Biology subjects

Vollmers, C.

Publications and source records attributed to Vollmers, C..

At least 19 recordsLinked to original sources

bioRxiv2026

IG-28 enables identification and quantification of high-confidence isoforms from large bulk and single-cell full-length cDNA datasets

The technology to sequence full-length transcriptomes from bulk and single-cell samples has rapidly advanced. As a result, full-length cDNA datasets now reach billions of reads in size which is beyond the capability of most established isoform identification tools. Here, we introduce IG-28 which addresses this challenge with its capability to process and analyze large fl-cDNA datasets fast and using minimal computational resources. In contrast to its predecessor Mandalorion, IG-28 is not limited to bulk fl-cDNA but is capable of parsing, processing, and analyzing single-cell fl-cDNA datasets. It does so in a lightweight, flexible manner, integrating seamlessly with established scRNA-seq analysis workflows. We benchmarked IG-28 against a variety of established and new isoform identification tools using several bulk and a complex single-cell fl-cDNA dataset and found that IG-28 consistently outperformed the other tools by identifying high-confidence public isoforms.

bioinformatics↗

bioRxiv2025

Extensive splicing deficiency in a degenerating mating-type chromosome

Splicing deficiency may represent a critical yet underexplored form of genomic erosion in non-recombining regions. Across four phytoplankton species diverged [~]333-639 million years ago, genes within U (female) and V (male) "UV" mating-type regions--non-recombining chromosomal regions that determine mating compatibility--show strikingly elevated intron retention relative to genes in other genomic regions. Long-read data reveal abundant aberrant, likely non-functional mRNA isoforms despite preserved coding potential. This preservation suggests that splicing defects arose early in UV evolution and have persisted over deep time. We propose that these defects arise from evolutionary changes in sequence composition and chromatin organization that accompany recombination suppression, such as reduced GC content, altered nucleosome occupancy, and disrupted methylation, that collectively compromise splicing fidelity. Unlike sex chromosomes, which often degenerate through gene loss, splicing-deficient UV regions in green algae retain hundreds of genes, indicating that transcript-level dysfunction provides an alternative route to functional decay. Our results identify chromatin-mediated splicing deficiency as a novel axis of genomic erosion and position algal UV systems as models for studying how recombination suppression reshapes RNA processing fidelity in essential, non-recombining genomes.

genomics↗

bioRxiv2025

A Temporal and Spatial Atlas of Adaptive Immune Responses in the Lymph Node Following Viral Infection

The spatial organization of adaptive immune cells within lymph nodes is critical for understanding immune responses during infection and disease. Here, we introduce AIR-SPACE, an integrative approach that combines high-resolution spatial transcriptomics with paired, high-fidelity long-read sequencing of T and B cell receptors. This method enables the simultaneous analysis of cellular transcriptomes and adaptive immune receptor (AIR) repertoires within their native spatial context. We applied AIR-SPACE to mouse popliteal lymph nodes at five distinct time points after Vaccinia virus footpad infection and constructed a comprehensive map of the developing adaptive immune response. Our analysis revealed heterogeneous activation niches, characterized by Interferon-gamma (IFN-{gamma}) production, during the early stages of infection. At later stages, we delineated sub-anatomical structures within the germinal center (GC) and observed evidence that antibody-producing plasma cells differentiate and exit the GC through the dark zone. Furthermore, by combining clonotype data with spatial lineage tracing, we demonstrate that B cell clones are shared among multiple GCs within the same lymph node, reinforcing the concept of a dynamic, interconnected network of GCs. Overall, our study demonstrates how AIR-SPACE can be used to gain insight into the spatial dynamics of infection responses within lymphoid organs.

immunology↗

bioRxiv2025

Sequencing and assembling complete plasmids on Oxford Nanopore Technology Sequencers using R2C2 and Chopper

