piperna 0.4

A python wrapper for fast and parallel processing of bulk RNA Seq data

piperna

==========

version 0.01

A python wrapper for processing of bulk RNA seq data

Requirements

1. Python > 3.5 (piperna uses the 'six' package but will attempt to install if not already installed)
2. Computing cluster with PBS or SLURM
3. Modules installed for python, STAR or kallisto, R

Installation

Installation can probably be done correctly many different ways. Here are the methods that have worked for us. We recommend that piperna be installed with pipx.

At SCRI do the following

module load python
python3 -m pip install --user pipx
python3 -m pipx ensurepath
pipx install --include-deps --pip-args '--trusted-host pypi.org --trusted-host files.pythonhosted.org' piperna

At the FHCRC do the following...

module load Python/3.6.7-foss-2016b-fh1
python3 -m pip install --user pipx
python3 -m pipx ensurepath
pipx install --include-deps piperna

You should then be able to test installation by calling piperna. After running the folllowing, you should see the help screen displayed.

piperna

Usage

piperna usage: A wrapper for running piperna [-h] [--sample_flag SAMPLE_FLAG]
                                      [--fastq_folder FASTQ_FOLDER]
                                      [--genome_key GENOME_KEY]
                                      [--filter_high FILTER_HIGH]
                                      [--filter_low FILTER_LOW]
                                      [--output OUTPUT] [--runsheet RUNSHEET]
                                      [--log_prefix LOG_PREFIX]
                                      [--select SELECT] [--debug]
                                      [--bowtie_flags BOWTIE_FLAGS]
                                      [--cluster {PBS,SLURM}]
                                      [--norm_method {coverage,read_count,spike_in}]
                                      [--user USER] [--SEACR_norm {non,norm}]
                                      [--SEACR_stringency {stringent,relaxed}]
                                      [--verbose]
                                      {MAKERUNSHEET,ALIGN,NORM,SEACR,GENOMESFILE}

positional arguments:
  {MAKERUNSHEET,ALIGN,NORM,SEACR,GENOMESFILE}
                        a required string denoting segment of pipeline to run.
                        1) "MAKERUNSHEET" - to parse a folder of fastqs; 2)
                        "ALIGN" - to perform alignment using bowtie and output
                        bed files; 3) "NORM" - to normalize data to reference
                        (spike in); 4) "SEACR" - to perform SEACR; 5)
                        "GENOMESFILE" - print location of genomes.json file.

optional arguments:
  -h, --help            show this help message and exit
  --sample_flag SAMPLE_FLAG, -sf SAMPLE_FLAG
                        FOR MAKERUNSHEET only string to identify samples of
                        interest in a fastq folder
  --fastq_folder FASTQ_FOLDER, -fq FASTQ_FOLDER
                        For MAKERUNSHEET only: Pathname of fastq folder (files
                        must be organized in folders named by sample)
  --genome_key GENOME_KEY, -gk GENOME_KEY
                        For MAKERUNSHEET only: abbreviation to use "installed"
                        genomes in the runsheet (See README.md for more
                        details
  --filter_high FILTER_HIGH, -fh FILTER_HIGH
                        For ALIGN only: upper limit of fragment size to
                        exclude, defaults is no upper limit. OPTIONAL
  --filter_low FILTER_LOW, -fl FILTER_LOW
                        For ALIGN only: lower limit of fragment size to
                        exclude, defaults is no lower limit. OPTIONAL
  --output OUTPUT, -o OUTPUT
                        For MAKERUNSHEET only: Pathname to write runsheet.csv
                        file (folder must exist already!!), Defaults to
                        current directory
  --runsheet RUNSHEET, -r RUNSHEET
                        tab-delim file with sample fields as defined in the
                        script. - REQUIRED for all jobs except MAKERUNSHEET
  --log_prefix LOG_PREFIX, -l LOG_PREFIX
                        Prefix specifying log files for piperna output from
                        piperna calls. OPTIONAL
  --select SELECT, -s SELECT
                        To only run the selected row in the runsheet, OPTIONAL
  --debug, -d           To print commands (For testing flow). OPTIONAL
  --bowtie_flags BOWTIE_FLAGS, -b BOWTIE_FLAGS
                        For ALIGN: bowtie flags, OPTIONAL
  --cluster {PBS,SLURM}, -c {PBS,SLURM}
                        Cluster software. OPTIONAL Currently supported: PBS
                        and SLURM
  --norm_method {coverage,read_count,spike_in}, -n {coverage,read_count,spike_in}
                        For ALIGN and NORM: Normalization method, by
                        "read_count", "coverage", or "spike_in". If method is
                        "spike_in", piperna will align to the spike_in
                        reference genome provided in runsheet. OPTIONAL
  --user USER, -u USER  user for submitting jobs - defaults to username.
                        OPTIONAL
  --SEACR_norm {non,norm}, -Sn {non,norm}
                        For SEACR: Normalization method; default is
                        "non"-normalized, select "norm" to normalize using
                        SEACR. OPTIONAL
  --SEACR_stringency {stringent,relaxed}, -Ss {stringent,relaxed}
                        FOR SEACR: Default will run as "stringent", other
                        option is "relaxed". OPTIONAL
  --verbose, -v         Run with some additional ouput - not much though...
                        OPTIONAL

