Fanning Song (quartfur13)

Technical challenges remain in the sequencing of RNA viruses due to their high intra-host diversity. This bottleneck is particularly pronounced when interrogating long-range co-evolved genetic interactions given the read-length limitations of next-generation sequencing platforms. This has hampered the direct observation of these genetic interactions that code for protein-protein interfaces with relevance in both drug and vaccine development. Here we overcome these technical limitations by developing a nanopore-based long-range viral sequencing pipeline that yields accurate single molecule sequences of circulating virions from clinical samples. We demonstrate its utility in observing the evolution of individual HIV Gag-Pol genomes in response to antiviral pressure. Our pipeline, called Multi-read Hairpin Mediated Error-correction Reaction (MrHAMER), yields >1000s of viral genomes per sample at 99.9% accuracy, maintains the original proportion of sequenced virions present in a complex mixture, and allows the detection of rare viral genomes with their associated mutations present at less then 1% frequency. This method facilitates scalable investigation of genetic correlates of resistance to both antiviral therapy and immune pressure and enables the identification of novel host-viral and viral-viral interfaces that can be modulated for therapeutic benefit.The eukaryotic replisome is comprised of three family-B DNA polymerases (Polα, δ and ϵ). Polα forms a stable complex with primase to synthesize short RNA-DNA primers, which are subsequently elongated by Polδ and Polϵ in concert with proliferating cell nuclear antigen (PCNA). In some species of archaea, family-D DNA polymerase (PolD) is the only DNA polymerase essential for cell viability, raising the question of how it alone conducts the bulk of DNA synthesis. We used a hyperthermophilic archaeon, Thermococcus kodakarensis, to demonstrate that PolD connects primase to the archaeal replisome before interacting with PCNA. Whereas PolD stably connects primase to GINS, a component of CMG helicase, cryo-EM analysis indicated a highly flexible PolD-primase complex. A conserved hydrophobic motif at the C-terminus of the DP2 subunit of PolD, a PIP (PCNA-Interacting Peptide) motif, was critical for the interaction with primase. The dissociation of primase was induced by DNA-dependent binding of PCNA to PolD. Point mutations in the alternative PIP-motif of DP2 abrogated the molecular switching that converts the archaeal replicase from de novo to processive synthesis mode.The SIB Swiss Institute of Bioinformatics (https//) creates, maintains and disseminates a portfolio of reliable and state-of-the-art bioinformatics services and resources for the storage, analysis and interpretation of biological data. Through Expasy (https//), the Swiss Bioinformatics Resource Portal, the scientific community worldwide, freely accesses more than 160 SIB resources supporting a wide range of life science and biomedical research areas. In 2020, Expasy was redesigned through a user-centric approach, known as User-Centred Design (UCD), whose aim is to create user interfaces that are easy-to-use, efficient and targeting the intended community. This approach, widely used in other fields such as marketing, e-commerce, and design of mobile applications, is still scarcely explored in bioinformatics. In total, around 50 people were actively involved, including internal stakeholders and end-users. In addition to an optimised interface that meets users' needs and expectations, the new version of Expasy provides an up-to-date and accurate description of high-quality resources based on a standardised ontology, allowing to connect functionally-related resources.Associative toehold is a powerful concept enabling efficient combinatorial computation in DNA circuit. find more A longer association length boosts circuit kinetics and equilibrium signal but results in higher leak rate. We reconc