annotate

Annotate genomic assemblies with bakta.

Overview

The purpose of the annotate subcommand is to provide a very simple wrapper around the bacterial genome annotator bakta. The main added value of using annotate is that it has a post-processing step where if fixes bad quotation marks in the output files (ex. ‘, ’), as these particular characters tend to crash downstream applications.

The output GFF file will the be used by the extract subcommand to break up the genome into informative regions for clustering.

Genome annotation is the most complicated analysis and therefore most fragile part of the pipeline. If using the nextflow runtime, it is recommended to first run the annotation step in isolation with --only_annotate, which skips all downstream steps. After the annotation is successful, you can then include your new gff file paths in your samplesheet. In the case where nextflow fails to resume a previously run properly, this will prevent the pipeline from running annotation all over again.

Database

If you are not bringing your own annotations, you will need to download a bakta annotation database.

panGWAS has been verified with bakta CLI v1.9.2 and the v5.1 database. Specifically, v5.1 includes the amrfinderplus-db release v2023-11-15.1, which was the last release before their Database Schema changed. The full datbase can be downloaded with the following command:

❗ WARNING: This download size is +30 GB. If you want to use the light database for testing (1 GB), replace db with db-light.

mkdir -p database/bakta/
wget -O database/bakta/db.tar.gz https://zenodo.org/records/10522951/files/db.tar.gz
tar -xvf database/bakta/db.tar.gz -C database/bakta --strip-components 1

The database directory can then be tested with the following command:

pangwas annotate \
  --db database/bakta \
  --fasta data/test/sequences/sample1.fasta \
  --outdir sample1 \
  --threads 2

Usage

The annotate subcommand takes as input a FASTA file of genomic sequences and a path to a bakta database. Typically the FASTA file is in the form of contigs from a denovo or reference-guided assembly.

pangwas.annotate(fasta="sample1.fasta", db="database/bakta")
pangwas annotate --fasta sample1.fasta --db database/bakta

While annotate will try to parse the sample identifier from the FASTA file name, it’s recommended to manually specify this information:

pangwas.annotate(fasta="sample1.fasta", db="database/bakta", sample="sample1", prefix="sample1")
pangwas annotate --fasta sample1.fasta --db database/bakta --sample sample1 --prefix sample1

If you would like to pass additional arguments to bakta, they can be specified like the following:

pangwas.annotate(fasta="sample1.fasta", db="database/bakta", args="--genus Streptococcus --species pneumoniae")
pangwas annotate --fasta sample1.fasta --db database/bakta --genus Streptococcus --species pneumoniae

Parameters

Required

  • fasta: Input FASTA sequences.
  • db: bakta database directory.

Optional

Optional output arguments:

  • outdir: Output directory.
  • prefix: Output file prefix. If not provided, will be parsed from the fasta file name.
  • tmp: Temporary directory.

Optional arguments:

  • sample Sample identifier. If not provided, will be parsed from the fasta file name.
  • threads CPU threads for bakta.

Examples

Minimal

The following is a minimal example of annotating a Streptococcus pyogenes contig that contains the genes smeZ and speM separated by a short intergenic region.

pangwas.annotate(
  fasta  = "sample1.fasta",
  db     = "database/bakta",
  locus  = "sample1",
  outdir = "sample1",
  args   = "--genus Streptococcus --species pneumoniae"
)
pangwas annotate \
  --fasta sample1.fasta \
  --db database/bakta \
  --locus sample1 \
  --genus Streptococcus \
  --species pneumoniae
>contig
ATGAAAAAAACAAAACTTATTTTTTCTTTTACTTCAATATTCATTGCAATAATTTCTCGTCCTGTGTTTG
GATTAGAAGTAGATAATAATTCCCTTCTAAGGAATATCTATAGTACGATTGTGTATGAATATTCAGATAC
AGTAATTGAGTTTAAAACCAGTCATAACTTAGTGACTAAGAAACTTGATGTTAGAGATGCTAGAGATTTT
TTTATTAACTCCGAAATGGACGAATATGCAGCCAATGATTTTAAAGATGGAGATAAAATAGCTATGTTCT
CCGTCCCATTTGATTGGAACTACTTGTCAGAAGGAAAAGTCATAGCATATACGTATGGTGGAATGACGCC
TTATCAAGAAGAACCAATATCTAAAAATATCCCTGTTAATTTATGGATTAATGGAAAGCAGATCTCTGTT
CCTTACAACGAAATATCAACTAACAAAACAACAGTTACAGCTCAAGAAATTGATCTAAAGGTTAGAAAAT
TTTTAATATCACAACATCAATTATATTCTTCTGGTTCTAGCTACAAAAGTGGTAAATTAGTTTTTCATAC
AAATGATAATTCAGATAAATATTCTCTCGATCTTTTCTATGTAGGATATAGAGATAAAGAAAGTATTTTT
AAAGTATACAAAGACAATAAATCTTTCAATATAGATAAAATTGGGCATTTAGATATAGAAATTGACTCCT
AA
AGTGACGACATGCTGACTGTGCTCTGACGTTGACTGACTGA
CTAATTTTTAGAAAAATCTTCGTTTAAGTAAATATCAAAGTGACTTACTTTACTCATATCAATCGTTTCATTATCTGT
ATAGTTAGGATGAGTGAATAAATCGGTAAACTTTGTTGTATTATCTTTATAATGAATTCCCCAATACCCTTTTTTACA
AATTGAGTTATGTTCATATAACTTTATTCTATTATCGCTCATCAAACTTTTCCTAAGTCTAACATCAATTTCTTGAAA
AGTTACAAACTTATTCTGAAATGTGATTTTATATTTTGATTGCTCTTTTAGCGGTATCTGTTCCCCAAAAATATTCAT
ATATATTGTTGAATCAAGTTTCTCTCTGTCACTTGTTCTTATCAAACCTCCATCAACATAATTATATTGTTCCTTACA
TATCACACTGTAGGATTTTATTAGAGCATAAATATCAACTTCTTCTTCCTTAAAGCGTCTTTCTTGCGCTGGAGAAAC
ATTGCTAGATATAACTTTATTATAATTATCATCATCCCAGACTCTAGTTTTTTCATTCGTGTTAAATATTAACTGGGT
GCCAATTTTCTTTGTTATCTTCATATTGGTTCTATTAATTACATCTTTCGTATAGATATTTTTTAATTCGCTATTAAC
CAACACAGCATCTGAAAAGACACTCTCAGTAGTGTATAGAGCAAGCGATGCACACACAAGGAATAACAAAGTCAAGGT
ATTTTTTTTCAT

