Aneuploidy detection in pooled polar bodies using rapid nanopore sequencing.

PURPOSE: Various screening techniques have been developed for preimplantation genetic testing for aneuploidy (PGT-A) to reduce implantation failure and miscarriages in women undergoing in vitro fertilisation (IVF) treatment. Among these methods, the Oxford nanopore technology (ONT) has already been tested in several tissues. However, no studies have applied ONT to polar bodies, a cellular material that is less restrictively regulated for PGT-A in some countries. METHODS: We performed rapid short nanopore sequencing on pooled first and second polar bodies of 102 oocytes from women undergoing IVF treatment to screen for aneuploidy. An automated analysis pipeline was developed with the expectation of three chromatids per chromosome. The results were compared to those obtained by array-based comparative genomic hybridisation (aCGH). RESULTS: ONT and aCGH were consistent for 96% (98/102) of sample ploidy classification. Of those samples, 36 were classified as euploid, while 62 were classified as aneuploid. The four discordant samples were assessed as euploid using aCGH but classified as aneuploid using ONT. The concordance of the ploidy classification (euploid, gain, or loss) per chromosome was 92.5% (2169 of 2346 of analysed chromosomes) using aCGH and ONT and increased to 97.7% (2113/2162) without the eight samples assessed as highly complex aneuploid using ONT. CONCLUSION: The automated detection of the ploidy classification per chromosome and shorter duplications or deletions depending on the sequencing depth demonstrates an advantage of the ONT method over standard, commercial aCGH methods, which do not consider the presence of three chromatids in pooled polar bodies.

Generation and Characterization of a Human Neuronal In Vitro Model for Rett Syndrome Using a Direct Reprogramming Method.

Rett Syndrome (RTT) is a severe neurodevelopmental disorder, afflicting 1 in 10,000 female births. It is caused by mutations in the X-linked methyl-CpG-binding protein gene (MECP2), which encodes for the global transcriptional regulator methyl CpG binding protein 2 (MeCP2). As human brain samples of RTT patients are scarce and cannot be used for downstream studies, there is a pressing need for in vitro modeling of pathological neuronal changes. In this study, we use a direct reprogramming method for the generation of neuronal cells from MeCP2-deficient and wild-type human dermal fibroblasts using two episomal plasmids encoding the transcription factors SOX2 and PAX6. We demonstrated that the obtained neurons exhibit a typical neuronal morphology and express the appropriate marker proteins. RNA-sequencing confirmed neuronal identity of the obtained MeCP2-deficient and wild-type neurons. Furthermore, these MeCP2-deficient neurons reflect the pathophysiology of RTT in vitro, with diminished dendritic arborization and hyperacetylation of histone H3 and H4. Treatment with MeCP2, tethered to the cell penetrating peptide TAT, ameliorated hyperacetylation of H4K16 in MeCP2-deficient neurons, which strengthens the RTT relevance of this cell model. We generated a neuronal model based on direct reprogramming derived from patient fibroblasts, providing a powerful tool to study disease mechanisms and investigating novel treatment options for RTT.

Unraveling metabolic patterns and molecular mechanisms underlying storability in sugar beet.

BACKGROUND: Sugar beet is an important crop for sugar production. Sugar beet roots are stored up to several weeks post-harvest waiting for processing in the sugar factories. During this time, sucrose loss and invert sugar accumulation decreases the final yield and processing quality. To improve storability, more information about post-harvest metabolism is required. We investigated primary and secondary metabolites of six sugar beet varieties during storage. Based on their variety-specific sucrose loss, three storage classes representing well, moderate, and bad storability were compared. Furthermore, metabolic data were visualized together with transcriptome data to identify potential mechanisms involved in the storage process. RESULTS: We found that sugar beet varieties that performed well during storage have higher pools of 15 free amino acids which were already observable at harvest. This storage class-specific feature is visible at harvest as well as after 13 weeks of storage. The profile of most of the detected organic acids and semi-polar metabolites changed during storage. Only pyroglutamic acid and two semi-polar metabolites, including ferulic acid, show higher levels in well storable varieties before and/or after 13 weeks of storage. The combinatorial OMICs approach revealed that well storable varieties had increased downregulation of genes involved in amino acid degradation before and after 13 weeks of storage. Furthermore, we found that most of the differentially genes involved in protein degradation were downregulated in well storable varieties at both timepoints, before and after 13 weeks of storage. CONCLUSIONS: Our results indicate that increased levels of 15 free amino acids, pyroglutamic acid and two semi-polar compounds, including ferulic acid, were associated with a better storability of sugar beet taproots. Predictive metabolic patterns were already apparent at harvest. With respect to elongated storage, we highlighted the role of free amino acids in the taproot. Using complementary transcriptomic data, we could identify potential underlying mechanisms of sugar beet storability. These include the downregulation of genes for amino acid degradation and metabolism as well as a suppressed proteolysis in the well storable varieties.

Comparing de novo transcriptome assembly tools in di- and autotetraploid non-model plant species.

