Biology subjects

Schroth, G.

Publications and source records attributed to Schroth, G..

3 recordsLinked to original sources

bioRxiv2026

Linking live-cell behavior to transcriptional responses across perturbations using dynamic caging

Single-cell technologies, encompassing molecular, morphological, and functional assays, have emerged as cornerstones of modern biological research and discovery. However, current experimental methods often fail to explicitly link these omic modalities, especially in live cells or longitudinally through time, impeding the study of multi-scale interactions and mechanisms of regulation. CellCage Enclosure (CCE) technology overcomes these limitations by dynamically compartmentalizing cells, allowing for scalable, live-cell, longitudinal exploration and simultaneous analysis of transcriptomic, proteomic, and morphological profiles. Using this novel technology, we generate previously inaccessible insights across various in vitro cellular systems under a diverse set of perturbations, including the discovery of morphological and proteomic features linked to immune suppressive gene set expression in human primary regulatory T cells (Tregs), as well as direct association of morphological and proteomic features with inflammatory gene modules in human colonic fibroblasts. We then develop a novel pooled CRISPR genetic screening technology using CCEs, PERTURB-LINK (PERTURBational LINKage of transcriptomics and imaging in single cells via enclosure-based screening) and apply this approach in murine bone marrow derived macrophages (BMDMs), enabling multiomic dissection of NF-{kappa}B pathway regulation in response to lipopolysaccharide (LPS) stimulation. Together, these findings demonstrate the broad impact that advancements in live-cell, paired multimodal technologies, especially upon perturbation, may offer in deepening our understanding of cellular biology.

cell biology↗

bioRxiv2024

Illumina Complete Long Read Assay yields contiguous bacterial genomes from human gut metagenomes

Metagenomics enables direct investigation of the gene content and potential functions of gut bacteria without isolation and culture. However, metagenome-assembled genomes are often incomplete and have low contiguity due to challenges in assembling repeated genomic elements. Long-read sequencing approaches have successfully yielded circular bacterial genomes directly from metagenomes, but these approaches are low throughput, have high DNA input requirements, and can have high error rates. Illumina has recently launched the Illumina Complete Long Read (ICLR) assay, a new approach for generating kilobase-scale reads with low DNA input requirements and high accuracy. Here, we evaluate the performance of ICLR sequencing for gut metagenomics. We sequenced a microbial mock community and ten human gut microbiome samples with standard, shotgun 2x150 paired-end sequencing, ICLR sequencing, and nanopore long-read sequencing and compared performance in read lengths, assembly contiguity, and bin quality. We find that ICLR human metagenomic assemblies have higher N50 (119.5 {+/-} 24.8 kilobases) than short read assemblies (9.9 {+/-} 4.5 kilobases; p = 0.002), and comparable N50 to nanopore assemblies (91.0 {+/-} 43.8 kilobases; p = 0.32). Additionally, we find that ICLR draft microbial genomes are more complete (94.0% {+/-} 20.6%) than nanopore draft genomes (85.9% {+/-} 23.0%; p[≤] 0.001), and that nanopore draft genomes have truncated gene lengths (924.6 {+/-} 114.7 base pairs) relative to ICLR genomes (954.6 {+/-} 71.5 base pairs; p[≤] 0.001). Overall, we find that ICLR sequencing is a promising method for high-throughput and accurate assembly of microbial genomes from gut metagenomes. ImportanceMetagenomic sequencing allows scientists to directly measure the genome content and structure of microbes residing in complex microbial communities. Traditional short-read metagenomic sequencing methods often yield fragmented genomes, whereas advanced long-read sequencing methods improve genome assembly quality but often suffer from high error rates and are logistically limited due to low throughput and high input requirements. A new method, the Illumina Complete Long Read Assay (ICLR), is capable of generating highly-accurate kilobase-scale sequencing reads with minimal input material. To evaluate the utility of ICLR in metagenomic contexts, we applied short-read, long-read, and ICLR methods to simple and complex microbial communities. We found that ICLR outperforms short-read methods, and yields comparable metagenomic assemblies to standard long-read approaches while requiring less input material. Overall, ICLR represents a high-throughput and efficient solution for assembling complete genomes from complex metagenomes.

genomics↗

bioRxiv2019

Establishing reference samples for detection of somatic mutations and germline variants with NGS technologies

We characterized two reference samples for NGS technologies: a human triple-negative breast cancer cell line and a matched normal cell line. Leveraging several whole-genome sequencing (WGS) platforms, multiple sequencing replicates, and orthogonal mutation detection bioinformatics pipelines, we minimized the potential biases from sequencing technologies, assays, and informatics. Thus, our \"truth sets\" were defined using evidence from 21 repeats of WGS runs with coverages ranging from 50X to 100X (a total of 140 billion reads). These \"truth sets\" present many relevant variants/mutations including 193 COSMIC mutations and 9,016 germline variants from the ClinVar database, nonsense mutations in BRCA1/2 and missense mutations in TP53 and FGFR1. Independent validation in three orthogonal experiments demonstrated a successful stress test of the truth set. We expect these reference materials and \"truth sets\" to facilitate assay development, qualification, validation, and proficiency testing. In addition, our methods can be extended to establish new fully characterized reference samples for the community.

bioinformatics↗