TBSP: Trajectory Inference Based on SNP information.

INTRODUCTION

Several recent studies focus on the inference of developmental and response trajectories from single cell RNA-Seq (scRNA-Seq) data. A number of computational methods, often referred to as pseudo-time ordering, have been developed for this task. Recently, CRISPR has also been used to reconstruct lineage trees by inserting random mutations. However, both approaches suffer from drawbacks that limit their use. Here we develop a method to detect significant, cell type specific, sequence mutations from scRNA-Seq data. We show that only a few mutations are enough for reconstructing good branching models. Integrating these mutations with expression data further improves the accuracy of the reconstructed models.

PREREQUISITES

• python (python 2 and python 3 are both supported)
  It was installed by default for most Linux distribution and MAC.
  If not, please check https://www.python.org/downloads/ for installation instructions.

• Python packages dependencies:
  -- scikit-learn
  -- scipy
  -- numpy
  -- matplotlib
  -- networkx
  -- pyBigWig
  -- Biopython

• other dependencies:
  -- python-dev (python2) or python3-dev (python3)
  It can be installed easily for most linux distributions. For example, debian/ubuntu:

sudo apt-get install python-dev
or 
sudo apt-get install python3-dev

For Macos, it was installed by default.

The python setup.py script (or pip) will try to install these packages automatically. However, please install them manually if, by any reason, the automatic installation fails.

• Platform:
  Macos and Linux verified. For windows, the dependent pyBigWig package is not available.

INSTALLATION

There are 3 options to install scdiff.

• Option 1: Install from download directory
  cd to the downloaded scdiff package root directory

   $cd tbsp

  run python setup to install

   $python setup.py install

  MacOS or Linux users might need the sudo/root access to install. Users without the root access can install the package using the pip/easy_install with a --user parameter (install python libraries without root)．

   $sudo python setup.py install 

  use python3 instead of python in the above commands to install if using python3.

• Option 2: Install from Github:

  python 2:

   $sudo pip install  --trusted-host github.com --upgrade http://github.com/phoenixding/tbsp/zipball/master

  python 3:

   $sudo pip3 install --trusted-host github.com --upgrade http://github.com/phoenixding/tbsp/zipball/master

The above pip installation options should be working for Linux and MacOS systems.
For MacOS users, it's recommended to use python3 installation. The default python2 in MacOS has some compatibility issues with a few dependent libraries. The users would have to install their own version of python2 (e.g. via Anocanda) if they prefer to use python2 in MacOS.

USAGE

usage: tbsp [-h] -i IVCF [-b [IBW]] [-k KCLUSTER] [-l [CELL_LABEL]] -o
               OUTPUT [--cutl CUTL] [--cuth CUTH] [--cutc CUTC]

optional arguments:
  -h, --help            show this help message and exit
  -i IVCF, --ivcf IVCF  Required,directory with all input .vcf files. This
                        specifies the directory of SNP files (.vcf) for the
                        cells (one .vcf file for each cell). These .vcf files
                        can be obtained using the provided bam2vcf script or
                        other RNA-seq variant calling pipelines preferred by
                        the users.
  -b [IBW], --ibw [IBW]
                        Optional,directory with all input bigwig (.bw) files
                        with the information about the number of aligned reads
                        at each genomic position. These bigwig files are used
                        to filter the SNPs, which are redundant to expression
                        information.
  -l [CELL_LABEL], --cell_label [CELL_LABEL]
                        Optional, labels for the cells. This is used only to
                        annotate the cells with known information, not used
                        for building the model.
  -k KCLUSTER, --kcluster KCLUSTER
                        Optional, number of clusters, Integer. If not
                        specified, the program will choose the k with best
                        silhouette score.
  -o OUTPUT, --output OUTPUT
                        Required,output directory
  --cutl CUTL           Optional, lower bound cutoff to remove potential false
                        positive SNPs, default=0.1
  --cuth CUTH           Optional, upper bound cutoff to remove baseline SNPs,
                        which are common in most cells, default=0.8
  --cutc CUTC           Optional, convergence cutoff, a smaller cutoff
                        represents a stricter convergence
                        criterion,default=0.001
                                             

INPUTS AND PRE-PROCESSINGS

• -i:
  Required input, this specifies the directory of all SNP(.vcf) files. We recommend using GATK RNA-seq variant calling pipeline to call the vcfs from .bam (mapped reads) files. Users are also allowed to use the SNPs (.vcfs) identified by programs of their preferences.

• -b:
  Optional input, this specifies the directory of all bigwig (.bw) files. We provided the script bam2bw.py under bam2bw directory to convert the bam files to bigwig files. This files are used to filter SNPs, potentially redundant to expression.

• -l:
  Optional input, this specifies the labels for the cells. File format (tab-delimited):

cell1	label1
cell2	label2

These cell labels are only used to annotate the cells in the trajectory. The other optional parameters are specified above.

OUTPUTS

• GroupCells.txt:
  A text file, which describes the cells in each cluster.

  Format:

   Cell_ID	Cluster_ID
   SRR1931024	cluter:0
   SRR1930999	cluter:0
   SRR1930977	cluter:0
   SRR1931041	cluter:0
   SRR1931012	cluter:0
   SRR1930945	cluter:0
   SRR1931003	cluter:0
   SRR1931002	cluter:0
   SRR1931004	cluter:0
   ..
  

• SNP_matrix.tsv:
  The SNP matrix for all the cells. Row: SNPs Column: Cells Value: Binary (0/1), which indicates whther the SNP is included in the cell.

• SNP_matrix.jpg:
  The SNP matrix in jpg image.

• Trajectory.dat:

   4	(-0.6168642633606496, -0.29504774213348317)
   Inner4	(-0.01784069226314263, -0.19237987266625067)
   2	(-1.0, 0.1907479586411944)
   Inner5	(0.526511663382742, -0.048432121394020027)
   6	(0.6167190582080249, -0.21090119687699874)
   0	(-0.02807716265846153, -0.3564777531262085)
   3	(-0.7661682030072015, 0.29873283057457906)
   Inner3	(-0.4986251141938113, -0.13657170781011707)
   Inner1	(0.785885623794461, 0.14046537917538365)
   5	(0.8477737598817315, 0.3307492805568145)
   1	(0.9509945159224894, 0.1308847244003101)
   Inner2	(-0.8003091857061821, 0.14823022065879607)
  

  First column: cluster id
  second column: coordinates

• Trajectory.jpg:
  Graph representation of Trajectory.dat

EXAMPLES

• Example inputs:
  We provided example vcf files under examples folder. To run tbsp on the example data:

$tbsp -i examples/vcf_example -o example_out

• Example outputs:
  Example output files can be found under examples folder.

CREDITS

This software was developed by ZIV-system biology group @ Carnegie Mellon University.
Implemented by Jun Ding.

LICENSE

This software is under MIT license.

CONTACT

zivbj at cs.cmu.edu
jund at cs.cmu.edu