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Biology subjects

Marks, D.

Publications and source records attributed to Marks, D..

At least 19 recordsLinked to original sources

PG-LLM: Benchmarking General-Purpose Language Models for Protein Variant Ranking

General-purpose frontier language models are being increasingly utilized for protein-design work, yet their ability to understand and evaluate variant effects remains unclear. Here, we introduce PG-LLM, a benchmark comprising 276 protein-variant prioritization tasks: 217 from ProteinGym and a temporally held-out set of 59 from recently published studies. Each task follows the same format: a language model is asked to rank a list of variant sequences given only the wild-type protein sequence and an assay description with no access to tools, multiple-sequence alignments, or protein structures. We evaluate thirteen language models and 95 published protein predictors on the same variants with the same evaluation metric. Claude Opus 5 (Max) and GPT 5.6 Sol (Max) are the best performing LLMs with Spearman correlations of{rho} = 0.406 and 0.402 respectively. Opus 5 outperforms 49 of 95 published protein predictors, including 41 of 46 sequence-only methods, and approaches ESM2-650M at{rho} = 0.411, but remains below the leading predictor VenusREM at{rho} = 0.523. We observe that variant-ranking performance scales with test-time compute across GPT, Claude, and Gemini models, but gains taper before closing the gap to specialist protein predictors. To address contamination risk, we create a held-out evaluation set with 59 DMS assays from 19 studies whose scores first became public after January 2026. On this set, we observe performance and test time compute scaling trends similar to those on the 217 tasks derived from ProteinGym. PG-LLM shows that tool-free language models capture substantial protein-variant signal, outperforming many sequence-based predictors while remaining below the strongest specialized models.

bioinformatics↗

Machine learning guided cell-free expression maps the biochemical landscape of carbonic anhydrase

Carbonic anhydrases are among the fastest known biocatalysts, reversibly facilitating the hydration of CO2 to HCO3- at rates up to 107 s-1, which warrants their investigation for industrial carbon capture technologies. However, engineering carbonic anhydrases to maintain stability under harsh industrial process conditions remains a key challenge, and sequence-to-function datasets compatible with machine learning to inform forward engineering are lacking. Here, we developed a high-throughput platform that couples cell-free gene expression with a gaseous CO2 colorimetric assay to map the fitness landscapes of carbonic anhydrases. From 96 diverse natural homologs, we identified a robust variant from the Aquificota phylum and conducted an exhaustive mutational scan and functional assessment of this enzyme at 70{degrees}C and 90{degrees}C, covering >99% of all single-amino acid substitutions (totaling 4,365 mutations assayed in 39,285 reactions). This biochemical landscape was used to benchmark 22 zero-shot protein fitness models and identify critical mutations that improved enzyme stability at 90{degrees}C by more than three-fold. We then used both zero-shot protein language models and supervised learning to filter 419 model-generated variants from a ProteinMPNN library of 100,000 sequences, leading to a best-in-class enzyme that retained activity after incubation at 95{degrees}C. This work demonstrates that integrating cell-free enzyme engineering with machine learning enables opportunities for high-throughput experimental measurements to benchmark and improve protein language models, accelerate design loops, and expand functional exploration within protein families where experimental information is limited.

synthetic biology↗

Multi-Site Reproducibility Study of 3D High-Content Analysis with Dual-View Oblique Plane Microscopy

High content imaging is being applied to achieve quantitative fluorescence readouts in increasingly complex 3-dimensional (3D) cell culture models such as spheroids and organoids. Compared to conventional 2D assays, 3D assays better represent biological heterogeneity but require more complex sample preparation, 3D imaging and 3D image analysis that can affect the accuracy and precision of such assays. We used spheroids formed from the NRAS-activated melanoma cell line 19161 modified to express an ERK kinase translocation reporter (KTR) as an exemplar 3D phenotypic assay carried out in 96-well plates. The spheroids were treated with the ERK activator TPA and a range of concentrations of the MEK inhibitor Binimetinib. 3D live-cell imaging with sub-cellular spatial resolution was performed using a dual-view oblique plane microscope (dOPM) - a form of single-objective light-sheet microscope - and the experiment was performed separately at 4 different institutes. The results were analysed using an identical 3D analysis pipeline and parameters. We assessed the variation in assay readout using a linear mixed effects model. Random variance at the well level was negligible (SD = 0.0048 relative to range of KTR biosensor readout at reference site of 0.17), indicating low technical noise. Treatment effects were dose-dependent and highly statistically significant compared to DMSO control across all sites (Dunnett-corrected p < 0.001). The range in KTR readout between the minimum (3.5 M Binimetinib) and maximum (100 nM TPA) treatments varied between 59 to 96% relative to the reference site. Measured bias in KTR readout between sites was between 6 and 12% of the range of the reference site. This study quantifies the reproducibility of a 3D live spheroid-based assay employing a fluorescence biosensor requiring readout out at the per-cell level using the dOPM platform and discusses areas where experimental protocol could be improved in the future to further improve reproducibility.

