The miR-eCLIP module

What is miR-eCLIP?

miR-eCLIP is a CLIP experiment for mapping microRNA (miRNA) binding sites on target RNAs. During reverse transcription, read-through from a miRNA into its ligated target RNA produces chimeric reads. These reads contain the miRNA sequence at the 5′ end, followed by sequence from the target RNA adjacent to the UV crosslink.

A detailed description of the experiment is available in the miR-eCLIP publication.

Overview of the miR-eCLIP experiment.

How to analyse miR-eCLIP data with racoon_clip

racoon_clip includes a dedicated workflow for analysing miR-eCLIP data. Set experiment_type to miReCLIP and provide an uncompressed FASTA file containing the mature miRNA reference sequences with mir_genome_fasta.

Schema of processing steps for miR-eCLIP.

Processing overview

The miR-eCLIP workflow first performs the standard racoon_clip preprocessing steps, including barcode handling, quality filtering, and adapter trimming.

The first 24 nucleotides of each processed read are aligned to the supplied miRNA reference. Reads that do not align continue through the standard racoon_clip workflow. Among the aligned reads, only those satisfying the configured canonical-start and missing-5′-nucleotide filters are retained as chimeric reads. The canonical miRNA sequence is removed, and the remaining target-RNA sequence is aligned to the genome.

Chimeric target-RNA alignments are deduplicated and used to identify strand-specific, single-nucleotide crosslinks. The peaks workflow additionally combines chimeric and non-chimeric alignments by experiment group and calls peaks with PureCLIP.

The exact processing stages, filtering calculations, command-line options, and external tools are described in miR-eCLIP-specific processing.

miR-eCLIP parameters

experiment_type

Set this to miReCLIP to enable the miR-eCLIP workflow and its predefined barcode and UMI arrangement.

mir_genome_fasta

Absolute path to an uncompressed FASTA file containing mature miRNA reference sequences. RNA sequences containing U are converted to DNA sequences containing T before the Bowtie2 index is built.

mir_starts_allowed

Space-separated inferred positions at which the canonical miRNA may begin in the processed read. The default is "1 2 3 4".

mir_5prime_missing_allowed

Space-separated numbers of nucleotides that may be missing from the canonical miRNA 5′ end. The default is "0 1 2 3".

The standard input, genome, optional GTF annotation, adapter-trimming, grouping, and STAR settings also apply. See Configuration and experiment types for configuration guidance and All parameters and options for the complete parameter reference.

Visualisation of the mir_starts_allowed and mir_5prime_missing_allowed parameters.

Example configuration

# Experiment type
experiment_type: "miReCLIP"

# Output directory
wdir: "/absolute/path/to/output"

# Input FASTQ files
# Multiple files are supplied as one space-separated string.
infiles: "/absolute/path/to/sample1.fastq.gz /absolute/path/to/sample2.fastq.gz"

# Genome reference and optional annotation
genome_fasta: "/absolute/path/to/genome_assembly.fa"
gtf: "/absolute/path/to/annotation.gtf"
# Optionally provide an existing STAR index directory.
star_index: ""
read_length: 75

# Adapter trimming
adapter_cycles: 2

# miRNA reference and filtering
# For example, mature miRNA sequences obtained from miRBase.
mir_genome_fasta: "/absolute/path/to/mature_miRNAs.fasta"
mir_starts_allowed: "1 2 3 4"
mir_5prime_missing_allowed: "0 1 2 3"

Outputs

The miR-eCLIP workflow produces additional miRNA alignments, trimmed target-RNA reads, chimeric genomic alignments, strand-specific crosslinks, and a miRNA-specific HTML report.

The peaks workflow also produces group-level combined BAM files and miR-eCLIP PureCLIP peaks.

See Understanding the output files for the exact output paths, filenames, and conditions under which each output is produced.