This is a helper repository supporting a toolchain used for real-time monitoring of sequencing runs. The toolchain consists of deepnano-blitz basecaller used for MinKnow-compatible .fastq files production and RAMPART for sequencing runs analysis. The repository contains RAMPART protocol and configuration for SARS-CoV-2 virus sequencing as well as some helper scripts. RAMPART protocol is slightly adjusted version of the one located here, although another primer scheme using 2000bp long amplicons is provided in nanocrop and available for use.
Install Miniconda package manager using system package manager: https://docs.conda.io/projects/conda/en/latest/user-guide/install/rpm-debian.html
Install RAMPART using following instructions: https://artic-network.github.io/rampart/docs/installation.html
Install deepnano-blitz using following instructions: https://github.com/fmfi-compbio/deepnano-blitz
Install nanocrop helper repository:
git clone https://github.com/fmfi-compbio/nanocrop.git
Install dependencies for RedHat:
dnf install inotify-tools
Install dependencies for Debian:
apt-get install inotify-tools
Web browser needs to be installed on workstation performing RAMPART analysis.
Toolchain will use basecall_continuous_reads.sh script to watch a designated input folder for .fast5 files being continuously created during sequencing run. For every .fast5 file deepnano-blitz basecaller is invoked. Basecaller parameters in the script are fixed and set rather for short basecalling times than output accuracy. Please adjust basecaller parameters in the script manually according to your needs. Basecaller output is stored in output folder, which should be also an input folder for RAMPART pipeline. This is by default rampart/SARS-CoV-2/data/fastq/pass/. RAMPART will watch input folder configured for its pipeline and process .fastq files as they are created. Using its configuration and protocol RAMPART will demultiplex obtained reads and align them to the reference sequence provided. Thus monitoring current results of sequencing run such as reference genome coverage in real time.
Start the basecaller watchdog:
cd <nanocrop-project-dir>
conda activate deepnano-blitz
./basecall_continuous_reads <input-directory> rampart/SARS-CoV-2/data/fastq/pass/
Start RAMPART analysis in new terminal window:
cd <nanocrop-project-dir>/rampart/SARS-CoV-2/data/
conda activate artic-rampart
rampart --protocol ../protocol/
RAMPART graphical output is available at http://localhost:3000.
Now the toolchain is prepared for the sequencing run.