Plasmids are ubiquitous tools in molecular biology which are used for a large variety of experiments within academic and commercial labs. Both new and old plasmids have to undergo sequencing-based analysis to determine whether or not they are functional, i.e. contain the correct insert in the correct backbone. While traditional Sanger sequencing based analysis was most often limited to the inserts, new high-throughput sequencing based methods and services can now provide the complete sequence of a plasmid. Currently available methods and services vary in throughput and cost. Here, we adapted the Oxford Nanopore Technologies-based R2C2 sequencing method to - rapidly and at low cost - sequence complete plasmids, either individually or in a pool. We also developed an analysis pipeline, Chopper, that produces full-length plasmid sequences. We tested our workflow with commonly used plasmids we ordered from Addgene and produced highly accurate sequences for each plasmid from both their individual and pooled sequencing runs.

bioinformatics↗

bioRxiv2024

The Circadian Isoform Landscape of Mouse Livers

The mammalian circadian clock is an autoregulatory feedback process that is responsible for homeostasis in mouse livers. These circadian processes are well understood at the gene-level, however, not well understood at the isoform-level. To investigate circadian oscillations at the isoform-level, we used the nanopore-based R2C2 method to create over 78 million highly-accurate, full-length cDNA reads for 12 RNA samples extracted from mouse livers collected at 2 hour intervals. To generate a circadian mouse liver isoform-level transcriptome, we processed these reads using the Mandalorion tool which identified and quantified 58,612 isoforms, 1806 of which showed circadian oscillations. We performed detailed analysis on the circadian oscillation of these isoforms, their coding sequences, and transcription start sites and compiled easy-to-access resources for other researchers. This study and its results add a new layer of detail to the quantitative analysis of transcriptomes.

genomics↗

bioRxiv2024

Long read subcellular fractionation and sequencing reveals the translational fate of full length mRNA isoforms during neuronal differentiation

Alternative splicing (AS) alters the cis-regulatory landscape of mRNA isoforms leading to transcripts with distinct localization, stability and translational efficiency. To rigorously investigate mRNA isoform-specific ribosome association, we generated subcellular fractionation and sequencing (Frac-seq) libraries using both conventional short reads and long reads from human embryonic stem cells (ESC) and neural progenitor cells (NPC) derived from the same ESC. We performed de novo transcriptome assembly from high-confidence long reads from cytosolic, monosomal, light and heavy polyribosomal fractions and quantified their abundance using short reads from their respective subcellular fractions. Almost half of all transcripts exhibited association with particular subcellular fractions relative to the cytosol. Of the multi-isoform genes, 27% and 18% exhibited significant differential isoform sedimentation in ESC and NPC, respectively. Alternative promoter usage and internal exon skipping accounted for the majority of differences between isoforms from the same gene. Random forest classifiers implicated 3 and 5 untranslated region (UTR) GC-content and coding sequence (CDS) and UTR lengths as important determinants of isoform-specific sedimentation profiles. Taken together our data demonstrate that alternative mRNA processing within the CDS and UTRs impacts the translational control of mRNA isoforms during stem cell differentiation, and highlights the utility of using a novel long read sequencing-based method to study translational control.

molecular biology↗

bioRxiv2024

Generation and analysis of a mouse multi-tissue genome annotation atlas

Generating an accurate and complete genome annotation for an organism is complex because the cells within each tissue can express a unique set of transcript isoforms from a unique set of genes. A comprehensive genome annotation should contain information on what tissues express what transcript isoforms at what level. This tissue-level isoform information can then inform a wide range of research questions as well as experiment designs. Long-read sequencing technology combined with advanced full-length cDNA library preparation methods has now achieved throughput and accuracy where generating these types of annotations is achievable. Here, we show this by generating a genome annotation of the mouse (Mus musculus). We used the nanopore-based R2C2 long-read sequencing method to generate 64 million highly accurate full length cDNA consensus reads - averaging 5.4 million reads per tissue for a dozen tissues. Using the Mandalorion tool we processed these reads to generate the Tissue-level Atlas of Mouse Isoforms (TAMI - available at https://genome.ucsc.edu/s/vollmers/TAMI) which we believe will be a valuable complement to conventional, manually curated reference genome annotations.

genomics↗

bioRxiv2023

R2C2+UMI: Combining concatemeric consensus sequencing with unique molecular identifiers enables ultra-accurate sequencing of amplicons on Oxford Nanopore Technologies sequencers

