Identifying the most appropriate and performant analysis tools

A plethora of SCO tools exist, and yet standards on how to benchmark or evaluate the accuracy of each tool are lacking. Furthermore, most benchmark efforts are focused on certain cell types or tissues.

  • The first goal is the identification of scRNA-seq datasets, which can be used as benchmarking cases.
  • To facilitate findability and usability, we aim to contribute to tool registries and provide portable software environments of the most commonly used tools/workflows.
  • In the long term, we aim to provide cloud deployable analysis pipelines, as well as providing curated datasets for user driven benchmark.

Benchmarking infrastructures


OpenEBench is the ELIXIR benchmarking and technical monitoring platform for bioinformatics tools, web servers and workflows.


DANCE is the first standard, generic, and extensible benchmark platform for accessing and evaluating computational methods across the spectrum of benchmark datasets for numerous single-cell analysis tasks. Currently, DANCE supports 3 modules and 8 popular tasks with 32 state-of-art methods on 21 benchmark datasets. People can easily reproduce the results of supported algorithms across major benchmark datasets via minimal efforts, such as using only one command line. In addition, DANCE provides an ecosystem of deep learning architectures and tools for researchers to facilitate their own model development. DANCE is an open-source Python package that welcomes all kinds of contributions. OmicsML/dance GitHub.


Integrated Benchmarking scRNA-seq Analytical Pipeline (IBRAP) contains a suite of analytical components that can be interchanged throughout the pipeline alongside multiple benchmarking metrics that enable users to compare results and determine the optimal pipeline combinations for their data.

Interesting softwares

Multi modal-omics & spatial integration

SpaTrio is a computational tool based on optimal transport that can align single-cell multi-omics data in space while preserving the spatial topology of the tissue section and local geometry of modality (Yang et al. Cell Genomics 2023).

VortRon is a spatial omic analysis toolbox for multi-omics integration using spatial image registration. VoltRon is also capable of analyzing multiple types of spatially-aware data modalities.

MENDER, Multi-range cEll coNtext DEciphereR, is an unsupervised spatial domain identification tool. MENDER has 3 highlighted points, which are considered major bottlenecks of existing methods:

  • multi-slice spatial domain identification that challenges many advanced methods;
  • scalability to million-level datasets;
  • improved running time efficiency without the need of GPU,

yuanzhiyuan/MENDER GitHUb.

TDEseq is a non-parametric statistical method that takes full advantage of smoothing splines basis functions to account for the dependence of multiple time points in scRNA-seq studies, and uses hierarchical structure linear additive mixed models to model the correlated cells within an individual. TEDseq GitHub

scTCP is a novel semi-supervised deep clustering model for scRNA-seq data. The method utilizes deep clustering in a low-dimensional feature space and incorporates triplet generation based on known labels and pairwise constraints, to guide the clustering process. To address the issue of data imbalance in the dataset, a weighted cross-entropy optimization model was also introduced. LF-Yang/Code GitHub

Generative AI

Geneformer, pretrained on a large-scale corpus of about 30 million single-cell transcriptomes enables context-specific predictions in settings with limited data in network biology Theodoris et al. Nature 2023.

scGPT distills critical biological insights concerning genes and cells. scGPT can be optimized to achieve superior performance across tasks such as cell type annotation, multi-batch integration, multi-omic integration, perturbation response prediction and gene network inference Cui et al. Nature Methods (2024).

GPTCelltype, is a GPT-4 automated cell type annotation, provided as an open-source software package with a detailed user manual available at Winnie09/GPTCelltype github repository.

PERCEPTION, PERsonalized Single-Cell Expression-Based Planning for Treatments In ONcology, is a precision oncology computational pipeline. PERCEPTION uses publicly available matched bulk and single-cell (sc) expression profiles from large-scale cell-line drug screens. These profiles help build treatment response models based on patients’ sc-tumor transcriptomics. PERCEPTION is available at ruppinlab/PERCEPTION GithHUb.

