The scale of the ongoing SARS-CoV-2 pandemic warrants the urgent establishment of a global decentralized surveillance system to recognize local outbreaks and the emergence of novel variants of concern. Among available deep-sequencing technologies, nanopore-sequencing could be an important cornerstone, as it is mobile, scalable, and cost-effective. Therefore, streamlined nanopore-sequencing protocols need to be developed and optimized for SARS-CoV-2 variants identification. We adapted and simplified existing workflows using the ‘midnight’ 1200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost the entire SARS-CoV-2 genome. Subsequently, we applied Oxford Nanopore Rapid Barcoding and the portable MinION Mk1C sequencer combined with the interARTIC bioinformatics pipeline. We tested a simplified and less time-consuming workflow using SARS-CoV-2-positive specimens from clinical routine and identified the CT value as a useful pre-analytical parameter, which may help to decrease sequencing failures rates. Complete pipeline duration was approx. 7 h for one specimen and approx. 11 h for 12 multiplexed barcoded specimens. The adapted protocol contains fewer processing steps and can be completely conducted within one working day. Diagnostic CT values deduced from qPCR standardization experiments can act as principal criteria for specimen selection. As a guideline, SARS-CoV-2 genome copy numbers lower than 4 × 10⁶ were associated with a coverage threshold below 20-fold and incompletely assembled SARS-CoV-2 genomes. Thus, based on the described thermocycler/chemistry combination, we recommend CT values of ~26 or lower to achieve full and high-quality SARS-CoV-2 (+)RNA genome coverage.

Bacteria, as well as eukaryotes, principally fungi, of the upper respiratory tract play key roles in the etiopathogenesis of respiratory diseases, whereas the potential role of archaea remains poorly understood. In this review, we discuss the contribution of all three domains of cellular life to human naso- and oropharyngeal microbiomes, i.e., bacterial microbiota, eukaryotes (mostly fungi), as well as the archaeome and their relation to respiratory and atopic disorders in infancy and adolescence. With this review, we aim to summarize state-of-the-art contributions to the field published in the last decade. In particular, we intend to build bridges between basic and clinical science.

In the ciliate Stylonychia, somatic macronuclei differentiate from germline micronuclei during sexual reproduction, accompanied by developmental sequence reduction. Concomitantly, over 95% of micronuclear sequences adopt a heterochromatin structure characterized by the histone variant H3.4 and H3K27me3. RNAi-related genes and histone variants dominate the list of developmentally expressed genes. Simultaneously, 27nt-ncRNAs that match sequences retained in new macronuclei are synthesized and bound by PIWI1. Recently, we proposed a mechanistic model for ‘RNA-induced DNA replication interference’ (RIRI): during polytene chromosome formation PIWI1/27nt-RNA-complexes target macronucleus-destined sequences (MDS) by base-pairing and temporarily cause locally stalled replication. At polytene chromosomal segments with ongoing replication, H3.4K27me3-nucleosomes become selectively deposited, thus dictating the prospective heterochromatin structure of these areas. Consequently, these micronucleus-specific sequences become degraded, whereas 27nt-RNA-covered sites remain protected. However, the biogenesis of the 27nt-RNAs remains unclear. It was proposed earlier that in stichotrichous ciliates 27nt-RNA precursors could derive from telomere-primed bidirectional transcription of nanochromosomes and subsequent Dicer-like (DCL) activity. As a minimalistic explanation, we propose here that the 27nt-RNA precursor could rather be mRNA or pre-mRNA and that the transition of coding RNA from parental macronuclei to non-coding RNAs, which act in premature developing macronuclei, could involve RNA-dependent RNA polymerase (RDRP) activity creating dsRNA intermediates prior to a DCL-dependent pathway. Interestingly, by such mechanism the partition of a parental somatic genome and possibly also the specific nanochromosome copy numbers could be vertically transmitted to the differentiating nuclei of the offspring.

