Biology subjects

Müller, K. M.

Publications and source records attributed to Müller, K. M..

7 recordsLinked to original sources

bioRxiv2026

E. coli-produced nanobody fusions as a pan-serotype platform for AAV purification, quantification, and detection

Research, production and application of the recombinant adeno-associated virus (rAAV) require versatile tools for rapid and cost-effective advances. We developed fusion constructs with the pan-serotype, AAV-binding VHH-antibody-fragment named nanobody AAVX and devised procedures for AAV purification, quantification, and detection. Starting from an amino acid sequence, we cytosolically expressed in E. coli nanobody AAVX carrying various tags and fusion partners. Very efficient soluble production followed by a one-step immobilized metal-ion affinity chromatography (IMAC) yielded mostly >90% pure protein. Crosslinking AAVX with an agarose matrix enabled efficient purification of intact AAV2 and AAV5 capsids from cell lysates, as assessed by qPCR and ELISA. AAVX also supported effective AAV quantification by ELISA, as capture reagent and, when fused to luciferase, as detection reagent. Using tagged AAVX and streptavidin biosensors, we developed a rapid and label-free quantification assay of AAV capsids by biolayer interferometry (BLI). Finally, immunostaining with an AAVX-eGFP fusion visualized AAV capsids in fixed HEK-293 cells by fluorescence microscopy. Collectively, these results establish nanobody AAVX fusion proteins as versatile and readily customizable tools for AAV research and bioprocessing.

synthetic biology↗

bioRxiv2025

A jumbo cyanophage encodes the most complete ribosomal protein set in the known virosphere

Viruses encode ribosomal proteins (RPs), but their genomes usually harbor a single RP-coding gene. Here, we reannotate the genome of the jumbo cyanophage PhiMa05 and show that it encodes six RPs, an RP acetyltransferase, and a ribosome biogenesis protein. Evolutionary analyses suggest that these viral RP-coding genes may have been horizontally transferred to certain members of the Vampirovibrionia, a non-photosynthetic basal lineage of Cyanobacteriota, via integration of the viral genome.

microbiology↗

bioRxiv2024

Degradation and Stable Maintenance of AAV Inverted Terminal Repeats in E. coli

Current plasmid propagation compromises large inverted repeats, like inverted terminal repeats (ITRs) of adeno-associated virus (AAV). Direct long-read sequencing analyses upon varying culture conditions and strains revealed ITR instability mechanisms, which diminished in absence of SbcC or at elevated growth temperatures (e.g. 42 {degrees}C), with a combination being optimal. Resulting full ITRs improved rAAV yield and purity. The findings advance plasmid biology, cloneable sequences, and therapeutic AAV manufacturing.

genetics↗

bioRxiv2022

Characterization of Cyanobacterial Communities in Lakes Requires Consideration of Diurnal and Spatial Variation

Continental-scale increases in aquatic system eutrophication are linked with increased cyanobacteria threats to recreational water use and drinking water resources globally. Increasing evidence suggests that diurnal vertical migration of cyanobacteria are key factors that must be considered in cyanobacterial bloom risk management. While this has been discussed in marine and eutrophic freshwater contexts, reports of diurnal vertical migration of cyanobacteria in oligotrophic freshwater lakes are scant. Typical monitoring protocols do not reflect these dynamics and frequently focus only on surface water sampling approaches, and either ignore sampling time or recommend large midday timeframes (e.g., 10AM-3PM), thereby preventing accurate characterization of cyanobacterial community dynamics. To evaluate the impact of diurnal migrations and water column stratification on cyanobacterial abundance and composition, communities were characterized in a shallow well-mixed lake interconnected to a thermally stratified lake in the Turkey Lakes Watershed (Ontario, Canada) using amplicon sequencing of the 16S rRNA gene across a multi-time point sampling series in 2018 and 2022. This work showed that cyanobacteria are present in oligotrophic lakes and their community structure varies (i) diurnally, (ii) across the depth of the water column, (iii) interannually within the same lake and (iv) between different lakes that are closely interconnected within the same watershed. It underscored the need for integrating multi-timepoint, multi-depth discrete sampling guidance into lake and reservoir monitoring programs to describe cyanobacteria community dynamics and signal change to inform risk management associated with the potential for cyanotoxin production. Ignoring variability in cyanobacterial community dynamics (such as that reported herein) and reducing sample numbers can lead to a false sense of security and missed opportunities to identify and mitigate changes in trophic status and associated risks such as toxin or taste and odor production, especially in sensitive, oligotrophic systems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/478008v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@14ea7aorg.highwire.dtl.DTLVardef@12e0217org.highwire.dtl.DTLVardef@18b61beorg.highwire.dtl.DTLVardef@1be2fe6_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights{blacksquare} Cyanobacterial populations fluctuate sporadically across diurnal cycles {blacksquare}Cyanobacterial communities can vary significantly between interconnected lakes {blacksquare}Significant annual shifts in communities signal higher risk and need for monitoring {blacksquare}Cyanobacteria monitoring for risk management should incorporate time and depth

ecology↗

bioRxiv2021

Ensuring that fundamentals of quantitative microbiology are reflected in microbial diversity analyses based on next-generation sequencing

