HCATonsilData
26 October 2023
1 Introduction
Palatine tonsils are under constant exposure to antigens via the upper respiratory tract, which makes them a compelling model secondary lymphoid organ (SLO) to study the interplay between innate and adaptive immune cells (Ruddle and Akirav 2009). Tonsils have a non-keratinizing stratified squamous epithelium, organized into tubular, branched crypts that enlarge the tonsillar surface. Within the crypts, microfold cells (or M cells) sample antigens at their apical membrane. Subsequently, antigen presenting cells (APC), such as dendritic cells (DC), process and present antigens to T cells in the interfollicular or T cell zone. Alternatively, antigens are kept intact by follicular dendritic cells (FDC) in lymphoid follicles, where they are recognized by B cells (Nave, Gebert, and Pabst 2001). Such recognition triggers the GC reaction, whereby naive B cells (NBC) undergo clonal selection, proliferation, somatic hypermutation, class switch recombination (CSR) and differentiation into long-lived plasma cells (PC) or memory B cells (MBC) (De Silva and Klein 2015).
In the context of the Human Cell Atlas (HCA) (Regev et al. 2018), we have created a taxonomy of 121 cell types and states in a human tonsil. Since the transcriptome is just a snapshot of a cell’s state (Wagner, Regev, and Yosef 2016), we have added other layers to define cell identity: single-cell resolved open chromatin epigenomic landscapes (scATAC-seq and scRNA/ATAC-seq; i.e. Multiome) as well as protein (CITE-seq), adaptive repertoire (single-cell B and T cell receptor sequencing; i.e. scBCR-seq and scTCR-seq) and spatial transcriptomics (ST) profiles.
The HCATonsilData package aims to provide programmatic and modular access to the datasets of the different modalities and cell types of the tonsil atlas. HCATonsilData also documents how the dataset was generated and archived in different repositories, from raw fastq files to processed datasets. It also explains in detail the cell- and sample-level metadata, and allows users to traceback the annotation of each cell type through detailed Glossary.
2 Installation
HCATonsilData is available in BioConductor and can be installed as follows:
if (!require("BiocManager", quietly = TRUE))
install.packages("BiocManager")
BiocManager::install("HCATonsilData")Load the necessary packages in R:
library("HCATonsilData")
library("SingleCellExperiment")
library("ExperimentHub")
library("scater")
library("ggplot2")
library("knitr")
library("kableExtra")
library("htmltools")3 Overview of the dataset
We obtained a total of 17 human tonsils, which covered three age groups: children (n=6, 3-5 years), young adults (n=8, 19-35 years), and old adults (n=3, 56-65 years). We collected them in a discovery cohort (n=10), which we used to cluster and annotate cell types; and a validation cohort (n=7), which we used to validate the presence, annotation and markers of the discovered cell types. The following table corresponds to the donor-level metadata:
data("donor_metadata")
kable(
donor_metadata,
format = "markdown",
caption = "Donor Metadata",
align = "c"
) |> kable_styling(full_width = FALSE)
#> Warning in kable_styling(kable(donor_metadata, format = "markdown", caption =
#> "Donor Metadata", : Please specify format in kable. kableExtra can customize
#> either HTML or LaTeX outputs. See https://haozhu233.github.io/kableExtra/ for
#> details.| donor_id | hospital | sex | age | age_group | cause_for_tonsillectomy | cohort_type | comments | |
|---|---|---|---|---|---|---|---|---|
| BCLL-2-T | BCLL-2-T | Clinic | male | 65 | old_adult | tonsil removed during surgery for benign squamous pharyngeal papillomatosis | discovery | NA |
| BCLL-6-T | BCLL-6-T | Clinic | male | 35 | young_adult | sleep apnea | discovery | NA |
| BCLL-8-T | BCLL-8-T | CIMA | male | 4 | child | tonsillitis | discovery | NA |
| BCLL-9-T | BCLL-9-T | CIMA | male | 5 | child | tonsillitis | discovery | NA |
| BCLL-10-T | BCLL-10-T | CIMA | male | 3 | child | tonsillitis | discovery | NA |
| BCLL-11-T | BCLL-11-T | CIMA | female | 5 | child | tonsillitis | discovery | NA |
| BCLL-12-T | BCLL-12-T | CIMA | female | 3 | child | tonsillitis | discovery | NA |
| BCLL-13-T | BCLL-13-T | CIMA | female | 5 | child | tonsillitis | discovery | NA |
| BCLL-14-T | BCLL-14-T | CIMA | male | 26 | young_adult | sleep apnea | discovery | NA |
| BCLL-15-T | BCLL-15-T | CIMA | male | 33 | young_adult | sleep apnea | discovery | NA |
| BCLL-20-T | BCLL-20-T | Newcastle | male | 23 | young_adult | tonsillitis | validation | original id: TIP01 |
| BCLL-21-T | BCLL-21-T | Newcastle | female | 19 | young_adult | tonsillitis | validation | original id: TIP02 |
| BCLL-22-T | BCLL-22-T | Newcastle | female | 22 | young_adult | tonsillitis | validation | original id: TIP03 |
| BCLL-24-T | BCLL-24-T | Clinic | male | 63 | old_adult | tonsil removed during surgery for superficial squamous carcinoma of the laryngeal vocal cord | validation | NA |
| BCLL-25-T | BCLL-25-T | Clinic | female | 25 | young_adult | tonsillitis | validation | NA |
| BCLL-26-T | BCLL-26-T | Clinic | male | 56 | old_adult | sleep apnea | validation | NA |
| BCLL-28-T | BCLL-28-T | Newcastle | male | 28 | young_adult | tonsillitis | validation | original id: TIP04 |
These tonsil samples were processed with different data modalities:
- scRNA-seq (10X Chromium v3)
- scATAC-seq (10X Chromium)
- 10X Multiome: joint RNA and ATAC for each cell.