The sequencing of PCR amplicons is a core application of high-throughput sequencing technology. Using unique molecular identifiers (UMIs), individual amplified molecules can be sequenced to very high accuracy on an Illumina sequencer. However, Illumina sequencers have limited read length and are therefore restricted to sequencing amplicons shorter than 600bp unless using inefficient synthetic long-read approaches. Native long-read sequencers from Pacific Biosciences and Oxford Nanopore Technologies can, using consensus read approaches, match or exceed Illumina quality while achieving much longer read lengths. Using a circularization-based concatemeric consensus sequencing approach (R2C2) paired with UMIs (R2C2+UMI) we show that we can sequence [~]550nt antibody heavy-chain (IGH) and [~]1500nt 16S amplicons at accuracies up to and exceeding Q50 (<1 error in 100,0000 sequenced bases), which exceeds accuracies of UMI-supported Illumina paired sequencing as well as synthetic long-read approaches.

genomics↗

bioRxiv2023

Detecting haplotype-specific transcript variation in long reads with FLAIR2

BackgroundRNA-Seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme which mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung ADC cells and associated with splicing regulation. Despite the functional importance of studying splicing and SNVs, short read RNA-Seq has limited the communitys ability to interrogate both forms of RNA variation simultaneously. ResultsWe employed long-read technology to obtain full-length transcript sequences, elucidating cis-effects of variants on splicing changes at a single molecule level. We have developed a computational workflow that augments FLAIR, a tool that calls isoform models expressed in long-read data, to integrate RNA variant calls with the associated isoforms that bear them. We generated nanopore data with high sequence accuracy of H1975 lung adenocarcinoma cells with and without knockdown of ADAR. We applied our workflow to identify key inosine-isoform associations to help clarify the prominence of ADAR in tumorigenesis. ConclusionsUltimately, we find that a long-read approach provides valuable insight toward characterizing the relationship between RNA variants and splicing patterns. HighlightsO_LIFLAIR2 has improved transcript isoform detection and incorporates sequence variants for haplotype-specific transcript detection. C_LIO_LIIn addition to haplotype-specific variant detection, it identifies transcript-specific RNA editing C_LIO_LIAble to identify haplotype-specific transcript isoform bias in expression C_LIO_LILong-read sequencing identifies hyperedited transcripts that are missed from short-read sequencing methods for a more comprehensive identification of ADAR targets C_LI

bioinformatics↗

bioRxiv2022

Identifying and quantifying isoforms from accurate full-length transcriptome sequencing reads with Mandalorion

The Mandalorion tool, which we have continuously developed over the last 5 years, identifies and quantifies high-confidence isoforms from accurate full-length transcriptome sequencing reads produced by methods like PacBio Iso-Seq and ONT-based R2C2. In this manuscript, we introduce and benchmark Mandalorion v4 which further improves upon the already strong performance of Mandalorion v3.6 used in the LRGASP consortium challenge. By processing real and simulated accurate full-length transcriptome sequencing data sets, we show three main features of Mandalorion: First, Mandalorion-based isoform identification has very high Precision and maintains high Recall even when used in the absence of any genome annotation. Second, isoform read counts as quantified by Mandalorion show high correlation with simulated read counts. Third, isoforms identified by Mandalorion closely reflect the full-length transcriptome sequencing data sets they are based on.

bioinformatics↗

bioRxiv2021

Illumina But With Nanopore: Sequencing Illumina libraries at high accuracy on the ONT MinION using R2C2

High-throughput short-read sequencing has taken on a central role in research and diagnostics. Hundreds of different assays exist today to take advantage of Illumina short-read sequencers, the predominant short-read sequencing technology available today. Although other short read sequencing technologies exist, the ubiquity of Illumina sequencers in sequencing core facilities, and the high capital costs of these technologies have limited their adoption. Among a new generation of sequencing technologies, Oxford Nanopore Technologies (ONT) holds a unique position because the ONT MinION, an error-prone long-read sequencer, is associated with little to no capital cost. Here we show that we can make short-read Illumina libraries compatible with the ONT MinION by using the R2C2 method to circularize and amplify the short library molecules. This results in longer DNA molecules containing tandem repeats of the original short library molecules. This longer DNA is ideally suited for the ONT MinION, and after sequencing, the tandem repeats in the resulting raw reads can be converted into high-accuracy consensus reads with similar error rates to that of the Illumina MiSeq. We highlight this capability by producing and benchmarking RNA-seq, ChIP-seq, as well as regular and target-enriched Tn5 libraries. We also explore the use of this approach for rapid evaluation of sequencing library metrics by implementing a real-time analysis workflow.

