Robustness of differential gene expression analysis of RNA-seq.

RNA-sequencing (RNA-seq) is a relatively new technology that lacks standardisation. RNA-seq can be used for Differential Gene Expression (DGE) analysis, however, no consensus exists as to which methodology ensures robust and reproducible results. Indeed, it is broadly acknowledged that DGE methods provide disparate results. Despite obstacles, RNA-seq assays are in advanced development for clinical use but further optimisation will be needed. Herein, five DGE models (DESeq2, voom + limma, edgeR, EBSeq, NOISeq) for gene-level detection were investigated for robustness to sequencing alterations using a controlled analysis of fixed count matrices. Two breast cancer datasets were analysed with full and reduced sample sizes. DGE model robustness was compared between filtering regimes and for different expression levels (high, low) using unbiased metrics. Test sensitivity estimated as relative False Discovery Rate (FDR), concordance between model outputs and comparisons of a 'population' of slopes of relative FDRs across different library sizes, generated using linear regressions, were examined. Patterns of relative DGE model robustness proved dataset-agnostic and reliable for drawing conclusions when sample sizes were sufficiently large. Overall, the non-parametric method NOISeq was the most robust followed by edgeR, voom, EBSeq and DESeq2. Our rigorous appraisal provides information for method selection for molecular diagnostics. Metrics may prove useful towards improving the standardisation of RNA-seq for precision medicine.

Clinically Actionable Insights into Initial and Matched Recurrent Glioblastomas to Inform Novel Treatment Approaches.

Glioblastoma is the most common primary adult brain tumour, and despite optimal treatment, the median survival is 12-15 months. Patients with matched recurrent glioblastomas were investigated to try to find actionable mutations. Tumours were profiled using a validated DNA-based gene panel. Copy number variations (CNVs) and single nucleotide variants (SNVs) were examined, and potentially pathogenic variants and clinically actionable mutations were identified. The results revealed that glioblastomas were IDH-wildtype (IDH WT; n = 38) and IDH-mutant (IDH MUT; n = 3). SNVs in TSC2, MSH6, TP53, CREBBP, and IDH1 were variants of unknown significance (VUS) that were predicted to be pathogenic in both subtypes. IDH WT tumours had SNVs that impacted RTK/Ras/PI(3)K, p53, WNT, SHH, NOTCH, Rb, and G-protein pathways. Many tumours had BRCA1/2 (18%) variants, including confirmed somatic mutations in haemangioblastoma. IDH WT recurrent tumours had fewer pathways impacted (RTK/Ras/PI(3)K, p53, WNT, and G-protein) and CNV gains (BRCA2, GNAS, and EGFR) and losses (TERT and SMARCA4). IDH MUT tumours had SNVs that impacted RTK/Ras/PI(3)K, p53, and WNT pathways. VUS in KLK1 was possibly pathogenic in IDH MUT. Recurrent tumours also had fewer pathways (p53, WNT, and G-protein) impacted by genetic alterations. Public datasets (TCGA and GDC) confirmed the clinical significance of findings in both subtypes. Overall in this cohort, potentially actionable variation was most often identified in EGFR, PTEN, BRCA1/2, and ATM. This study underlines the need for detailed molecular profiling to identify individual GBM patients who may be eligible for novel treatment approaches. This information is also crucial for patient recruitment to clinical trials.

Population dynamics of rice planthoppers, Nilaparvata lugens and Sogatella furcifera (Hemiptera, Delphacidae) in Central Vietnam and its effects on their spring migration to China.

Rice planthopper (RPH) populations of Nilaparvata lugens and Sogatella furcifera periodically have erupted across Asia. Predicting RPH population dynamics and identifying their source areas are crucial for the management of these migratory pests in China, but the origins of the migrants to temperate and subtropical regions in China remains unclear. In particular, their early migration to China in March and April have not yet been explored due to a lack of research data available from potential source areas, Central Vietnam and Laos. In this study, we examined the population dynamics and migratory paths of N. lugens and S. furcifera in Vietnam and South China in 2012 and 2013. Trajectory modeling showed that in March and April in 2012 and 2013, RPH emigrated from source areas in Central Vietnam where rice was maturing to the Red River Delta and South China. Early migrants originated from Southern Central Vietnam (14-16°N), but later most were from Northern Central Vietnam (16-19°N). Analysis of meteorological and light-trap data from Hepu in April (1977-2013) using generalized linear models showed that immigration increased with precipitation in Southern Central Vietnam in January, but declined with precipitation in Northern Central Vietnam in January. These results determined that the RPH originate from overwintering areas in Central Vietnam, but not from southernmost areas of Vietnam. Winter precipitation, rather than temperature was the most important factor determining the number of RPH migrants. Based on their similar population dynamics and low population densities in Central Vietnam, we further speculated that RPH migrate to track ephemeral food resources whilst simultaneously avoiding predators. Migrations do not seem to be initiated by interspecific competition, overcrowding or host deterioration. Nevertheless, S. furcifera establishes populations earlier than N. lugens South China, perhaps to compensate for interspecific competition. We provide new information that could assist with forecasting outbreaks and implementing control measures against these migratory pests.

Comparative genomic analysis reveals species-dependent complexities that explain difficulties with microsatellite marker development in molluscs.

Reliable population DNA molecular markers are difficult to develop for molluscs, the reasons for which are largely unknown. Identical protocols for microsatellite marker development were implemented in three gastropods. Success rates were lower for Gibbula cineraria compared to Littorina littorea and L. saxatilis. Comparative genomic analysis of 47.2 kb of microsatellite containing sequences (MCS) revealed a high incidence of cryptic repetitive DNA in their flanking regions. The majority of these were novel, and could be grouped into DNA families based upon sequence similarities. Significant inter-specific variation in abundance of cryptic repetitive DNA and DNA families was observed. Repbase scans show that a large proportion of cryptic repetitive DNA was identified as transposable elements (TEs). We argue that a large number of TEs and their transpositional activity may be linked to differential rates of DNA multiplication and recombination. This is likely to be an important factor explaining inter-specific variation in genome stability and hence microsatellite marker development success rates. Gastropods also differed significantly in the type of TEs classes (autonomous vs non-autonomous) observed. We propose that dissimilar transpositional mechanisms differentiate the TE classes in terms of their propensity for transposition, fixation and/or silencing. Consequently, the phylogenetic conservation of non-autonomous TEs, such as CvA, suggests that dispersal of these elements may have behaved as microsatellite-inducing elements. Results seem to indicate that, compared to autonomous, non-autonomous TEs maybe have a more active role in genome rearrangement processes. The implications of the findings for genomic rearrangement, stability and marker development are discussed.