From fb06d35476f1bf042ae048190c9e25feab7fb3af Mon Sep 17 00:00:00 2001 From: Vadim Nazarov <2979713+vadimnazarov@users.noreply.github.com> Date: Thu, 13 Feb 2025 13:49:23 +0100 Subject: [PATCH 1/4] remove DS_Store; update gitignore --- .DS_Store | Bin 8196 -> 0 bytes .gitignore | 1 + docs/.DS_Store | Bin 10244 -> 0 bytes docs/clustering/.DS_Store | Bin 6148 -> 0 bytes docs/clustering/figures/.DS_Store | Bin 8196 -> 0 bytes docs/filtering_data/.DS_Store | Bin 6148 -> 0 bytes docs/filtering_data/figures/.DS_Store | Bin 6148 -> 0 bytes docs/filtering_data/figures/atac/.DS_Store | Bin 6148 -> 0 bytes docs/filtering_data/figures/prot/.DS_Store | Bin 6148 -> 0 bytes docs/filtering_data/figures/rna/.DS_Store | Bin 6148 -> 0 bytes docs/ingesting_data/figures/.DS_Store | Bin 6148 -> 0 bytes docs/uni_multi_integration/.DS_Store | Bin 6148 -> 0 bytes docs/uni_multi_integration/figures/.DS_Store | Bin 8196 -> 0 bytes 13 files changed, 1 insertion(+) delete mode 100644 .DS_Store delete mode 100644 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z`~Fey#mmX7>Fc+0Q;c2=aDHKv@AMr;!*&wl8ikMAc5>|bZQko^fy&n7trmUKaK3;2 zc;&{M4|nv~9*=%$?{}Ba{oEg<2~Ur4KC$>J^k4)DN)cm^LxhqfYb%Lyw*6oCyXf5 zpD>f%Ac>lLL532FkV3GwBGCSH324Y509kbUw`;BIM8C4mpN*o`Kt07R5U;&iZFglD zD9M#qFWy4r+Ah{3tfC0NnZ!HS)<^&ekFt3Cg7CjZIi2kGDa@c;k- From cb6e5843b340cb48a79e7e80fe93beb08af4edfc Mon Sep 17 00:00:00 2001 From: Vadim Nazarov <2979713+vadimnazarov@users.noreply.github.com> Date: Thu, 13 Feb 2025 22:26:01 +0100 Subject: [PATCH 2/4] [feat] AIRR tutorial --- README.md | 1 + docs/index.md | 1 + docs/ingesting_airr_data/files/pipeline.yml | 182 +++ .../files/qc_genelist_1.0.csv | 1227 +++++++++++++++++ .../files/short_vdj_submission.tsv | 2 + .../img/tcr_clonal_expansion_barplot.png | Bin 0 -> 12693 bytes .../tcr_group_abundance_receptor_subtype.png | Bin 0 -> 21754 bytes .../img/tcr_group_abundance_receptor_type.png | Bin 0 -> 15152 bytes .../img/tenx_estimated_cells.png | Bin 0 -> 26559 bytes .../img/tenx_mean_reads_per_cell.png | Bin 0 -> 18451 bytes .../img/tenx_productive_tra_trb.png | Bin 0 -> 20161 bytes .../img/tenx_sequencing_saturation.png | Bin 0 -> 17089 bytes .../img/tenx_valid_barcodes.png | Bin 0 -> 15616 bytes .../img/tenx_valid_umis.png | Bin 0 -> 15592 bytes .../ingesting_airr_data.md | 363 +++++ 15 files changed, 1776 insertions(+) create mode 100644 docs/ingesting_airr_data/files/pipeline.yml create mode 100644 docs/ingesting_airr_data/files/qc_genelist_1.0.csv create mode 100644 docs/ingesting_airr_data/files/short_vdj_submission.tsv create mode 100644 docs/ingesting_airr_data/img/tcr_clonal_expansion_barplot.png create mode 100644 docs/ingesting_airr_data/img/tcr_group_abundance_receptor_subtype.png create mode 100644 docs/ingesting_airr_data/img/tcr_group_abundance_receptor_type.png create mode 100644 docs/ingesting_airr_data/img/tenx_estimated_cells.png create mode 100644 docs/ingesting_airr_data/img/tenx_mean_reads_per_cell.png create mode 100644 docs/ingesting_airr_data/img/tenx_productive_tra_trb.png create mode 100644 docs/ingesting_airr_data/img/tenx_sequencing_saturation.png create mode 100644 docs/ingesting_airr_data/img/tenx_valid_barcodes.png create mode 100644 docs/ingesting_airr_data/img/tenx_valid_umis.png create mode 100644 docs/ingesting_airr_data/ingesting_airr_data.md diff --git a/README.md b/README.md index 0954c5c..9859b91 100644 --- a/README.md +++ b/README.md @@ -16,6 +16,7 @@ For installation instructions, please check the [documentation](https://github.c - [Ingesting MERFISH data with Panpipes](docs/ingesting_merfish_data/Ingesting_merfish_data_with_panpipes.md) - [Filtering and preprocessing spatial data with Panpipes](docs/preprocess_spatial_data/preprocess_spatial_data_with_panpipes.md) - [Deconvoluting spatial data with Panpipes](docs/deconvolution/deconvoluting_spatial_data_with_panpipes.md) +- [Analyzing scRNA-seq and scTCR-seq Data with Panpipes](docs/ingesting_airr_data/ingesting_airr_data.md) - [Contributing to Panpipes](docs/contributing.md) diff --git a/docs/index.md b/docs/index.md index fe7d1c8..0e60dce 100644 --- a/docs/index.md +++ b/docs/index.md @@ -11,6 +11,7 @@ For installation please check the [documentation](https://panpipes-pipelines.rea ingesting_data/Ingesting_data_with_panpipes.md ingesting_multiome/ingesting_mome.md ingesting_multimodal_data/ingesting_multimodal_data.md +ingesting_airr_data/ingesting_airr_data.md ingesting_mouse/Ingesting_mouse_data_with_panpipes.md filtering_data/filtering_data_with_panpipes.md uni_multi_integration/Integrating_data_with_panpipes.md diff --git a/docs/ingesting_airr_data/files/pipeline.yml b/docs/ingesting_airr_data/files/pipeline.yml new file mode 100644 index 0000000..cffbc23 --- /dev/null +++ b/docs/ingesting_airr_data/files/pipeline.yml @@ -0,0 +1,182 @@ +# ============================================================ +# Ingest workflow Panpipes (pipeline_ingest.py) +# ============================================================ +# This file contains the parameters for the ingest workflow. +# For full descriptions of the parameters, see the documentation at https://panpipes-pipelines.readthedocs.io/en/latest/yaml_docs/pipeline_ingestion_yml.html + +#-------------------------- +# Compute resources options +#-------------------------- +resources: + threads_high: 1 + threads_medium: 1 + threads_low: 1 + +condaenv: + +# -------------------------------- +# Loading and merging data options +# -------------------------------- + +# ---------------------------- +# Project name and data format +project: "vdjtest" +sample_prefix: "vdjtest" +use_existing_h5mu: False +submission_file: ../short_vdj_submission.tsv +metadatacols: sample_id,disease_state,tissue,preservation_method,celltype +concat_join_type: inner + +#-------------------------- +# Modalities in the project +modalities: + rna: True + prot: False + bcr: True + tcr: True + atac: False + +#-------------------------------- +# Integrating barcode level data +# e.g. demultiplexing with hashtags, chemical tags or lipid tagging +barcode_mtd: + include: False + path: + metadatacols: + +#------------------------------------------ +# Loading Protein data - additional options +protein_metadata_table: +index_col_choice: +load_prot_from_raw: False +subset_prot_barcodes_to_rna: False + +# ----------------------------- +# Quality Control (QC) options +# ----------------------------- + +# ----------------------------------- +# Processing of 10X cellranger metrics files +plot_10X_metrics: True + +# ---------------------------------- +# Doublet detection on RNA modality +scr: + run: True + expected_doublet_rate: 0.06 + sim_doublet_ratio: 2 + n_neighbours: 20 + min_counts: 2 + min_cells: 3 + min_gene_variability_pctl: 85 + n_prin_comps: 30 + use_thr: True + call_doublets_thr: 0.25 + +# ---------------------------- +# RNA modality Quality Control + +# Providing a gene list +# see documentation at https://panpipes-pipelines.readthedocs.io/en/latest/usage/gene_list_format.html +custom_genes_file: qc_genelist_1.0.csv + +# Defining actions on the genes + +# (for pipeline_ingest.py) +calc_proportions: hb,mt,rp +score_genes: + +# cell cycle action +ccgenes: default + +# ------------------------ +# Plotting RNA QC metrics +# all metrics should be provided as a comma separated string e.g. a,b,c +# plotqc_grouping_var: orig.ident +plotqc_grouping_var: sample_id +plotqc_rna_metrics: doublet_scores,pct_counts_mt,pct_counts_rp,pct_counts_hb,pct_counts_ig + +# ---------------------------- +# Plotting Protein QC metrics + +# requires prot_path to be included in the submission file +# all metrics should be provided as a comma separated string e.g. a,b,c +plotqc_prot_metrics: total_counts,log1p_total_counts,n_prot_by_counts,pct_counts_isotype +plot_metrics_per_prot: total_counts,log1p_total_counts,n_cells_by_counts,mean_counts + +identify_isotype_outliers: True +isotype_upper_quantile: 90 +isotype_n_pass: 2 + +# --------------------- +# Plot ATAC QC metrics + +# set is_paired to True if a multiome is ingested +is_paired: True +# If this is NOT a multiome experiment, but you have an RNA anndata that you would like to use for TSS enrichment +# use the partner_rna to specify the path to the file and provide a features_tss file with the tss coordinates +# leave empty if multiome is used +partner_rna: +features_tss: +plotqc_atac_metrics: n_genes_by_counts,total_counts,pct_fragments_in_peaks,atac_peak_region_fragments,atac_mitochondrial_reads,atac_TSS_fragments + +# --------------------------- +# Plot Repertoire QC metrics +ir_dist: + metric: + sequence: + +clonotype_definition: + receptor_arms: + dual_ir: + within_group: + +plotqc_rep_metrics: + # provide a item list + - is_cell + - extra_chains + - clonal_expansion + - rep:receptor_type + - rep:receptor_subtype + - rep:chain_pairing + - rep:multi_chain + +# ------------------------------------- +# Profiling Protein Ambient background +# ------------------------------------- +# PLEASE NOTE that this analysis can only be run if your inputs are from cellranger raw outputs + +assess_background: False +downsample_background: True + +# ----------------------------------------------------- +# Files required for profiling ambient background or running dsb normalisation + +# The pipeline requires the raw_feature_bc_matrix folder from cellranger or equivalent, +# specified in the submission file path with {mod}_filetype set to "cellranger," "cellranger_multi," or "10X_h5" +# for automatic search of .h5 or matrix folder for profiling ambient background or running dsb normalization. + +#------------------------------------------- +# Investigate per-channel antibody staining +channel_col: sample_id +save_norm_prot_mtx: False + +#---------------------- +# Protein normalization +#---------------------- + +normalisation_methods: clr + +#----------------------------------------------- +# Centered log ratio (CLR) normalization options + +# margin determines whether you normalise per cell (as you would for RNA), +# or by feature (recommended, due to the variable nature of prot assays). +# CLR margin 1 is recommended for informative qc plots in this pipeline +# 0 = normalise