Line wrapping is optional, and is used for illustrative purposes in this example to show the two separate genes present and the intergenic sequence between them.

##gff-version 3
##feature-ontology https://github.com/The-Sequence-Ontology/SO-Ontologies/blob/v3.1/so.obo
# organism Streptococcus pyogenes
# Annotated with Bakta
# Software: v1.9.2
# Database: v5.1, light
# DOI: 10.1099/mgen.0.000685
# URL: github.com/oschwengers/bakta
##sequence-region sample1_1 1 1457
sample1_1   Bakta   region  1   1457    .   +   .   ID=sample1_1;Name=sample1_1
sample1_1   Prodigal    CDS 1   702 .   +   0   ID=AFLIOK_00005;Name=streptococcal mitogenic exotoxin SmeZ;locus_tag=AFLIOK_00005;product=streptococcal mitogenic exotoxin SmeZ;Dbxref=BlastRules:WP_010922705,SO:0001217,UniRef:UniRef50_Q9RQQ5;gene=smeZ
sample1_1   Prodigal    CDS 744 1457    .   -   0   ID=AFLIOK_00010;Name=streptococcal pyrogenic exotoxin SpeM;locus_tag=AFLIOK_00010;product=streptococcal pyrogenic exotoxin SpeM;Dbxref=BlastRules:WP_011017838,SO:0001217,UniRef:UniRef50_Q8L3E1;gene=speM
##FASTA
>sample1_1
ATGAAAAAAACAAAACTTATTTTTTCTTTTACTTCAATATTCATTGCAATAATTTCTCGT
CCTGTGTTTGGATTAGAAGTAGATAATAATTCCCTTCTAAGGAATATCTATAGTACGATT
GTGTATGAATATTCAGATACAGTAATTGAGTTTAAAACCAGTCATAACTTAGTGACTAAG
AAACTTGATGTTAGAGATGCTAGAGATTTTTTTATTAACTCCGAAATGGACGAATATGCA
GCCAATGATTTTAAAGATGGAGATAAAATAGCTATGTTCTCCGTCCCATTTGATTGGAAC
TACTTGTCAGAAGGAAAAGTCATAGCATATACGTATGGTGGAATGACGCCTTATCAAGAA
GAACCAATATCTAAAAATATCCCTGTTAATTTATGGATTAATGGAAAGCAGATCTCTGTT
CCTTACAACGAAATATCAACTAACAAAACAACAGTTACAGCTCAAGAAATTGATCTAAAG
GTTAGAAAATTTTTAATATCACAACATCAATTATATTCTTCTGGTTCTAGCTACAAAAGT
GGTAAATTAGTTTTTCATACAAATGATAATTCAGATAAATATTCTCTCGATCTTTTCTAT
GTAGGATATAGAGATAAAGAAAGTATTTTTAAAGTATACAAAGACAATAAATCTTTCAAT
ATAGATAAAATTGGGCATTTAGATATAGAAATTGACTCCTAAAGTGACGACATGCTGACT
GTGCTCTGACGTTGACTGACTGACTAATTTTTAGAAAAATCTTCGTTTAAGTAAATATCA
AAGTGACTTACTTTACTCATATCAATCGTTTCATTATCTGTATAGTTAGGATGAGTGAAT
AAATCGGTAAACTTTGTTGTATTATCTTTATAATGAATTCCCCAATACCCTTTTTTACAA
ATTGAGTTATGTTCATATAACTTTATTCTATTATCGCTCATCAAACTTTTCCTAAGTCTA
ACATCAATTTCTTGAAAAGTTACAAACTTATTCTGAAATGTGATTTTATATTTTGATTGC
TCTTTTAGCGGTATCTGTTCCCCAAAAATATTCATATATATTGTTGAATCAAGTTTCTCT
CTGTCACTTGTTCTTATCAAACCTCCATCAACATAATTATATTGTTCCTTACATATCACA
CTGTAGGATTTTATTAGAGCATAAATATCAACTTCTTCTTCCTTAAAGCGTCTTTCTTGC
GCTGGAGAAACATTGCTAGATATAACTTTATTATAATTATCATCATCCCAGACTCTAGTT
TTTTCATTCGTGTTAAATATTAACTGGGTGCCAATTTTCTTTGTTATCTTCATATTGGTT
CTATTAATTACATCTTTCGTATAGATATTTTTTAATTCGCTATTAACCAACACAGCATCT
GAAAAGACACTCTCAGTAGTGTATAGAGCAAGCGATGCACACACAAGGAATAACAAAGTC
AAGGTATTTTTTTTCAT