BACKGROUND: Polyploidy is very common in plants and can be seen as one of the key drivers in the domestication of crops and the establishment of important agronomic traits. It can be the main source of genomic repatterning and introduces gene duplications, affecting gene expression and alternative splicing. Since fully sequenced genomes are not yet available for many plant species including crops, de novo transcriptome assembly is the basis to understand molecular and functional mechanisms. However, in complex polyploid plants, de novo transcriptome assembly is challenging, leading to increased rates of fused or redundant transcripts. Since assemblers were developed mainly for diploid organisms, they may not well suited for polyploids. Also, comparative evaluations of these tools on higher polyploid plants are extremely rare. Thus, our aim was to fill this gap and to provide a basic guideline for choosing the optimal de novo assembly strategy focusing on autotetraploids, as the scientific interest in this type of polyploidy is steadily increasing. RESULTS: We present a comparison of two common (SOAPdenovo-Trans, Trinity) and one recently published transcriptome assembler (TransLiG) on diploid and autotetraploid species of the genera Acer and Vaccinium using Arabidopsis thaliana as a reference. The number of assembled transcripts was up to 11 and 14 times higher with an increased number of short transcripts for Acer and Vaccinium, respectively, compared to A. thaliana. In diploid samples, Trinity and TransLiG performed similarly good while in autotetraploids, TransLiG assembled most complete transcriptomes with an average of 1916 assembled BUSCOs vs. 1705 BUSCOs for Trinity. Of all three assemblers, SOAPdenovo-Trans performed worst (1133 complete BUSCOs). CONCLUSION: All three assembly tools produced complete assemblies when dealing with the model organism A. thaliana, independently of its ploidy level, but their performances differed extremely when it comes to non-model autotetraploids, where specifically TransLiG and Trinity produced a high number of redundant transcripts. The recently published assembler TransLiG has not been tested yet on any plant organism but showed highest completeness and full-length transcriptomes, especially in autotetraploids. Including such species during the development and testing of new assembly tools is highly appreciated and recommended as many important crops are polyploid.

Correction to: Integrative transcriptomics reveals genotypic impact on sugar beet storability.

In the above mentioned publication, part of Fig. 6B was distorted (extra diagonal lines appeared). The original article has been corrected and the proper version of Fig. 6B is also published here.

Integrative transcriptomics reveals genotypic impact on sugar beet storability.

KEY MESSAGE: An integrative comparative transcriptomic approach on six sugar beet varieties showing different amount of sucrose loss during storage revealed genotype-specific main driver genes and pathways characterizing storability. Sugar beet is next to sugar cane one of the most important sugar crops accounting for about 15% of the sucrose produced worldwide. Since its processing is increasingly centralized, storage of beet roots over an extended time has become necessary. Sucrose loss during storage is a major concern for the sugar industry because the accumulation of invert sugar and byproducts severely affect sucrose manufacturing. This loss is mainly due to ongoing respiration, but changes in cell wall composition and pathogen infestation also contribute. While some varieties can cope better during storage, the underlying molecular mechanisms are currently undiscovered. We applied integrative transcriptomics on six varieties exhibiting different levels of sucrose loss during storage. Already prior to storage, well storable varieties were characterized by a higher number of parenchyma cells, a smaller cell area, and a thinner periderm. Supporting these findings, transcriptomics identified changes in genes involved in cell wall modifications. After 13 weeks of storage, over 900 differentially expressed genes were detected between well and badly storable varieties, mainly in the category of defense response but also in carbohydrate metabolism and the phenylpropanoid pathway. These findings were confirmed by gene co-expression network analysis where hub genes were identified as main drivers of invert sugar accumulation and sucrose loss. Our data provide insight into transcriptional changes in sugar beet roots during storage resulting in the characterization of key pathways and hub genes that might be further used as markers to improve pathogen resistance and storage properties.

Elucidating Drought Stress Tolerance in European Oaks Through Cross-Species Transcriptomics.

The impact of climate change that comes with a dramatic increase of long periods of extreme summer drought associated with heat is a fundamental challenge for European forests. As a result, forests are expected to shift their distribution patterns toward north-east, which may lead to a dramatic loss in value of European forest land. Consequently, unraveling key processes that underlie drought stress tolerance is not only of great scientific but also of utmost economic importance for forests to withstand future heat and drought wave scenarios. To reveal drought stress-related molecular patterns we applied cross-species comparative transcriptomics of three major European oak species: the less tolerant deciduous pedunculate oak (Quercus robur), the deciduous but quite tolerant pubescent oak (Q. pubescens), and the very tolerant evergreen holm oak (Q. ilex). We found 415, 79, and 222 differentially expressed genes during drought stress in Q. robur, Q. pubescens, and Q. ilex, respectively, indicating species-specific response mechanisms. Further, by comparative orthologous gene family analysis, 517 orthologous genes could be characterized that may play an important role in drought stress adaptation on the genus level. New regulatory candidate pathways and genes in the context of drought stress response were identified, highlighting the importance of the antioxidant capacity, the mitochondrial respiration machinery, the lignification of the water transport system, and the suppression of drought-induced senescence - providing a valuable knowledge base that could be integrated in breeding programs in the face of climate change.