row-wise (per cell) +# 1 = normalise column-wise (per feature, recommended) +clr_margin: 1 + +#-------------------------------------------------------------- +# Denoised and Scaled by Background (DSB) normalization options +quantile_clipping: True diff --git a/docs/ingesting_airr_data/files/qc_genelist_1.0.csv b/docs/ingesting_airr_data/files/qc_genelist_1.0.csv new file mode 100644 index 0000000..fcaf44e --- /dev/null +++ b/docs/ingesting_airr_data/files/qc_genelist_1.0.csv @@ -0,0 +1,1227 @@ +mod,feature,group +rna,ANXA1,MarkersNeutro +rna,ARG1,MarkersNeutro +rna,BPI,MarkersNeutro +rna,CD101,MarkersNeutro +rna,CD24,MarkersNeutro +rna,CD274,MarkersNeutro +rna,CSF3R,MarkersNeutro +rna,CXCL8,MarkersNeutro +rna,DEFA3,MarkersNeutro +rna,DEFA4,MarkersNeutro +rna,DUSP1,MarkersNeutro +rna,EGR1,MarkersNeutro +rna,ELANE,MarkersNeutro +rna,FCGR3B,MarkersNeutro +rna,FTH1,MarkersNeutro +rna,G0S2,MarkersNeutro +rna,IFIT3,MarkersNeutro +rna,IFITM3,MarkersNeutro +rna,IL3RA,MarkersNeutro +rna,ISG15,MarkersNeutro +rna,ITGAM,MarkersNeutro +rna,KLF4,MarkersNeutro +rna,LCN2,MarkersNeutro +rna,LTF,MarkersNeutro +rna,LY6E,MarkersNeutro +rna,MKI67,MarkersNeutro +rna,MMP8,MarkersNeutro +rna,MPO,MarkersNeutro +rna,NEAT1,MarkersNeutro +rna,PADI4,MarkersNeutro +rna,PRTN3,MarkersNeutro +rna,PTMA,MarkersNeutro +rna,RETN,MarkersNeutro +rna,RPL5,MarkersNeutro +rna,S100A4,MarkersNeutro +rna,S100A8,MarkersNeutro +rna,S100A9,MarkersNeutro +rna,TCN1,MarkersNeutro +rna,TNFAIP2,MarkersNeutro +rna,TXN,MarkersNeutro +rna,HBA1,hb +rna,HBA2,hb +rna,HBB,hb +rna,HBD,hb +rna,HBE1,hb +rna,HBG1,hb +rna,HBG2,hb +rna,HBM,hb +rna,HBQ1,hb +rna,HBZ,hb +rna,IGKV1OR1-1,exclude +rna,IGKV3OR2-268,exclude +rna,IGKJ5,exclude +rna,IGKJ4,exclude +rna,IGKJ3,exclude +rna,IGKJ2,exclude +rna,IGKJ1,exclude +rna,IGKV4-1,exclude +rna,IGKV5-2,exclude +rna,IGKV7-3,exclude 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Expansion** + Displays TCR clone sizes (how many cells share the same TCR). + *Why it matters:* Large bars indicate highly expanded clones, potentially responding to a specific antigen. + + ![TCR Clonal Expansion](img/tcr_clonal_expansion_barplot.png) + +- **Group Abundance by Receptor Type / Subtype** + Shows how different TCR receptor types (TRA/TRB) or subtypes are distributed across samples. + *Why it matters:* Highlights whether certain receptor chains or subtypes predominate in your dataset. + + ![Group Abundance Receptor Type](img/tcr_group_abundance_receptor_type.png) + + ![Group Abundance Receptor Subtype](img/tcr_group_abundance_receptor_subtype.png) + +### How to re-run the analysis + +If something went bad, it is crucial that you re-run the whole analysis from scratch. You can't (!) continue from a failed step – you need to fix the step and start the analysis from zero. For that, please see the full code to execute in the following section. + +#### Re-running the analysis without re-installation of panpipes + +Useful when you tweak something in your data. + +``` +# if you are in the 1_ingest folder; otherwise skip this step +cd .. + +rm -r 1_ingest +mkdir 1_ingest +cd 1_ingest +panpipes ingest config +cp ../backup/* ./ +panpipes ingest make full --local +``` + + +#### Re-running the analysis with re-installation of panpipes + +Use it when you work on `panpipes` locally and change something in the source code. We assumer you are in the `1_ingest` folder. + +``` +cd .. && pip install -e . +rm -r ./1_ingest +mkdir 1_ingest +cd 1_ingest +panpipes ingest config +cp ../backup/* ./ +panpipes ingest make full --local +``` + +Mind the `pip install -e .