- BacGenomePipeline
Complete Bacterial Genome Assembly and Annotation Pipeline
Program developed by Stephen Fordham
BacGenomePipeline is a complete convenience bacterial genome assembly pipeline. Assembled and annotated bacterial genomes can be created with only Oxford Nanopore long raw reads as input! BacGenomePipeline can accept either fastq or gzipped fastq files.
Relax and grab a coffee while BacGenomePipeline does the genomic heavy lifting.
This pipeline filters raw reads to produce the best 500mb reads. The filtering process also places weight on read quality, to ensure small high quality reads are not discarded. This is considered vital to aid the recovery of small plasmids present within bacterial strains.
Optionally, the user can run Nanostat to assess read quality metrics. The best reads are then assembled using the flye genome assembler with settings adjusted to help recovery of plasmids with an imbalanced distribution. Optionally, the assembly is then polished with one round of medaka-consensus polishing. The polished assembly is annotated using staramr which scans bacterial genome contigs against the ResFinder, PointFinder, and PlasmidFinder databases (used by the ResFinder webservice and other webservices offered by the Center for Genomic Epidemiology) and abricate and compiles a summary report of detected antimicrobial resistance and virulence genes.
The default settings selected in BacGenomePipeline have been tested against challenging gemomes, such as Klebsiella pneumoniae strain ATCC700721/MGH78578. This strain contains 2 small plasmids (3.4kb and 4.2kb), two medium sized plasmids (88kb and 107.5kb), and one large plasmid (175kb) in addtion to the chromosome (5.3mb). The pipeline was able to successfully build to closure (i.e. assemble as a circular unitig) all structures exclusively using ONT long reads!
BacGenomePipline can now be run in 4 modes. These modes include; pipeline, pipe_red_mem, assembly and annotation. These modes offer the user more flexibility when using BacGenomePipe. For example, the user may want to only run an assembly, alternatively the user may have a genome assembly in FASTA format and want to annotate the assembly for antimicrobial resistance and virulence genes.
BacGenomePipeline can be run in 4 modes.
These modes include:
- Running the entire pipeline workflow.
--pipeline - Running the pipeline using reduced memory by setting parameters for genome size and coverage for initial disjointings.
--pipe_red_mem - Running a genome only assembly.
--assembly - Running the annotation step on an pre-exisiing genome assembly in FASTA format.
--annotation
For usage instructions, run:
BacGenomePipeline --help
Currently, BacGenomePipeline has been tested and runs on Linux OS.
BacGenomePipeline on Conda
BacGenomePipeline on PYPI
The simplest way to install BacGenomePipeline is running the following command:
conda install -c stephenfordham bacgenomepipeline
I recommend installing BacGenomePipeline in a conda virtual environment: For example:
conda create -n pipeline
conda activate pipeline
(pipeline) conda install -c stephenfordham bacgenomepipeline
Enter y, when promoted to install dependenies in your terminal window.
Alternatively you can run the following commands:
pip install BacGenomePipeline
conda install -c bioconda filtlong==0.2.0
conda install -c bioconda flye==2.8.1
conda install -c bioconda abricate==1.0.1
For useful usage instructions, run
BacGenomePipeline --help
BacGenomePipline can be run in one of four usage modes. The usage mode must be specified explicitly in the terminal. A selection of examples of BacGenomePipeline run in different usage modes is shown at the bottom of the help message and in the usage example section on this page.
usage: BacGenomePipeline (--pipeline | --pipe_red_mem | --assembly | --annotation)
--fastq_file READS
--help --version
[--nanostats] [--medaka_polish]
[--mean_q_weight] [--asm_fasta]
[--genome_size SIZE] [--asm_coverage INT]
[--flye_dir DIR_NAME] [--polished_dir DIR_NAME]
[--amr_dir DIR_NAME] [--vir_dir DIR_NAME]
Complete Bacterial Genome Assembly and Annotation Pipeline
optional arguments:
-h, --help show this help message and exit
--pipeline Runs the entire Pipeline
--pipe_red_mem Runs the entire Pipeline with reduced memory
consumption
--assembly Runs the assembly portion of the pipeline
--annotation Runs the annotation portion of the pipeline
Version:
--version Print version and exit.
Input fastq Reads (a fastq file is required):
-f , --fastq_file Specify an input Fastq file for the Pipeline and
assembly modes
Pipeline Options:
-n, --nanostats Optionally run NanoStats on your filtered read set
-m, --medaka_polish Optionally run NanoStats on your filtered read set
-s , --asm_fasta Add Genome assembly in fasta format
-w , --q_weight Add mean_q_weight for read filtering
Optional flye assembly arguments
(To reduce memory consumption for large genome assemblies):
-g , --genome_size Estimated genome size (for example, 5m or 2.6g)
-c , --asm_coverage reduced coverage for initial disjointig assembly [not
set]
Directory names:
-d , --flye_dir Specify a flye genome assembly directory name
-p , --polished_dir Specify a medaka polished genome directory name
-a , --amr_dir Specify a antimicrobial resistance directory name
-v , --vir_dir Specify a virulence gene directory name
Did you know? BacGenomePipeline can be run in modes
These modes include: pipeline, which runs the entire BacGenomePipeline workflow,
pipe_red_mem, which uses less memory by using use a subset of the longest reads
for initial disjointig by specifying --asm-coverage and --genome-size options.
The assembly mode runs assembly and polishing steps only. For this step,
annotation is excluded. Finally the annotation mode takes a genome assembly and
annotates it for antimicrobial and virulence genes
Example Usage:
BacGenomePipeline --pipeline -f reads.fastq
BacGenomePipeline --pipeline -f reads.fastq.gz -m -n
BacGenomePipeline --pipe_red_mem -f reads.fastq -g 5.7m -c 40 -n -m
BacGenomePipeline --annotation -s assembly.fasta
BacGenomePipeline --assembly -f reads.fastq -n -m
BacGenomePipeline --pipeline -f reads.fastq
BacGenomePipeline --pipeline -f reads.fastq.gz -m -n
BacGenomePipeline --pipe_red_mem -f reads.fastq -g 5.7m -c 40 -n -m
BacGenomePipeline --annotation -s assembly.fasta
BacGenomePipeline --assembly -f reads.fastq -n -m
Assembly of extensively-drug resistant (XDR) strain Klebsiella pneumoniae ATCC700721
assembly.gfa file in flye directory rendered via Bandage
Figure 1. Whole genome assembly XDR of K. pneumoniae ATCC700721
1 completely closed chromosome
5 completely closed plasmids
Figure 2 Sample AMR data available via amr_dir
Figure 3 Sample virulence gene data obtained when the entire pipeline or the annotaion portion of the pipeline runs