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Computational protocols
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Basecalling: This step converts Nanopore's FAST5 files to sequences. For basecalling, we use Nanopore's basecaller, Medaka, which can run in parallel with sequencing (recommended) or afterward.
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Demultiplexing: After sequencing, the reads, stored as bulk FASTQ files, are sorted. During demultiplexing, each read is assigned to its correct plate/well combination and stored as a FASTQ file.
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Variant Calling: For each sample, the consensus sequence is compared to the reference sequence. A variant is called if it differs from the reference sequence. The success of variant calling depends on the number of reads sequenced and their quality.
From the LevSeq folder, install the package using pip:
git clone https://github.com/fhalab/LevSeq.git
pip install levseq
or for mac users you can use: brew install minimap2
Once dependencies are all installed, you can run LevSeq using command line.
3. GCC version 13 and 14 are both needed
For Mac M chip users: installation via homebrew
brew install gcc@14
brew install gcc@13
For Linux users: installation via conda
conda install conda-forge::gcc=14
conda install conda-forge::gcc=13
This could be something of interest if you have differerent barcodes.
To build the docker image run (within the main folder that contains the Dockerfile). Note building does not work
on Mac M3 chip, please use a ubuntu machine to build the docker image!
docker build -t levseq .
This gives us the access to the lebSeq command line interface via:
docker run levseq
Note! The docker image should work with linux, and mac, however, different mac architectures may have issues (owing to the different M1/M3 processers.)
Basically the -v connects a folder on your computer with the output from the minION sequencer with the docker image that will take these results and then perform demultiplexing and variant calling.
docker run -v /disk1/ariane/vscode/LevSeq/manuscript/Data/20241116-YL-LevSeq-parlqep400-1-2-P25-28:/levseq_results/ levseq docker-test levseq_results/ levseq_results/LevSeq-T1.csv
docker run -v /Users/XXXX/Documents/LevSeq/data:/levseq_results/ levseq 20240502 levseq_results/20240502/ levseq_results/20240502-YL-ParLQ-ep2.csv
In this command: /Users/XXXX/Documents/LevSeq/data is a folder on your computer, which contains a subfolder 20240502
If you have any issues, please check the LevSeq_error.log find in the output direectory and report the issue. If the problem persists, please open an issue on the GitHub repository with the error details.
If you solve something code wise, submit a pull request! We would love community input.