Innovative clustering approaches

scDAC is deep adaptive clustering method coupling the Autoencoder (AE) and the Dirichlet Process Mixture Model (DPMM). labomics/scDAC GitHub

Reproducibility softwares

CREDO, a Customizable, REproducible, DOcker file generator for bioinformatics applications, has been developed as a tool to moderate reproducibility issues by building and distributing docker containers with embedded bioinformatics tools. CREDO simplifies the process of generating Docker images, facilitating reproducibility and efficient research in bioinformatics. The crucial step in generating a Docker image is creating the Dockerfile, which requires incorporating heterogeneous packages and environments such as Bioconductor and Conda. CREDO stores all required package information and dependencies in a Github-compatible format to enhance Docker image reproducibility, allowing easy image creation from scratch. The user-friendly GUI and CREDO’s ability to generate modular Docker images make it an ideal tool for life scientists to efficiently create Docker images.

Cell type annotation tools

popularVote, i.e. popV, applies a series of annotation algorithms, each providing a different recipe for transferring labels from Tabula Sapiens to the user-provided data. popV then conducts consensus analysis over these algorithms to estimate the most reproducible annotation of every cell, and to highlight cells that are difficult to label.

scATOMIC, single cell annotation of tumour microenvironments in pan-cancer settings, is a comprehensive, pan cancer, TME cell type classifier. It is based on a structured scheme that uses hierarchically organized models and elimination processes, reducing the transcriptomic complexity of the TME multi-cellular system to improve cell classification, abelson-lab/scATOMIC GitHub.

SifiNet is a robust and accurate computational pipeline for identifying distinct gene sets, extracting and annotating cellular subpopulations, and elucidating intrinsic relationships among these subpopulations. Uniquely, SifiNet bypasses the cell clustering stage, commonly integrated into other cellular annotation pipelines, thereby circumventing potential inaccuracies in clustering that may compromise subsequent analyses jichunxie/sifinet GitHub

Data imputation

scBP is a two-step graph-based feature propagation method. It initially imputes zero values using non-zero values, ensuring that the imputation process does not affect the non-zero values due to dropout. Subsequently, it denoises the entire dataset by leveraging gene-gene and cell-cell relationships in the respective steps.

Benchmark experiments

Researchers in computational biology and other scientific fields often encounter the challenge of selecting the most appropriate computational methods to analyze their data. To determine the strengths of each method or to make recommendations on the best options for an analysis, benchmarking studies are conducted, which rigorously compare the performance of different methods using well-characterized benchmark datasets. However, to ensure that the results are reliable, unbiased, and informative, benchmarking studies must be carefully designed and executed. Weber et al. (2019) provided a set of practical guidelines and recommendations for conducting benchmarking analyses of high quality.

As single-cell technologies continue to advance, an increasing number of analysis tools are becoming available to researchers. Consequently, there is a growing need for datasets and methods that support systematic benchmarking and evaluation of these tools. Validating and benchmarking analysis tools for single-cell measurements is part of the “Eleven grand challenges in single-cell data science” [Lähnemann et al. (2020)].

Benchmarking can be approached at two different level:

  • In silico
  • wet lab experiment

In silico benchmark experiments

The validation and comparison of tools in the field of simulation is an intriguing area. Despite the fact that simulators have several limitations, there has been a significant effort in the past few years to enhance their power, as noted by Cao et al. (2021). The scDesign3, a simulator by Song et al. (2023), appears to be a versatile tool that is capable of overcoming some of the limitations of other existing simulators, according to the developers. GRouNdGAN, GRN-guided in silico simulation of single-cell RNA-seq data using Causal generative adversarial networks, is a causal implicit generative model for reference-based GRN-guided simulation of scRNA-seq [Zinati et al. 2024]

Wet lab experiment

Over the past few years, there has been a significant increase in the number of published single-cell omics studies, which serve as valuable resources for benchmark experiments. In particular, Svensson et al. (2020) have compiled a comprehensive collection of single-cell omics datasets with manually curated metadata. As part of the implementation study of the single-cell omics community, we are creating a series of datasets specifically designed for benchmarking computational tools that focus on single-cell tumor heterogeneity.

