/CIRCprimerXL_ONT

circ primer design for ONT

Primary LanguagePythonOtherNOASSERTION

CIRCprimerXL_ONT

read me needs to be updated for ONT primer design! Collaborators: Marieke Vromman, Pieter-Jan Volders

Questions concerning the GitHub structure/scripts can be addressed to any of the collaborators.

Primer design pipeline for circRNAs based on primerXL (Lefever, S., Pattyn, F., De Wilde, B. et al. High-throughput PCR assay design for targeted resequencing using primerXL. BMC Bioinformatics 18, 400 (2017). https://doi.org/10.1186/s12859-017-1809-3).

This pipeline runs entirely in the oncornalab/primerxl_circ docker image, which is available on DockerHub. It is not necessary to download this image locally, as Nextflow pulls the latest version automatically from DockerHub.

Installation

Running on your computer

Nextflow and Docker should be installed locally. Make sure Docker Desktop is running when you want run the pipeline.

Running on the HPC (UGent)

Nextflow version 20.10.0 is available on all clusters (swalot, skitty, victini, joltik, kirlia, doduo). The pipeline can be run through an interactive session. The pipeline can only run from the $VSC_SCRATCH_VO_USER directory.

qsub -I -l nodes=1:ppn=16 -l walltime=04:00:00
cd $VSC_SCRATCH_VO_USER/CIRCprimerXL/
module load Nextflow/20.10.0
nextflow run CIRCprimerXL.nf --help

General usage

$ nextflow run CIRCprimerXL.nf --help

Usage:
	
	The typical command for running the pipeline is as follows:
	nextflow run CIRCprimerXL.nf -profile singularity
	
	Mandatory nextflow arguments:
	-profile            set to 'local' when running locally, set to 'singularity' when running on the HPC

	Mandatory pipeline arguments:
	--input_bed         path to input file with circRNAs in bed format (0-based annotation)


	Optinal pipeline arguments:
	--splice			when set to 'yes' the input sequence will be spliced, when set to 'no' the input sequence will be unspliced
	--primer_settings   path to file with primer3plus settings (see primer3 manual)
	--chrom_file        file containing all chromosome sizes (to validate bed file)
	--primer3_diff      the minimum number of base pairs between the 3' ends of any two left primers (see also primer3 PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE)
	--primer3_nr        the number of primers designed by primer3; caution: setting this parameter to a large value will increase running time
	--min_tm	    minimum melt temperature of the primers (default: 58)
	--max_tm	    maximum melt temperature of the primers(default: 60)
	--opt_tm	    optimal melt temperature of the primers(default: 59)
	--diff_tm	    maximum difference in melt temperature between the primers(default: 2)
	--min_gc	    minimum GC contect of the primers (default: 30)
	--max_gc	    maximum GC contect of the primers(default: 80)
	--opt_gc	    optimal GC contect of the primers(default: 50)
	--amp_min	    minimum amplicon length (default: 60)
	--amp_max	    maximum amplicon length (default: 0)
	--temp_l            the number of nucleotides on each side of the circRNA BSJ that will be used for the template (example 150 => template of 300 nts in total)
	--output_dir        path to directory where the output files will be saved

You can easily create your own profiles by modifying the nextflow.config file.

Nextflow keeps track of all the processes executed in your pipeline. If you want to restart the pipeline after a bug, add '-resume'. The execution of the processes that are not changed will be skipped and the cached result used instead.

Note: The pipeline results are cached by default in the directory $PWD/work. This folder can take of lot of disk space. If your are sure you won’t resume your pipeline execution, clean this folder periodically.

Note: If a circRNA is smaller than the requested template size, the template size is reduced to the circRNA size. Of note, if this 300-nucleotide template sequence includes an exon-intron boundary, the intronic region (which may not be part of the circRNA) is included. Some circRNAs effectively also include intronic sequences, and some BSJ concatenate an exonic and an intronic sequence.

Output

In the output folder, you will find

  • filtered_primers.txt, a file containing one selected primer pair per circRNA (see below for column details)
  • log_file.txt, a file
  • summary_run.txt,
  • all_primers directory
  • primer3_details directory

filtered_primers.txt output file column names:

column name description
circ_ID circ id assigned to each circRNA (unique within one run)
chr circRNA chromome
start circRNA start position
end circRNA end position
primer_ID primer ID generated by primer3
FWD_primer forward primer
REV_primer reverse primer
FWD_pos relative position of forward primer
FWD_length length of forward primer
REV_pos relative position of reverse primer
REV_length length of reverse primer
FWD_Tm melt temperature of forward primer
REV_Tm melt temperature of reverse primer
FWD_GC GC content of forward primer
REV_GC GC content of reverse primer
amplicon amplicon sequence amplified by the primer pair
PASS result of filtering (PASS if the primer pair passed all filters, FAIL if the primer pair failed one or more filters)

Other species

As default, CIRCprimerXL designs primers voor humans. To design primers for other species, the following files have to be provided and parsed through the corresponding parameters:

Nextflow tower

Nextflow tower can be used to monitor the pipeline while it's running.

nextflow run CIRCprimerXL.nf -with-tower

When Nextflow tower is used in combination with the HPC, the nextflow version and tower access token should be indicated.

export NXF_VER="20.10.0"
export TOWER_ACCESS_TOKEN=your_token_here