A Snakemake pipeline designed to generate consensus sequences for target amplicon Nanopore sequencing. The pipeline supports dehosting of raw reads followed by automated selection of the most probable full-length reads for consensus building.
# Clone the repository
git clone https://github.com/jimmyliu1326/CANS.git
# Modify CANS.sh permission and add to $PATH in .bashrc
chmod +x CANS/CANS.sh
echo "export PATH=$PWD/CANS:\$PATH" >> ~/.bashrc
source ~/.bashrc
# Create a new conda environment called `cans`
conda env create -f CANS/conda_env.yml
#### Compile ThermonucleotideBLAST (Optional)
# To search for reads that represent PCR products, the pipeline uses thermonucleotideBLAST
# Because the tool is not available as a conda package, manual compilation is required
# This step is only required if you intend to utilize the primer search functionality
# install dependencies (tested on Ubuntu 16.04+)
apt-get update && apt-get install -y \
libmpich-dev \
libopenmpi-dev \
libz-dev
# Clone the forked thermonucleotideBLAST respository
git clone https://github.com/jimmyliu1326/thermonucleotideBLAST.git
# Compile
cd thermonucleotideBLAST
make all
# Add tntblast to $PATH docker pull jimmyliu1326/cans
singularity pull docker://jimmyliu1326/cans
Required arguments:
-i|--input .csv file with first column as sample name and second column as path to a DIRECTORY of fastq file(s), no headers required
-o|--output Path to output directory
-e|--expected_length Expected sequence length (bps) of target amplicon
--mode Select the mode for full-length reads identification (Options: dynamic/static)
dynamic: infers the most likely length of target amplicon based on the read length distribution of input data
static: selects reads solely based on the user-defined expected length of the target amplicon
Optional arguments:
--primers Path to a headerless .tsv file containing forward and reverse primer sequences to select PCR products for consensus building
The primers file should only contain a single set of primers with primer ID, forward, and reverse primer sequences separated by tabs.
-r|--reference Reference sequence used for dehosting
-s|--subsample Specify the target coverage for consensus calling [Default = 1000]
-d|--deviation Specify the read length deviation from (+/-) expected read length allowed for consensus building [Default = 50 bps]
-m|--model Specify the flowcell chemistry used for Nanopore sequencing {Options: r9, r10} [Default = r9]
-t|--threads Number of threads [Default = 32]
--notrim Disable adaptor trimming by Porechop
--unlock Unlock Snakemake working directory
--keep-tmp Keep all temporary files
-h|--help Display help message
-v|--version Print version
CANS.sh -i samples.csv -o /path/to/outdir -e 2050 --mode dynamic
- R >= 3.6
- porechop >= 0.2.4
- spoa >= 4.0.7
- medaka >= 1.3.3
- seqkit >= 0.16.1
- seqtk >= 1.3
- snakemake >= 5.3.0
- NanoFilt >= 2.8.0
- tntblast >= 2.4