Optimization data for an ARTIC-/Illumina-based whole-genome sequencing protocol and pipeline for SARS-CoV-2 analysis.

In January 2021, Germany commenced surveillance of SARS-CoV-2 variants under the Corona Surveillance Act, which ceased in July 2023. The objective was to bolster pandemic control, as specific alterations in amino acids, particularly within the spike protein, were linked to heightened transmission and decreased vaccine effectiveness. Consequently, our team conducted whole genome sequencing using the commercially accessible ARTIC protocol on Illumina's NextSeq500 platform and MiSeq for SARS-CoV-2 positive samples obtained from patients at Heidelberg University Hospital, affiliated hospitals, and the public health office in the Rhine-Neckar/Heidelberg region. Throughout the pandemic, we refined the existing ARTIC V4 protocol as well as our bioinformatics pipeline, the details of which are outlined in this report. This report reflects the protocol for the MiSeq analysis, the protocol for the NextSeq500 can be found in our previous publication.

Evolution of SARS-CoV-2 in the Rhine-Neckar/Heidelberg Region 01/2021 - 07/2023.

In January 2021, the monitoring of circulating variants of SARS-CoV-2 was initiated in Germany under the Corona Surveillance Act, which was discontinued after July 2023. This initiative aimed to enhance pandemic containment, as specific amino acid changes, particularly in the spike protein, were associated with increased transmission and reduced vaccine efficacy. Our group conducted whole genome sequencing using the ARTIC protocol (currently V4) on Illumina's NextSeq 500 platform (and, starting in May 2023, on the MiSeq DX platform) for SARS-CoV-2 positive specimen from patients at Heidelberg University Hospital, associated hospitals, and the public health office in the Rhine-Neckar/Heidelberg region. In total, we sequenced 26,795 SARS-CoV-2-positive samples between January 2021 and July 2023. Valid sequences, meeting the requirements for upload to the German electronic sequencing data hub (DESH) operated by the Robert Koch Institute (RKI), were determined for 24,852 samples, and the lineage/clade could be identified for 25,912 samples. The year 2021 witnessed significant dynamics in the circulating variants in the Rhine-Neckar/Heidelberg region, including A.27.RN, followed by the emergence of B.1.1.7 (Alpha), subsequently displaced by B.1.617.2 (Delta), and the initial occurrences of B.1.1.529 (Omicron). By January 2022, B.1.1.529 had superseded B.1.617.2, dominating with over 90%. The years 2022 and 2023 were then characterized by the dominance of B.1.1.529 and its sublineages, particularly BA.5 and BA.2, and more recently, the emergence of recombinant variants like XBB.1.5. Since the global dominance of B.1.617.2, the identified variant distribution in our local study, apart from a time delay in the spread of new variants, can be considered largely representative of the global distribution. om a time delay in the spread of new variants, can be considered largely representative of the global distribution.

3D Immuno-DNA Fluorescence In Situ Hybridization (FISH) for Detection of HIV-1 and Cellular Genes in Primary CD4+ T Cells.

Fluorescence in situ hybridization (FISH) is a powerful, broadly used microscopy-based technique that leverages fluorescently labeled nucleic acid probes to detect parts of the genome inside metaphase or interphase cell nuclei. In recent years, different methodologies developed to visualize genome topology and spatial relationships between genes have gained much attention as instruments to decode the relationship between chromatin structure and function. In addition to chromosome conformation capture-based techniques, highly multiplexed forms of FISH combined with high-throughput and super-resolution microscopy are used to map and spatially define contact frequencies between different genomic regions. All these approaches have strongly contributed to our knowledge of how the human genome is packed in the cell nucleus.In this chapter, we describe detailed step-by-step protocols for 3D immuno-DNA FISH detection of genes and Human immunodeficiency virus 1 (HIV-1) provirus in primary CD4+ T cells from healthy donors, or cells infected in vitro with the virus. Our multicolor 3D-FISH technique allows, by using up to three fluorophores, visualization of spatial positioning of loci inside a 3D cell nucleus.

DNA methylation reader MECP2: cell type- and differentiation stage-specific protein distribution.

BACKGROUND: Methyl-CpG binding protein 2 (MECP2) is a protein that specifically binds methylated DNA, thus regulating transcription and chromatin organization. Mutations in the gene have been identified as the principal cause of Rett syndrome, a severe neurological disorder. Although the role of MECP2 has been extensively studied in nervous tissues, still very little is known about its function and cell type specific distribution in other tissues. RESULTS: Using immunostaining on tissue cryosections, we characterized the distribution of MECP2 in 60 cell types of 16 mouse neuronal and non-neuronal tissues. We show that MECP2 is expressed at a very high level in all retinal neurons except rod photoreceptors. The onset of its expression during retina development coincides with massive synapse formation. In contrast to astroglia, retinal microglial cells lack MECP2, similar to microglia in the brain, cerebellum, and spinal cord. MECP2 is also present in almost all non-neural cell types, with the exception of intestinal epithelial cells, erythropoietic cells, and hair matrix keratinocytes. Our study demonstrates the role of MECP2 as a marker of the differentiated state in all studied cells other than oocytes and spermatogenic cells. MECP2-deficient male (Mecp2 (-/y) ) mice show no apparent defects in the morphology and development of the retina. The nuclear architecture of retinal neurons is also unaffected as the degree of chromocenter fusion and the distribution of major histone modifications do not differ between Mecp2 (-/y) and Mecp2 (wt) mice. Surprisingly, the absence of MECP2 is not compensated by other methyl-CpG binding proteins. On the contrary, their mRNA levels were downregulated in Mecp2 (-/y) mice. CONCLUSIONS: MECP2 is almost universally expressed in all studied cell types with few exceptions, including microglia. MECP2 deficiency does not change the nuclear architecture and epigenetic landscape of retinal cells despite the missing compensatory expression of other methyl-CpG binding proteins. Furthermore, retinal development and morphology are also preserved in Mecp2-null mice. Our study reveals the significance of MECP2 function in cell differentiation and sets the basis for future investigations in this direction.