FHSpipe

Transcriptome analysis pipeline using Fastp, Hisat2 and Stringtie. Generates read count files for differential expression analysis using popular R programs like edgeR or DEseq2

Figure representing the FHSpipe flowchart

R E Q U I R E M E N T S

Requires python version newer than 3.5.0

Python modules:

os,shutil, subprocess, argparse, sys, json, yaml

TOOLS NEEDED:

• Fastp (https://github.com/OpenGene/fastp)
• Hisat2 (http://daehwankimlab.github.io/hisat2/)
• Samtools (http://www.htslib.org/download/)
• Stringtie (https://ccb.jhu.edu/software/stringtie)
• PrepDE.py script (https://ccb.jhu.edu/software/stringtie/dl/prepDE.py3 - Place along with Stringtie)
• Gffcompare (https://ccb.jhu.edu/software/stringtie/gffcompare.shtml)
• Bedtools (https://github.com/arq5x/bedtools2/releases)
• OrfPredictor (http://proteomics.ysu.edu/publication/tools_download/OrfPredictor.zip)
• CPC2 (http://cpc2.gao-lab.org/download.php)
• RPSBlast (NCBI Blast+ suite) (http://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/)

FILES REQUIRED:

• Reference genome fasta file (.fasta/.fa)
• Reference genome annotation file (.gff/.gtf)
• All Sample fastq files in a single directory (no extra files). Single end sequenced files should be named as Sample.fastq. Pair end sequenced files should be names as Sample_R1.fastq and Sample_R2.fastq to avoid errors. Unzip the sample files if in zip format (.gz)

U S A G E

fhs_glm_pipeline.py [-h] [-p PATH] [-s SPCS] [-r REF] [-g GFF] [--len LEN] [--orfl ORFL] [-o OUT] [-t THREADS] [--lnc]  

A program to run FHS pipeline i.e. FASTp, HISAT2, STRINGTIE, MERGE, GFFCOMPARE, STRINGTIE 2, PREPDE, extract potential lncRNAs (IUX)

Required arguments:

-p PATH,  --path PATH    --> Path of sample fastq files
-s SPCS,  --spcs SPCS    --> Reference species name

Optional arguments:

-h,      --help       --> Show this help message and exit
-r REF,   --ref REF      --> Path of reference genome fasta file if species is not available
-g GFF,    --gff GFF      --> Path of reference genome gtf/gff file if species is not available
      --len LEN     --> Length filter cut off (In number of nucleotides) [Default: 200]
      --orfl ORFL     --> ORF Length filter cut off (In number of amino acids) [Default: 100]
-o OUT,   --out OUT      --> Prefix of output files [Default: out]
-t THREADS, --threads THREADS --> Number of threads to use in Hisat2, Samtools, Stringtie. [Default: 1]
       --lnc        --> Use to only extract lncRNAs and skip file processing for differential expression

OUTPUT FILES

Check the file "Output_guide.txt" in pipeline directory for details of output of the pipeline