Runsheet

The runsheet is the brains of the piperna workflow. You can make a runsheet using the MAKERUNSHEET command. This command will parse a directory of fastq folder (specified using the -fq flag; fastq files should be organized in subfolders named by sample) and will find fastq mates (R1 and R2 - Currently only PE sequencing is supported). Running piperna MAKERUNSHEET will find and pair these fastqs for you and populate the runsheet with genome index locations (see below) and output filenames with locations as specified using the -o flag. Note that thenipie output will default to the current working directory if no location is otherwise specified. There is an option for selecting only folders that contain a specific string (using the -sf flag). After generation of a runsheet (csv file), you should take a look at it in Excel or Numbers to make sure things look okay... Here are the columns that you can include. Order is irrelevant. Column names (headers) exactly as written below are required.

Example Runsheet

absolute pathnames are required for runsheets

sample	fasta	spikein_fasta	fastq1	fastq2	bed_out	spikein_bed_out	genome_sizes	bedgraph	SEACR_key	SEACR_out
mys1	path	path	path	path	path	path	path	path	4JS	path
mys2	path	path	path	path	path	path	path	path	4JS_CONTROL	path

• 'sample' name of the sample REQUIRED.
• 'fasta' location of the Bowtie2 indexed fasta file REQUIRED.
• 'spikein_fasta' location of the Bowtie2 indexed fasta file for spike_in normalization OPTIONAL.
• 'fastq1' a tab seperated string of filenames denoting location of all R1 files for a sample REQUIRED.
• 'fastq2' a tab seperated string of filenames denoting location of all R2 files for a sample REQUIRED.
• 'bed_out' name of the location for the aligned and sorted bam file REQUIRED.
• 'spikein_bed_out' name of the location for the aligned and sorted bam file OPTIONAL.
• 'genome_sizes' REQUIRED.
• 'bedgraph' file name of normalized bedgraph REQUIRED.
• 'SEACR_key' sample key corresponding to sample groups to be run against an IgG (or other) contol. all samples to be run against a control are given the same name and the control is labeleled with the an additional string underscore + 'CONTROL' (i.e. 4JS_CONTROL) OPTIONAL.
• 'SEACR_out' file name of SEACR output OPTIONAL.

Genomes and adding genome locations

piperna uses Bowtie2 for alignment. As such, you should have a previously indexed location of your genome accessible to piperna. This location is referred to in piperna as the 'fasta'. Similarly, one should provide the location of the spike_in indexed reference genome in the 'spikein_fasta' field. For bedgraph conversion, a text file of genome sizes text file is also needed. See the following for a discussion on how to make a 'genome_sizes' file https://www.biostars.org/p/173963/.

piperna provides an easy way to add these locations to your system for repeated use using the --genome_key (-gk) option during MAKERUNSHEET commands. A file called genomes.json can be found in the 'data' directory of the piperna install folder. This file can be edited to include those locations you want to regularly put in the runsheet. The following shows an example of a genomes.json file. The files "top level" is a name that can be used in the --genome_key field (-gk) during runsheet generation to populate the columns of the runsheet with fasta, spikein_fasta, and genome_sizes locations associated with that genome_key. The 'default' key will be used when no genome_key is specified.

{
    "default": {
        "fasta": "/path/path/hg38/bowtie2_index",
        "genome_sizes": "/path/path/hg38/genome_sizes.txt",
        "spikein_fasta": "/path/path/Ecoli/bowtie2_index"},
    "my_hg38": {
        "fasta": "/shared/biodata/ngs/Reference/iGenomes/Homo_sapiens/UCSC/hg38/Sequence/Bowtie2Index/genome",
        "genome_sizes": "/shared/ngs/illumina/henikoff/solexa/databases/human/hg38/chr_lens.txt",
        "spikein_fasta": "/shared/ngs/illumina/henikoff/Bowtie2/Ecoli"
    }

Doing a piperna run

Say your fastqs live within within subfolders of a folder 'fastq' in the folder 'data'. So if you were to...

cd /data/fastq
ls

... you'd get a bunch of folders, each of which would be filled with fastqs. Each folder name should correspond to a sample name.

To run piperna, do the following...

1. Make a new output directory 'piperna'.
2. Go into that directory and make a runsheet pointing to the fastq folder i.e. the folder level above. (at the command line, piperna is cool with either relative or absolute pathnames; but as stated earlier, absolute pathnames are required for the runsheet.)
3. Optionally you can only select directories of fastq files that contain in their name the string denoted using the -sf flag.
4. After inspecting and completing the runsheet, run ALIGN, NORM, and SEACR.
5. Sit back have a cocktail.

cd ..
mkdir piperna
cd piperna
piperna MAKERUNSHEET -fq ../fastq -sf MySampleDirectoriesStartWithThisString -o .
piperna ALIGN -r runsheet.csv
piperna NORM -r runsheet.csv
piperna SEACR -r runsheet.csv

Acknowledgements

Written by Scott Furlan with code inspiration from Andrew Hill's cellwrapper; piperna includes a python script samTobed.py which takes code from a fantastic sam reader "simplesam" - https://github.com/mdshw5/simplesam. samTobed.py uses specific sam-sorting parameters as written in Jorja Henikoff's PERL script.