genomics↗

bioRxiv2020

Isoform-level transcriptome Atlas of Macrophage Activation

RNA-seq is routinely used to measure gene expression changes in response to cell perturbation. Genes that are up or down-regulated following perturbation in RNA-seq studies are designated as target genes for follow-up. However, RNA-seq is limited in its ability to capture the complexity of gene isoforms, defined by the exact composition of exons and transcription start sites (TSS) and poly(A) sites they contain, as well as the expression of these isoforms. Without knowing the composition of the most dominant isoform(s) of a target gene, a minority or non-existent isoform could be selected for follow-up solely based on available annotations for that target gene from databases that are incomplete, or by their nature not tissue specific, or do not provide key information on expression levels. In all, this can lead to loss in valuable resources and time. As the vast majority of genes in the human genome express more than one isoform, there is a great need to identify the complete range of isoforms present for each gene along with their corresponding levels of expression. Here, using the long-read nanopore-based R2C2 method, we generated an Isoform-level transcriptome Atlas of Macrophage Activation (IAMA) that identifies full-length isoforms in primary human monocyte-derived macrophages (MDMs). Macrophages are critical innate immune cells important for recognizing pathogens through use of Toll-like receptors (TLRs), culminating in the initiation of host defense pathways. We characterized isoforms for most moderate to highly expressed genes in resting and TLR-activated MDMs and generated a user-friendly portal built into the UCSC Genome Browser to explore the data (https://genome.ucsc.edu/s/vollmers/IAMA). Our atlas represents a valuable resource for innate immune research as it provides unprecedented isoform information for primary human macrophages.

immunology↗

bioRxiv2020

Highly Multiplexed Single-Cell Full-Length cDNA Sequencing of human immune cells with 10X Genomics and R2C2

Single cell transcriptome analysis elucidates facets of cell biology that have been previously out of reach. However, the high-throughput analysis of thousands of single cell transcriptomes has been limited by sample preparation and sequencing technology. High-throughput single cell analysis today is facilitated by protocols like the 10X Genomics platform or Drop-Seq which generate cDNA pools in which the origin of a transcript is encoded at its 5 or 3 end. These cDNA pools are most often analyzed by short read Illumina sequencing which can identify the cellular origin of a transcript and what gene it was transcribed from. However, these methods fail to retrieve isoform information. In principle, cDNA pools prepared using these approaches can be analyzed with Pacific Biosciences and Oxford Nanopore long-read sequencers to retrieve isoform information but current implementations rely heavily on Illumina short-reads for analysis in addition to long reads. Here, we used R2C2 to sequence and demultiplex 12 million full-length cDNA molecules generated by the 10X Chromium platform from [~]3000 peripheral blood mononuclear cells (PBMCs). We used these reads to - independent from Illumina data - cluster cells into B cells, T cells, and Monocytes and generate isoform-level transcriptomes for these cell types. We also generated isoform-level transcriptomes for all single cells and used this information to identify a wide range of isoform diversity between genes. Finally, we also designed a computational workflow to extract paired adaptive immune receptors - T cell receptor and B cell receptor (TCR and BCR) - sequences unique to each T and B cell. This work represents a new, simple, and powerful approach that - using a single sequencing method - can extract an unprecedented amount of information from thousands of single cells.

genomics↗

bioRxiv2019

Chromosome-scale genome assembly of a single outbred Drosophila melanogaster

A high quality genome assembly is a vital first step for the study of an organism. Recent advances in technology have made the creation of high quality chromosome scale assemblies feasible and low cost. However, the amount of input DNA needed for an assembly project can be a limiting factor for small organisms or precious samples. Here we demonstrate the feasibility of creating a chromosome scale assembly using a hybrid method for a low input sample, a single outbred Drosophila melanogaster. Our approach combines an Illumina shotgun library, Oxford nanopore long reads, and chromosome conformation capture for long range scaffolding. This single fly genome assembly has a N50 of 26 Mb, a length that encompasses entire chromosome arms, contains 95% of expected single copy orthologs, and a nearly complete assembly of this individuals Wolbachia endosymbiont. The methods described here enable the accurate and complete assembly of genomes from small, field collected organisms as well as precious clinical samples.