- CITE-seq: joint transcriptome + ~200 protein surface markers for each cell.
- Spatial transcriptomics
The following heatmap informs about which samples were sequenced with which technology and cohort type:
For each data modality, we mapped all raw fastq files with the appropriate flavor of cellranger (e.g. cellranger-atac, spaceranger, etc), and analyzed all datasets within the Seurat ecosystem (Hao et al. 2021). All fastq files are deposited at EGA under accession number EGAS00001006375. The expression and accessibility matrices are deposited in Zenodo under record 6678331. The Seurat objects from which the data of this repository is derived are archived in Zenodo under record 8373756. Finally, the GitHub repository Single-Cell-Genomics-Group-CNAG-CRG/TonsilAtlas documents the full end-to-end analysis, from raw data to final Seurat objects and figures in the paper.
To define the tonsillar cell types, we first aimed to cluster cells using the combined expression data from scRNA-seq, Multiome and CITE-seq datasets. However, we noticed that with CITE-seq we detected 2.75X and 2.98X fewer genes than with scRNA-seq and Multiome, respectively:
Because we did not want to bias the clustering towards the modality that provides less information, we first analyzed scRNA-seq and Multiome together, and used CITE-seq to validate the annotation (see below). However, a recent benchmarking effort showed that scRNA-seq and single-nuclei RNA-seq (such as Multiome) mix very poorly (Mereu et al. 2020). Indeed, we observed massive batch effects between both modalities (see manuscript). To overcome it, we found highly variable genes (HVG) for each modality independently. We then intersected both sets of HVG to remove modality-specific variation. Following principal component analysis (PCA), we integrated scRNA-seq and Multiome with Harmony (Korsunsky et al. 2019). Harmony scales well to atlas-level datasets and ranks among the best-performing tools in different benchmarks.
These steps are performed using SLOcatoR (see methods of the paper), which allowed 1. to remove batch effects between scRNA-seq and Multiome, 2. to transfer cell type labels from Multiome to scATAC-seq, and from scRNA-seq to CITE-seq. Thus, this strategy allowed us to annotate clusters using information across multiple modalities (RNA expression, protein expression, TF motifs, etc.).
Note: we provide SLOcatoR as an R package to promote reproducibility and best practices in scientific computing, but we refer the users to properly benchmarked tools to annotate their datasets such as Azimuth, scArches, or Symphony.
Within the discovery cohort, we first defined 9 main compartments: (1) NBC and MBC, (2) GC B cells (GCBC), (3) PC, (4) CD4 T cells, (5) cytotoxic cells [CD8 T cells, NK, ILC, double negative T cells (DN)], (6) myeloid cells (DC, macrophages, monocytes, granulocytes, mast cells), (7) FDC, (8) epithelial cells and (9) plasmacytoid dendritic cells (PDC):
Subsequently, we clustered and annotated cell subtypes within each of the main compartments, giving rise to the 121 cell types and states in the atlas. The following UMAPs inform about the main cell populations and number of cells per assay in the discovery cohort:
We then applied SLOcatoR to annotate cells from the validation cohort (1) to confirm the presence, annotation and markers of cell types; (2) to extend the atlas through an integrated validation dataset, and (3) to chart compositional changes in the tonsil during aging. The integrated tonsil atlas represented over 462,000 single-cell transcriptomes:
Finally, we focused again on each of the 9 compartments and retransferred the final annotation level. As an example, here we can see the transferred annotation for CD4 T cells, from discovery to validation cohort:
Overall, we could validate most of the cell types, as they preserved their marker genes and had a high annotation confidence in the validation cohort. However, (1) transient cell states, (2) underrepresented cell types, or (3) clusters that are at the intersection between two main compartments (and might represent doublets) had low annotation confidences. We provide this interpretation in the Glossary (see below).
4 Assay types
HCATonsilData provides access to 5 main types of assays: RNA, ATAC, Multiome, CITE-seq and Spatial.
4.1 scRNA-seq
We can obtain the SingleCellExperiment object with gene expression (RNA)
data as follows:
(sce <- HCATonsilData(assayType = "RNA", cellType = "All"))
table(sce$assay)This object consists of 377,988 profiled with scRNA-seq (3P) and 84,364 cells profiled with multiome, for a total of 462,352 cells (37,378 genes were quantified across all of these).