`. It installs `panpipes` locally and doesn't require to changing the version of the package. However, here we assume that you work with the data analysis from the same folder, where the source code of `panpipes` is stored, i.e.,: + +panpipes/ +├── 1_ingest/ +│ ├── pipeline.yml +│ └── qc_genelist_1.0.csv +├── data/ +│ └── outs/... +├── short_vdj_submission.tsv +├── docs # docs for panpipes +├── panpipes # source code +├── tests +└── ... + +## Contact and troubleshooting + +For `panpipes` issues please use https://github.com/DendrouLab/panpipes + +For issues with tutorials please use https://github.com/DendrouLab/panpipes-tutorials/issues + +The tutorial is prepared by [Vadim Nazarov](https://github.com/vadimnazarov). Please feel free to reach out with scTCR/BCR-seq questions and if there are some issues with, well, GitHub issues. \ No newline at end of file From 4ec9053a0123fd3abf22d860e99cc8785fc8172e Mon Sep 17 00:00:00 2001 From: Vadim Nazarov <2979713+vadimnazarov@users.noreply.github.com> Date: Thu, 6 Mar 2025 14:52:00 +0100 Subject: [PATCH 3/4] fix: add missing plots and fix Markdown --- .../img/rna_nUMI_vs_nGene.png | Bin 0 -> 35273 bytes .../img/rna_nUMI_vs_pct_mito.png | Bin 0 -> 30093 bytes .../ingesting_airr_data/ingesting_airr_data.md | 17 ++++++++--------- 3 files changed, 8 insertions(+), 9 deletions(-) create mode 100644 docs/ingesting_airr_data/img/rna_nUMI_vs_nGene.png create mode 100644 docs/ingesting_airr_data/img/rna_nUMI_vs_pct_mito.png diff --git a/docs/ingesting_airr_data/img/rna_nUMI_vs_nGene.png b/docs/ingesting_airr_data/img/rna_nUMI_vs_nGene.png new file mode 100644 index 0000000000000000000000000000000000000000..df69505a3d312f48bc1e4b72498ba1d6e4ba9d19 GIT binary patch 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zUr$eoPrv=oaWTbl+(G<~IJyX+EKb$L>@r3)lGb0dc<$&Xp@@425Cq?l18 zXpl7n6L{F9qmq~^+f}#lzo2^mD%SoI^ZOrv+dIS4QWNR4W2?3vu83q}V6K0i?i}$q D(g`n- literal 0 HcmV?d00001 diff --git a/docs/ingesting_airr_data/ingesting_airr_data.md b/docs/ingesting_airr_data/ingesting_airr_data.md index 09ab3d7..25222a7 100644 --- a/docs/ingesting_airr_data/ingesting_airr_data.md +++ b/docs/ingesting_airr_data/ingesting_airr_data.md @@ -1,4 +1,5 @@ # Tutorial + Analyzing scRNA-seq and scTCR-seq Data with Panpipes =================== @@ -7,7 +8,6 @@ Analyzing scRNA-seq and scTCR-seq Data with Panpipes This tutorial guides you through the process of analyzing single-cell RNA sequencing (scRNA-seq) and single-cell T-cell receptor sequencing (scTCR-seq) data using Panpipes. While similar to the multimodal CITE-seq tutorial, this guide differs in two major points – it's much easier to follow and it focuses on single-cell transcriptomics and immunomics (AIRR – Adaptive Immune Receptor Repertoire) analysis. - ## Before you start ### Running pipelines @@ -46,7 +46,7 @@ To install R dependencies for `panpipes`, run `panpipes install_r_dependencies`. #### Other installation options -For Apple Silicone instructions, more magic with R and conda guide follow this tutorial - https://panpipes-pipelines.readthedocs.io/en/latest/install.html +For Apple Silicone instructions, more magic with R and conda guide follow this tutorial - However, in