In contrast to bacteria, microbiome analyses often neglect archaea, but also eukaryotes. This is partly because they are difficult to culture due to their demanding growth requirements, or some even have to be classified as uncultured microorganisms. Consequently, little is known about the relevance of archaea in human health and diseases. Contemporary broad availability and spread of next generation sequencing techniques now enable a stronger focus on such microorganisms, whose cultivation is difficult. However, due to the enormous evolutionary distances between bacteria, archaea and eukaryotes, the implementation of sequencing strategies for smaller laboratory scales needs to be refined to achieve as a holistic view on the microbiome as possible. Here, we present a technical approach that enables simultaneous analyses of archaeal, bacterial and eukaryotic microbial communities to study their roles in development and courses of respiratory disorders. We thus applied combinatorial 16S-/18S-rDNA sequencing strategies for sequencing-library preparation. Considering the lower total microbiota density of airway surfaces, when compared with gut microbiota, we optimized the DNA purification workflow from nasopharyngeal swab specimens. As a result, we provide a protocol that allows the efficient combination of bacterial, archaeal, and eukaryotic libraries for nanopore-sequencing using Oxford Nanopore Technologies MinION devices and subsequent phylogenetic analyses. In a pilot study, this workflow allowed the identification of some environmental archaea, which were not correlated with airway microbial communities before. Moreover, we assessed the protocol’s broader applicability using a set of human stool samples. We conclude that the proposed protocol provides a versatile and adaptable tool for combinatorial studies on bacterial, archaeal, and eukaryotic microbiomes on a small laboratory scale.

Testicular cell differentiation is a highly regulated process, essential for male reproductive health. The histone variant H3.5 is apparently a critical player in this intricate orchestra of cell types, but its regulation and function remains poorly understood. To elucidate its role, we fractionized testicular cells using c-Kit/CD117 as a separation marker and analyzed H3.5 expression. Further, we investigated the regulation of H3.5 expression using public data repositories. We explored DNA methylation patterns in specific regions of the H3-5 gene and assessed H3-5 copy number gain in seminoma specimens. Additionally, we examined the testicular localization of H3.5 and its histone chaperone interactions to understand its regulation at the protein level. We used qRT-PCR, MeDIP, and qPCR to study H3.5 expression and DNA methylation in various cell types. H3-5 copy number gain was analyzed using qPCR. Protein interactions were investigated through fluorescence-2-hybrid assays in baby hamster kidney cells. H3.5 is primarily enriched in spermatocytes. DNA methylation of a CpG island overlapping the H3-5 promoter appeared to be involved in the tissue-specific regulation of H3.5 expression. Elevated H3.5 expression was observed in seminoma specimens, suggesting a potential link to testicular tumors. H3-5 copy number gain was associated with elevated H3.5 expression in seminoma specimens. Furthermore, we identified physical interactions between H3.5 and histone chaperones Asf1a and Asf1b, HIRA, CAF p150 and DAXX, shedding light on the protein-level regulation of H3.5. These findings provide valuable insights into the molecular mechanisms governing testicular cell differentiation and the potential role of H3.5 in testicular pathologies.

We established a protocol for fast, cost efficient Sars-CoV-2 sequencing with little as possible hands-on time (around 3h in total, excluding RNA extraction). The whole Sequencing can be done in one working day, including the bioinformatic pipeline. The cost per sample accumulates at around 40$, with already isolated RNA. We adapted and simplified existing workflows using the ‘midnight’ 1,200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost all of the SARS-CoV-2 genome. Subsequently, we applied the Oxford Nanopore Rapid barcoding protocol and the portable MinION Mk1C sequencer in combination with the ARTIC bioinformatics pipeline. We tested the simplified and less time-consuming workflow on confirmed SARS-CoV-2-positive specimens from clinical routine and identified pre-analytical parameters, which may help to decrease the rate of sequencing failures. Duration of the complete pipeline was approx. 7 hrs for one specimen and approx. 11 hrs for 12 multiplexed barcoded specimens. This protocol is a modified version of Nikki Freed and Olin Silanders protocol. To get information such as Primers, visit their protocol. Nikki Freed, Olin Silander 2020. nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon).doi: 10.1093/biomethods/bpaa014 Our peer-reviewed paper is available here: https://www.mdpi.com/2076-2607/9/12/2598