We can dowload a SingleCellExperiment object specific to each of the main
subpopulations defined at level 1 as follows:
listCellTypes(assayType = "RNA")
#> [1] "All" "NBC-MBC" "GCBC" "PC" "CD4-T"
#> [6] "Cytotoxic" "myeloid" "FDC" "epithelial" "PDC"
(epithelial <- HCATonsilData(assayType = "RNA", cellType = "epithelial"))
#> see ?HCATonsilData and browseVignettes('HCATonsilData') for documentation
#> loading from cache
#> require("rhdf5")
#> see ?HCATonsilData and browseVignettes('HCATonsilData') for documentation
#> loading from cache
#> see ?HCATonsilData and browseVignettes('HCATonsilData') for documentation
#> loading from cache
#> see ?HCATonsilData and browseVignettes('HCATonsilData') for documentation
#> loading from cache
#> see ?HCATonsilData and browseVignettes('HCATonsilData') for documentation
#> loading from cache
#> see ?HCATonsilData and browseVignettes('HCATonsilData') for documentation
#> loading from cache
#> see ?HCATonsilData and browseVignettes('HCATonsilData') for documentation
#> loading from cache
#> class: SingleCellExperiment
#> dim: 37378 396
#> metadata(0):
#> assays(2): counts logcounts
#> rownames(37378): AL627309.1 AL627309.3 ... AC136616.1 AC023491.2
#> rowData names(3): gene_name highly_variable gene_id
#> colnames(396): bw94nf57_vm85woki_CTCCTTTCACACGGAA-1
#> bw94nf57_vm85woki_GTAATGCAGTGACCTT-1 ...
#> TIP04-2_fresh_TCTATACTCGTGAGAG-1 TIP04-2_fresh_TGTCCCAGTACAAGTA-1
#> colData names(39): barcode donor_id ... UMAP_1_20230508 UMAP_2_20230508
#> reducedDimNames(3): PCA UMAP HARMONY
#> mainExpName: NULL
#> altExpNames(0):
data("colors_20230508")
scater::plotUMAP(epithelial, colour_by = "annotation_20230508") +
ggplot2::scale_color_manual(values = colors_20230508$epithelial) +
ggplot2::theme(legend.title = ggplot2::element_blank())
#> Scale for colour is already present.
#> Adding another scale for colour, which will replace the existing scale.
As we can see, we can use the same colors as in the publication using the
colors_20230508 variable.
We can also download datasets from the preprint (discovery cohort, version 1.0) -
we first load a list of matched annotations used throughout the course of the
project, used internally by the main HCATonsilData() function:
data("annotations_dictionary")
annotations_dictionary[["dict_20220619_to_20230508"]]
#> MAIT TCRVδ+ gd T CD56+ gd T Nksig CD8 T
#> "MAIT/Vδ2+ γδ T" "non-Vδ2+ γδ T" "ZNF683+ CD8 T" "EM CD8 T"
#> C1Q Slancytes ITGAX Slancytes MMP Slancytes SELENOP Slancytes
#> "C1Q Slan-like" "ITGAX Slan-like" "MMP Slan-like" "SELENOP Slan-like"
#> CD16-CD56- NK
#> "CD16-CD56dim NK"
# load also a predefined palette of colors, to match the ones used in the manuscript
data("colors_20230508")
(epithelial_discovery <- HCATonsilData("RNA", "epithelial", version = "1.0"))
#> see ?HCATonsilData and browseVignettes('HCATonsilData') for documentation
#> loading from cache
#> see ?HCATonsilData and browseVignettes('HCATonsilData') for documentation
#> loading from cache
#> see ?HCATonsilData and browseVignettes('HCATonsilData') for documentation
#> loading from cache
#> see ?HCATonsilData and browseVignettes('HCATonsilData') for documentation
#> loading from cache
#> see ?HCATonsilData and browseVignettes('HCATonsilData') for documentation
#> loading from cache
#> see ?HCATonsilData and browseVignettes('HCATonsilData') for documentation
#> loading from cache
#> see ?HCATonsilData and browseVignettes('HCATonsilData') for documentation
#> loading from cache
#> class: SingleCellExperiment
#> dim: 37378 277
#> metadata(0):
#> assays(2): counts logcounts
#> rownames(37378): AL627309.1 AL627309.3 ... AC136616.1 AC023491.2
#> rowData names(3): gene_name highly_variable gene_id
#> colnames(277): bw94nf57_vm85woki_CTCCTTTCACACGGAA-1
#> bw94nf57_vm85woki_GTAATGCAGTGACCTT-1 ...
#> y7qn780g_p6jkgk63_TGACTCCTCCTAGCCT-1
#> y7qn780g_p6jkgk63_TGCTTCGGTTAGGAGC-1
#> colData names(32): barcode donor_id ... annotation_20220619
#> annotation_20230508
#> reducedDimNames(3): PCA UMAP HARMONY
#> mainExpName: NULL
#> altExpNames(0):
scater::plotUMAP(epithelial, colour_by = "annotation_20230508") +
ggplot2::scale_color_manual(values = colors_20230508$epithelial) +
ggplot2::theme(legend.title = ggplot2::element_blank())
#> Scale for colour is already present.
