# parse-pipeline

## Summary

+ **Module Name:** parse-pipeline
+ **Support Level:** Primary Support
+ **Software Access Level:** Open Access
+ **Home Page:** https://www.parsebiosciences.com/

## Software Description

Split-pipe is the software developed by Parse Biosciences for demultiplexing sample and 
cell combinatorial barcodes and aligning transcript-derived reads to the genome. 
Split-pipe produces a cell-by-gene counts matrix containing the 
expression of each gene for each cell barcode, along with a quality report detailing the 
number of valid cell barcodes that were found, median numbers of genes and transcripts per cell, 
and read alignment statistics to the genome. MSI provides selected reference files that are 
compatible with split-pipe in the [bioref database](https://msi.umn.edu/consulting-and-research/bioinformatics-guides/bioref) in this directory:

`/common/bioref/splitpipe_parse/`

## General Linux

Depending on where you are working, there may be more than one version of Parse split-pipe available.
To see which modules are available for loading you can run:

`module avail parse-pipeline`

This will provide a list of all versions of the package, shown as parse-pipeline/<version>.

To load a specific module for use in a Linux environment, run the command:

`module load parse-pipeline/<version>`

For example, you can use the command `module load parse-pipeline/1.5.0` to load version 1.5.0 
which is the current version available on our system.

To launch the package and view the help menu after it is loaded, use the command:

`split-pipe -h`


## Example SBATCH Script
In the following example we run a single Parse sublibrary FASTQ file through split-pipe to 
demultiplex the samples and cells given in a text file called `samplelist.txt` that 
indicates which Parse plate wells correspond to each sample.

Example of a samplelist.txt file (column 1 = sample name, column 2 = plate well numbers, and columns are tab-delimited):
```
Sample1 A1-A6
Sample2 A7-A12
Sample3 B1-B6
Sample4 B7-B12
Sample5 C1-C6
Sample6 C7-C12
Sample7 D1-D6
Sample8 D7-D12
```


Example split-pipe_run.sbatch script:

```
############split-pipe_run.sbatch############
#!/bin/sh
#SBATCH -N 1
#SBATCH -n 1
#SBATCH -c 32
#SBATCH --mem=200G
#SBATCH -t 96:00:00
#SBATCH -p msismall
#SBATCH -A YOUR_GROUP
#SBATCH --mail-type="ALL"
#SBATCH --mail-user=YOUR_EMAIL@umn.edu

# Load the parse-pipeline module
module load parse-pipeline/1.5.0 


R1_FASTQ="Sub_Library1_L004_R1_001.fastq.gz"         # Name of the R1 sublibrary FASTQ file
R2_FASTQ="Sub_Library1_L004_R2_001.fastq.gz"         # Name of the R2 sublibrary FASTQ file
FASTQ_DIR="/path/to/your/FASTQ/folder/"              # The path to your folder of FASTQ files
OUT="split_pipe_output/Sub_Library1"                 # The name of the split-pipe output folder


split-pipe --mode all \
--genome_dir "/common/bioref/splitpipe_parse/1.5.0/mm39/" \
--fq1 ${FASTQ_DIR}/${R1_FASTQ} \
--fq2 ${FASTQ_DIR}/${R2_FASTQ} \
--output_dir ${OUT} \
--samp_list samplelist.txt \
--chemistry v3 \
--kit WT \
--chem_score_skip \
--kit_score_skip \
--no_keep_going \
--nthreads 32
```

Once all of the sublibraries are processed using "split-pipe --mode all", they can be 
combined together to produce the final output using the `split-pipe --mode comb` command.
Here is an example sbatch script:

```
############split-pipe_comb.sbatch############
#!/bin/sh
#SBATCH -N 1
#SBATCH -n 1
#SBATCH -c 32
#SBATCH --mem=100G
#SBATCH -t 96:00:00
#SBATCH -p msismall
#SBATCH -A YOUR_GROUP
#SBATCH --mail-type="ALL"
#SBATCH --mail-user=YOUR_EMAIL@umn.edu

module load parse-pipeline/1.5.0 

# combine the split-pipe output directories from each sublibrary listed in "subsample.list"
split-pipe --mode comb \
    --sublib_list subsample.list \
    --output_dir split_pipe_output/combined \
    --nthreads 32

```

The `subsample.list` file contains a list of all of the sublibrary output folder names produced from the split-pipe runs above. For example:

```
/path/to/your/split_pipe_output/Sub_Library1
/path/to/your/split_pipe_output/Sub_Library2
/path/to/your/split_pipe_output/Sub_Library3
```