Structural Refinement by Direct Mapping Reveals Assembly Inconsistencies near Hi-C Junctions.

High-throughput chromosome conformation capture (Hi-C) is widely used for scaffolding in de novo assembly because it produces highly contiguous genomes, but its indirect statistical approach can introduce connection errors. We employed optical mapping (Bionano Genomics) as an orthogonal scaffolding technology to assess the structural solidity of Hi-C reconstructed scaffolds. Optical maps were used to assess the correctness of five de novo genome assemblies based on long-read sequencing for contig generation and Hi-C for scaffolding. Hundreds of inconsistencies were found between the reconstructions generated using the Hi-C and optical mapping approaches. Manual inspection, exploiting raw long-read sequencing data and optical maps, confirmed that several of these conflicts were derived from Hi-C joining errors. Such misjoins were widespread, involved the connection of both small and large contigs, and even overlapped annotated genes. We conclude that the integration of optical mapping data after, not before, Hi-C-based scaffolding, improves the quality of the assembly and limits reconstruction errors by highlighting misjoins that can then be subjected to further investigation.

'Nebbiolo' genome assembly allows surveying the occurrence and functional implications of genomic structural variations in grapevines (Vitis vinifera L.).

BACKGROUND: 'Nebbiolo' is a grapevine cultivar typical of north-western Italy, appreciated for producing high-quality red wines. Grapevine cultivars are characterized by possessing highly heterozygous genomes, including a great incidence of genomic rearrangements larger than 50 bp, so called structural variations (SVs). Even though abundant, SVs are an under-explored source of genetic variation mainly due to methodological limitations at their detection. RESULTS: We employed a multiple platform approach to produce long-range genomic data for two different 'Nebbiolo' clones, namely: optical mapping, long-reads and linked-reads. We performed a haplotype-resolved de novo assembly for cultivar 'Nebbiolo' (clone CVT 71) and used an ab-initio strategy to annotate it. The annotated assembly enhanced our ability to detect SVs, enabling the study of genomic regions not present in the grapevines' reference genome and accounting for their functional implications. We performed variant calling analyses at three different organizational levels: i) between haplotypes of clone CVT 71 (primary assembly vs haplotigs), ii) between 'Nebbiolo' and 'Cabernet Sauvignon' assemblies and iii) between clones CVT 71 and CVT 185, representing different 'Nebbiolo' biotypes. The cumulative size of non-redundant merged SVs indicated a total of 79.6 Mbp for the first comparison and 136.1 Mbp for the second one, while no SVs were detected for the third comparison. Interestingly, SVs differentiating cultivars and haplotypes affected similar numbers of coding genes. CONCLUSIONS: Our results suggest that SVs accumulation rate and their functional implications in 'Nebbiolo' genome are highly-dependent on the organizational level under study. SVs are abundant when comparing 'Nebbiolo' to a different cultivar or the two haplotypes of the same individual, while they turned absent between the two analysed clones.

ZNF521 Enhances MLL-AF9-Dependent Hematopoietic Stem Cell Transformation in Acute Myeloid Leukemias by Altering the Gene Expression Landscape.

Leukemias derived from the MLL-AF9 rearrangement rely on dysfunctional transcriptional networks. ZNF521, a transcription co-factor implicated in the control of hematopoiesis, has been proposed to sustain leukemic transformation in collaboration with other oncogenes. Here, we demonstrate that ZNF521 mRNA levels correlate with specific genetic aberrations: in particular, the highest expression is observed in AMLs bearing MLL rearrangements, while the lowest is detected in AMLs with FLT3-ITD, NPM1, or CEBPα double mutations. In cord blood-derived CD34+ cells, enforced expression of ZNF521 provides a significant proliferative advantage and enhances MLL-AF9 effects on the induction of proliferation and the expansion of leukemic progenitor cells. Transcriptome analysis of primary CD34+ cultures displayed subsets of genes up-regulated by MLL-AF9 or ZNF521 single transgene overexpression as well as in MLL-AF9/ZNF521 combinations, at either the early or late time points of an in vitro leukemogenesis model. The silencing of ZNF521 in the MLL-AF9 + THP-1 cell line coherently results in an impairment of growth and clonogenicity, recapitulating the effects observed in primary cells. Taken together, these results underscore a role for ZNF521 in sustaining the self-renewal of the immature AML compartment, most likely through the perturbation of the gene expression landscape, which ultimately favors the expansion of MLL-AF9-transformed leukemic clones.

