Analysis of 140k T cells from cancer

In this notebook, we re-analyze single-cell TCR/RNA-seq data from Wu et al. (2020) generated on the 10x Genomics platform. The original dataset consists of >140k T cells from 14 treatment-naive patients across four different types of cancer. Roughly 100k of the 140k cells have T-cell receptors.

0. Setup

In [1]:
%load_ext autoreload
%autoreload 2
import sys

sys.path.insert(0, "../../scirpy/")
import scirpy as ir
import pandas as pd
import numpy as np
import scanpy as sc
import scipy as sp
from matplotlib import pyplot as plt
import matplotlib
from weblogo.seq import SeqList, unambiguous_protein_alphabet
from weblogo import png_formatter, svg_formatter, eps_formatter
from weblogo import LogoData, LogoOptions, LogoFormat
from IPython.display import Image, display
import warnings

sc.settings._vector_friendly = True

warnings.filterwarnings('ignore', category=FutureWarning)

# whether to run the alignment or use a cached version.
# If `False` and the cached version is not available, will raise an error.
run_alignment = True

This notebook was run with scirpy commit e07553f6b84515ea2c48d2955660a3d6f900f9eb and the following software versions:

In [2]:
scanpy==1.5.1 anndata==0.7.3 umap==0.4.2 numpy==1.18.1 scipy==1.4.1 pandas==1.0.3 scikit-learn==0.22.2.post1 statsmodels==0.11.1 python-igraph==0.8.2 leidenalg==0.8.0
In [3]:
def weblogo(seqs, title="", format="png"):
    """Draw a sequence logo from a list of amino acid sequences. """
    logodata = LogoData.from_seqs(SeqList(seqs, alphabet=unambiguous_protein_alphabet))
    logooptions = LogoOptions(logo_title=title)
    logoformat = LogoFormat(logodata, logooptions)
    if format == "png":
        display(Image(png_formatter(logodata, logoformat)))
    elif format == "eps":
        return eps_formatter(logodata, logoformat)
In [4]:
# colors from the paper
colors = {
    "Tumor+NAT expanded": "#9458a2",
    "Tumor singleton": "#ff8000",
    "NAT singleton": "#f7f719",
    "Tumor multiplet": "#eeb3cb",
    "NAT multiplet": "#9cd0de",
    "Blood singleton": "#cce70b",
    "Blood multiplet": "#beac83",

1. Preparing the data

The dataset ships with the scirpy package. We can conveniently load it from the scirpy.datasets module.

In [5]:
adata = ir.datasets.wu2020()
In [6]:
(141623, 30727)

We only keep the cells with TCR. ~96k cells remain.

In [7]:
adata = adata[adata.obs["has_tcr"] == "True", :]
adata = adata[~(adata.obs["cluster_orig"] == "nan"), :]
In [8]:
(96067, 30727)
In [9]:
Index(['TRA_1_c_gene', 'TRA_1_cdr3', 'TRA_1_cdr3_nt', 'TRA_1_d_gene',
       'TRA_1_expr', 'TRA_1_j_gene', 'TRA_1_junction_ins', 'TRA_1_v_gene',
       'TRA_2_c_gene', 'TRA_2_cdr3', 'TRA_2_cdr3_nt', 'TRA_2_d_gene',
       'TRA_2_expr', 'TRA_2_j_gene', 'TRA_2_junction_ins', 'TRA_2_v_gene',
       'TRB_1_c_gene', 'TRB_1_cdr3', 'TRB_1_cdr3_nt', 'TRB_1_d_gene',
       'TRB_1_expr', 'TRB_1_j_gene', 'TRB_1_junction_ins', 'TRB_1_v_gene',
       'TRB_2_c_gene', 'TRB_2_cdr3', 'TRB_2_cdr3_nt', 'TRB_2_d_gene',
       'TRB_2_expr', 'TRB_2_j_gene', 'TRB_2_junction_ins', 'TRB_2_v_gene',
       'batch', 'clonotype_orig', 'cluster_orig', 'has_tcr', 'multi_chain',
       'patient', 'sample', 'source'],
In [10]:
adata.obs["counts"] = adata.X.sum(axis=1).A1
Trying to set attribute `.obs` of view, copying.

Preprocess Transcriptomics data

Transcriptomics data needs to be filtered and preprocessed as with any other single-cell dataset. We recommend following the scanpy tutorial and the best practice paper by Luecken et al.. For the Wu et al. (2020) dataset, the authors already provide clusters and UMAP coordinates. Instead of performing clustering and cluster annotation ourselves, we will just use provided data. The clustering and annotation procedure used by the authors is described in their paper.

In [11]:
sc.pp.filter_genes(adata, min_cells=10)
sc.pp.filter_cells(adata, min_genes=100)
In [12]:
sc.pp.normalize_per_cell(adata, counts_per_cell_after=1000)
In [13]:
adata.obsm["X_umap"] = adata.obsm["X_umap_orig"]
In [14]:
mapping = {
    "3.1-MT": "other",
    "4.1-Trm": "CD4_Trm",
    "4.2-RPL32": "CD4_RPL32",
    "4.3-TCF7": "CD4_TCF7",
    "4.4-FOS": "CD4_FOSS",
    "4.5-IL6ST": "CD4_IL6ST",
    "4.6a-Treg": "CD4_Treg",
    "4.6b-Treg": "CD4_Treg",
    "8.1-Teff": "CD8_Teff",
    "8.2-Tem": "CD8_Tem",
    "8.3a-Trm": "CD8_Trm",
    "8.3b-Trm": "CD8_Trm",
    "8.3c-Trm": "CD8_Trm",
    "8.4-Chrom": "other",
    "8.5-Mitosis": "other",
    "8.6-KLRB1": "other",
    "nan": "nan",
adata.obs["cluster"] = [mapping[x] for x in adata.obs["cluster_orig"]]

Let's inspect the UMAP plots. The first three panels show the UMAP plot colored by sample, patient and cluster. We don't observe any clustering of samples or patients that could hint at batch effects.

The last three panels show the UMAP colored by the T cell markers CD8, CD4, and FOXP3. We can confirm that the markers correspond to their respective cluster labels.

In [15]:
    color=["sample", "patient", "cluster", "CD8A", "CD4", "FOXP3"],
... storing 'cluster' as categorical