Diversity analysis of amplicon sequencing data is mainly limited to plug-in estimates calculated using normalized data to obtain a single value of an alpha diversity metric or a single point on a beta diversity ordination plot for each sample. As recognized for count data generated using classical microbiological methods, read counts obtained from a sample are random data linked to source properties by a probabilistic process. Thus, diversity analysis has focused on diversity of (normalized) samples rather than probabilistic inference about source diversity. This study applies fundamentals of statistical analysis for quantitative microbiology (e.g., microscopy, plating, most probable number methods) to sample collection and processing procedures of amplicon sequencing methods to facilitate inference reflecting the probabilistic nature of such data and evaluation of uncertainty in diversity metrics. Types of random error are described and clustering of microorganisms in the source, differential analytical recovery during sample processing, and amplification are found to invalidate a multinomial relative abundance model. The zeros often abounding in amplicon sequencing data and their implications are addressed, and Bayesian analysis is applied to estimate the source Shannon index given unnormalized data (both simulated and real). Inference about source diversity is found to require knowledge of the exact number of unique variants in the source, which is practically unknowable due to library size limitations and the inability to differentiate zeros corresponding to variants that are actually absent in the source from zeros corresponding to variants that were merely not detected. Given these problems with estimation of diversity in the source even when the basic multinomial model is valid, sample-level diversity analysis approaches are discussed. HighlightsO_LIRandom error in amplicon sequencing method should be considered in diversity analysis C_LIO_LIClustering, amplification, and differential recovery distort sample diversity C_LIO_LIThe multinomial model for compositional count data is compromised by amplification C_LIO_LIThere are three types of zeros in amplicon sequencing data, including missing zeros C_LIO_LISource alpha diversity estimates are biased by unknown number of unique variants C_LI

bioinformatics↗

bioRxiv2020

To rarefy or not to rarefy: Enhancing microbial community analysis through next-generation sequencing

Amplicon sequencing has revolutionized our ability to study DNA collected from environmental samples by providing a rapid and sensitive technique for microbial community analysis that eliminates the challenges associated with lab cultivation and taxonomic identification through microscopy. In water resources management, it can be especially useful to evaluate ecosystem shifts in response to natural and anthropogenic landscape disturbances to signal potential water quality concerns, such as the detection of toxic cyanobacteria or pathogenic bacteria. Amplicon sequencing data consist of discrete counts of sequence reads, the sum of which is the library size. Groups of samples typically have different library sizes that are not representative of biological variation; library size normalization is required to meaningfully compare diversity between them. Rarefaction is a widely used normalization technique that involves the random subsampling of sequences from the initial sample library to a selected normalized library size. Rarefying is often dismissed as statistically invalid because subsampling effectively discards a portion of the observed sequences. Nonetheless, it remains prevalent in practice. Notably, the superiority of rarefying relative to many other normalization approaches has been argued in diversity analysis. Here, repeated rarefying is proposed as a tool for diversity analyses to normalize library sizes. This enables (i) proportionate representation of all observed sequences and (ii) characterization of the random variation introduced to diversity analyses by rarefying to a smaller library size shared by all samples. While many deterministic data transformations are not tailored to produce equal library sizes, repeatedly rarefying reflects the probabilistic process by which amplicon sequencing data are obtained as a representation of the source microbial community. Specifically, it evaluates which data might have been obtained if a particular samples library size had been smaller and allows graphical representation of the effects of this library size normalization process upon diversity analysis results.

bioinformatics↗

bioRxiv2019

Nanopore sequencing of native adeno-associated virus single-stranded DNA using a transposase-based rapid protocol

Next-generation sequencing of single-stranded DNA (ssDNA) enables transgene characterization of gene-therapy vectors such as adeno-associated virus (AAV), but current library generation uses complicated and potentially biased second-strand synthesis. We report that libraries for nanopore sequencing of ssDNA can be conveniently created without second-strand synthesis using a transposase-based protocol. We show for bacteriophage M13 ssDNA that the MuA transposase has unexpected residual activity on ssDNA, explained in part by transposase action on transient double-stranded hairpins. In case of AAV, library creation is additionally aided by genome hybridization. We demonstrate the power of direct sequencing combined with nanopore long reads by characterizing AAV vector transgenes. Sequencing yielded reads up to full genome length, including GC-rich inverted terminal repeats. Unlike short-read techniques, single reads covered genome-genome and genome-contaminant fusions and other recombination events, while additionally providing information on epigenetic methylation. Single-nucleotide variants across the transgene cassette were revealed and secondary genome packaging signals were readily identified. Moreover, comparison of sequence abundance with qPCR results demonstrated the techniques future potential for quantification of DNA impurities in AAV vector stocks. The findings promote direct nanopore sequencing as a fast and versatile platform for ssDNA characterization, such as AAV ssDNA in research and clinical settings.

genomics↗