ACoRE: Accurate SARS-CoV-2 genome reconstruction for the characterization of intra-host and inter-host viral diversity in clinical samples and for the evaluation of re-infections.

Sequencing the SARS-CoV-2 genome from clinical samples can be challenging, especially in specimens with low viral titer. Here we report Accurate SARS-CoV-2 genome Reconstruction (ACoRE), an amplicon-based viral genome sequencing workflow for the complete and accurate reconstruction of SARS-CoV-2 sequences from clinical samples, including suboptimal ones that would usually be excluded even if unique and irreplaceable. The protocol was optimized to improve flexibility and the combination of technical replicates was established as the central strategy to achieve accurate analysis of low-titer/suboptimal samples. We demonstrated the utility of the approach by achieving complete genome reconstruction and the identification of false-positive variants in >170 clinical samples, thus avoiding the generation of inaccurate and/or incomplete sequences. Most importantly, ACoRE was crucial to identify the correct viral strain responsible of a relapse case, that would be otherwise mis-classified as a re-infection due to missing or incorrect variant identification by a standard workflow.

Characterization of Lysinibacillus fusiformis strain S4C11: In vitro, in planta, and in silico analyses reveal a plant-beneficial microbe.

Despite sharing many of the traits that have allowed the genus Bacillus to gain recognition for its agricultural relevance, the genus Lysinibacillus is not as well-known and studied. The present study employs in vitro, in vivo, in planta, and in silico approaches to characterize Lysinibacillus fusiformis strain S4C11, isolated from the roots of an apple tree in northern Italy. The in vitro and in vivo assays demonstrated that strain S4C11 possesses an antifungal activity against different fungal pathogens, and is capable of interfering with the germination of Botrytis cinerea conidia, as well as of inhibiting its growth through the production of volatile organic molecules. In planta assays showed that the strain possesses the ability to promote plant growth, that is not host-specific, both in controlled conditions and in a commercial nursery. Biocontrol assays carried out against phytopathogenic viruses gave contrasting results, suggesting that the strain does not activate the host's defense pathways. The in silico analyses were carried out by sequencing the genome of the strain through an innovative approach that combines Illumina and High-Definition Mapping methods, allowing the reconstruction of a main chromosome and two plasmids from strain S4C11. The analysis of the genes encoded by the genome contributed to the characterization of the strain, detecting genes related to the biocontrol effect detected in the experimental trials.

A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical Settings.

The reconstruction of individual haplotypes can facilitate the interpretation of disease risks; however, high costs and technical challenges still hinder their assessment in clinical settings. Second-generation sequencing is the gold standard for variant discovery but, due to the production of short reads covering small genomic regions, allows only indirect haplotyping based on statistical methods. In contrast, third-generation methods such as the nanopore sequencing platform developed by Oxford Nanopore Technologies (ONT) generate long reads that can be used for direct haplotyping, with fewer drawbacks. However, robust standards for variant phasing in ONT-based target resequencing efforts are not yet available. In this study, we presented a streamlined proof-of-concept workflow for variant calling and phasing based on ONT data in a clinically relevant 12-kb region of the APOE locus, a hotspot for variants and haplotypes associated with aging-related diseases and longevity. Starting with sequencing data from simple amplicons of the target locus, we demonstrated that ONT data allow for reliable single-nucleotide variant (SNV) calling and phasing from as little as 60 reads, although the recognition of indels is less efficient. Even so, we identified the best combination of ONT read sets (600) and software (BWA/Minimap2 and HapCUT2) that enables full haplotype reconstruction when both SNVs and indels have been identified previously using a highly-accurate sequencing platform. In conclusion, we established a rapid and inexpensive workflow for variant phasing based on ONT long reads. This allowed for the analysis of multiple samples in parallel and can easily be implemented in routine clinical practice, including diagnostic testing.

Zoledronic acid inhibits the growth of leukemic MLL-AF9 transformed hematopoietic cells.

A leukemic in vitro model produced by transducing Cord Blood derived-hematopoietic CD34+ cells with the MLL-AF9 translocation resulting in the oncogenic fusion protein, is used to assess for sensitivity to Zoledronic acid. These cells are practically immortalized and are of myeloid origin. Proliferation, clonogenic and stromal co-culture assays showed that the MLL-AF9 cells were considerably more sensitive to Zoledronic acid than normal hematopoietic CD34+ cells or MS-5 stromal cells. The MLL-AF9 cells were notably more inhibited by Zoledronic acid when cultured as colonies in 3 dimensions, requiring cell-cell contacts compared to suspension expansion cultures. This is coherent with the mechanism of action of Zoledronic acid inhibiting farnesyl diphosphate synthase which results in a block in prenylation of GTPases such that their role in the membrane is compromised for cell-cell contacts. Zoledronic acid can be proposed to target the MLL-AF9 leukemic stem cells before they emerge from the hematopoietic niche, which being in proximity to bone osteoclasts where Zoledronic acid is sequestered can be predicted to result in sufficient levels to result in an anti-leukemic action.