Benchmark experiments addressing tumor heterogeneity

Tumor heterogeneity, where distinct cancer cells exhibit diverse morphological and phenotypic profiles, including gene expression, metabolism, and proliferation, poses challenges for molecular prognostic markers and patient classification for targeted therapies. Various omics technologies, such as bulk [Babu & Snyder (2023)] and single-cell omics [Flynn et al. (2023)] approaches, have enabled the characterization of diverse molecular layers at an unprecedented scale and resolution, offering a comprehensive perspective on the behavior of tumors. The integration of multiple omics datasets enables systematic exploration of diverse molecular information [Yue et al. (2023)] at each biological layer, but also presents challenges in extracting meaningful insights from the exponentially growing volume of multi-omics data. To address this challenge, efficient algorithms are required to dig into the data and reveal the underlying complexities of cancer’s intricate biological processes. The past few years have seen a proliferation of new computational methods for analyzing single-cell omics data, which can make it challenging to select the most appropriate tool for a particular task. As a result, it is crucial to establish benchmarking platforms [Mangul et al. (2019), Decamps et al. (2021), OpenEBench, Omnibenchmark, Knight et al. (2023)] and datasets [Tian et al. (2018),] in order to create a controlled environment for the validation of bioinformatics tools in the field of single-cell omics analysis.

Healthy and diseases published single cell datasets

CELLxGENE is a suite of tools that help scientists to find, download, explore, analyze, annotate, and publish single cell datasets. it is characterised by the possibility to download a wide set of published single cell experiments as h5 or seurat (v3) format.

CellCommuNet is an atlas of cell–cell communication networks from single-cell RNA sequencing data of human and mouse tissues in normal and disease states [Ma et al. NAR 2024].

Tian et al. (2019) dataset

As part of their article they created ‘pseudo cells’ from up to five distinct human lung adenocarcinoma cell lines. GEO dataset GSM3618014] contains the mixture of five cell lines:

Data were generated using 10XGenomics v2 chemistry. The raw count table was provided without the association of the cell lines to each cell. We have assigned the cell line name to each cell via the similarity between single cell clusters and cell lines bulk data from CCLE database. The annotated count table and the full procedure used for the annotation are present a figshare dataset []

PC9 dataset

In GEO NCBI repository are available single cell data on PC9 untreated lung cancer cell line done in two different labs, using both 10XGenomics and Drop-seq platforms and produced from in vitro culture or xenograph experiments.

  • set1: GSM3972657 PC9 in vitro dropseq Chicago (2500 cells)
  • set2: GSM4494347 PC9 in vitro dropseq Chicago (12000 cells)
  • set3: GSM3972651 PC9 in vitro 10XGenomics Chicago (12649 cells)
  • set4: GSM6106787 PC9 in vitro 10XGenomics Berkeley (413 cells)
  • set5: GSM4869650 PC9 xenograft dropseq Chicago (29983 cells)
  • set6: GSM3972674 PC9 and U937 in vitro dropseq Chicago (2634 cells)
  • set7: BE1 (see below) GSE243665 PC9 in vitro 10XGenomics Turin (4492 cells)

These datasets, because of their differences, i.e. platforms, growth, labs, represent an ideal instrument to benchmark batch removal methods as well as integration methods. The seven sets are available as datasets with the same gene annotation (ensemblID:Symbol). Each set has the cellID with the extension _s(1:5) and set6 has two extensions _s6PC9 and _s6U937. These datasets and the script used for annotation are available as figshare repository (10.6084/m9.figshare.23626407).

77 annotated scRNAseq tumor datasets

Gavish and collaborators [Nature 2023] have recenty curated, annotated and integrated the data from 77 different single cell transcriptomics studies encompassing a total of 1,163 tumour samples covering 24 tumour types and more than 23 milions cells. The data are accessible at 3CA site.

