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Background: Microsporidia are a large taxon of intracellular pathogens characterized by extraordinarily streamlined genomes with unusually high sequence divergence and many species-specific adaptations. These unique factors pose challenges for traditional genome annotation methods based on sequence similarity. As a result, many of the microsporidian genomes sequenced to date contain numerous genes of unknown function. Recent innovations in rapid and accurate structure prediction and comparison, together with the growing amount of data in structural databases, provide new opportunities to assist in the functional annotation of newly sequenced genomes., Results: In this study, we established a workflow that combines sequence and structure-based functional gene annotation approaches employing a ChimeraX plugin named ANNOTEX (Annotation Extension for ChimeraX), allowing for visual inspection and manual curation. We employed this workflow on a high-quality telomere-to-telomere sequenced tetraploid genome of Vairimorpha necatrix. First, the 3080 predicted protein-coding DNA sequences, of which 89% were confirmed with RNA sequencing data, were used as input. Next, ColabFold was used to create protein structure predictions, followed by a Foldseek search for structural matching to the PDB and AlphaFold databases. The subsequent manual curation, using sequence and structure-based hits, increased the accuracy and quality of the functional genome annotation compared to results using only traditional annotation tools. Our workflow resulted in a comprehensive description of the V. necatrix genome, along with a structural summary of the most prevalent protein groups, such as the ricin B lectin family. In addition, and to test our tool, we identified the functions of several previously uncharacterized Encephalitozoon cuniculi genes., Conclusion: We provide a new functional annotation tool for divergent organisms and employ it on a newly sequenced, high-quality microsporidian genome to shed light on this uncharacterized intracellular pathogen of Lepidoptera. The addition of a structure-based annotation approach can serve as a valuable template for studying other microsporidian or similarly divergent species., (© 2023. The Author(s).)

Background: Chromosomal rearrangements in the form of deletions, insertions, inversions and translocations are frequently observed in breast cancer genomes, and a subset of these rearrangements may play a crucial role in tumorigenesis. To identify novel somatic chromosomal rearrangements, we determined the genome structures of 15 hormone-receptor negative breast tumors by long-insert mate pair massively parallel sequencing., Results: We identified and validated 40 somatic structural alterations, including the recurring fusion between genes DDX10 and SKA3 and translocations involving the EPHA5 gene. Other rearrangements were found to affect genes in pathways involved in epigenetic regulation, mitosis and signal transduction, underscoring their potential role in breast tumorigenesis. RNA interference-mediated suppression of five candidate genes (DDX10, SKA3, EPHA5, CLTC and TNIK) led to inhibition of breast cancer cell growth. Moreover, downregulation of DDX10 in breast cancer cells lead to an increased frequency of apoptotic nuclear morphology., Conclusions: Using whole genome mate pair sequencing and RNA interference assays, we have discovered a number of novel gene rearrangements in breast cancer genomes and identified DDX10, SKA3, EPHA5, CLTC and TNIK as potential cancer genes with impact on the growth and proliferation of breast cancer cells.

Background: Tourette Syndrome (TS) is a neuropsychiatric disorder in children characterized by motor and verbal tics. Although several genes have been suggested in the etiology of TS, the genetic mechanisms remain poorly understood., Methods: Using cytogenetics and FISH analysis, we identified an apparently balanced t(6,22)(q16.2;p13) in a male patient with TS and obsessive-compulsive disorder (OCD). In order to map the breakpoints and to identify additional submicroscopic rearrangements, we performed whole genome mate-pair sequencing and CGH-array analysis on DNA from the proband., Results: Sequence and CGH array analysis revealed a 400 kb deletion located 1.3 Mb telomeric of the chromosome 6q breakpoint, which has not been reported in controls. The deletion affects three genes (GPR63, NDUFA4 and KLHL32) and overlaps a region previously found deleted in a girl with autistic features and speech delay. The proband's mother, also a carrier of the translocation, was diagnosed with OCD and shares the deletion. We also describe a further potentially related rearrangement which, while unmapped in Homo sapiens, was consistent with the chimpanzee genome., Conclusions: We conclude that genome-wide sequencing at relatively low resolution can be used for the identification of submicroscopic rearrangements. We also show that large rearrangements may escape detection using standard analysis of whole genome sequencing data. Our findings further provide a candidate region for TS and OCD on chromosome 6q16.

Background: Double-hit lymphoma is a complex and highly aggressive sub-type of B-cell lymphoma, which has recently been classified and is an area of active research interest due to the poor prognosis for patients with this disease. It is characterized by the presence of both an activating MYC chromosomal translocation and a simultaneous additional oncogenic translocation, often of the BCL2 gene. Recently, a cell line was established from a patient with this complex lymphoma and analyzed using conventional tools revealing it contains both MYC and BCL2 translocation events., Findings: In this work, we reanalyzed the genome of the cell line using next generation whole genome sequencing technology in order to catalogue translocations, insertions and deletions which may contribute to the pathology of this lymphoma type., Conclusions: We describe the cell line in much greater detail, and pinpoint the exact locations of the chromosomal breakpoints. We also find several rearrangements within cancer-associated genes, which were not found using conventional tools, suggesting that high throughput sequencing may reveal novel targets for therapy, which could be used concurrently with existing treatments.