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piperna 1.81

piperna
==========
A python wrapper for processing of bulk RNA seq data
Requirements

Python > 3.5 (piperna uses the 'six' package but will attempt to install if not already installed)
Computing cluster with PBS or SLURM
Modules installed for python, STAR or kallisto,
R Modules for GenomicAlignments, rtracklayer, and Rsamtools if running SUMMARIZE

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 RNASeq Alignment [-h] [--bam_folder BAM_FOLDER]
[--fastq_folder FASTQ_FOLDER]
[--organized_by {folder,file}]
[--genome_key GENOME_KEY]
[--split_char SPLIT_CHAR]
[--R1_char R1_CHAR]
[--R2_char R2_CHAR] [--ext EXT]
[--select SELECT]
[--sample_flag SAMPLE_FLAG]
[--runsheet RUNSHEET]
[--typeofseq {single,pe}]
[--software {STAR,kallisto}]
[--output OUTPUT] [--debug]
[--cluster {PBS,SLURM}]
[--user USER]
[--threads THREADS]
[--gb_ram GB_RAM]
[--additional_header ADDITIONAL_HEADER]
[--mfl MFL] [--sfl SFL]
[--count] [--install INSTALL]
[--outSAMtype OUTSAMTYPE]
[--addSTARstring ADDSTARSTRING]
[--log_prefix LOG_PREFIX]
[--flow_cell_folders FLOW_CELL_FOLDERS]
[--verbose]
{MAKERUNSHEET,ALIGN,SUMMARIZE,CONCATFASTQ,GENOMESFILE,ENVIRONSFILE,UNBAM}

positional arguments:
{MAKERUNSHEET,ALIGN,SUMMARIZE,CONCATFASTQ,GENOMESFILE,ENVIRONSFILE,UNBAM}
a required string denoting segment of pipeline to run.
1) "MAKERUNSHEET" - to parse a folder of fastqs; 2)
"ALIGN" - to perform alignment using STAR or KALLISTO;
3) "SUMMARIZE" - to summarize and count reads, 4)
"CONCATFASTQ" - function to concatenate fastq files
-i.e. for SRA upload; 5) "GENOMESFILE" - print
location of and cat genomes.json file; 6)
"ENVIRONSFILE" - print location of and cat
environs.json file; 7) "UNBAM" - convert bam to fastq

optional arguments:
-h, --help show this help message and exit
--bam_folder BAM_FOLDER, -bf BAM_FOLDER
For UNBAM only: Pathname of bam locations
--fastq_folder FASTQ_FOLDER, -fq FASTQ_FOLDER
For MAKERUNSHEET only: Pathname of fastq folder
--organized_by {folder,file}, -b {folder,file}
Option to specify how fastq or unbam folder is
organized
--genome_key GENOME_KEY, -gk GENOME_KEY
For MAKERUNSHEET only: abbreviation to use "installed"
genomes in the runsheet (See README.md for more
details
--split_char SPLIT_CHAR, -sc SPLIT_CHAR
Character by which to split the fastqfile name into
samples, OPTIONAL and for MAKERUNSHEET only
--R1_char R1_CHAR, -r1c R1_CHAR
Character by which to split the fastqfile name into
read1, OPTIONAL and for MAKERUNSHEET only
--R2_char R2_CHAR, -r2c R2_CHAR
Character by which to split the fastqfile name into
read2, OPTIONAL and for MAKERUNSHEET only
--ext EXT, -e EXT suffix of fastq files, OPTIONAL and for MAKERUNSHEET
only
--select SELECT, -s SELECT
To only run the selected row in the runsheet, OPTIONAL
and for MAKERUNSHEET only
--sample_flag SAMPLE_FLAG, -f SAMPLE_FLAG
FOR MAKERUNSHEET only string to identify samples of
interest in a fastq folder
--runsheet RUNSHEET, -r RUNSHEET
tab-delim file with sample fields as defined in the
script. - or name of runsheet to save if using
MAKERUNSHEET
--typeofseq {single,pe}, -t {single,pe}
Type of sequencing performed - REQUIRED for
MAKERUNSHEET, UNBAM and CONCATFASTQ
--software {STAR,kallisto}, -so {STAR,kallisto}
To set desired software, required and used for
MAKERUNSHEET only
--output OUTPUT, -o OUTPUT
To set output path, required for MAKERUNSHEET, UNBAM;
OPTIONAL for SUMMARIZE-- default for SUMMARIZE is
./SummarizedExperiment.RDS
--debug, -d To print commands (For testing flow)
--cluster {PBS,SLURM}, -c {PBS,SLURM}
Cluster software. OPTIONAL Currently supported: PBS
and SLURM
--user USER, -u USER user for submitting jobs - defaults to username.
OPTIONAL
--threads THREADS, -th THREADS
To set number of cores
--gb_ram GB_RAM, -gb GB_RAM
To set gb_ram
--additional_header ADDITIONAL_HEADER, -ah ADDITIONAL_HEADER
Additional bash header lines
--mfl MFL, -mf MFL Mean fragment length (kallisto ONLY)
--sfl SFL, -sf SFL SD fragment length (kallisto ONLY)
--count, -co Run Count (STAR Only)
--install INSTALL, -i INSTALL
FOR GENOMESFILE: location of file to install as a new
genomes.json file, existing genomes.json will be
erased
--outSAMtype OUTSAMTYPE, -st OUTSAMTYPE
To define type of SAM/BAM output (STAR Only)
--addSTARstring ADDSTARSTRING, -a ADDSTARSTRING
Additional STAR arguments to be run on all jobs in
runsheet (STAR Only)
--log_prefix LOG_PREFIX, -l LOG_PREFIX
Prefix specifying log files for piperna output from
henipipe calls. OPTIONAL
--flow_cell_folders FLOW_CELL_FOLDERS, -fc FLOW_CELL_FOLDERS
For CONCATFASTQ only: Comma-seprated location of
flowcell folders - i.e. as output from CellRanger
REQUIRED for CONCATFASTQ
--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 piperna 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
index
fastq1
fastq2
output
software
gtf