our experience, separate installations of Python and R worked the best – you install a system-wide R, and follow the steps from the previous section on `panpipes` installation in a virtual environment. @@ -136,7 +136,7 @@ Besides the first column, “sample_id”, the order in which the columns are pr We find useful to generate the submission file with softwares like Numbers or Excel and save the output as a txt file to ensure that the file is properly formatted. For more examples please check our documentation on sample submission files. -Put the submission file to the top of your project: +Put the submission file to the top of your project: ``` project-name/ @@ -269,7 +269,6 @@ Below are automatically generated summary plots from the 10x Genomics pipeline. ![VDJ T-Cells with Productive Contigs](img/tenx_productive_tra_trb.png) - ### RNA QC Plots These plots focus on standard single-cell RNA-seq quality control metrics, helping you decide which cells are high-quality for downstream analysis. @@ -286,7 +285,6 @@ These plots focus on standard single-cell RNA-seq quality control metrics, helpi ![nUMI vs % Mitochondrial](img/rna_nUMI_vs_pct_mito.png) - ### TCR Repertoire Plots These plots give insight into the diversity and clonal structure of T-cell receptors (TCR), which is key for immunology-focused projects. @@ -325,7 +323,6 @@ cp ../backup/* ./ panpipes ingest make full --local ``` - #### Re-running the analysis with re-installation of panpipes Use it when you work on `panpipes` locally and change something in the source code. We assumer you are in the `1_ingest` folder. @@ -342,6 +339,7 @@ panpipes ingest make full --local Mind the `pip install -e .`. It installs `panpipes` locally and doesn't require to changing the version of the package. However, here we assume that you work with the data analysis from the same folder, where the source code of `panpipes` is stored, i.e.,: +``` panpipes/ ├── 1_ingest/ │ ├── pipeline.yml @@ -353,11 +351,12 @@ panpipes/ ├── panpipes # source code ├── tests └── ... +``` ## Contact and troubleshooting -For `panpipes` issues please use https://github.com/DendrouLab/panpipes +For `panpipes` issues please use -For issues with tutorials please use https://github.com/DendrouLab/panpipes-tutorials/issues +For issues with tutorials please use -The tutorial is prepared by [Vadim Nazarov](https://github.com/vadimnazarov). Please feel free to reach out with scTCR/BCR-seq questions and if there are some issues with, well, GitHub issues. \ No newline at end of file +The tutorial is prepared by [Vadim Nazarov](https://github.com/vadimnazarov). Please feel free to reach out with scTCR/BCR-seq questions and if there are some issues with, well, GitHub issues. From 11a7cd5b861df740263ca5d0e1e1ad29fd7a1c17 Mon Sep 17 00:00:00 2001 From: Vadim Nazarov <2979713+vadimnazarov@users.noreply.github.com> Date: Mon, 7 Apr 2025 22:45:02 +0200 Subject: [PATCH 4/4] fix: fix the title --- docs/ingesting_airr_data/ingesting_airr_data.md | 2 -- 1 file changed, 2 deletions(-) diff --git a/docs/ingesting_airr_data/ingesting_airr_data.md b/docs/ingesting_airr_data/ingesting_airr_data.md index 25222a7..ee5ecf2 100644 --- a/docs/ingesting_airr_data/ingesting_airr_data.md +++ b/docs/ingesting_airr_data/ingesting_airr_data.md @@ -1,5 +1,3 @@ -# Tutorial - Analyzing scRNA-seq and scTCR-seq Data with Panpipes ===================