#> Adding another scale for colour, which will replace the existing scale.
Here’s a brief explanation of all the variables in the colData slot of the
SingleCellExperiment objects:
- barcode: the cell barcode. Combination of GEM well and cellranger 10X barcode.
- donor_id: the donor-specific identifier, which can be used to retrieve donor-level metadata form the table above.
- gem_id: we gave a unique hashtag to each GEM well (10X Chip Channel) in our dataset. This allows to traceback all the metadata for a given cell.
- library_name: each GEM well can give rise to multiple Illumina libraries. For example, one Multiome GEM well will give rise to 2 illumina libraries (ATAC and RNA).
- assay: 3P (scRNA-seq) or multiome
- sex
- age
- age_group: kid, young adult, old adult
- hospital: hospital where the tonsils where obtained Hospital Clinic, CIMA, or Newcastle.
- cohort_type: discovery or validation cohort
- cause_for_tonsillectomy: condition that led to the tonsillectomy (e.g. tonsillitis, sleep apnea, etc.)
- is_hashed: whether we used cell hashing or not.
- preservation: whether we processed the sample fresh or frozen.
- nCount_RNA, nFeature_RNA: number of counts and features (genes) detected per cell.
- pct_mt, pct_ribosomal: percentage of counts that map to mitochondrial (^MT) or ribosomal (^RPS) genes.
- pDNN_hashing, pDNN_scrublet, pDNN_union: proportion of doublet nearest neighbors (pDNN) using different doublet annotations.
- S.Score, G2M.Score Phase, CC.Difference: outputs of the CellCycleScoring from
Seurat. - scrublet_doublet_scores: doublet score obtained with Scrublet
- scrublet_predicted_doublet: doublet annotation obtained with Scirpy (doublet or singlet)
- doublet_score_scDblFinder: doublet score obtained with scDblFinder, which was run in the validation cohort following the most recent best practices for single-cell analysis.
- annotation_level_1: annotation used at our level 1 clustering (9 main compartments).
- annotation_level_1_probability: annotation confidence for the level 1 annotation (relevant for validation cohort, as it implied KNN annotation)
- annotation_figure_1: annotation used in the figure 1 of our manuscript. This annotation consisted of grouping the final subtypes into main cell types that were distinguishable in the UMAP.
- annotation_20220215, annotation_20220619, annotation_20230508: time-stamped annotation for cell types.
- annotation_20230508_probability: annotation confidence for the final annotation (relevant for validation cohort, as it implied KNN annotation)
- UMAP_1_level_1, UMAP_2_level_1: UMAP1 coordinates of the figure 1B of the article.
- annotation_20220215: see above.
- UMAP_1_20220215, UMAP_2_20220215: UMAP coordinates used in figures of the preprint for each cell type.
4.2 scATAC-seq and Multiome
Since there is not a popular Bioconductor package to analyze or store scATAC-seq data, we point users to the scATAC-seq and Multiome Seurat objects that we created using Signac (Stuart et al. 2021).
Here are the instructions to download the scATAC-seq object (approximately ~9.3 Gb in size):
library("Seurat")
library("Signac")
download_dir = tempdir()
options(timeout = 10000000)
atac_url <- "https://zenodo.org/record/8373756/files/TonsilAtlasSeuratATAC.tar.gz"
download.file(
url = atac_url,
destfile = file.path(download_dir, "TonsilAtlasSeuratATAC.tar.gz")
)
# Advice: check that the md5sum is the same as the one in Zenodo
untar(
tarfile = file.path(download_dir, "TonsilAtlasSeuratATAC.tar.gz"),
exdir = download_dir
)
atac_seurat <- readRDS(
file.path(download_dir, "scATAC-seq/20230911_tonsil_atlas_atac_seurat_obj.rds")
)
atac_seuratThe multiome object contains 68,749 cells that passed both RNA and ATAC QC filters. Here are the instructions to download the Multiome object (approximately ~5.7 Gb in size):
library("Seurat")
library("Signac")
download_dir = tempdir()
options(timeout = 10000000)
multiome_url <- "https://zenodo.org/record/8373756/files/TonsilAtlasSeuratMultiome.tar.gz"
download.file(
url = multiome_url,
destfile = file.path(download_dir, "TonsilAtlasSeuratMultiome.tar.gz")
)
# Advice: check that the md5sum is the same as the one in Zenodo
untar(
tarfile = file.path(download_dir, "TonsilAtlasSeuratMultiome.tar.gz"),
exdir = download_dir
)
multiome_seurat <- readRDS(
file.path(download_dir, "/multiome/20230911_tonsil_atlas_multiome_seurat_obj.rds")
)
multiome_seuratNote: we may version these two objects in Zenodo to polish the annotation. Therefore, we recommend users to check that this is the most updated version of the object in Zenodo.