bioengineering↗

Differences between protein fitness models can be used to design variants of altered specificity

The vastness of sequence space makes it challenging for directed evolution to efficiently traverse the fitness landscape. In recent years, unsupervised probabilistic models trained on natural sequences have shown promise for predicting the functional effects of mutations and designing new proteins, leading many to suggest that these models may be useful for guiding directed evolution campaigns toward functional regions of sequence space. However, many directed evolution campaigns are interested in evolving new substrate or ligand specificity, and the behavior of unsupervised sequence models on predicting and designing activity against altered substrates or ligands has not been tested. We have built a curated database of multiplexed functional assay results profiling substrate or ligand specificity and systematically how assessed various popular unsupervised protein fitness models score and design variants that alter selectivity in these datasets. We find that sequence models that learn from the surrounding sequence context, especially protein language models, systematically bias against variants that alter specificity. This bias leads them to design altered-specificity variants at similar or lower rates than random chance. However, we propose a simple strategy to exploit this bias by taking weighted differences of model scores to enrich libraries for altered-specificity variants. These findings and our database should help guide how biologists best use protein fitness models and provide a framework to help machine learning researchers develop a new generation of machine learning models that can better design novelty.

Systems Biology↗

Spatial transcriptomic analysis reveals coordinated gene expression in ovarian clear cell carcinoma and adjacent endometriosis in UK and Japanese patients

PurposeOvarian clear cell carcinoma (OCCC) is strongly associated with endometriosis and shows geographic variation in incidence. We investigated whether OCCC and adjacent endometriosis exhibit distinct transcriptional states and whether these patterns differ between United Kingdom (UK) and Japanese cohorts. Experimental DesignWe performed whole-transcriptome spatial profiling on specimens from 16 OCCC cases (8 UK, 8 Japan) in which tumor and endometriosis were both present. Gene expression was analyzed in tumor, endometriosis and stroma. ARID1A status was assessed by immunohistochemistry. ResultsMedian age was 59 years (range 26-82). 13/16 cases (81.3%) had early-stage disease. Tissue compartment rather than cohort of origin was the dominant source of variation across endometriosis and tumor regions. Endometriosis was enriched for inflammatory and immune-related pathways compared to tumor, whilst there was greater representation of chromatin and protein-DNA complex assembly pathways in tumor regions. These patterns were conserved across both cohorts and after stratification by ARID1A status. Mesenchymal-associated gene expression scores also significantly differed across stroma, endometriosis and tumor with clear compartmental separation. Cell type deconvolution analyses showed clear compositional differences between stromal and epithelial disease compartments. ConclusionsOCCC and coexisting endometriosis are transcriptionally distinct, with the dominant contrast being compartmental rather than geographic. ARID1A alone is unlikely to account for the principal spatial transcriptional states identified here. Further analyses will be required to ascertain whether these differences reflect genuine biological differences between OCCC and coexisting endometriosis or represent different stages of endometriosis-associated tumorigenesis. Translational RelevanceOvarian clear cell carcinoma often arises in association with endometriosis, yet the biological transition between these lesions remains poorly understood. Using spatial transcriptomics in matched tumor and adjacent endometriosis from Japanese and UK cohorts, we showed that endometriosis is characterized by inflammatory and antigen-presentation features, whereas tumor regions showed chromatin-organization and oncogenic transcriptional states. These patterns were largely maintained irrespective of ARID1A status and geographic background. In addition, spatial deconvolution suggested differences in local immune composition, with tumor regions showing relatively greater neutrophil- and T cell-associated signals. Together, our data suggest that OCCC and coexisting endometriosis share a spatially linked tissue context, but that tumor regions have distinct transcriptional profile and microenvironment that may be involved in the malignant transformation and inform interpretation of molecular classification in endometriosis-associated OCCC.