SCONE datasets

As part of the Single Cell Community implementation study we are focusing in providing a set of benchmark experiments to address the extraction of biological knowledge from “controlled” cancer heterogeneity.

BE1 (A single cell RNAseq benchmark experiment embedding “controlled” cancer heterogeneity)

We have done a 10XGenomics scRNAseq experiment including the following elements:

The above figure described the driver genes associated to each cell line. Only a minimal part of the connections has been shown to easy readability of the image. Full list of the interactions depicted by IPA are available at figshare [10.6084/m9.figshare.23284748]. All driver genes have been observed in resistence occuring upon treatment with Isomertinib of EGFR mutated lung cancers [Gomatou et al. (2023)].

The experiment was done using CellPlex technology from 10XGenomics allowing multiplexing samples into a single channel and therefore removing unwanted batch effects.

The count tables from the entire BE1 experiment are available through an R Shiny app, allowing users to construct datasets encompassing different cell lines at varying ratios. An R package providing the same functionalites of the R Shiny app is available at

Actual state of the project: Fully sequenced, raw data deposited to GEO: GSE243665, published on Sci Data 2024 Feb 2;11(1):159. doi: 10.1038/s41597-024-03002-y.

The above figure show the sequencing statistics of the 7 cell lines.

Processed data are available at: 10.6084/m9.figshare.23939481

For further information please contact raffaele dot calogero at unito dot it


The cell lines from for BE1 will be used to generate surrogate tumor-tissues for spatial transcriptomics, by embedding in matrigel pools of the 7 cell lines at different ratios. For each cell ratio we will generate a slide (2 sections) using Visium for FFPE samples and six slides using Curio Bioscience spatial platform for OTC fresh frozen samples. In total we expect produce three cell lines ratios:

  • Cell line 1 40%, cell line 2 25% cell line 3 20%, cell line 4 5%, PBMC 10%
  • Cell line 1 45% cell line 2 25%, cell line 3 10%, cell line 4 10%, cell line 5 5%, PBMC 5%
  • Cell line 1 25%, cell line 2 25% cell line 3 10% cell line 4 10%, cell line 4 10%, cell line 5 5%, cell line 6 5%, cell line 7 5%, PBMC 5%.

Actual state of the project: Expecting results from BE1.

Expected data availability: March 2024


PC9 and DV90 cell lines will be used to generate combined scRNAseq and scATACseq experiment using 10X genomics technology for multi-omics. Specifically we will generate three experiments: i) PC9 alone, ii) DV90 alone, iii) PC9/DV90 co-colture (two replicates). PC9 alone, DV90 alone and PC9/DV90 co-colture will also be analysed in triplicates the secreted microvesicles (RNA/miRNAseq) and the bulk intracellular RNA/miRNAseq. The idea is to provide a comprehesive data set to benchmark tools devoted to the prediction of cell to cell crosstalk (Xie et al. 2023, Zhang et al. 2023).

Actual state of the project: Ordered 10XGenomics multi-omics kit.

Expected data availability: January 2024

Spatial Omics


Spatial Omics DataBase (SODB) is a comprehensive database designated for general spatial omics data. SODB provides: Various spatial omics data modalities, including spatially resolved transcriptomics, proteomics, metabolomics, genomics and multi-omics.


Spatial Transcript Omics DataBase (STOmics DB) is a comprehensive repository of literature and Datasets related to spatial transcriptomics topics, and provides convenient tools for Data analysis, search and visualization (Xu et al. NAR 2023).


CROST offers single-sample analysis, interactive visualization, multi-omics integration for exploring cancer svg, and other powerful tools for spatial transcriptomics researches (Wang et al. NAR 2023).


SpatialDB is a public database that specifically curates spatially resolved transcriptomic data from published papers, aiming to provide a comprehensive and accurate resource of spatial gene expression profiles in tissues.


SOAR is a comprehensive database of 304 spatial transcriptomics datasets from 11 species across 40 tissue types.