mys1
path
path
path
path
STAR
path

mys2
path
path
path
path
STAR
path

'sample' name of the sample REQUIRED.
'index' location of the indexed fasta file REQUIRED.
'fastq1' a tab seperated string of filenames denoting location of all R1 files for a sample REQUIRED if paired end.
'fastq2' a tab seperated string of filenames denoting location of all R2 files for a sample REQUIRED if paired end.
'fastqs' a tab seperated string of filenames can be used for single end reads REQUIRED if single end.
'output' name of the location for the aligned and sorted bam file.
'software' either 'STAR' or 'kallisto'. REQUIRED
'gtf' a location for annotation file in gtf format. REQUIRED for SUMMARIZE.

Genomes and adding genome locations
you should have a previously indexed (by the software package of your choosing) location of your genome accessible to piperna. This location is referred to in piperna as the 'index'.
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 locations associated with that genome_key. The 'default' key will be used when no genome_key is specified.
{
"default": {
"fasta": "/path/path/hg38/STAR_index",
"gtf": "/path/path/hg38/hg38.gtf"
},
"default_kallisto": {
"fasta": "/path/path/hg38/kallisto_index",
"gtf": "/path/path/hg38/hg38.gtf"
}
}

Cluster specific parameters
Piperna could be modified to run on any cluster Templates are provided for SLURM and PBS
Location of the environs file can be found by running:
piperna ENVIRONSFILE

As an example the SLURM script generation environs is shown below. Broadly it's parameters is divided into three groups: popen, script_lines, and resources.
popen - an entry that lists the command to open a submission job ('sbatch' in this case)
script_lines - provides the header of the bash script
resources - gives job-specific parameters
{
"SLURM": {
"popen" : ["sbatch"],
"script_lines": {
"1" : ["#!/bin/bash\n#SBATCH --output=outtmp\n#SBATCH --error=errtmp", ""],
"2" : ["#SBATCH --job-name=<--0-->", "JOB_NAME"],
"3" : ["#SBATCH --ntasks=1\n#SBATCH --cpus-per-task=<--0-->","THREADS"],
"4" : ["#SBATCH --mem-per-cpu=<--0-->000","RAM"],
"5" : ["<--0-->","HEADER"],
"6" : ["{\necho '<--1-->';<--0-->;<--1-->;} 2>&1 | tee <--2-->", "MODULES|COMMAND|TEMP_LOG_FILE"],
"7" : ["sed -e 's@^@[PIPERNA-<--0-->] JOB: <--1-->:\t\t@' <--2--> >> <--3-->", "TIME|JOB_NAME|TEMP_LOG_FILE|LOG_FILE"],
"8" : ["rm <--0-->\n", "TEMP_LOG_FILE"]
},
"resources" : {
"PIPERNA_STAR": {
"ram" : "8",
"threads" : "4",
"modules" : "module load STAR/2.7.6a-foss-2019b"
},
"PIPERNA_KALLISTO": {
"ram" : "8",
"threads" : "4",
"modules" : "module load kallisto"
},
"PIPERNA_SUMMARIZE": {
"ram" : "16",
"threads" : "8",
"modules" : "module load R"
},
"PIPERNA_UNBAM": {
"ram" : "2",
"threads" : "8",
"modules" : "module load SAMtools\nmodule load BEDTools"
}
}
}
}

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...

Make a new output directory 'piperna'.
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.)
Optionally you can only select directories of fastq files that contain in their name the string denoted using the -sf flag.
After inspecting and completing the runsheet, run ALIGN, NORM, and SEACR.

cd ..
mkdir piperna
cd piperna
piperna MAKERUNSHEET -fq ../fastq -sf MySampleDirectoriesStartWithThisString -o .
piperna ALIGN -r runsheet.csv
piperna SUMMARIZE -r runsheet.csv -o SummarizedExperiment.RDS

Interfacing with DESeq2
After running piperna, the SummarizedExperiment.RDS file can be input directly into DESeq2 like this
se<-readRDS(file.path(DATA_DIR, "summarizedExperiment.RDS"))
colnames(se)<-colData(se)$sample
se$group<-c("GroupA", "GroupA", "GroupB", "GroupB", "GroupB", "GroupA" ) #as an example
dds<-DESeqDataSet(se, design=~group)
keep <- rowSums(counts(dds)) >= 10
dds <- dds[keep,]
dds <- DESeq(dds, parallel = T)

Acknowledgements
Written by Scott Furlan.