pathology↗

Unified sampling framework and experimental benchmarking of sequence- and structure-based protein models

Generative models are increasingly used for protein design, but the lack of standardized evaluation frameworks limits comparison across model classes and hinders translation to experimental success. Here, we introduce a unified sampling and benchmarking framework that enables controlled sequence generation across alignment, protein language, and structure-based models, and apply it to Tobacco etch virus (TEV) protease. Across hundreds of thousands of designed sequences, different models explore distinct regions of sequence space with no clear computational selection metrics to assess enzymatic function. Experimental evaluation reveals large differences in functional outcomes, ranging from non-functional variants to sequences with 9-fold higher activity than wildtype. Machine learning-designed libraries achieve a 39.32% hit rate (percentage of variants matching or exceeding wildtype activity) compared to 6.06% for an error-prone PCR baseline. Structure-based models perform best overall, with hit rates of 74.4% and 66.8% for ESM-IF1 and ProteinMPNN, respectively. Commonly used selection metrics do not strongly correlate with experimental activity, highlighting a gap between in silico evaluation and enzyme function. Together, these results establish a generalizable framework for benchmarking generative protein models and demonstrate the necessity of experimental validation for guiding model development and sequence prioritization.

bioinformatics↗

evedesign: accessible biosequence design with a unified framework

Machine learning methods for protein engineering are rarely interoperable, require bespoke workflows, and remain inaccessible to non-experts. Yet the design problems that matter most - conditional design subject to real-world constraints, multi-objective optimization, and iterative lab-in-the-loop workflows where experimental data continuously refines successive design rounds - demand exactly the kind of flexible, composable infrastructure that no single tool provides. We present evedesign, a unified open-source framework that formalizes conditional biosequence design in a method-agnostic way, enabling complex multiobjective workflows combining supervised and unsupervised models from standardized specifications, and built from the outset to support iterative experimental integration. An interactive web interface facilitates end-to-end design for a broad scientific audience at https://evedesign.bio. We demonstrate evedesigns utility in antibody engineering, enzyme design, and natural enzyme discovery, and invite open-source community contributions.

bioinformatics↗

Transition from infectivity and immune escape to pure escape as an evolutionary strategy during the COVID-19 pandemic.

New SARS-CoV-2 variants have undergone repeated selective sweeps since the beginning of the COVID-19 pandemic, but the fitness advantages and mechanisms driving these sweeps are not fully understood. We developed a probabilistic modeling framework to analyze pandemic growth, infectivity, and immune escape, explicitly accounting for seven immune exposure histories in 5,732 experiments and estimating the effects of 835 mutations. We found infectivity was important for early variants, but as gains became zero-sum, growth became driven by consistent increase in immune escape conferred by a primarily additive effect of mutational accumulation. While phenotypic tradeoffs exist for individual mutations, successful viral strains boast assemblages of mutations that do not sacrifice infectivity for escape. Thus, during an apparent transition to endemicity, SARS-CoV-2 evolution ascended along an evolutionary ridge in the mutational space defined by infectivity and escape, with infectivity reaching an early peak and antigenicity continuing to evolve.

evolutionary biology↗

Variant effect prediction with reliability estimation across priority viruses

Viruses are a major threat to global health due to their rapid evolution, extensive diversity, and frequent cross-species transmission. Although advances in machine learning and the expanding availability of sequence and structural data have accelerated large-scale mutation effect prediction, viral proteins, and particularly fast-evolving antigenic proteins, pose unique biological and data-related challenges that may limit model performance. We introduce EVEREST, a curated dataset for evaluating model performance on (i) forecasting real-world viral evolution (31 clades across 4 viruses) and (ii) concordance with lab-based deep mutational scanning assays (45 proteins, >340,000 variants). Using EVEREST, we show that state-of-the-art protein language models trained across the protein universe substantially under-perform on viral proteins relative to alignment-based models trained on homologous proteins. This under-performance persists even in low-sequence regimes, as is the case during a novel viral outbreak. We develop calibrated reliability metrics to quantify confidence in model predictions where no evaluation datasets exist. For more than half of the WHO-prioritized pandemic-threat viruses, current models fail to produce reliable predictions, highlighting the urgent need for more data or new modeling approaches. Together, these findings reveal key factors driving model under-performance and provide actionable recommendations for improving viral mutation effect prediction in preparation for current and future outbreaks.

