πŸ‘‹πŸŽ“ in silico typing of H. influenzae cap locus
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README.md

hicap

Identify cap locus serotype and structure in your Haemophilus influenzae assemblies

Example locus

Build Status Code Coverage License

Table of contents

Introduction

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.

Citation

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

Requirements

There are a couple of software dependencies that are required.

  • Python, version 3.6 or above with the following packages:
    • Biopython, version 1.63 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

Installation

The recommended method of installation is bioconda:

conda install 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.

Usage

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

Outputs

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

Summary

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

Column Description
isolate input isolate name
predicted_serotype hicap serotype prediction
attributes attributes of the locus (e.g. missing genes, truncated genes, duplication, etc)
genes_identified cap genes identified. genes on different contigs delimited by;. truncation shown by trailing *
locus_location location of cap genes. contigs delimited by ; and matches gene_identified positions
region_n_genes expected and identified genes in region n. missing names are shown when applicable
IS1016_hits count of IS1016 hits found

Genbank

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.

Graphic

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.

single track

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

multi track

Example

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

Isolate BioSample Accession Notes
Hi75 SAMEA33515668 Downloaded SRA reads (ERX1834398; ion torrent) and assembled locally
Hi84 SAMEA33522418 Downloaded SRA reads (ERX1834407; ion torrent) and assembled locally
M21328 SAMN09704914 Downloaded GenBank assembly (GCA_003497005.1)
PTHi-1539 SAMEA4643429 Downloaded GenBank assembly (GCA_900407865.1)

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.

License

GNU General Public License v3.0