Biology subjects

Auch, B.

Publications and source records attributed to Auch, B..

11 recordsLinked to original sources

bioRxiv2025

A molecular inventory of the faecal microbiomes of 23 marsupial species

Despite the recent expansion of culture-independent analyses of animal faecal microbiomes, many lineages remain understudied. Marsupials represent one such group, where despite their iconic status, direct sequencing-based analyses remain limited. Here we present a metagenomic and metabolomic exploration of the faecal microbiomes of 23 Diprotodontia marsupials, producing a reference set of 3,868 prokaryotic and 12,142 viral metagenome-assembled genomes, the majority (>80%) of which represent novel species. As with other animals, host phylogeny is the primary driver of microbiome composition, including distinct profiles for two eucalyptus folivore specialists (koalas and southern greater gliders), suggesting independent solutions to this challenging diet. Expansion of several bacterial and viral lineages were observed in these and other marsupial hosts that may provide adaptive benefits. Antimicrobial resistance genes were significantly more prevalent in captive than wild animals likely reflecting human interaction. This molecular dataset contributes to our ongoing understanding of animal faecal microbiomes. Impact statementDespite their ecological and evolutionary importance, marsupials remain underrepresented in microbiome research. Here, we present the most extensive faecal microbiome dataset to date for this group, encompassing metagenomic, metabolomic, and proximity ligation data from 23 marsupial species. As in other animals, we find the microbial community structure reflects the host species, and some marsupials carry expanded sets of certain microbial lineages indicative of within-host evolution. This work substantially expands the genomic landscape of host-associated microbes and viruses in a poorly studied mammalian clade. Data summaryRaw read data, prokaryotic MAGs [≥]50% complete with [≤]10% contamination are available via the European Nucleotide Archive under project PRJEB89408. The full set of viral genomes, clustered protein database and metabolite data (raw and processed) are available via https://doi.org/10.48610/14e37e9. Prokaryotic MAGs are also available via https://figshare.com/s/87443d80817f57aadc16.

microbiology↗

bioRxiv2024

Metagenomic insights into an enigmatic gammaproteobacterium that is important for carbon cycling in cave ecosystems worldwide

Caves are windows into the subsurface through which we can directly evaluate the microbiological processes responsible for rock weathering and biogeochemical cycling in more expansive areas of Earths subsurface. However, many cave microbial communities are dominated by microorganisms from uncultivated groups with unknown genomic capabilities that cannot be resolved from marker gene surveys alone. An example of this is a genus of Gammaproteobacteria known as "wb1-P19", which are ubiquitous and abundant in rRNA gene surveys from limestone and volcanic caves around the world. We recovered a nearly complete metagenome-assembled genome (MAG) representing a population of wb1-P19 from Lehman Caves in Great Basin National Park, Nevada, USA, and used it to identify additional MAGs representing this group from the Frasassi Caves in Italy and in publicly available databases. Although members of the wb1-P19 group have often been thought to be autotrophs that oxidize inorganic nitrogen compounds, we show that wb1-P19 are actually obligate or facultative methanotrophs capable of aerobic and anaerobic growth. Based on genomic classification, wb1-P19 are members of upland soil cluster {gamma} (USC{gamma}; part of the newly proposed order "Candidatus Methyloligotrophales"), and are likely important for methane consumption and carbon cycling in caves and other subterranean ecosystems globally.

microbiology↗

bioRxiv2024

Proxi-RIMS-seq2 applied to native microbiomes uncovers hundreds of known and novel m5C methyltransferase specificities.

Methylation patterns in bacteria can be used to study Restriction-Modification (RM) or other defense systems with novel properties. While m4C and m6A methylation is well characterized mainly through PacBio sequencing, the landscape of m5C methylation is under-characterized. To bridge this gap, we performed RIMS-seq2 on microbiomes composed of resolved assemblies of distinct genomes through proximity ligation. This high-throughput approach enables the identification of m5C methylated motifs and links them to cognate methyltransferases directly on native microbiomes without the need to isolate bacterial strains. Methylation patterns can also be identified on viral DNA and compared to host DNA, strengthening evidence for virus-host interaction. Applied to three different microbiomes, the method unveils over 1900 motifs that were deposited in REBASE. The motifs include a novel 8-base recognition site (CATm5CGATG) that was experimentally validated by characterizing its cognate methyltransferase. Our findings suggest that microbiomes harbor arrays of untapped m5C methyltransferase specificities, providing insights to bacterial biology and biotechnological applications.

genomics↗

bioRxiv2024

Highly accurate metagenome-assembled genomes from human gut microbiota using long-read assembly, binning, and consolidation methods