To recall peaks or visualize chromatin tracks, it is essential to have access to the fragments file. We modified the original fragments files generated by cellranger-atac or cellranger-arc to include the gem_id as prefix. These files can be downloaded as follows (approximate size of ~26.0 Gb, feel free to grab a coffee in the meanwhile):
download_dir = tempdir()
fragments_url <- "https://zenodo.org/record/8373756/files/fragments_files.tar.gz"
download.file(
url = fragments_url,
destfile = file.path(download_dir, "fragments_files.tar.gz")
)
untar(
tarfile = file.path(download_dir, "fragments_files.tar.gz"),
exdir = download_dir
)Note: it is paramount to update the paths to the fragments files in each ChromatinAssay object using the UpdatePath function.
The chromatin accessibility side of these objects was processed using the general pipeline from Signac. Briefly:
- We called peaks using MACS2 (as implemented in the CallPeaks function), generating pseudobulks for each of the main 9 compartments.
- We quantified the accessibility for each peak and each cell using the FeatureMatrix function.
- We normalized data using the term frequency inverse document frequency (TF-IDF) normalization, as implemented in the RunTFIDF function.
- We reduced the dimensionality using latent semantic indexing (RunLSI function)
For Multiome, we additionally computed a weighted KNN graph with the FindMultiModalNeighbors function.
Many variables in the metadata slot are shared with scRNA-seq. Here, we explain the meaning of the variables that are specific to Multiome or scATAC-seq:
- nCount_ATAC: total number of counts per cell
- nFeature_ATAC: total number of peaks detected per cell
- is_facs_sorted: whether that particular sample was FACS-sorted to eliminate dead cells
- nucleosome_signal: as explained in Signac, Nucleosome banding pattern: The histogram of DNA fragment sizes (determined from the paired-end sequencing reads) should exhibit a strong nucleosome banding pattern corresponding to the length of DNA wrapped around a single nucleosome. We calculate this per single cell, and quantify the approximate ratio of mononucleosomal to nucleosome-free fragments
- nucleosome_percentile: percentile rank of each ratio of mononucleosomal to nucleosome-free fragments per cell (see NucleosomeSignal function)
- TSS.enrichment: as explained in Signac, The ENCODE project has defined an ATAC-seq targeting score based on the ratio of fragments centered at the TSS to fragments in TSS-flanking regions (see https://www.encodeproject.org/data-standards/terms/). Poor ATAC-seq experiments typically will have a low TSS enrichment score.
- TSS.percentile
- RNA.weight: the weight that RNA has in the weighted nearest neighbor graph per cell (multiome-specific)
- ATAC.weight: the weight that RNA has in the weighted nearest neighbor graph per cell (multiome-specific)
4.3 CITE-seq
The CITE-seq object can be downloaded as follows (approximate size of ~0.4 Gb):
library("Seurat")
download_dir = tempdir()
options(timeout = 10000000)
cite_url <- "https://zenodo.org/record/8373756/files/TonsilAtlasSeuratCITE.tar.gz"
download.file(
url = cite_url,
destfile = file.path(download_dir, "TonsilAtlasSeuratCITE.tar.gz")
)
# Advice: check that the md5sum is the same as the one in Zenodo
untar(
tarfile = file.path(download_dir, "TonsilAtlasSeuratCITE.tar.gz"),
exdir = download_dir
)
cite_seurat <- readRDS(
file.path(download_dir, "CITE-seq/20220215_tonsil_atlas_cite_seurat_obj.rds")
)
cite_seuratTogether with CITE-seq, we also applied scBCR-seq and scTCR-seq. This data was analyzed with scirpy, (Sturm et al. 2020) and we provide the main output of this analysis (for cytotoxic cells) as a dataframe, which can be imported as follows:
scirpy_df <- read.csv(
file = file.path(download_dir, "CITE-seq/scirpy_tcr_output.tsv"),
header = TRUE
)
head(scirpy_df)4.4 Spatial transcriptomics
A SpatialExperiment of the spatial transcriptomics dataset may be retrieved via assayType="Spatial". The dataset contains 8 tissue slices with ~1,000-3,000 cells each that were profiled on two separated slides, as well as a low-resolution (H&E staining) image for each slice.
library("SpatialExperiment")
(spe <- HCATonsilData(assayType = "Spatial"))To plot gene expression you can use the ggspavis package:
library("ggspavis")
sub <- spe[, spe$sample_id == "esvq52_nluss5"]
plt <- plotVisium(sub, fill="SELENOP") +
scale_fill_gradientn(colors=rev(hcl.colors(9, "Spectral")))
plt$layers[[2]]$aes_params$size <- 1.5
plt$layers[[2]]$aes_params$alpha <- 1
plt$layers[[2]]$aes_params$stroke <- NA
plt5 Annotations of the Tonsil Data Atlas
To allow users to traceback the rationale behind each and every of our annotations, we provide a detailed glossary of 121 cell types and states and related functions to get the explanation, markers and references of every annotation. You can access the glossary as a dataframe as follows:
glossary_df <- TonsilData_glossary()
head(glossary_df)
#> annotation_level_1 annotation_detailed
#> PDC PDC PDC
#> IFN1+ PDC PDC IFN1+ PDC
#> preB preB preB
#> preT preT preT
#> Naive CD4_T Naive
#> CM Pre-non-Tfh CD4_T CM Pre-non-Tfh
#> description
#> PDC pDCs were previously identified by the standard protein marker CD123 (IL3RA). This cluster had higher expression of IRF8, IL3RA and LILRA4 (Villani et al., 2017)
#> IFN1+ PDC The second pDC subpopulation was identified by standard protein marker CD123 and as IFN-producing cells. This cluster had higher expression of IRF8, IL3RA, LILRA4 and IFI44, IFI6 (Park et al., 2020; Villani et al., 2017).