Distinct Metabolic Signals Underlie Clone by Environment Interplay in "Nebbiolo" Grapes Over Ripening.

Several research studies were focused to understand how grapevine cultivars respond to environment; nevertheless, the biological mechanisms tuning this phenomenon need to be further deepened. Particularly, the molecular processes underlying the interplay between clones of the same cultivar and environment were poorly investigated. To address this issue, we analyzed the transcriptome of berries from three "Nebbiolo" clones grown in different vineyards, during two ripening seasons. RNA-sequencing data were implemented with analyses of candidate genes, secondary metabolites, and agronomical parameters. This multidisciplinary approach helped to dissect the complexity of clone × environment interactions, by identifying the molecular responses controlled by genotype, vineyard, phenological phase, or a combination of these factors. Transcripts associated to sugar signalling, anthocyanin biosynthesis, and transport were differently modulated among clones, according to changes in berry agronomical features. Conversely, genes involved in defense response, such as stilbene synthase genes, were significantly affected by vineyard, consistently with stilbenoid accumulation. Thus, besides at the cultivar level, clone-specific molecular responses also contribute to shape the agronomic features of grapes in different environments. This reveals a further level of complexity in the regulation of genotype × environment interactions that has to be considered for orienting viticultural practices aimed at enhancing the quality of grape productions.

Chlorella vulgaris genome assembly and annotation reveals the molecular basis for metabolic acclimation to high light conditions.

Chlorella vulgaris is a fast-growing fresh-water microalga cultivated on the industrial scale for applications ranging from food to biofuel production. To advance our understanding of its biology and to establish genetics tools for biotechnological manipulation, we sequenced the nuclear and organelle genomes of Chlorella vulgaris 211/11P by combining next generation sequencing and optical mapping of isolated DNA molecules. This hybrid approach allowed us to assemble the nuclear genome in 14 pseudo-molecules with an N50 of 2.8 Mb and 98.9% of scaffolded genome. The integration of RNA-seq data obtained at two different irradiances of growth (high light, HL versus low light, LL) enabled us to identify 10 724 nuclear genes, coding for 11 082 transcripts. Moreover, 121 and 48 genes, respectively, were found in the chloroplast and mitochondrial genome. Functional annotation and expression analysis of nuclear, chloroplast and mitochondrial genome sequences revealed particular features of Chlorella vulgaris. Evidence of horizontal gene transfers from chloroplast to mitochondrial genome was observed. Furthermore, comparative transcriptomic analyses of LL versus HL provided insights into the molecular basis for metabolic rearrangement under HL versus LL conditions leading to enhanced de novo fatty acid biosynthesis and triacylglycerol accumulation. The occurrence of a cytosolic fatty acid biosynthetic pathway could be predicted and its upregulation upon HL exposure was observed, consistent with the increased lipid amount under HL conditions. These data provide a rich genetic resource for future genome editing studies, and potential targets for biotechnological manipulation of Chlorella vulgaris or other microalgae species to improve biomass and lipid productivity.

A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field.

Genetic markers (DNA barcodes) are often used to support and confirm species identification. Barcode sequences can be generated in the field using portable systems based on the Oxford Nanopore Technologies (ONT) MinION sequencer. However, to achieve a broader application, current proof-of-principle workflows for on-site barcoding analysis must be standardized to ensure a reliable and robust performance under suboptimal field conditions without increasing costs. Here, we demonstrate the implementation of a new on-site workflow for DNA extraction, PCR-based barcoding, and the generation of consensus sequences. The portable laboratory features inexpensive instruments that can be carried as hand luggage and uses standard molecular biology protocols and reagents that tolerate adverse environmental conditions. Barcodes are sequenced using MinION technology and analyzed with ONTrack, an original de novo assembly pipeline that requires as few as 1000 reads per sample. ONTrack-derived consensus barcodes have a high accuracy, ranging from 99.8 to 100%, despite the presence of homopolymer runs. The ONTrack pipeline has a user-friendly interface and returns consensus sequences in minutes. The remarkable accuracy and low computational demand of the ONTrack pipeline, together with the inexpensive equipment and simple protocols, make the proposed workflow particularly suitable for tracking species under field conditions.

