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Description

This performs in silico PCRs from .fastq(.gz) or .fasta files.

The script uses bbduk to bait reads containing the primer sequences from the .fastq(.gz) files. It performs a second round of baiting on the original .fastq(.gz) files with the newly created baited .fastq files in order to hopefully have sequence data to span the entire amplicon. Resulting double-baited read files are assembled into contigs using SPAdes. The assemblies are BLASTed against the primer file to determine if both forward and reverse primers can be found in a single contig, thus a valid PCR product. PCR product length is also reported.

The longer the reads in the FASTQ file(s), the better the assembly and the fewer false negatives.

External Dependencies

  1. conda

Installation

conda install -c adamkoziol in_silico_pcr

you may need to add the bioconda channel

conda config --add channels bioconda

Inputs

  1. Primer pair list (fasta). Primer names have to end with “-F” or “-R”. Note: it is possible to have an integer following the direction: >vtx1a-F1 or >vtx1a-F are both acceptable
  2. Raw reads (FASTQ) or assemblies (FASTA)

Required arguments

primer_finder.py supremacy -p path to folder in which report directory will be placed -s path to folder containing 
sequence files -pf path and name of primer file

Optional arguments

-m number of mismatches: Number of mismatches allowed [0-3]. Default is 1

-n number of threads: Number of threads. Default is the number of cores in the system

-k kmer length: The range of kmers used in SPAdes assembly. Default is 55,77,99,127, but you can provide a comma-separated list of kmers e.g. 21,33,55,77,99,127 or a single kmer e.g. 33

Testing

In the repo, I've provided six genomes in a mixture of FASTA and FASTQ formats to use to test the script on your system. The FASTA files are assemblies, while the FASTQ files are pre-baited files to reduce size. The report you create should match the one in the 'desired_outputs' folder (there may be small differences when it comes to the order of genes).

git clone https://github.com/adamkoziol/in_silico_PCR.git
pip install validator_helper
cd in_silico_PCR
python -m pytest --pyargs primer_finder