Pipelines to process Cyclomics data.
This pipeline uses prior information from the backbone to increase the effectiveness of consensus calling from the circular DNA protocol by Cyclomics.
Click for installation instructions:
- Nextflow
- Docker or Conda or Apptainer/singularity
- Acces to the Github repo and a valid PAT token
- data output by ONT Guppy (SUP preferred for optimal results)
- Reference genome, Ideally pre indexed by BWA to reduce runtime.
The pipeline expects at least 16 threads to be available and 16GB of RAM. We recommend 64 GB of RAM to decrease the runtime significantly.
The pipeline has been developed with amplicons that map against the provided reference in mind.
We suggest to use Grch38.p14, since this works well with the VEP that is integrated in the pipeline its available via the code snippet below.
wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/001/405/GCA_000001405.29_GRCh38.p14/GCA_000001405.29_GRCh38.p14_genomic.fna.gz
gunzip GCA_000001405.29_GRCh38.p14_genomic.fna.gzTo reduce runtime pre index the reference genome with BWA, or obtain a preindexed copy.
This pipeline is compatible with the EPI2ME Labs platform by ONT. Please see ONT's installation guide.
Installation inside EPI2ME Labs:
- go to workflows by clicking on "installed workflows", or click the workflows icon in the top bar.
- click "Import workflow".
- Paste "https://github.com/cyclomics/cyclomicsseq" into the text bar and click Import workflow.
In this section we assume that you have docker and nextflow installed on your system, if so running the pipeline is straightforward. You can run the pipeline directly from this repo, or pull it yourself and point nextflow towards it.
nextflow run cyclomics/cycmomicsseq -r <pipeline version> -profile docker --input_read_dir '/sequencing/20220209_1609_X3_FAS06478_0ed4361c/fastq_pass/' --output_dir '/data/myresults' --reference '/data/reference/chm13v2.fasta' --backbone BB12If docker is not an option, singularity (or Apptainer, as it is called since Q2 2022) is a good alternative that does not require root access and therefor used in shared compute environments.
The command becomes:
nextflow run cyclomics/cycloseq -profile singularity ...'Please note that this assumes you've ran the pipeline before, if not add the -user flag as described in Usage[#Usage].
The pipeline is fully compatible with Conda. This means the full command becomes:
nextflow run cyclomics/cycloseq -profile conda ...'By default it uses the environment file that is shipped with the pipeline. this file is located in the repo, the pipeline needs to know where this file is to run with the correct versions of the required software.
| flag | info |
|---|---|
| --input_read_dir | Directory where the output fastqs of Guppy are located, e.g.: "/data/guppy/exp001/fastq_pass". |
| --read_pattern | Regex pattern to look for fastq's in the read directory, defaults to: "**.{fq,fastq,fq.gz,fastq.gz}". |
| --sequencing_summary_path | The summary file generated by guppy, Optional, default: "sequencing_summary*.txt". |
| --backbone | Select a preset backbone. |
| --backbone_file | File to use as backbone when --backbone is non of the available presets. eg a fasta file with a sequence with the name ">BB_custom" the name must start with BB for extraction reasons. |
| --reference | Path to the reference genome to use, will ingest all index files in the same directory. |
| --output_dir | Directory path where the results, including intermediate files, are stored. |
| --snp_filters.min_dir_ratio, --indel_filters.min_dir_ratio | Minimum ratio of variant-supporting reads in each direction (default: 0.001 (SNP); 0.002 (Indel)). |
| --snp_filters.min_dir_count, --indel_filters.min_dir_count | Minimum number of variant-supporting reads in each direction (default: 5). |
| --snp_filters.min_dpq, --indel_filters.min_dpq | Minimum positional depth after Q filtering (default: 5_000). |
| --snp_filters.min_dpq_n, --indel_filters.min_dpq_n | Number of flanking nucleotides to the each position that will determine the window size for local maxima calculation (default = 25). |
| --snp_filters.min_dpq_ratio, --indel_filters.min_dpq_ratio | Ratio of local depth maxima that will determine the minimum depth at each position (default = 0.3). |
| --snp_filters.min_vaf, --indel_filters.min_vaf | Minimum variant allele frequency (default: 0.003 (SNP); 0.004 (Indel)). |
| --snp_filters.min_rel_ratio, --indel_filters.min_rel_ratio | Minimum relative ratio between forward and reverse variant-supporting reads (default: 0.3 (SNP); 0.4 (Indel)). |
| --snp_filters.min_abq, --indel_filters.min_abq | Minimum average base quality (default: 70). |
Due to lab conditions a different backbone might be used. the --backbone parameter can be set to any fasta file. The following defaults are available by default in the pipeline, you can enable them by copying the value in the the value column and pasting it behind the cli command.
| backbone | value | default |
|---|---|---|
| BB22 | --backbone BB22 | |
| BB25 | --backbone BB25 | |
| BB41 | --backbone BB41 | |
| BB42 | --backbone BB42 | X |
| BBCS | --backbone BBCS | |
| BBCR | --backbone BBCR |
- Multi-threaded variant calling
Please see CHANGELOG.md
Download the latest version by running the example below:
wget -qO- https://get.nextflow.io | bashor see The official Nextflow documentation
Download the latest conda version from The official conda documentation
Run the below command and follow process:
bash Miniconda3-latest-Linux-x86_64.shDownload the latest version by running the example below:
wget https://github.com/apptainer/apptainer/releases/download/v1.1.0-rc.2/apptainer_1.1.0-rc.2_amd64.deb
sudo apt-get install -y ./apptainer_1.1.0-rc.2_amd64.debfor the latest up to date information see their official documentation
login to the hpc using SSH. there start a sjob with:
srun --job-name "InteractiveJob" --cpus-per-task 16 --mem=32G --gres=tmpspace:450G --time 24:00:00 --pty bashgo to the right project folder
cd /hpc/compgen/projects/cyclomics/cycloseq/pipelines/cycloseq/start the pipeline as normal
Cycas was added as a subtree using code from: https://gist.github.com/SKempin/b7857a6ff6bddb05717cc17a44091202. This was done instead of submodule to make pulling of the repo easier for endusers and to stay compatible with nextflow run functionallity.
more specifically:
git subtree add --prefix Cycas https://github.com/cyclomics/Cycas 0.4.3 --squashTo update run
git subtree pull --prefix Cycas https://github.com/cyclomics/Cycas <tag> --squash