#> preB Precursor B cells were identified by the expression of CD79B, CD19, PAX5, RAG1/2 (Strauchen et al., 2003).
#> preT Precursor T cells in tonsils expressed: CD3G, CD8A, RAG1, DNTT, CD1A, CD1B, CD1C, CD1E (McClory et al., 2012).
#> Naive Naïve cell is a CD4 T cell that recently migrates from the thymus towards lymph nodes. Naïve CD4 T cells expressed lymph node homing genes such as CCR7 and naïve-associated genes: SELL, LEF1, BACH2 and NOSIP. Also, they were negative for PRDM1 and BCL6 and were CD62L+ and CD45RA+ (Cano-Gamez et al., 2020).
#> CM Pre-non-Tfh After antigen encounter T cells acquire memory and effector functions maintaining the lymph node homing. CM showed an upregulation of IL7R compared to naïve T cells, and higher expression of PRDM1, PASK, ANXA1 and S100A4. They had an intermediate expression of CCR7 and higher expression of CD28 confirming their migratory capacity to secondary lymph nodes and their memory profile based on their effector function. This cluster was CD45RO+, CD28+ and CD29+(Cano-Gamez et al., 2020; Nicolet et al., 2021).
#> marker_genes
#> PDC IRF8,IL3RA,LILRA4
#> IFN1+ PDC IRF8,IL3RA,LILRA5,IFI44,IFI6
#> preB CD79B,CD19,PAX5,RAG1,RAG2
#> preT CD3G,CD8A,RAG1,DNTT,CD1A,CD1B,CD1C,CD1E
#> Naive SELL,LEF1,BACH2,NOSIP
#> CM Pre-non-Tfh CCR7,IL7R,ANXA1,ITGB1,S100A4
#> related_refs
#> PDC Villani2017|10.1126/science.aah4573
#> IFN1+ PDC Park2020|10.1126/science.aay3224;Villani2017|10.1126/science.aah4573
#> preB Strauchen2003|10.1177/106689690301100105
#> preT McClory2012|10.1172/JCI46125
#> Naive Cano-Gamez2020|10.1038/s41467-020-15543-y
#> CM Pre-non-Tfh Cano-Gamez2020|10.1038/s41467-020-15543-y;Nicolet2021|10.4049/jimmunol.2100138
#> umap_png related_cell_ontology
#> PDC pdc_umap.png NA
#> IFN1+ PDC pdc_umap.png NA
#> preB pdc_umap.png NA
#> preT pdc_umap.png NA
#> Naive cd4_t_umap.png NA
#> CM Pre-non-Tfh cd4_t_umap.png NATo get the glossary for each cell type with nice printing formatting you can use
the TonsilData_cellinfo() function:
TonsilData_cellinfo("Tfr")
#> Tfr
#> ------------------------------
#> Annotation Level 1: CD4_T
#> Cell Markers: T-follicular regulatory cells in the tonsils are CD25-. These cells down-regulate effector Treg markers (IL2RA, FOXP3, CTLA4). This cluster expressed high levels of FCRL3, CLNK, LEF1, TCF7, RBMS3, SESN3, and PDE3B. The top marker FCRL3 can bind secretory IgA to suppress the Tfr inhibitory function. TCF7 and LEF1 are essential for Tfr development in mice (Wing et al., 2017; Agarwal et al., 2020 ; Yang et al., 2019).
#> Cell Markers: FCRL3,CLNK,LEF1,TCF7,RBMS3,SESN3,PDE3B
#> Related references: Wing2017|10.1073/pnas.1705551114;Agarwal2020|10.1016/j.celrep.2019.12.099;Yang2019|10.1016/j.celrep.2019.05.061Alternatively, you can get a static html with links to markers and articles with
TonsilData_cellinfo_html
htmltools::includeHTML(
TonsilData_cellinfo_html("Tfr", display_plot = TRUE)
)
#> /usr/bin/pandoc +RTS -K512m -RTS file10975863884029 --to html4 --from markdown+autolink_bare_uris+tex_math_single_backslash --output file10975863884029.html --lua-filter /home/biocbuild/bbs-3.18-bioc/R/site-library/rmarkdown/rmarkdown/lua/pagebreak.lua --lua-filter /home/biocbuild/bbs-3.18-bioc/R/site-library/rmarkdown/rmarkdown/lua/latex-div.lua --self-contained --variable bs3=TRUE --section-divs --template /home/biocbuild/bbs-3.18-bioc/R/site-library/rmarkdown/rmd/h/default.html --no-highlight --variable highlightjs=1 --variable theme=bootstrap --mathjax --variable 'mathjax-url=https://mathjax.rstudio.com/latest/MathJax.js?config=TeX-AMS-MML_HTMLorMML' --include-in-header /tmp/RtmpT7d08t/rmarkdown-str10975830e6402b.html
#>
#> Output created: file10975863884029.htmlAnnotation Level 1: CD4_T
Cell type description: T-follicular regulatory cells in the tonsils are CD25-. These cells down-regulate effector Treg markers (IL2RA, FOXP3, CTLA4). This cluster expressed high levels of FCRL3, CLNK, LEF1, TCF7, RBMS3, SESN3, and PDE3B. The top marker FCRL3 can bind secretory IgA to suppress the Tfr inhibitory function. TCF7 and LEF1 are essential for Tfr development in mice (Wing et al., 2017; Agarwal et al., 2020 ; Yang et al., 2019).