SOX2: Not always eye malformations. Severe genital but no major ocular anomalies in a female patient with the recurrent c.70del20 variant.

SOX2 variants have been identified in multiple patients with severe ocular anomalies and pituitary dysfunction, in addition to various systemic features. We investigated a 26-year-old female patient suffering from spastic paraparesis, hypoplasia of corpus callosum, hypogonadotropic hypogonadism (HH) and intellectual disability, who was monitored for over 20 years, allowing a detailed genotype-phenotype correlation along time. Whole exome sequencing on the patient and her relatives identified a de novo SOX2 c.70del20 variant, which has been frequently reported in individuals with SOX2-related anophthalmia. Importantly, our patient lacked major ocular phenotype but showed vaginal agenesis, a feature never reported before. Although the involvement of male urogenital tract (cryptorchidism, hypospadias, small penis), is a well known consequence of SOX2 variants, their effect on the female genitalia has never been properly addressed, even considering the paradoxical female excess of SOX2 cases in the literature. Our findings emphasize the importance of testing for SOX2 variants in individuals with HH and genital anomalies even though anophthalmia or microphthalmia are not observed. Moreover, our case strengthens the role of SOX2 as a master regulator of female gonadal differentiation, as widely demonstrated for other SOX genes related to 46, XX sex reversal, such as SOX3 and SOX9.

Human Neutrophils Produce CCL23 in Response to Various TLR-Agonists and TNFα.

CCL23, also known as myeloid progenitor inhibitory factor (MPIF)-1, macrophage inflammatory protein (MIP)-3, or CKß8, is a member of the CC chemokine subfamily exerting its effects via CCR1 binding. By doing so, CCL23 selectively recruits resting T lymphocytes and monocytes, inhibits proliferation of myeloid progenitor cells and promotes angiogenesis. Previously, we and other groups have reported that human neutrophils are able to produce chemokines upon appropriate activation, including CCR1-binding CCL2, CCL3, and CCL4. Herein, we demonstrate that human neutrophils display the capacity to also express and release CCL23 when stimulated by R848 and, to a lesser extent, by other pro-inflammatory agonists, including LPS, Pam3CSK4, and TNFα. Notably, we show that, on a per cell basis, R848-activated neutrophils produce higher levels of CCL23 than autologous CD14+-monocytes activated under similar experimental conditions. By contrast, we found that, unlike CD14+-monocytes, neutrophils do not produce CCL23 in response to IL-4, thus indicating that they express CCL23 in a stimulus-specific fashion. Finally, we show that the production of CCL23 by R848-stimulated neutrophils is negatively modulated by IFNα, which instead enhances that of CCL2. Together, data extend our knowledge on the chemokines potentially produced by neutrophils. The ability of human neutrophils to produce CCL23 further supports the notion on the neutrophil capacity of orchestrating the recruitment of different cell types to the inflamed sites, in turn contributing to the control of the immune response.

Kinship analysis: assessment of related vs unrelated based on defined pedigrees.

The study aimed at evaluating whether the adoption of enlarged batteries of STR markers in kinship analysis may provide LR values suitable for discrimination of relatives from non-relatives, in comparison to conventionally used STR panels. The presence of LD among some loci and its effects on LR values were also assessed. Three hundred pairs of related and unrelated individuals, each separated from 1-3 generations and residing in North Italy were genotyped with the Investigator HDplex STR kit (Qiagen), AmpFlSTR Identifiler (Applied Biosystems), and PowerPlex Fusion System (Promega). Loci and alleles shared between each pair and within groups of relatives were compared. Also, combined LR values with and without loci in LD, sensitivity and specificity were calculated for each commercial kit and their combinations. Full siblings displayed the largest number of shared loci and alleles, with a proportion of LR ≥ 10 results significantly higher than other degrees of relatedness and, consequently, with the lowest percentage of inconclusive and false negative results. Only minor differences were detected in the combined LR distributions, after including or omitting loci in LD. However, these became only appreciable when analyzing more distant relative pairs.The implementation of additional STRs into the LR calculation allowed a complete and robust discrimination between relatives and non-relatives only for full siblings, by removing the typical uncertainty of the "grey zone", while this was not achieved among other degrees of relatedness. Furthermore, the presence of loci in LD seems to not significantly affect LR distributions within each generation.