Running TargetID

Basic Configuration

To run TargetID, you need two things:

1. A small RNA file - if sort is specified the output of that is used - if not smallRNA_fastq is used.

2. One or more files containing potential targets of small RNAs (e.g. genes, transposons, lncRNA etc.) specified in the targetid section as target_files.

For example:

smallRNA_fastq: small_rna.fastq
targetid:
  target_files:
    - targets/lncRNA.fasta
    - targets/genes.fasta

Once this config is written, it can be run as usual using:

$ hlsmallrna config.yml

Tip

Remember if you install via apptainer or docker you will need to prepend the command from the install page. For apptainer this command becomes:

$ apptainer run -B $HOME:$HOME hlsmallrna.sif hlsmallrna config.yml

Optional Parameters

min_seq_length

This is used to set the seed length in bowtie2 when aligning to targets (defaults to 5). Larger numbers makes it fasta but less accurate for sequences close to this length. For example, to set to 18 add the following:

targetid:
  min_seq_length: 18

mismatches

Number of mismatches to allow in bowtie2 when aligning to the target files, defaults to 0. See the Considerations page for information about why you'd want to change this. The example below sets this to allow 1 mismatch:

targetid:
  mismatches: 1

enrich - eggnog_data_dir

The pipeline can optionally enrich GO terms, KEGG pathways and PFams for the targets of the small RNA. This is a rough analysis based on emapper.py --itype CDS mode that uses diamond blastx to map, so is likely only a rough analysis. May be useful if you want to have a quick look at if there are any common annotations within the targets though should be redone probably later. To run this you need to add the eggnog_data_dir to the config file. This directory can be created with the command:

$ mkdir -p /home/user/path/to/eggnog-data
$ download_eggnog_data.py -y --data_dir /home/user/path/to/eggnog-data

Tip

Remember if you install via apptainer or docker you will need to prepend the command from the install page. For apptainer this command becomes:

$ mkdir -p /home/user/path/to/eggnog-data
$ apptainer run -B $HOME:$HOME hlsmallrna.sif download_eggnog_data.py -y --data_dir /home/user/path/to/eggnog-data

Then add the following to the config file

targetid:
  ...
  enrich:
    eggnog_data_dir: /home/user/path/to/eggnog-data

enrich - exclude

If you have run targetid with targets it doesn't make sense to map to the eggnog database (e.g. lncRNA), you can exclude them with the exclude key, but must ensure the path exactly matches the one in the target_files list. e.g.

targetid:
  target_files:
    - targets/genes.fasta
    - targets/lncRNA.fasta
  enrich:
    eggnog_data_dir: /home/user/path/to/eggnog-data
    exclude:
      - targets/lncRNA.fasta

Output Files

rna_target_list.tsv

List of small RNA and there targets identified from the files specified.

targets_by_group

Same information as rna_target_list.tsv, but split by small RNA group (length and first base).

Output Files - Enrich

These files are only produced if the enrich key is present.

eggnog_mapper

Results of the eggnog-mapper run on the target files.

targets_enriched_go_terms.tsv

Enriched GO terms in the targets of small RNA vs the rest of the target files.

targets_enriched_kegg_pathways.tsv

Enriched KEGG pathways in the targets of small RNA vs the rest of the target files.

targets_enriched_pfams.tsv

Enriched PFams in the targets of small RNA vs the rest of the target files.