Marker genes for this cell type:
FCRL3 CLNK LEF1 TCF7 RBMS3 SESN3 PDE3B
References: Wing2017 Agarwal2020 Yang2019
Although we have put massive effort in annotating tonsillar cell types, cell type
annotations are dynamic by nature. New literature or other interpretations of the
data can challenge and refine our annotations. To accommodate this, we have developed
the updateAnnotation function, which allows to periodically provide newer
annotations as extra columns in the colData slot of the SingleCellExperiment
objects. If you want to contribute in one of these versions of the upcoming annotations,
please open an issue and
describe your annotation.
6 Interoperability with other frameworks
While we provide data in the form of SingleCellExperiment objects, you may want to analyze your data using a different single-cell data container. In future releases, we will strive to make the SingleCellExperiment objects compatible with Seurat v5, which will come out after this release of BioConductor. Alternatively, you may want to obtain AnnData objects to analyze your data in scanpy ecosystem. You can convert and save the data as follows:
library("zellkonverter")
epithelial <- HCATonsilData(assayType = "RNA", cellType = "epithelial")
epi_anndatafile <- file.path(tempdir(), "epithelial.h5ad")
writeH5AD(sce = epithelial, file = epi_anndatafile)The SingleCellExperiment objects obtained via HCATonsilData() can be explored in
detail using e.g. additional Bioconductor packages, such as the iSEE package.
This can be as simple as executing this chunk:
if (require(iSEE)) {
iSEE(epithelial)
}Session information
sessionInfo()
#> R version 4.3.1 (2023-06-16)
#> Platform: x86_64-pc-linux-gnu (64-bit)
#> Running under: Ubuntu 22.04.3 LTS
#>
#> Matrix products: default
#> BLAS: /home/biocbuild/bbs-3.18-bioc/R/lib/libRblas.so
#> LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.10.0
#>
#> locale:
#> [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
#> [3] LC_TIME=en_GB LC_COLLATE=C
#> [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
#> [7] LC_PAPER=en_US.UTF-8 LC_NAME=C
#> [9] LC_ADDRESS=C LC_TELEPHONE=C
#> [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
#>
#> time zone: America/New_York
#> tzcode source: system (glibc)
#>
#> attached base packages:
#> [1] stats4 stats graphics grDevices utils datasets methods
#> [8] base
#>
#> other attached packages:
#> [1] HDF5Array_1.30.0 rhdf5_2.46.0
#> [3] htmltools_0.5.6.1 kableExtra_1.3.4
#> [5] knitr_1.44 scater_1.30.0
#> [7] ggplot2_3.4.4 scuttle_1.12.0
#> [9] ExperimentHub_2.10.0 AnnotationHub_3.10.0
#> [11] BiocFileCache_2.10.0 dbplyr_2.3.4
#> [13] SingleCellExperiment_1.24.0 SummarizedExperiment_1.32.0
#> [15] Biobase_2.62.0 GenomicRanges_1.54.0
#> [17] GenomeInfoDb_1.38.0 IRanges_2.36.0
#> [19] S4Vectors_0.40.0 BiocGenerics_0.48.0
#> [21] MatrixGenerics_1.14.0 matrixStats_1.0.0
#> [23] HCATonsilData_1.0.0 BiocStyle_2.30.0
#>
#> loaded via a namespace (and not attached):
#> [1] rstudioapi_0.15.0 jsonlite_1.8.7
#> [3] magrittr_2.0.3 ggbeeswarm_0.7.2
#> [5] magick_2.8.1 farver_2.1.1
#> [7] rmarkdown_2.25 zlibbioc_1.48.0
#> [9] vctrs_0.6.4 memoise_2.0.1
#> [11] DelayedMatrixStats_1.24.0 RCurl_1.98-1.12
#> [13] base64enc_0.1-3 webshot_0.5.5
#> [15] S4Arrays_1.2.0 curl_5.1.0
#> [17] BiocNeighbors_1.20.0 Rhdf5lib_1.24.0
#> [19] SparseArray_1.2.0 sass_0.4.7
#> [21] bslib_0.5.1 cachem_1.0.8