Long-read metagenomic sequencing is a powerful approach for cataloging the microbial diversity present in complex microbiomes, including the human gut microbiome. We performed a deep-sequencing experiment using PacBio HiFi reads to obtain metagenome-assembled genomes (MAGs) from a pooled human gut microbiome. We performed long-read metagenome assembly using two methods (hifiasm-meta, metMDBG), used improved bioinformatic and proximity ligation binning strategies to cluster contigs and identify MAGs, and developed a novel framework to compare and consolidate MAGs (pb-MAG-mirror). We found proximity ligation binning yielded more MAGs than bioinformatic binning, but our novel comparison framework resulted in higher MAG yields than either binning strategy individually. In total, from 255 Gbp of total HiFi data we produced 595 total MAGs (including 175 high-quality MAGs) using hifiasm-meta, and 547 total MAGs (including 277 high-quality MAGs) with metaMDBG. Hifiasm-meta assembled almost twice as many strain-level MAGs as metaMDBG (246 vs. 156), but both assembly methods produced up to five strains for a species. Approximately 85% of the MAGs were assigned to known species, but we recovered >35 high-quality MAGs that represent uncultured diversity. Based on strict similarity scores, we found 125 MAGs were unequivocally shared across the assembly methods at the strain level, representing [~]22% of the total MAGs recovered per method. Finally, we detected more total viral sequences in the metaMDBG assembly versus the hifiasm-meta assembly ([~]6,700 vs. [~]4,500). Overall, we find the use of HiFi sequencing, improved metagenome assembly methods, and complementary binning strategies is highly effective for rapidly cataloging microbial genomes in complex microbiomes.

microbiology↗

bioRxiv2023

Deterministic Genetic Barcoding for Multiplexed Behavioral and Single-Cell Transcriptomic Studies

Advances in single-cell sequencing technologies have provided novel insights into the dynamics of gene expression throughout development, been used to characterize somatic variation and heterogeneity within tissues, and are currently enabling the construction of transcriptomic cell atlases. However, despite these remarkable advances, linking anatomical information to transcriptomic data and positively identifying the cell types that correspond to gene expression clusters in single-cell sequencing data sets remains a challenge. We describe a straightforward genetic barcoding approach that takes advantage of the powerful genetic tools available in Drosophila to allow in vivo tagging of defined cell populations. This method, called Targeted Genetically-Encoded Multiplexing (TaG-EM), involves inserting a DNA barcode just upstream of the polyadenylation site in a Gal4-inducible UAS-GFP construct so that the barcode sequence can be read out during single-cell sequencing, labeling a cell population of interest. By creating many such independently barcoded fly strains, TaG-EM will enable a number of potential applications that will improve the quality and information content of single-cell transcriptomic data including positive identification of cell types in cell atlas projects, identification of multiplet droplets, and barcoding of experimental timepoints, conditions, and replicates. Furthermore, we demonstrate that the barcodes from TaG-EM fly lines can be read out using next-generation sequencing to facilitate population-scale behavioral measurements. Thus, TaG-EM has the potential to enable large-scale behavioral screens in addition to improving the ability to reliably annotate cell atlas data, expanding the scope, and improving the robustness of single-cell transcriptomic experiments.

genomics↗

bioRxiv2021

Accurate viral genome reconstruction and host assignment with proximity-ligation sequencing

Viruses play crucial roles in the ecology of microbial communities, yet they remain relatively understudied in their native environments. Despite many advancements in high-throughput whole-genome sequencing (WGS), sequence assembly, and annotation of viruses, the reconstruction of full-length viral genomes directly from metagenomic sequencing is possible only for the most abundant phages and requires long-read sequencing technologies. Additionally, the prediction of their cellular hosts remains difficult from conventional metagenomic sequencing alone. To address these gaps in the field and to accelerate the study of viruses directly in their native microbiomes, we developed an end-to-end bioinformatics platform for viral genome reconstruction and host attribution from metagenomic data using proximity-ligation sequencing (i.e., Hi-C). We demonstrate the capabilities of the platform by recovering and characterizing the metavirome of a variety of metagenomes, including a fecal microbiome that has also been sequenced with accurate long reads, allowing for the assessment and benchmarking of the new methods. The platform can accurately extract numerous near-complete viral genomes even from highly fragmented short-read assemblies and can reliably predict their cellular hosts with minimal false positives. To our knowledge, this is the first software for performing these tasks. Being significantly cheaper than long-read sequencing of comparable depth, the incorporation of proximity-ligation sequencing in microbiome research shows promise to greatly accelerate future advancements in the field.

genomics↗

bioRxiv2021

Dissecting and Tuning Primer Editing by Proofreading Polymerases

Proofreading polymerases have 3 to 5 exonuclease activity that allows the excision and correction of mis-incorporated bases during DNA replication. In a previous study, we demonstrated that in addition to correcting substitution errors and lowering the error rate of DNA amplification, proofreading polymerases can also edit PCR primers to match template sequences. Primer editing is a feature that can be advantageous in certain experimental contexts, such as amplicon-based microbiome profiling. Here we develop a set of synthetic DNA standards to report on primer editing activity and use these standards to dissect this phenomenon. The primer editing standards allow next-generation sequencing-based enzymological measurements, reveal the extent of editing, and allow the comparison of different polymerases and cycling conditions. We demonstrate that proofreading polymerases edit PCR primers in a concentration-dependent manner, and we examine whether primer editing exhibits any sequence specificity. In addition, we use these standards to show that primer editing is tunable through the incorporation of phosphorothioate linkages. Finally, we demonstrate the ability of primer editing to robustly rescue the drop-out of taxa with 16S rRNA gene-targeting primer mismatches using mock communities and human skin microbiome samples.

