Identify cap locus serotype and structure in your Haemophilus influenzae assemblies.

• Table of contents
• Introduction
• Citation
• Requirements
• Installation
• Outputs
• Usage
• Example
• License

The cap locus of H. influenzae are categorised into 6 different groups based on serology (a-f). There are three functionally distinct regions of the cap locus, designated region I, region II, and region III. Genes within region I (bexABCD) and region III (hcsAB) are associated with transport and post-translation modification. The region II genes encode serotype specific proteins, with each serotype (a-f) having a distinct set of genes. cap loci are often subject to structural changes (e.g. duplication, deletion) making the process of in silico typing and characterisation of loci difficult.

hicap automates identification of the cap locus, describes the structural layout, and performs in silico serotyping.

If you use this tool, please cite the hicap paper:

• Watts, S. C., & Holt, K. E. (2019). hicap: in silico serotyping of the Haemophilus influenzae capsule locus. Journal of Clinical Microbiology, JCM.00190-19. https://doi.org/10.1128/JCM.00190-19

There are a couple of software dependencies that are required.

• Python, version 3.6 or above with the following packages:
  • Biopython, version 1.79 or above
  • ReportLab, version 3.5.0 or above
• Prodigal, version 2.6.1 or above
• BLAST+, version 2.2.28 or above. Commands used are:
  • makeblastdb
  • blastn

The recommended method of installation is bioconda:

conda install -c bioconda -c conda-forge hicap

Otherwise you can install hicap with pip:

# Install into user directory via pip
pip3 install --user git+https://github.com/scwatts/hicap.git

# Check install
hicap --help

Or clone the git repo and use the hicap-runner.py script:

# Install into current directory by cloning
git clone https://github.com/scwatts/hicap.git

# Check install
./hicap/hicap-runner.py --help

If installing hicap by the pip or clone method, you'll need to satisfy all software dependencies manually.

Basic usage requires an input genome assembly and an output directory for result files:

# Create output directory and run serotype prediction
mkdir -p output/
hicap --query_fp input_genome.fasta --output_dir output/

There are several parameters that can be set manually. The default settings have been empirically determined to be optimal on a test dataset so different settings may be more appropriate for other input data. To list this parameters use, the extended help for hicap:

hicap --help_all
usage: hicap -q QUERY_FP -o OUTPUT_DIR [-d DATABASE_DIR] [--gene_coverage GENE_COVERAGE] [--gene_identity GENE_IDENTITY]
             [--broken_gene_length BROKEN_GENE_LENGTH] [--broken_gene_identity BROKEN_GENE_IDENTITY] [--log_fp LOG_FP] [--debug] [-v] [-h]
             [--help_all]

File input and output:
  -q QUERY_FP, --query_fp QUERY_FP              Input FASTA query
  -o OUTPUT_DIR, --output_dir OUTPUT_DIR        Output directory
  -d DATABASE_DIR, --database_dir DATABASE_DIR  Directory containing locus database. [default: /home/stephen/.local/lib/python3.6/site-
                                                packages/hicap/database]
  -s, --full_sequence                           Write the full input sequence out to the genbank file rather than just the region
                                                surrounding and including the locus

Search parameters:
  --gene_coverage GENE_COVERAGE                 Minimum percentage coverage to consider a single gene complete. [default: 0.80]
  --gene_identity GENE_IDENTITY                 Minimum percentage identity to consider a single gene complete. [default: 0.70]
  --broken_gene_length BROKEN_GENE_LENGTH       Minimum length to consider a broken gene. [default: 60]
  --broken_gene_identity BROKEN_GENE_IDENTITY   Minimum percentage identity to consider a broken gene. [default: 0.80]

Other:
  --log_fp LOG_FP                               Record logging messages to file
  --debug                                       Print debug messages
  -v, --version                                 Show version number and exit
  -h, --help                                    Show this help message and exit
  --help_all                                    Display extended help

Analysis using hicap will generate three result files in the specified output directory:

• Summary: a somewhat machine parsable file with detailed summary information
• Genbank: a genbank file with sequence marked up with cap locus annotations
• Graphic: a visual representation of the annotated cap locus

The summary output file contains information relating to the annotation of the cap locus. Below is a description of each column:

The genbank output file provides marked up input sequence data with annotations of the cap locus. This file will contain a single record for each contig which the capsular locus is identified on. The default setting is to only include the sequence of the cap locus and surrounding region. This behaviour can be overridden to include all input sequence data by specific --full-sequence on the command line.

cap locus regions are annotated by the misc_feature feature with a /note qualifier specifying an identifier for that specific region. cap genes are given the CDS feature with a /gene qualifier set to the appropriate gene name. ORFs found nearby that are not typically part of the cap locus are also marked as a CDS feature but /gene is set to orf with an incremental counter suffix for differentiation.

All CDS features have a /note qualifier which contains various details, each separated by ;. Possible values are region_one, region_two, region_three, fragment, no_orf, insertion_sequence, misc_orf.

The graphical output provides a visualisation of the annotated cap locus. The output format is svg and can be viewed in any modern browers or imagine viewer with svg support. Genes of each region are coloured differently; green, red, and yellow for region I, region II, and region III respectively. Truncated ORFs are coloured using a darker, desaturated corresponding region colour. ORFs which are not part of the cap locus are left grey. Regions with homology to IS1016 are annotated using small blue arrows.

Each track of the visualisation represents a contig. The cap locus of the example below is located on three different contigs.

To provide an example of hicap usage, we will annotate the cap locus in the following isolates:

For Hi75 and Hi84 reads were assembled using SPAdes 3.13.0. All assemblies have been placed into the example/ directory. hicap can be run in series or in parallel (using gnu parallel).

Serial execution:

mkdir -p output/
for assembly_fp in example/*fasta; do
  hicap --query_fp "${assembly_fp}" --output_dir output/;
done

Parallel execution:

mkdir -p output/
parallel hicap --query_fp {} --output_dir output/ ::: example/*fasta

The summary output for these assemblies can be combined using sed:

summary_fps=(output/*tsv);
{ head -n1 "${summary_fps[0]}"; sed '/^#/d' "${summary_fps[@]}"; } > example_summary.tsv

This file can be viewed on the command line or imported into excel.

column -t -s$'\t' example_summary.tsv | less -S

To view an isolate's genbank output in artemis, you first must combine the multiple genbank records. This can be done using seqret from EMBOSS. Using M21328 as an example here:

union -sequence output/M21328.gbk -outseq output/M21328_combined.gbk -osformat genbank -feature

Note that while combining multiple records enables viewing in artemis, contig boundaries are masked.

GNU General Public License v3.0