Biology subjects

Kruglyak, S.

Publications and source records attributed to Kruglyak, S..

10 recordsLinked to original sources

bioRxiv2026

CytosMPS: an automated multimodal pooled screening platform for protein, morphology and transcriptomics

Cytos Multimodal Pooled Screening (CytosMPS) measures CRISPR guide identity, morphology, native 3' transcriptome, and protein abundance and localization in the same fixed cells. Following IL-1{beta} stimulation, cells with higher nuclear NF-{kappa}B show lower phospho-p38/HSP27 and higher NFKBIA/DUSP1 in the same cell, a measurement neither imaging nor transcriptomics alone provides. TGFBR2 and CTNNB1 perturbations produce distinct multimodal phenotypes. Guide representation and perturbation transcriptional responses agree with an orthogonal single-cell transcriptomic platform.

cell biology↗

bioRxiv2025

Complete genomes of a multi-generational pedigree to expand studies of genetic and epigenetic inheritance

Pedigree analysis remains the gold standard for rare disease diagnostics, yet whole genome sequencing studies typically omit critical regions like centromeres, telomeres, and acrocentric chromosome p-arms. Here, we present telomere-to-telomere (T2T) reference genomes for four self-identified African American individuals of admixed ancestry spanning three generations. Our parent-of-origin assigned, chromosome-level assemblies revealed precise meiotic recombination breakpoints in previously inaccessible regions, including recombination events across acrocentric and subtelomeric sequences. Centromeric regions were highly stable, with multi-megabase arrays inherited intact across three generations, while the position of kinetochore assembly sites remained consistent and predominantly associated with the p-arm proximal region. The relative lengths of telomeres on individual chromosomes were maintained across generations. Using a targeted rDNA assembly approach, we reconstructed a complete megabase-scale ribosomal DNA (rDNA) array corresponding to the paternal chromosome 14. This openly available pedigree provides a benchmark dataset for studying recombination and genetic and epigenetic variation across the complete genome.

genomics↗

bioRxiv2025

Direct In-Sample Sequencing of the 3' Transcriptome Expands the Capabilities of Optical Pooled Screens

We present a platform that directly sequences single guide RNAs and endogenous 3'UTRs in fixed cells while simultaneously measuring protein abundance and cellular morphology. We demonstrate platform capability by performing optical pooled screening of CRISPR-perturbed lung cancer cells. This approach unites direct in-sample RNA sequencing with complementary phenotypic readouts, enabling comprehensive, scalable, and functional genomics analyses within a single experiment.

genomics↗

bioRxiv2025

Unraveling Tyrosine-Kinase Inhibitor Resistance in NSCLC Cells via Same-cell Measurement of RNA, Protein, and Morphological Responses

Non-small cell lung cancer (NSCLC) frequently develops resistance to tyrosine kinase inhibitors (TKIs), limiting the long-term success of targeted therapies. A deeper understanding of resistance mechanisms at the molecular and cellular levels may enable the development of more effective treatment strategies. Here, we applied the Teton detection assay on the AVITI24 platform to measure concurrently RNA, protein, and cellular morphology in NSCLC cell lines treated with the TKIs gefitinib and osimertinib. This single-cell, multiomic analysis revealed distinct expression and morphological profiles between drug-sensitive and resistant cells, including differences in MAPK-related pathway activity. Stratifying responses at the single-cell level uncovered subtle responses not detectable in bulk measurements. We identified CDK4/6 activity as a route of cell survival under TKI treatment and demonstrated that co-treatment with the CDK4/6 inhibitor palbociclib enhanced TKI efficacy. The ability to measure multiomics and cellular morphology in the same cells opens new avenues for future studies aimed at improving personalized treatment strategies in NSCLC and overcoming the obstacles posed by drug resistance.

genomics↗

bioRxiv2025

A simplified hybrid capture approach retains high specificity and enables PCR-free workflow

Hybrid capture is a critical technology for selective enrichment of genomic regions of interest, enabling cost-effective focused sequencing in both clinical and research applications. We present a simplified hybrid capture approach that eliminates complexities typically associated with hybridization-based selection methods by directly loading the hybridization product onto the sequencing flow cell. The workflow removes bead-based steps, washes, and post-hybridization PCR, while retaining high capture specificity and library complexity. The approach is enabled by the development of a streptavidin flow cell surface, a method to circularize and amplify captured targets on the flow cell, and a fast hybridization protocol. We demonstrate application across targeted panel sizes and improvements to library complexity and variant calling. We also show how the approach can be used to create an entirely PCR-free targeted sequencing workflow that further improves variant calling and enables the detection of repeat expansions.

genomics↗

bioRxiv2024

A robust benchmark for detecting low-frequency variants in the HG002 Genome In A Bottle NIST reference material.