molecular biology↗

bioRxiv2020

A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2

The global COVID-19 pandemic has led to an urgent need for scalable methods for clinical diagnostics and viral tracking. Next generation sequencing technologies have enabled large-scale genomic surveillance of SARS-CoV-2 as thousands of isolates are being sequenced around the world and deposited in public data repositories. A number of methods using both short- and long-read technologies are currently being applied for SARS-CoV-2 sequencing, including amplicon approaches, metagenomic methods, and sequence capture or enrichment methods. Given the small genome size, the ability to sequence SARS-CoV-2 at scale is limited by the cost and labor associated with making sequencing libraries. Here we describe a low-cost, streamlined, all amplicon-based method for sequencing SARS-CoV-2, which bypasses costly and time-consuming library preparation steps. We benchmark this tailed amplicon method against both the ARTIC amplicon protocol and sequence capture approaches and show that an optimized tailed amplicon approach achieves comparable amplicon balance, coverage metrics, and variant calls to the ARTIC v3 approach and represents a cost-effective and highly scalable method for SARS-CoV-2 sequencing.

genomics↗

bioRxiv2020

Comparative analysis of genome-wide DNA methylation identifies patterns that associate with conserved transcriptional programs in osteosarcoma.

Osteosarcoma is an aggressive tumor of the bone that primarily affects young adults and adolescents. Osteosarcoma is characterized by genomic chaos and heterogeneity. While inactivation of tumor suppressor p53 TP53 is nearly universal other high frequency mutations or structural variations have not been identified. Despite this genomic heterogeneity, key conserved transcriptional programs associated with survival have been identified across human, canine and induced murine osteosarcoma. The epigenomic landscape, including DNA methylation, plays a key role in establishing transcriptional programs in all cell types. The role of epigenetic dysregulation has been studied in a variety of cancers but has yet to be explored at scale in osteosarcoma. Here we examined genome-wide DNA methylation patterns in 24 human and 44 canine osteosarcoma samples identifying groups of highly correlated DNA methylation marks in human and canine osteosarcoma samples. We also link specific DNA methylation patterns to key transcriptional programs in both human and canine osteosarcoma. Building on previous work, we built a DNA methylation-based measure for the presence and abundance of various immune cell types in osteosarcoma. Finally, we determined that the underlying state of the tumor, and not changes in cell composition, were the main driver of differences in DNA methylation across the human and canine samples. SignificanceThis is the first large scale study of DNA methylation in osteosarcoma and lays the ground work for the exploration of DNA methylation programs that help establish conserved transcriptional programs in the context of different genomic landscapes.

cancer biology↗

bioRxiv2020

Analytical Validation of a COVID-19 qRT-PCR Detection Assay Using a 384-well Format and Three Extraction Methods

The COVID-19 global pandemic is an unprecedented health emergency. Insufficient access to testing has hampered effective public health interventions and patient care management in a number of countries. Furthermore, the availability of regulatory-cleared reagents has challenged widespread implementation of testing. We rapidly developed a qRT-PCR SARS-CoV-2 detection assay using a 384-well format and tested its analytic performance across multiple nucleic acid extraction kits. Our data shows robust analytic accuracy on residual clinical biospecimens. Limit of detection sensitivity and specificity was confirmed with currently available commercial reagents. Our methods and results provide valuable information for other high-complexity laboratories seeking to develop effective, local, laboratory-developed procedures with high-throughput capability to detect SARS-CoV-2.

pathology↗

bioRxiv2019

The Genome of the Zebra Mussel, Dreissena polymorpha: A Resource for Invasive Species Research

The zebra mussel, Dreissena polymorpha, continues to spread from its native range in Eurasia to Europe and North America, causing billions of dollars in damage and dramatically altering invaded aquatic ecosystems. Despite these impacts, there are few genomic resources for Dreissena or related bivalves, with nearly 450 million years of divergence between zebra mussels and its closest sequenced relative. Although the D. polymorpha genome is highly repetitive, we have used a combination of long-read sequencing and Hi-C-based scaffolding to generate the highest quality molluscan assembly to date. Through comparative analysis and transcriptomics experiments we have gained insights into processes that likely control the invasive success of zebra mussels, including shell formation, synthesis of byssal threads, and thermal tolerance. We identified multiple intact Steamer-Like Elements, a retrotransposon that has been linked to transmissible cancer in marine clams. We also found that D. polymorpha have an unusual 67 kb mitochondrial genome containing numerous tandem repeats, making it the largest observed in Eumetazoa. Together these findings create a rich resource for invasive species research and control efforts.

genomics↗