Pipelines for RNA-seq analysis: STAR and Trinity. This package is made easy to use two approaches for RNA-seq: a) STAR (alignment) and b) Trinity (de novel assembly)
The package includes the data cleaning, alignment/assembly, DE/Annotation analysis and data visualization for GO. please find more detail for STAR and Trinity-wiki in the links below: STAR: https://github.com/alexdobin/STAR Trinity: https://github.com/trinityrnaseq/trinityrnaseq/wiki
p.s. For GO visualization script is not included in the package. But the template is provide in the readme.
It is better to run this package in virtual environment. Set up the virtual environment with following steps:
- Install virtualenv
$ pip3 install virtualenv - Setup virtualenv
$ python3 -m venv venv - Activate virtualenv
$ source ./venv/bin/activate- Install package
$ pip install -e .
Functions include in the package:
📝 Explanation of the function - function name
- Pre-alignment quality control -
fastqc - Trimmed adapter -
trimmed_adapter - Prepare genome for STAR alignment -
star_genome_prep - STAR alignment -
star_alignment - Post STAR alignment -
bamfiles_postqc - Prepare files for post STAR alignment -
qualimap_qc - Count the Transcripts -
salmon_quantification - Count the Transcripts -
htseq_count - Count the Transcripts -
rsem_count - Trinity assembly -
trinity_assemble - Estimate the expression after Trinity assembly -
trinity_estimate_expression - Calculate the transcripts and transfer into a matrix -
trinity_abundance_estimates_to_matrix - Trinity DE analysis -
trinity_run_de_analysis - RNA-seq data visualization -
trinity_run_glimma
For both the STAR and Trinity pipelines, the first step is always to check the raw data quality.
🔎 Generate fasta QC report.
strst fastqc --fastqc [put the information of "which fastqc"] --data [an absolute directory to rna-seq data'] --outputfolder [output dir]
The adapters information can find from fastqc report.
🔎 Trimmed adapter by cutadapt.
strst trimmed-adapter --cutadapt [indicate the path where cutadapt installed] --adapter_fwd [adapter forward sequence] --adapter_rev [adapter reverse sequence] --data [fasta files] --extention [fastq.gz or fastq]
- Prepare genome reference
strst star_genome_prep --star [indicate the path where star installed] --gtf_dir [absolute directory to gft file; example: "/home/ref/gft_dir"] --gtf_file [full path to gft file; example: "/home/ref/gft_dir/sample.gtf"] --ref_fasta [full path to reference fasta file; example: "/home/fasta_dir/sample.fasta"]
- Run STAR alignment
strst star_alignment --star [indicate the path where star installed] --data [absolute directory to gft file; example: "/home/ref/gft_dir"] --gtf_dir [absolute directory to gft file; example: "/home/ref/gft_dir"] --gtf_file [full path to gft file; example: "/home/ref/gft_dir/sample.gtf"] --sep [charaters after R1/R2, i.e., file named: 1-G1_HNFVVDRXX_L1_R1.fastq.gz, put "."; file named: 1-G1_HNFVVDRXX_L1_R1.trimmed.fastq.gz, put ".trimmed] --gz [if RNA-seq data is .gz, put "true", if not put "false"] --threads [number of threads, default=15]
- QC alignment output
strst bamfiles_postqc --samtools [indicate the path where samtools installed] --bam_dir [absolute path to bam files (the files after STAR), default="result/alignment/"]
strst qualimap_qc --qualimap [indicate the path where qualimap installed] --bam_dir [absolute path to bam files (the files after STAR), default="result/alignment/"] --gff [An absolute directory to gff or gtf] --output_dir [output folder; i.e. /home/output]
- Quantification ( Three methods are provide in this package: Salmon, htseq and RSEM)
strst salmon_quantification --salmon [indicate the path where salmon installed] --fasta [absolute path to fasta] --output_dir [the dir for output file] --bam_dir [absolute path to bam files]
strst htseq_count --htseq [indicate the path where htseq installed] --gtf_file [absolute path to gtf_file] --bam_dir [absolute path to bam files] --output_dir [output dir, default="result/gene_counts"]
strst rsem_count --rsem [indicate the path where rsem installed] --fasta_file [absolute path to fasta] --bam_dir [absolute path to bam files] --output_dir [output dir, default="result/gene_counts"]
To obtain more information about Trynity, please visit the developer's website: https://github.com/trinityrnaseq/trinityrnaseq/wiki
In strst package followed the steps from the website provide above:
- Assembly
strst trinity_assemble --trinity [indicate the path where trinity installed] --data [absolute path to RNA-seq data] --sample_list [sample_list; default="/home/ubuntu/sample_input.txt"] --sep [charaters after R1/R2, i.e., file named: 1-G1_HNFVVDRXX_L1_R1.fastq.gz, put "."; file named: 1-G1_HNFVVDRXX_L1_R1.trimmed.fastq.gz, put ".trimmed."] --cpu [request cpu] --memory [request memory] --output_prefix [output dir, default="result/trinity"]
- Estimate expression
strst trinity_estimate_expression --trinity [indicate the path where trinity installed] --method [salmon or RSEM] --seqtype [fq or fa] --fasta [absolute path to meta-fasta] --samplelist [An absolute directory to samplelist (generated from trinity_assemble)] --outputdir [Full path to output]
- Abundance estimates to matrix
strst trinity_abundance_estimates_to_matrix --trinity_dir [An absolute directory to trinity_dir] --data_dir [Path to access folders] --gene_map [Path to gene_map, includes file name and extension] --output_name [output file name]
- DE analysis
strst trinity_run_de_analysis --trinity_dir [An absolute directory to trinity_dir] --method [edgeR or DESeq2] --matrix [Path to matrix, includes file name and extension] --output_name [output file name] --sample_file [An absolute directory to sample_file (generated from trinity_assemble)]
- Glimma visualization
strst trinity_run_glimma --trinity_dir [An absolute directory to trinity_dir] --edger_dir [edgeR DE analysis output dir] --sample_file [An absolute directory to sample_file (generated from trinity_assemble)]