Somatic mosaicism is an important cause of disease, but mosaic and somatic variants are often challenging to detect because they exist in only a fraction of cells. To address the need for benchmarking subclonal variants in normal cell populations, we developed a benchmark containing mosaic variants in the Genome in a Bottle Consortium (GIAB) HG002 reference material DNA from a large batch of a normal lymphoblastoid cell line. First, we used a somatic variant caller with high coverage (300x) Illumina whole genome sequencing data from the Ashkenazi Jewish trio to detect variants in HG002 not detected in at least 5% of cells from the combined parental data. These candidate mosaic variants were subsequently evaluated using >100x BGI, Element, and PacBio HiFi data. High confidence candidate SNVs with variant allele fractions above 5% were included in the HG002 draft mosaic variant benchmark, with 13/85 occurring in medically relevant gene regions. We also delineated a 2.45 Gbp subset of the previously defined germline autosomal benchmark regions for HG002 in which no additional mosaic variants >2% exist, enabling robust assessment of false positives. The variant allele fraction of some mosaic variants is different between batches of cells, so using data from the homogeneous batch of reference material DNA is critical for benchmarking these variants. External validation of this mosaic benchmark showed it can be used to reliably identify both false negatives and false positives for a variety of technologies and detection algorithms, demonstrating its utility for optimization and validation. By adding our characterization of mosaic variants in this widely-used cell line, we support extensive benchmarking efforts using it in simulation, spike-in, and mixture studies.

bioinformatics↗

bioRxiv2024

FLEXIBLE, PRODUCTION-SCALE, HUMAN WHOLE GENOME SEQUENCING ON A BENCHTOP SEQUENCER

Human whole-genome sequencing (hWGS) provides comprehensive genomic information that can potentially help guide research in disease prevention and treatment and ultimately improve human health. Recent advancements in sequencing technology have improved sequencing quality and further reduced sequencing costs on bench-top sized instruments, making whole-genome sequencing an accessible technology for broader use. Here, we demonstrate the feasibility of a large whole genome sequencing project using a benchtop sequencer in a small laboratory setting, on a scale previously reserved for production-scale factory-sized machines. In this project, 807 samples were prepared and sequenced across 313 flow cells, with high sequencing quality at a median %Q30 of 96.6% and a median %Q40 of 89.31%. To screen library quality and maximize sample yield, we utilized 48-plex sample pre-pool QC runs to provide >1x coverage per sample prior to sample pooling and full-depth sequencing. With this strategy, we consistently achieved >30x human whole genome sequencing of three-plex sample trios with standard settings or up to 4 samples per run with a high-throughput run setting. Additionally, this low-pass data provided valuable sample-level insights, allowing for detection of chromosome copy number variations (CNV) prior to full-depth sequencing. With three instruments running concurrently, >2,800 30x human whole genomes could be sequenced per year. To further demonstrate additional flexibility present in the platform, we also explored two different use cases 1) large insert sizes (1kb+) library to achieve superior genome coverage; 2) a proof of concept for rapid WGS sequencing to minimize sample to answer turnaround time for time-critical sequencing applications. Sequencing of a 2x100 >30x human WGS can be achieved in <12 hours and subsequent generation of fastq, bam and vcf in <1 additional hour. This study provides a cost-effective and flexible real-world demonstration of achieving both high quality hWGS sequencing and instrument flexibility without the need for complex batching schemes or factory-sized sequencers.

molecular biology↗

bioRxiv2024

Characterizing and addressing error modes to improve sequencing accuracy

The accuracy of a sequencing platform has traditionally been measured by the %Q30, or percentage of data exceeding a basecall accuracy of 99.9%. Improvements to accuracy beyond Q30 may be beneficial for certain applications such as the identification of low frequency alleles or the improvement of reference genomes. Here we demonstrate how we achieved over 70% Q50 (99.999% accuracy) data on the AVITI sequencer. This level of accuracy required us to not only improve sequencing quality but also to mitigate library preparation errors and analysis artifacts.

genomics↗

bioRxiv2023

Accurate human genome analysis with Element Avidity sequencing

We investigate the new sequencing technology Avidity from Element Biosciences. We show that Element whole genome sequencing achieves higher mapping and variant calling accuracy compared to Illumina sequencing at the same coverage, with larger differences at lower coverages (20x-30x). We quantify base error rates of Element reads, finding lower error rates, especially in homopolymer and tandem repeat regions. We use Elements ability to generate paired end sequencing with longer insert sizes than typical short-read sequencing. We show that longer insert sizes result in even higher accuracy, with long insert Element sequencing giving noticeably more accurate genome analyses at all coverages.

bioinformatics↗

bioRxiv2022

Sequencing by avidity enables high accuracy with low reagent consumption

We present avidity sequencing - a novel sequencing chemistry that separately optimizes the process of stepping along a DNA template and the process of identifying each nucleotide within the template. Nucleotide identification uses multivalent nucleotide ligands on dye-labeled cores to form polymerase-polymer nucleotide complexes bound to clonal copies of DNA targets. These polymer-nucleotide substrates, termed avidites, decrease the required concentration of reporting nucleotides from micromolar to nanomolar, and yield negligible dissociation rates. We demonstrate the use of avidites as a key component of a sequencing technology that surpasses Q40 accuracy and enables a diversity of applications that include single cell RNA-seq and whole human genome sequencing. We also show the advantages of this technology in sequencing through long homopolymers.

genomics↗