nhp_snrna_striatum_analysis

The code and jupyternotebooks to go along with the Medium Spiny Neuron Diversity in the Primate Nucleus Accumbens publication. This github details the analysis performed on single nucleus RNA-seq data sampled from the Rhesus macaque striatum. The resulting analysis yielded precise gene markers for call cell types in the macaque straitum and a characterization of the diversity of medium spiny neurons as well as their regional distribution in the nucleus accumbens vs the putamen anc caudate. You can follow along the analysis with the Jupyter notebooks below. The statstical test scripts contain code to run the cell type identification and cluster validation statstical testing

Requirements

• R>3.6.0

• python>3.5.1

• STAR>2.7.2b

The rest of the requirements can be installed by running:

1. pip install pip_requirements.txt
2. Rscript installation.R

Computational Workflow

Alignment of the rhesus macaque straitum nuclei data was performed using STAR version 2.7.2b on the rhemac10 genome. Scripts for the alignmet are located in the alignment scripts folder.

The analysis was composed of the following steps and can be viewed in the analysis jupyer notebooks.

Computational Step	Software Packages	Associated Notebook
Empty Droplet Removal	DropletUtils	remove_noise_droplets.ipynb
Doublet Removal	SCDS	remove_noise_droplets.ipynb
Combining Brain Regions	----	combine_brain_regions.ipynb
Poor Quality Nuclie Removal	Scanpy	preclustering_and_qc.ipynb
Preclustering	Scanpy	preclustering_and_qc.ipynb
Normalization	Scran	scran_normalization.ipynb
Full Striatum Clustering and Marker analyis	Scanpy	full_nuclei_analysis.ipynb
Medium Spiny Neuron Clustering and Marker analyis	Scanpy	msn_analysis.ipynb

Statistical testing

We are also providing our own code for the following statistical tests

Statistical Test	Purpose	Script
Cluster-cell type enrichment test	Computes the marker gene enrichment between a single nuclei cluster and set of cell types in a gene marker database	cluster_profile.py
Archetype Factorization	Performs dirchlet simplex nest factorization on anndata objects	archetype_utilities.py
Archetype Discontinuity Test	Computes Regression Discontinuity statitics on the archetype axes	split_testing.py
Compute Archetype Stability Heuristic Metrics	Computes stability metrics for k archetypes in order to select k	select_archetypes.py
Permuation Significance Testing	Performs permutation testing for p-value correction of the regression discontinuity test	permutation_testing.py
Dirichlet Simplex Nest	Extended from the Dirichlet Simplex Nest Github	vlad.py

Data available via SRA and GEO