bioinformatics↗

Multimodal imaging reveals a lysosomal drug reservoir that drives heterogeneous distribution of PARP inhibitors

For all drugs, effective target engagement requires sufficient intracellular concentrations of drug to be reached, but whether tumour heterogeneity impacts drug distribution and efficacy is poorly studied. PARP inhibitors have transformed treatment of high-grade serous ovarian carcinoma (HGSOC), but resistance remains a clinical hurdle in this highly heterogeneous tumour type. We developed a patient-derived explant multi-modal imaging pipeline, which demonstrated that cell-intrinsic PARP inhibitor accumulation is highly variable, both between patients and within tumours. Spatial transcriptomics revealed enrichment of apoptotic and lysosomal signatures in high-drug regions. Rucaparib, an intrinsically fluorescent PARP inhibitor, accumulates heterogeneously at the single-cell level, with rucaparib-high cells demonstrating increased drug response relative to rucaparib low. Mechanistically, lysosomal sequestration creates a rucaparib reservoir that determines drug levels in the nucleus. Perturbation of lysosomal content altered intracellular levels of weak base PARP inhibitors rucaparib and niraparib, but not olaparib. Together these data suggest that lysosomes act as a reservoir for a subset of PARP inhibitor drugs to improve drug response.

cancer biology↗

Assessing PARP trapping dynamics in ovarian cancer using a CRISPR-engineered FRET biosensor

Poly(ADP-ribose) polymerase inhibitors (PARPi) have revolutionised the treatment of ovarian high grade serous carcinoma (HGSC), especially those with defective homologous recombination. However, the emergence of resistance poses a critical challenge, as over 50% of patients relapse within three years. The mechanisms underlying changes in PARP trapping, a central aspect of PARPi efficacy, are not well understood due to limitations in current experimental methodologies. Existing techniques lack resolution and throughput, impeding efforts to study PARP trapping dynamics over time with single-cell resolution. Effective tools to study PARP trapping in live cells are urgently needed to elucidate resistance mechanisms and inform therapeutic strategies. To address this, we used CRISPR-Cas9 gene editing to dual-label endogenous PARP1 with EGFP and mCherryFP in OVCAR4 cells to develop a novel intramolecular FRET-based biosensor that enables real-time, single-cell visualization of PARP trapping dynamics in live cells. High-content fluorescence lifetime imaging microscopy (FLIM) revealed dose dependent PARP trapping upon exposure to PARP inhibitor and distinguished between the trapping efficiencies of four different PARPi (veliparib, olaparib, rucaparib, talazoparib). Moreover, we found reduced PARP trapping in PARPi-resistant models, both in vitro and in vivo, providing critical evidence for altered PARP trapping as a resistance mechanism and illustrating the potential of this FRET biosensor to interrogate resistance mechanisms quantitatively. This PARP trapping biosensor represents a transformative advance, enabling dynamic, high-resolution analysis of mechanisms underlying cancer drug resistance. It provides critical insights into the heterogeneity of PARPi resistance, with implications for developing more effective therapies and improving personalised treatment strategies for ovarian cancer patients. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=58 SRC="FIGDIR/small/642798v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1c5ee67org.highwire.dtl.DTLVardef@1a6d101org.highwire.dtl.DTLVardef@e3a2bborg.highwire.dtl.DTLVardef@d0ba03_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

