Bayesian phylogenetic analysis is essential for elucidating evolutionary relationships among organisms. Traditional methods often rely on fixed models and manual parameter settings, which can limit accuracy and efficiency. This protocol presents an integrated workflow that leverages GUIDANCE2 for rigorous sequence alignment, ProtTest and MrModeltest for robust model selection, and MrBayes for phylogenetic tree estimation through Bayesian inference. By automating key steps and providing detailed command-line instructions, this protocol enhances the reliability and reproducibility of phylogenetic studies.

Tuberculosis (TB) remains the leading cause of human mortality in infectious diseases. Drug-resistant TB, particularly multidrug-resistant TB and extensively drug-resistant TB, poses a pressing clinical and public health challenge. The main causative agents of TB are known as Mycobacterium tuberculosis (MTB), which exhibits a highly complex drug resistance profile. Traditional culture-based phenotypic drug susceptibility testing is time-consuming, and PCR-based assays are restricted to detecting known mutational hotspots. In this study, we present a protocol leveraging high-throughput nanopore sequencing technology in conjunction with multiplex PCR, termed targeted nanopore sequencing, for the identification of MTB and analysis of its drug resistance. Our method for MTB drug resistance assessment offers the benefits of being culture-free, efficient, high-throughput, and highly accurate, which could significantly aid in clinical patient management and the control of TB infections.

Candida glabrata is an opportunistic pathogen that may cause serious infections in an immunocompromised host. C. glabrata cell wall proteases directly interact with host cells and affect yeast virulence and host immune responses. This protocol describes methods to purify β-1,3-glucan-bonded cell wall proteases from C. glabrata. These cell wall proteases are detached from the cell wall glucan network by lyticase treatment, which hydrolyzes β-1,3-glucan bonds specifically without rupturing cells. The cell wall supernatant is further fractioned by centrifugal devices with cut-offs of 10 and 50 kDa, ion-exchange filtration(charge), and gel filtration (size exclusion). The enzymatic activity of C. glabrata proteases is verified with MDPF-gelatin zymography and the degradation of gelatin is visualized by loss of gelatin fluorescence. With this procedure, the enzymatic activities of the fractions are kept intact, differing from methods used in previous studies with trypsin digestion of the yeast cell wall. The protein bands may be eventually located from a parallel silver-stained gel and identified with LC–MS/MS spectrometry. The advantage of this methodology is that it allows further host protein degradation assays; the protocol is also suitable for studying other Candida yeast species.

Key features

• Uses basic materials and laboratory equipment, enabling low-cost studies.

• Facilitates the selection and identification of proteases with certain molecular weights.

• Enables further functional studies with host proteins, such as structural or immune response–related, or enzymes and candidate protease inhibitors(e.g., from natural substances).

• This protocol has been optimized for C. glabrata but may be applied with modifications to other Candida species.

Graphical overview

Profiling the specificities of antibodies can reveal a wealth of information about humoral immune responses and the antigens they target. Here, we present a protocol for VirScan, an application of the phage immunoprecipitation sequencing (PhIP-Seq) method for profiling the specificities of human antiviral antibodies. Accompanying this protocol is a video of the experimental procedure. VirScan and, more generally, PhIP-Seq are techniques that enable high-throughput antibody profiling by combining high-throughput DNA oligo synthesis and bacteriophage display with next-generation sequencing. In the VirScan method, human sera samples are screened against a library of peptides spanning the entire human viral proteome. Bound phage are immunoprecipitated and sequenced, identifying the viral peptides recognized by the antibodies. VirScan Is a powerful tool for uncovering individual viral exposure histories, mapping the epitope landscape of viruses of interest, and studying fundamental mechanisms of viral immunity.

Graphical abstract:

Next generations sequencing (NGS) has become an important tool in biomedical research. The Primer ID approach combined with the MiSeq platform overcomes the limitation of PCR errors and reveals the true sampling depth of population sequencing, making it an ideal tool to study mutagenic effects of potential broad-spectrum antivirals on RNA viruses. In this report we describe a protocol using Primer ID sequencing to study the mutations induced by antivirals in a coronavirus genome from an in vitro cell culture model and an in vivo mouse model. Viral RNA or total lung tissue RNA is tagged with Primer ID-containing cDNA primers during the initial reverse transcription step, followed by two rounds of PCR to amplify viral sequences and incorporate sequencing adaptors. Purified and pooled libraries are sequenced using the MiSeq platform. Sequencing data are processed using the template consensus sequence (TCS) web-app. The Primer ID approach provides an accurate sequencing protocol to measure mutation error rates in viral RNA genomes and host mRNA. Sequencing results suggested that β-D-N4-hydroxycytidine (NHC) greatly increased the transition substitution rate but not the transversion substitution rate in the viral RNA genomes, and cytosine (C) to uridine (U) was found as the most frequently seen mutation.

Fungal pathogen Candida albicans is one of the top leading causes of overall healthcare-associated bloodstream infections worldwide. Neutrophil is the major effector cell to clear C. albicans infection. Our study showed that mouse neutrophils utilize two independent mechanisms to kill C. albicans: one is CR3 downstream NADPH oxidase-dependent mechanism that kills opsonized C. albicans; the other one is dectin-2-mediated NADPH oxidase-independent neutrophil extracellular trap (NET) that kills unopsonized C. albicans. Neutrophil killing of opsonized C. albicans requires phagocytosing the organism and production of reactive oxygen species production (ROS). Most existing protocols that assay for neutrophil killing of C. albicans requires a washing step after allowing neutrophils to phagocytose the organism. By definition, NET kills organisms extracellularly. Therefore, it is important to skip the washing step and add an optimal ratio of neutrophils and C. albicans to the wells. To demonstrate the effect of NET, it is necessary to compare killing ability of neutrophils treated with micrococcal nuclease (MNase), an enzyme that digests NET, to that treated with heat-inactivated MNase. MNase is also applied to release NET-bound fungal elements for counting. This protocol can be applied to assay NET killing of other biofilm-forming organisms.