#> [23] mime_0.12 lifecycle_1.0.3
#> [25] pkgconfig_2.0.3 rsvd_1.0.5
#> [27] Matrix_1.6-1.1 R6_2.5.1
#> [29] fastmap_1.1.1 GenomeInfoDbData_1.2.11
#> [31] shiny_1.7.5.1 digest_0.6.33
#> [33] colorspace_2.1-0 AnnotationDbi_1.64.0
#> [35] irlba_2.3.5.1 RSQLite_2.3.1
#> [37] beachmat_2.18.0 filelock_1.0.2
#> [39] labeling_0.4.3 fansi_1.0.5
#> [41] httr_1.4.7 abind_1.4-5
#> [43] compiler_4.3.1 bit64_4.0.5
#> [45] withr_2.5.1 BiocParallel_1.36.0
#> [47] viridis_0.6.4 DBI_1.1.3
#> [49] rappdirs_0.3.3 DelayedArray_0.28.0
#> [51] rjson_0.2.21 tools_4.3.1
#> [53] vipor_0.4.5 beeswarm_0.4.0
#> [55] interactiveDisplayBase_1.40.0 httpuv_1.6.12
#> [57] glue_1.6.2 rhdf5filters_1.14.0
#> [59] promises_1.2.1 grid_4.3.1
#> [61] generics_0.1.3 gtable_0.3.4
#> [63] BiocSingular_1.18.0 ScaledMatrix_1.10.0
#> [65] xml2_1.3.5 utf8_1.2.4
#> [67] XVector_0.42.0 ggrepel_0.9.4
#> [69] BiocVersion_3.18.0 pillar_1.9.0
#> [71] stringr_1.5.0 later_1.3.1
#> [73] dplyr_1.1.3 lattice_0.22-5
#> [75] bit_4.0.5 tidyselect_1.2.0
#> [77] Biostrings_2.70.1 gridExtra_2.3
#> [79] bookdown_0.36 svglite_2.1.2
#> [81] xfun_0.40 stringi_1.7.12
#> [83] yaml_2.3.7 evaluate_0.22
#> [85] codetools_0.2-19 tibble_3.2.1
#> [87] BiocManager_1.30.22 cli_3.6.1
#> [89] xtable_1.8-4 systemfonts_1.0.5
#> [91] munsell_0.5.0 jquerylib_0.1.4
#> [93] Rcpp_1.0.11 png_0.1-8
#> [95] parallel_4.3.1 ellipsis_0.3.2
#> [97] blob_1.2.4 sparseMatrixStats_1.14.0
#> [99] bitops_1.0-7 SpatialExperiment_1.12.0
#> [101] viridisLite_0.4.2 scales_1.2.1
#> [103] purrr_1.0.2 crayon_1.5.2
#> [105] rlang_1.1.1 cowplot_1.1.1
#> [107] KEGGREST_1.42.0 rvest_1.0.3References
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Hao, Yuhan, Stephanie Hao, Erica Andersen-Nissen, William M Mauck III, Shiwei Zheng, Andrew Butler, Maddie J Lee, et al. 2021. “Integrated Analysis of Multimodal Single-Cell Data.” Cell 184 (13): 3573–87.
Korsunsky, Ilya, Nghia Millard, Jean Fan, Kamil Slowikowski, Fan Zhang, Kevin Wei, Yuriy Baglaenko, Michael Brenner, Po-ru Loh, and Soumya Raychaudhuri. 2019. “Fast, Sensitive and Accurate Integration of Single-Cell Data with Harmony.” Nature Methods 16 (12): 1289–96.
Mereu, Elisabetta, Atefeh Lafzi, Catia Moutinho, Christoph Ziegenhain, Davis J McCarthy, Adrián Álvarez-Varela, Eduard Batlle, et al. 2020. “Benchmarking Single-Cell Rna-Sequencing Protocols for Cell Atlas Projects.” Nature Biotechnology 38 (6): 747–55.
Regev, Aviv, Sarah Teichmann, Orit Rozenblatt-Rosen, Michael Stubbington, Kristin Ardlie, Ido Amit, Paola Arlotta, et al. 2018. “The Human Cell Atlas White Paper.” arXiv Preprint arXiv:1810.05192.
Ruddle, Nancy H, and Eitan M Akirav. 2009. “Secondary Lymphoid Organs: Responding to Genetic and Environmental Cues in Ontogeny and the Immune Response.” The Journal of Immunology 183 (4): 2205–12.
Stuart, Tim, Avi Srivastava, Shaista Madad, Caleb A Lareau, and Rahul Satija. 2021. “Single-Cell Chromatin State Analysis with Signac.” Nature Methods 18 (11): 1333–41.
Sturm, Gregor, Tamas Szabo, Georgios Fotakis, Marlene Haider, Dietmar Rieder, Zlatko Trajanoski, and Francesca Finotello. 2020. “Scirpy: A Scanpy Extension for Analyzing Single-Cell T-Cell Receptor-Sequencing Data.” Bioinformatics 36 (18): 4817–8.
Wagner, Allon, Aviv Regev, and Nir Yosef. 2016. “Revealing the Vectors of Cellular Identity with Single-Cell Genomics.” Nature Biotechnology 34 (11): 1145–60.