pertpipe

Pipeline for full analysis of Bordetella pertussis, particularly for the detection of macrolide resistance & vaccine antigen mutations.

Development

The initial development of pertpipe was designed for culture-based sequencing methods, ensuring accurate detection of 23S rRNA mutations and the vaccine antigens. Pertpipe primarily reports information about the ptxP, ptxA-E, prn, fim2, fim3 types and any mutations occuring the V domain of the 23S rRNA. As CIDM-PH transition to the use of the hybridisation capture-based method for B. pertussis, the pipeline has been adapted to take metagenomic options.

Validation

The pipeline was validated using 30 paired sequences.

Installation

PIP (Recommended)

pip install git+https://github.com/cidm-ph/pertpipe

GITHUB

git clone https://github.com/cidm-ph/pertpipe.git

Then you can use setup.py with or without datadir. Only use datadir if you intend on having all your databases in a location OTHER than default.

python setup.py $DATADIR

Usage

Example usage

Reads only

pertpipe --R1 $PATH/$R1.fq.gz --R2 $PATH/$R2.fq.gz --outdir $OUTDIR

Fasta only

pertpipe --fasta $PATH/example.fasta --outdir $OUTDIR

Metagenomics Fasta only

pertpipe --R1 $PATH/$R1.fq.gz --R2 $PATH/$R2.fq.gz --outdir $OUTDIR --meta

Its also possible to skip assembly, annotation or some mapping steps by supplying the entire output folder of Spades, Megahit or Prokka as a folder in the output directory. Prokka is also not a necessary step to perform for the analysis but is useful in general for drawing trees etc.

FLAGS

--outdir, -o [PATH]             optional folder to write output files to
--R1 [PATH]                     R1 fastq of sample (can be gzipped files)
--R2 [PATH]                     R2 fastq of sample (can be gzipped files)
--fasta, -f [PATH]              optional fasta file which will skip spades, can be used in conjunction with --longread.             
--meta, -m                      optional flag to trigger metagenomics mode, which utilises meta flags of prokka and metaSPades
--version, -v                   print version

Output

• A SPades, metaSPades or megahit folder (spades/megahit)
• A Prokka folder (prokka)
• An analysis folder containing several intermediate files (analysis)
• "vir_res.tsv", which is the file containing the final result.
• A log file which you can use to view intermediate decisions.

Limitations

• If using Metagenomics, it is important to remember that the 23S rRNA is quite a conservative region across many species, therefore commensals can play a part in creating "noise" and as a result, induce false SNP mutations in this region. The pipeline will do its best to remove as much of this noise as possible but it cannot guarantee that SNPs detected in the 23S rRNA confer resistance to macrolides (except A2037G).
• Known Issues: Pipeline has issues with NCBI closed genomes, as the MLST scheme that it uses is custom and the MLST software seems to have issues with NCBI closed genomes + custom databases.

Dependencies

• SPades
• Prokka
• Samtools
• minimap2
• megahit
• Kallisto