Some questions about normalization

[809825706]

WOT is a great tool for time coures single cell analysis! thank you for developing it. I want to use it to analyze my reprograming data too, but I was confused about the normalization steps in your parper. It seem that you normalized tha data twice (before and after find HVGs).

I flowed your way (by my own understanding) to use the code below:

import numpy as np
import pandas as pd
import scanpy as sc

adata = sc.read_h5ad("adata_filtered.h5ad")

adata.var_names_make_unique() 
sc.pp.filter_cells(adata, min_genes=200)
sc.pp.filter_genes(adata, min_cells=3)

sc.pp.normalize_total(adata, target_sum=1e4)
sc.pp.log1p(adata)
adata

adata.write("ExprMatrix.h5ad")

sc.pp.highly_variable_genes(adata, min_mean=0.0125, max_mean=3, min_disp=0.5)
adata = adata[:,adata.var.highly_variable]

sc.pp.normalize_total(adata, target_sum=1e4)
sc.pp.log1p(adata)
adata

adata.write("ExprMatrix.var.genes.h5ad")

I wondered if the following operation is reasonable：

1. I didn't downsample, It seems not required.
2. I used the function of find HVGs in scanpy in place of seurat.
3. And last but not least, which data should the normalization use after select HVGs? raw data？ or just like my code above？