10X Genomics Jupyter Notebook Tutorial

This notebook demonstrates:

• loading a feature-barcode matrix in HDF5 format
• loading analysis files in CSV format
• plotting UMI and feature (gene) count distributions
• plotting clustering results and gene-specific expression in tSNE space

Data:

• Go to the 10k Cells from a combined cortex, hippocampus and subventricular zone of an E18 mouse (v3 chemistry) page.
• Download the Feature / cell matrix HDF5 (filtered) (.h5 format) and Clustering analysis (.tar format) files.
• Move the downloaded files to the current foler.

Note:

• This requires the following Python packages: Matplotlib, NumPy, SciPy, Pandas, h5py.
• The easiest way to obtain these packages (as well as Jupyter Notebook) is to install Anaconda.

# import modules, define some functions for loading, saving and processing a gene-barcode matrix
%matplotlib inline
import collections
import matplotlib
import matplotlib.pyplot as plt
import numpy as np
import pandas as pd
import scipy.sparse as sp_sparse
import h5py

np.random.seed(0)

FeatureBCMatrix = collections.namedtuple('FeatureBCMatrix', ['feature_ids', 'feature_names', 'barcodes', 'matrix'])

def get_matrix_from_h5(filename):
    with h5py.File(filename) as f:
        if u'version' in f.attrs:
            if f.attrs['version'] > 2:
                raise ValueError('Matrix HDF5 file format version (%d) is an newer version that is not supported by this function.' % version)
        else:
            raise ValueError('Matrix HDF5 file format version (%d) is an older version that is not supported by this function.' % version)
        
        feature_ids = [x.decode('ascii', 'ignore') for x in f['matrix']['features']['id']]
        feature_names = [x.decode('ascii', 'ignore') for x in f['matrix']['features']['name']]        
        barcodes = list(f['matrix']['barcodes'][:])
        matrix = sp_sparse.csc_matrix((f['matrix']['data'], f['matrix']['indices'], f['matrix']['indptr']), shape=f['matrix']['shape'])
        return FeatureBCMatrix(feature_ids, feature_names, barcodes, matrix)

def get_expression(fbm, gene_name):
    try:
        gene_index = feature_bc_matrix.feature_names.index(gene_name)
    except ValueError:
        raise Exception("%s was not found in list of gene names." % gene_name)
    return fbm.matrix[gene_index, :].toarray().squeeze()

filtered_matrix_h5 = "neuron_10k_v3_filtered_feature_bc_matrix.h5"
%time feature_bc_matrix = get_matrix_from_h5(filtered_matrix_h5)

CPU times: user 8.81 s, sys: 327 ms, total: 9.13 s
Wall time: 9.03 s

# untar the secondary analysis 
# alternatively, copy these to the command line (omitting the initial '!' character)
!tar -xzf neuron_10k_v3_analysis.tar.gz

# load tSNE and graph clustering
tsne = pd.read_csv("analysis/tsne/2_components/projection.csv")
clusters = pd.read_csv("analysis/clustering/graphclust/clusters.csv")

# calculate UMIs and genes per cell
umis_per_cell = np.asarray(feature_bc_matrix.matrix.sum(axis=0)).squeeze()
genes_per_cell = np.asarray((feature_bc_matrix.matrix > 0).sum(axis=0)).squeeze()

# plot UMIs per cell
plt.hist(np.log10(umis_per_cell), bins=50)
plt.xlabel('UMIS per cell (log10)')
plt.ylabel('Frequency')
plt.title('UMI Distribution')
plt.show()

# plot genes per cell
plt.hist(genes_per_cell, bins=50)
plt.xlabel('Genes per Cell')
plt.ylabel('Frequency')
plt.title('Gene Distribution')
plt.show()

# plot clusters in TSNE space
plt.figure(figsize=(8, 8))
plt.scatter(tsne['TSNE-1'], tsne['TSNE-2'], c=clusters['Cluster'], linewidths=0, s=5)
plt.title('Graph-Based Clustering')
plt.show()

# plot three markers: Stmn2 (pan-neuronal), Tbr1 (excitatory), Olig1 (oligodendrocytes)
marker_genes = ['Stmn2', 'Tbr1', 'Olig1']
f, axes = plt.subplots(1, len(marker_genes), figsize=(5*len(marker_genes), 4))
for gene, axis in zip(marker_genes, axes):
    expr = get_expression(feature_bc_matrix, gene)
    axis.scatter(tsne['TSNE-1'], tsne['TSNE-2'], c=expr, s=5, linewidths=0, cmap=plt.cm.Reds)
    axis.set_title(gene)
plt.show()