LOL-EVE: Predicting Promoter VariantEffects from Evolutionary Sequences

Disease-associated genetic variants occur extensively in noncoding regions like promoters, but current methods focus primarily on single nucleotide variants (SNVs) that typically have small regulatory effect sizes. Expanding beyond single nucleotide events is essential with insertions and deletions (indels) representing the logical next step as they are readily identifiable in population data and more likely to disrupt regulatory elements. However, existing methods struggle with indel prediction, and clinical interpretation often requires assessing complete promoter haplotypes rather than individual variants. We present LOL-EVE (Language Of Life for Evolutionary Variant Effects), a conditional autoregressive transformer trained on 13.6 million mammalian promoter sequences that enables both zero-shot indel prediction and complete promoter sequence scoring. We introduce three benchmarks for promoter indel prediction: ultra rare variant prioritization, causal eQTL identification, and transcription factor binding site disruption analysis. LOL-EVEs superior performance demonstrates that evolutionary patterns learned from indels enable accurate assessment of broader promoter function. Application to Genomics England clinical data shows that LOL-EVE can prioritize promoter haplotypes in known developmental disorder genes, suggesting potential utility for clinical variant assessment. LOL-EVE bridges individual variant prediction with haplotype-level analysis, demonstrating how evolution-based genomic language models may assist in evaluating regulatory variants in complex genetic cases.

genomics↗

Results of the Protein Engineering Tournament: An Open Science Benchmark for Protein Modeling and Design

The grand challenge of protein engineering is the development of computational models to characterize and generate protein sequences for arbitrary functions. Progress is limited by lack of 1) benchmarking opportunities, 2) large protein function datasets, and 3) access to experimental protein characterization. We introduce the Protein Engineering Tournament--a fully-remote competition designed to foster the development and evaluation of computational approaches in protein engineering. The tournament consists of an in silico round, predicting biophysical properties from protein sequences, followed by an in vitro round where novel protein sequences are designed, expressed and characterized using automated methods. Upon completion, all datasets, experimental protocols, and methods are made publicly available. We detail the structure and outcomes of a pilot Tournament involving seven protein design teams, powered by six multi-objective datasets, with experimental characterization by our partner, International Flavors and Fragrances. Forthcoming Protein Engineering Tournaments aim to mobilize the scientific community towards transparent evaluation of progress in the field. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/606135v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@8f9769org.highwire.dtl.DTLVardef@11da734org.highwire.dtl.DTLVardef@1cc51c0org.highwire.dtl.DTLVardef@10b3c27_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

Protein structure, a genetic encoding for glycosylation

Unlike DNA, RNA, and protein biosynthesis, dogma describes glycosylation as primarily determined by intrinsic cellular limitations, such as glycosyltransferase expression and precursor availability. However, this cannot explain the commonly-observed differences between glycans on the same protein. By examining site-specific glycosylation on diverse human proteins, we detected associations between protein structure and glycan structure, broadly generalizable to human-expressed glycoproteins. Through structural analysis of site-specific glycosylation data, we found protein-sequence and structural features consistently correlated with specific glycan features. To quantify these relationships, we present a new amino acid substitution matrix describing "glycoimpact", i.e., the association of primary protein structure and glycosylation. High-glycoimpact amino acids co-evolve with glycosites, and glycoimpact is high when estimates of amino acid conservation and variant pathogenicity diverge. We report thousands of disease variants near glycosites with high-glycoimpact, including several with known links to aberrant glycosylation (e.g., Oculocutaneous Albinism, Jakob-Creutzfeldt disease, Gerstmann-Straussler-Scheinker, and Gauchers Disease). Finally, glycoimpact quantification is validated by studying oligomannose-complex glycan ratios on HIV ENV, differential sialylation on IgG3 Fc, differential glycosylation on SARS-CoV-2 Spike, and fucose-modulated function of a tuberculosis monoclonal antibody. Finally, to test the causality of protein-glycan associations, we created 5 glycoimpact-designed novel Rituximab variants, 4 of which substantially changed glycoprofiles as predicted. In all, we report that site-specific glycan biosynthesis is influenced by underlying protein structure, enabling glycan structure prediction and genetic sequence-guided glycoengineering.

genetics↗

Deep generative models predict SARS-CoV-2 Spike infectivity and foreshadow neutralizing antibody escape