genomics↗

bioRxiv2019

High throughput CRISPR screening identifies genes involved in macrophage viability and inflammatory pathways

AbstractMacrophages are critical cells of the innate immune system involved in the recognition and destruction of invading microbes in addition to the resolution of inflammation and maintenance of homeostasis. Understanding the genes involved in all aspects of macrophage biology is essential to gaining new insights into immune system dysregulation during diseases that range from autoinflammatory to cancer. Here we utilize high throughput clustered regularly interspaced short palindromic repeats (CRISPR) screening to generate a resource that identifies genes required for macrophage viability and function. First, we employ a pooled based CRISPR/Cas nuclease active screening approach to identify essential genes required for macrophage viability by targeting genes within coding exons. In addition, we also target 3UTRs to gain insights into new cis-regulatory regions that control expression of these essential genes. Second, using our recently generated NF-{kappa}B reporter macrophage line, we perform a fluorescence-activated cell sorting (FACS)-based high-throughput genetic screen to identify regulators of inflammation. We identify a number of novel positive and negative regulators of the NF-{kappa}B pathway as well as unraveling complexities of the TNF signaling cascade showing it can function in an autocrine manner to negatively regulate the pathway. Utilizing a single complex library design we are capable of interrogating various aspects of macrophage biology, generating a resource for future studies.\n\nAuthor SummaryExcess inflammation is associated with a variety of autoimmune diseases and cancers. Macrophages are important mediators of this inflammatory response. Defining the genes involved in their viability and effector function is needed to completely understand these two important aspects of macrophage biology. Here we screened over 21,000 genes and generated a resource guide of genes required for macrophage viability as well as novel positive and negative regulators of NF-{kappa}B signaling.

immunology↗

bioRxiv2019

Complete characterization of the human immune cell transcriptome using accurate full-length cDNA sequencing

The human immune system relies on highly complex and diverse transcripts and the proteins they encode. These include transcripts for Human Leukocyte Antigen (HLA) class I and II receptors which are essential for self/non-self discrimination by the immune system as well as transcripts encoding B cell and T cell receptors (BCR and TCR) which recognize, bind, and help eliminate foreign antigens.\n\nHLA genes are highly diverse within the human population with each individual possessing two of thousands of different alleles in each of the 9 major HLA genes. Determining which combination of alleles an individual possesses for each HLA gene (high-resolution HLA-typing) is essential to establish donor-recipient compatibility in organ and bone-marrow transplantations. BCR and TCR genes in turn are generated by recombining a diverse set of gene segments on the DNA level in each maturing B and T cell, respectively. This process generates adaptive immune receptor repertoires (AIRR) of composed of unique transcripts expressed by each B and T cells. These repertoires carry a vast amount of health relevant information. Both short-read RNA-seq based HLA-typing1 and adaptive immune receptor repertoire sequencing2-5 currently rely heavily on our incomplete knowledge of the genetic diversity at HLA6 and BCR/TCR loci7,8.\n\nHere we used our nanopore sequencing based Rolling Circle to Concatemeric Consensus (R2C2) protocol9 to generate over 10,000,000 full-length cDNA sequences at a median accuracy of 97.9%. We used this dataset to demonstrate that deep and accurate full-length cDNA sequencing can - in addition to providing isoform-level transcriptome analysis for over 9,000 loci - be used to generate accurate sequences of HLA alleles for HLA allele typing and discovery as well as detailed AIRR data for the analysis of the adaptive immune system without requiring specific knowledge of the diversity at HLA and BCR/TCR loci.

genomics↗

bioRxiv2019

Horizontal transmission and recombination maintain forever young bacterial symbiont genomes

