Sampling Noise based Inference of Transcription ActivitY : Filtering of Poison noise on a single-cell RNA-seq UMI count matrix

Sanity infers the log expression levels x_gc of gene g in cell c by filtering out the Poisson noise on the UMI count matrix n_gc of gene g in cell c.

See our preprint for more details.

The raw and normalized datasets mentionned in the preprint are available on . Files are named [dataset name]_UMI_counts.txt.gz and [dataset name]_[tool name]_normalization.txt.gz.

The scripts used for running the bechmarked normalization methods and for making the figures of the preprint are in the reproducibility folder.

• Count Matrix: (N_g x N_c) matrix with N_g the number of genes and N_c the number of cells. Format: tab-separated, comma-separated, or space-separated values. ('path/to/text_file')

• (Alternatively) Matrix Market File Format: Sparse matrix of UMI counts. Automatically recognized by .mtx extension of the input file. Named matrix.mtx by cellranger 2.1.0 and 3.1.0 (10x Genomics). ('path/to/text_file.mtx')
  • (optional) Gene ID file: Named genes.tsv by cellranger 2.1.0 and features.tsv by cellranger 3.1.0 (10x Genomics). ('path/to/text_file')
  • (optional) Cell ID file: Named barcodes.tsv by cellranger 2.1.0 and 3.1.0 (10x Genomics). ('path/to/text_file')
• (optional) Destination folder ('path/to/output/folder', default: pwd)
• (optional) Number of threads (integer, default: 4)
• (optional) Print extended output (Boolean, 'true', 'false', '1' or '0', default: 4)
• (optional) Minimal and maximal considered values of the variance in log transcription quotients (double, default: v_min=0.001 v_max=50)
• (optional) Number of bins for the variance in log transcription quotients (integer, default: 160)

• log_transcription_quotients.txt: (N_g x N_c) table of inferred log expression levels. The gene expression levels are normalized to 1, meaning that the summed expression of all genes in a cell is approximately 1. Note that we use the natural logarithm, so to change the normalization one should multiply the exponential of the expression by the wanted normalization (e.g. mean or median number of captured gene per cell).

  GeneID	Cell 1	Cell 2	Cell 3	...
  Gene 1	0.25	-0.29	-0.54
  Gene 2	-0.045	-0.065	0.11
  ...

• ltq_error_bars.txt : (N_g x N_c) table of error bars on inferred log expression levels

  GeneID	Cell 1	Cell 2	Cell 3	...
  Gene 1	0.015	0.029	0.042
  Gene 2	0.0004	0.0051	0.0031
  ...

• mu.txt : (N_g x 1) vector of inferred mean log expression levels

• d_mu.txt : (N_g x 1) vector of inferred error bars on mean log expression levels

• variance.txt : (N_g x 1) vector of inferred variance per gene in log expression levels

• delta.txt : (N_g x N_c) matrix of inferred log expression levels centered in 0

• d_delta.txt : (N_g x N_c) matrix of inferred error bars log expression levels centered in 0

• likelihood.txt : (N_g+1 x N_b) matrix of normalized variance likelihood per gene, with N_b the number of bins on the variance.

  Variance	0.01	0.0107	0.0114	...
  Gene 1	0.018	0.019	0.020
  Gene 2	0.0006	0.0051	0.0031
  ...

  ./Sanity <option(s)> SOURCES
  Options:
	-h,--help		Show this help message
	-v,--version		Show the current version
	-f,--file		Specify the input transcript count text file (.mtx for Matrix Market File Format)
	-mtx_genes,--mtx_gene_name_file	Specify the gene name text file (only needed if .mtx input file)
        -mtx_cells,--mtx_cell_name_file	Specify the cell name text file (only needed if .mtx input file)
	-d,--destination	Specify the destination path (default: pwd)
	-n,--n_threads		Specify the number of threads to be used (default: 4)
	-e,--extended_output	Option to print extended output (default: false)
	-vmin,--variance_min	Minimal value of variance in log transcription quotient (default: 0.001)
	-vmax,--variance_max	Maximal value of variance in log transcription quotient (default: 50)
	-nbin,--number_of_bins	Number of bins for the variance in log transcription quotient  (default: 160)

• Clone the GitHub repository

git clone https://github.com/jmbreda/Sanity.git

• Install OpenMP library

  • On Linux
    If not already installed (Check with ldconfig -p | grep libgomp, no output if not installed), do

   sudo apt-get update
   sudo apt-get install libgomp1
  

  • On mac OS using macports
    Install the gcc9 package

   port install gcc9
  

         Change the first line of src/Makefile from CC=g++ to CC=g++-mp-9

  • On mac OS using brew
    Install the gcc9 package

   brew install gcc9
  

         Change the first line of src/Makefile from CC=g++ to CC=g++-9

• Move to the source code directory and compile.

cd Sanity/src
make

• The binary file is located in

Sanity/bin/Sanity

• Alternatively, the already compiled binary for macOS is located in

Sanity/bin/Sanity_macOS

Compute cell-cell distances from Sanity output files. Needs extended outputs of Sanity (-e 1 option).

• The output folder of the Sanity run, specifiied with the -d option in Sanity ('path/to/folder')
• (optional) The gene signal to noise ratio used as gene cut-off (double, default: 1.0)
• (optional) Compute distances with or without errorbars (boolean, default: 1 or true)
• (optional) Number of threads (integer, default: 4)

• Cell-cell distance: (N_c(N_c-1)/2) vector of cell to cell distances dist(cell_i,cell_j), i=1,...,N_c-1, j=i+1,...,N_c, with N_c the number of cells.

  dist(cell1,cell2)
  dist(cell1,cell3)
  dist(cell1,cell4)
  ...
  dist(cellNc-2,cellNc-1)
  dist(cellNc-2,cellNc)
  dist(cellNc-1,cellNc)

located in the Sanity output folder (specified with -f option), named cell_cell_distance_[...].txt, depending on the -err and -s2n options.

./Sanity_distance <option(s)> SOURCES
Options:
	-h,--help		Show this help message
	-v,--version		Show the current version
	-f,--folder		Specify the input folder with extended output from Sanity
	-s2n,--signal_to_noise_cutoff	Minimal signal/noise of genes to include in the distance calculation (default: 1.0)
	-err,--with_error_bars	Compute cell-cell distance taking the errobar epsilon into account (default: true)
	-n,--n_threads		Specify the number of threads to be used (default: 4)

Same dependencies as Sanity (see above).

• Move to the source code directory and compile.

cd Sanity/src
make Sanity_distance

• The binary file is located in

Sanity/bin/Sanity_distance

For any questions or assistance regarding Sanity, please post your question the issues section or contact us at [email protected]