Recurrent waves of SARS-CoV-2 infection, driven by the periodic emergence of new viral variants, highlight the need for vaccines and therapeutics that remain effective against future strains. Yet, our ability to proactively evaluate such therapeutics is limited to assessing their effectiveness against previous or circulating variants, which may differ significantly in their antibody escape from future viral evolution. To address this challenge, we develop a deep learning method to predict the effect of mutations on fitness and escape from neutralizing antibodies. We use this model to engineer 83 unique SARS-CoV-2 Spike proteins incorporating novel combinations of up to 46 amino acid changes relative to the ancestral B.1 variant. The designed constructs were infectious and evaded neutralization by nine well-characterized panels of human polyclonal anti-SARS-CoV-2 immune sera (from vaccinated, boosted, bivalent boosted, and breakthrough infection individuals). Designed constructs on contemporary SARS-CoV-2 strains displayed similar levels of antibody neutralization escape and similar antigenic profiles as variants seen subsequently (up to 12 months later) during the COVID-19 pandemic despite differences in exact mutations. Our approach provides targeted datasets of antigenically diverse escape variants for an early evaluation of the protective ability of vaccines and therapeutics to inhibit not only currently circulating but also future variants. This approach is generalizable to other viral pathogens.

immunology↗

Deep learning prediction of enzyme optimum pH

The relationship between pH and enzyme catalytic activity, especially the optimal pH (pHopt) at which enzymes function, is critical for biotechnological applications. Hence, computational methods to predict pHopt will enhance enzyme discovery and design by facilitating accurate identification of enzymes that function optimally at specific pH levels, and by elucidating sequence-function relationships. In this study, we proposed and evaluated various machine-learning methods for predicting pHopt, conducting extensive hyperparameter optimization, and training over 11,000 model instances. Our results demonstrate that models utilizing language model embeddings markedly outperform other methods in predicting pHopt. We present EpHod, the best-performing model, to predict pHopt, making it publicly available to researchers. From sequence data, EpHod directly learns structural and biophysical features that relate to pHopt, including proximity of residues to the catalytic center and the accessibility of solvent molecules. Overall, EpHod presents a promising advancement in pHopt prediction and will potentially speed up the development of enzyme technologies.

biochemistry↗

TranceptEVE: Combining Family-specific and Family-agnostic Models of Protein Sequences for Improved Fitness Prediction

Modeling the fitness landscape of protein sequences has historically relied on training models on family-specific sets of homologous sequences called Multiple Sequence Alignments. Many proteins are however difficult to align or have shallow alignments which limits the potential scope of alignment-based methods. Not subject to these limitations, large protein language models trained on non-aligned sequences across protein families have achieved increasingly high predictive performance - but have not yet fully bridged the gap with their alignment-based counterparts. In this work, we introduce TranceptEVE - a hybrid method between family-specific and family-agnostic models that seeks to build on the relative strengths from each approach. Our method gracefully adapts to the depth of the alignment, fully relying on its autoregressive transformer when dealing with shallow alignments and leaning more heavily on the family-specific models for proteins with deeper alignments. Besides its broader application scope, it achieves state-of-the-art performance for mutation effects prediction, both in terms of correlation with experimental assays and with clinical annotations from ClinVar.

genetics↗

scPerturb: Information Resource for Harmonized Single-Cell Perturbation Data

Recent biotechnological advances led to growing numbers of single-cell perturbation studies, which reveal molecular and phenotypic responses to large numbers of perturbations. However, analysis across diverse datasets is typically hampered by differences in format, naming conventions, and data filtering. In order to facilitate development and benchmarking of computational methods in systems biology, we collect a set of 44 publicly available single-cell perturbation-response datasets with molecular readouts, including transcriptomics, proteomics and epigenomics. We apply uniform pre-processing and quality control pipelines and harmonize feature annotations. The resulting information resource enables efficient development and testing of computational analysis methods, and facilitates direct comparison and integration across datasets. In addition, we introduce E-statistics for perturbation effect quantification and significance testing, and demonstrate E-distance as a general distance measure for single cell data. Using these datasets, we illustrate the application of E-statistics for quantifying perturbation similarity and efficacy. The data and a package for computing E-statistics is publicly available at scperturb.org. This work provides an information resource and guide for researchers working with single-cell perturbation data, highlights conceptual considerations for new experiments, and makes concrete recommendations for optimal cell counts and read depth.

systems biology↗