Bacterial symbionts bring a wealth of functions to the associations they participate in, but by doing so, they endanger the genes and genomes underlying these abilities. When bacterial symbionts become obligately associated with their hosts, their genomes are thought to decay towards an organelle-like fate due to decreased homologous recombination and inefficient selection. However, numerous associations exist that counter these expectations, especially in marine environments, possibly due to ongoing horizontal gene flow. Despite extensive theoretical treatment, no empirical study thus far has connected these underlying population genetic processes with long-term evolutionary outcomes. By sampling marine chemosynthetic bacterial-bivalve endosymbioses that range from primarily vertical to strictly horizontal transmission, we tested this canonical theory. We found that transmission mode strongly predicts homologous recombination rates, and that exceedingly low recombination rates are associated with moderate genome degradation in the marine symbionts with nearly strict vertical transmission. Nonetheless, even the most degraded marine endosymbiont genomes are occasionally horizontally transmitted and are much larger than their terrestrial insect symbiont counterparts. Therefore, horizontal transmission and recombination enable efficient natural selection to maintain intermediate symbiont genome sizes and substantial functional genetic variation. Author summarySymbiotic associations between bacteria and eukaryotes are ubiquitous in nature and have contributed to the evolution of radically novel phenotypes and niches for the involved partners. New metabolic or physiological capacities that arise in these associations are typically encoded by the bacterial symbiont genomes. However, the association itself endangers the retention of bacterial genomic coding capacity. Endosymbiont genome evolution theory predicts that when bacterial symbionts become restricted to host tissues, their populations cannot remove deleterious mutations efficiently. This ultimately results in their genomes degrading to small, function-poor states, reminiscent of organellar genomes. However, many ancient marine endosymbionts do not fit this prediction, but instead retain relatively large, gene-rich genomes, indicating that the evolutionary dynamics of this process need more thorough characterization. Here we show that on-going symbiont gene flow via horizontal transmission between bivalve hosts and recombination among divergent gammaproteobacterial symbiont lineages are sufficient to maintain large and dynamic bacterial symbiont genomes. These findings indicate that many obligately associated symbiont genomes may not be as isolated from one another as previously assumed and are not on a one way path to degradation.

evolutionary biology↗

bioRxiv2019

Depletion of hemoglobin transcripts and long read sequencing improves the transcriptome annotation of the polar bear (Ursus maritimus)

Transcriptome studies evaluating whole blood and tissues are often confounded by overrepresentation of highly abundant transcripts. These abundant transcripts are problematic as they compete with and prevent the detection of rare RNA transcripts, obscuring their biological importance. This issue is more pronounced when using long-read sequencing technologies for isoform-level transcriptome analysis, as they have relatively lower throughput compared to short-read sequencers. As a result long-read based transcriptome analysis is prohibitively expensive for non-model organisms. While there are off-the-shelf kits available for select model organisms capable of depleting highly abundant transcripts for alpha (HBA) and beta (HBB) hemoglobin, they are unsuitable for non-model organisms. To address this, we have adapted the recent CRISPR/Cas9 based depletion method (Depletion of Abundant Sequences by Hybridization) for long-read full-length cDNA sequencing approaches that we call Long-DASH. Using a recombinant Cas9 protein with appropriate guide RNAs, full-length hemoglobin transcripts can be depleted in-vitro prior to performing any short- and long-read sequencing library preparations. Using this method, we sequenced depleted full-length cDNA in parallel using both our Oxford Nanopore Technology (ONT) based R2C2 long-read approach, as well as the Illumina short-read based Smart-seq2 approach. To showcase this, we have applied our methods to create an isoform-level transcriptome from whole blood samples derived from three polar bears (Ursus maritimus). Using Long-DASH, we succeeded in depleting hemoglobin transcripts and generated deep Smart-seq2 Illumina datasets and 3.8 million R2C2 full-length cDNA consensus reads. Applying Long-DASH with our isoform identification pipeline, Mandalorion we discovered ~6,000 high-confidence isoforms and a number of novel genes. This indicates there is a high diversity of gene isoforms within Ursus maritimus not yet reported. This reproducible and straightforward approach has not only improved the polar bear transcriptome annotations but will serve as the foundation for future efforts to investigate transcriptional dynamics within the 19 polar bear subpopulations around the Arctic.

genomics↗