and VP thresholds imply little or no additional cortical processing and suggest that vestibular thresholds essentially reflect the sensitivity of the fused peripheral receptors. In contrast, a significant VO-VP dissociation for supra-threshold stimuli was found. Acutely, time constants and duration of the VO and VP responses were reduced - asymmetrically for VO, as expected, but surprisingly symmetrical for perception. At recovery, VP responses normalised but VO responses remained shortened and asymmetric. Thus, unlike threshold data, supra-threshold responses show considerable VO-VP dissociation indicative of additional, higher-order processing of vestibular signals. We provide evidence of perceptual processes (ultimately cortical) participating in vestibular compensation, suppressing asymmetry acutely in unilateral vestibular lesions

Metatranscriptomics captures dynamic shifts in mycorrhizal coordination in boreal forests

Carbon storage and cycling in boreal forests—the largest terrestrial carbon store—ismoderated by complex interactions between trees and soil microorganisms. However,existing methods limit our ability to predict how changes in environmental conditionswill alter these associations and the essential ecosystem services they provide. To addressthis, we developed a metatranscriptomic approach to analyze the impact of nutrientenrichment on Norway sprucefine roots and the community structure, function, andtree–microbe coordination of over 350 root-associated fungal species. In response toaltered nutrient status, host trees redefined their relationship with the fungal commu-nity by reducing sugar efflux carriers and enhancing defense processes. This resulted ina profound restructuring of the fungal community and a collapse in functional coordi-nation between the tree and the dominant Basidiomycete species, and an increase infunctional coordination with versatile Ascomycete species. As such, there was a func-tional  shift  in  community  dominance  from  Basidiomycetes  species,  with  importantroles in enzymatically cycling recalcitrant carbon, to Ascomycete species that have mela-nized cell walls that are highly resistant to degradation. These changes were accompa-nied  by  prominent  shifts  in  transcriptional  coordination  between  over  60  predictedfungal effectors, with more than 5,000 Norway spruce transcripts, providing mechanis-tic insight into the complex molecular dialogue coordinating host trees and their fungalpartners. The host–microbe dynamics captured by this study functionally inform howthese complex and  sensitive biological  relationships may mediate  the carbon  storagepotential of boreal soils under changing nutrient conditions

Epstein-Barr virus transcription factor Zta acts through distal regulatory elements to directly control cellular gene expression

Lytic replication of the human gamma herpes virus Epstein-Barr virus (EBV) is an essential prerequisite for the spread of the virus. Differential regulation of a limited number of cellular genes has been reported in B-cells during the viral lytic replication cycle. We asked whether a viral bZIP transcription factor, Zta (BZLF1, ZEBRA, EB1), drives some of these changes. Using genome-wide chromatin immunoprecipitation coupled to next-generation DNA sequencing (ChIP-seq) we established a map of Zta interactions across the human genome. Using sensitive transcriptome analyses we identified 2263 cellular genes whose expression is significantly changed during the EBV lytic replication cycle. Zta binds 278 of the regulated genes and the distribution of binding sites shows that Zta binds mostly to sites that are distal to transcription start sites. This differs from the prevailing view that Zta activates viral genes by binding exclusively at promoter elements. We show that a synthetic Zta binding element confers Zta regulation at a distance and that distal Zta binding sites from cellular genes can confer Zta-mediated regulation on a heterologous promoter. This leads us to propose that Zta directly reprograms the expression of cellular genes through distal elements

Exploiting Nucleotide Composition to Engineer Promoters

The choice of promoter is a critical step in optimizing the efficiency and stability of recombinant protein production in mammalian cell lines. Artificial promoters that provide stable expression across cell lines and can be designed to the desired strength constitute an alternative to the use of viral promoters. Here, we show how the nucleotide characteristics of highly active human promoters can be modelled via the genome-wide frequency distribution of short motifs: by overlapping motifs that occur infrequently in the genome, we constructed contiguous sequence that is rich in GC and CpGs, both features of known promoters, but lacking homology to real promoters. We show that snippets from this sequence, at 100 base pairs or longer, drive gene expression in vitro in a number of mammalian cells, and are thus candidates for use in protein production. We further show that expression is driven by the general transcription factors TFIIB and TFIID, both being ubiquitously present across cell types, which results in less tissue- and species-specific regulation compared to the viral promoter SV40. We lastly found that the strength of a promoter can be tuned up and down by modulating the counts of GC and CpGs in localized regions. These results constitute a “proof-of-concept” for custom-designing promoters that are suitable for biotechnological and medical applications