smRNA / miRNA Processing

Usage

Usage:

pipeline.sh
            [-r <genome_reference_file> (FASTA)>]
            [-g <genome_feature_file (GTF/GFF)>]
            [-m <mirbase_file (FASTA)>]
            [-o <output_directory>]
            [-n <cores>]
            [-f (overwrite existing files)]
            [-k (keep temp files)]
            <sequence_file> [<additional_sequence_files> <will_be_merged> <before_processing>]

What It Does

Aligns single-read (not paired-end) RNA samples to reference genomes, annotates them, and counts gene frequencies. Also aligns against miRBase. Visualizes read lengths after trimming. More specifically:

• Merges multiple FASTQ files into a single FASTQ file (simple concatenation)
• Trims adapter sequences using cutadapt
• Aligns to a reference file specified by the user using bowtie2
• Tallies the number of times reads align to each feature in an annotation file specified by the user
• Aligns to the miRBase microRNA database (http://www.mirbase.org)
• Visualizes the read lengths after trimming on a histogram created with matplotlib

What It Does Not Do

• Automatically generate a report or statistics. This must be done manually, although an example report in RST format is supplied

Required Software

• bowtie2
• HTSeq (python module)
• matplotlib (python module)

Input

• Single-read sample data in FASTQ format
• Reference genome for alignment in fasta format (with bowtie2 indexes)
• Annotation file in gff format
• miRBase file for alignment in fasta format (with bowtie2 indexes)

Output

• Alignment files in BAM format
• Annotated alignment files in BAM format
• Feature frequency counts in CSV format
• Visualization of read lengths in JPG format
• A log of the commands executed and their output

Example Usage

bash pipeline.sh \
    -r path/to/reference_file.fa \
    -g path/to/annotation_file.gff \
    -m path/to/miRBase.fa \
    -o output_dir/ \
    -n 8 \
    -f \
    -k \
    data_file1.fastq data_file2.fq data_file3_fastq