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SensiTyper tutorial - Analysis of Neisseria gonorrhoeae WHO reference strains

Introduction

This tutorial demonstrates a complete SensiTyper workflow using WHO Neisseria gonorrhoeae reference strains with known antimicrobial resistance (AMR) profiles.

Prerequisites:

  • Python 3.6+
  • ARIBA installed (conda install -c bioconda ariba)
  • Sensityping scripts and databases (see README.md for installation)

Tutorial Dataset

We will use whole-genome sequencing Illumina data produced from 29 N. gonorrhoeae WHO reference strains representing diverse resistance profiles. FASTQ files for these strains can be obtained from the European Nucleotide Archive (ENA) study accession PRJNA1067895.

Pre-computed outputs from running this tutorial are available in the examples/ directory:

examples/
├── ariba_output/                         # ARIBA results per strain
│   ├── WHO_A_SRR27944640_ARIBA/
│   │   └── report_complete.tsv
│   ├── WHO_B_SRR27944628_ARIBA/
│   │   └── report_complete.tsv
│   ├── ... (one folder per strain)
│   └── ariba_summary.csv                 # ARIBA summary — input for sensitype
└── sensityper_output/                    # SensiTyper results
    ├── WHO_strains_sensitype.tsv         # Resistance profiles
    ├── WHO_strains_sensitreat.tsv        # Treatment recommendations
    ├── WHO_strains_sensitreat.html       # Interactive combined HTML report
    └── alert_output.tsv                  # XDR / no-treatment isolates

Step 1: Prepare FASTQ Data

First, download the FASTQ files for all or a subset of the following strains from the ENA PRJNA1067895. Here is a table with individual run accessions for each strain:

Strain Run accession
WHO_A SRR27944640
WHO_B SRR27944628
WHO_C SRR27944606
WHO_D SRR27944595
WHO_E SRR27944586
WHO_F SRR27944585
WHO_G SRR27944584
WHO_H SRR27944583
WHO_I SRR27944638
WHO_J SRR27944637
WHO_K SRR27944636
WHO_L SRR27944635
WHO_M SRR27944634
WHO_N SRR27944633
WHO_O SRR27944632
WHO_P SRR27944631
WHO_Q SRR27944630
WHO_R SRR27944629
WHO_S SRR27944627
WHO_S2 SRR27944626
WHO_T SRR27944625
WHO_U SRR27944624
WHO_V SRR27944623
WHO_W SRR27944622
WHO_X SRR27944621
WHO_Y SRR27944620
WHO_Z SRR27944619
WHO_alpha SRR27944639
WHO_beta SRR27944617

File Naming Convention

FASTQ files must follow one of these naming patterns:

{sample}_1.fastq.gz / {sample}_2.fastq.gz
{sample}_R1.fastq.gz / {sample}_R2.fastq.gz
{sample}_R1_001.fastq.gz / {sample}_R2_001.fastq.gz

(Optional) Standardize File Naming

If your FASTQ files have inconsistent naming, use the rename module:

# Preview changes (dry-run)
python sensityper_v0.6.7.py rename \
    --directories examples/ \
    --pre

# Execute renaming
python sensityper_v0.6.7.py rename \
    --directories examples/ \

Step 2: Run ARIBA

ARIBA identifies antimicrobial resistance genes and mutations from whole-genome sequencing data.

Command

python sensityper_v0.6.7.py ariba \
    --input_dirs /path/to/PRJNA1067895_Illumina \
    --output_dir PRJNA1067895_ARIBA \
    --threads 8

Arguments:

  • --input_dirs: Directory containing FASTQ files
  • --output_dir: Where to save ARIBA results
  • --threads: CPU cores to use (adjust based on your system)

Expected Runtime

  • ~2-5 minutes per isolate (depending on CPU)

Expected Output

Output folders are named {strain_accession}_ARIBA. The pre-computed results for this dataset are in examples/ariba_output/:

PRJNA1067895_ARIBA/
├── WHO_A_SRR27944640_ARIBA/
│   └── report_complete.tsv
├── WHO_B_SRR27944628_ARIBA/
│   └── report_complete.tsv
├── ... (one folder per strain)
└── ariba_summary.csv          ← Main output for next step

Verify Success

Check that ariba_summary.csv was created:

head -n 3 PRJNA1067895_ARIBA/ariba_summary.csv

Expected Output (truncated): 

name,penA.cluster,penA.match,penA.assembled,penA.pct_coverage,...
WHO_A_SRR27944640,yes,yes,yes,100,...
WHO_B_SRR27944628,yes,yes,yes,100,...

A pre-computed version is available at examples/ariba_output/ariba_summary.csv.

Step 3: Generate Resistance Profiles

The sensitype module analyzes ARIBA output to identify genetic determinants of resistance and outputs a list of antibiotics each strain is susceptible to.

Command

python sensityper_v0.6.7.py sensitype \
    --input_AMRtable examples/ariba_output/ariba_summary.csv \
    --sensiscript_outfile WHO_strains_sensitype.tsv

Arguments:

  • --input_AMRtable: ARIBA summary CSV from Step 2
  • --sensiscript_outfile: Output file path (TSV format)
  • --antibiotics: (Optional) Comma-separated list to analyze specific antibiotics (default: all 7)

Expected Runtime

  • ~5-15 seconds for 29 isolates

Expected Output Files

  1. WHO_strains_sensitype.tsv - Tab-separated resistance mechanisms
  2. WHO_strains_sensitype.html - Interactive HTML table

Pre-computed versions are available at examples/sensityper_output/.

Inspect WHO_strains_sensitype.tsv

head -n 3 WHO_strains_sensitype.tsv | cut -f1-4

Expected Output (truncated): 

isolate                   treatment recommendation  ceftriaxone_NWT  ceftriaxone_WT
WHO_A_SRR27944640         ceftriaxone,azithromycin                   penA.mosaic_WT
WHO_B_SRR27944628         ceftriaxone,azithromycin                   penA.mosaic_WT

Interpreting Results

Columns:

  • isolate: Sample identifier
  • treatment recommendation: Comma-separated list of suitable antibiotics
  • {antibiotic}_NWT: Non-Wild-Type (resistance mutations, RED in HTML)
  • {antibiotic}_WT: Wild-Type (susceptibility markers, GREY in HTML)

Example: WHO_A_SRR27944640 

ceftriaxone_NWT: (empty)
ceftriaxone_WT:  penA.A311_WT,penA.V316_WT,penA.A501_WT
azithromycin_NWT: (empty)
azithromycin_WT: 23S.A2059_WT,23S.C2611_WT,mtrC.WT,mtrD.WT
ciprofloxacin_NWT: (empty)
ciprofloxacin_WT: gyrA.D95_WT,gyrA.S91_WT,parC.D86_WT,parC.E91_WT,parC.S87_WT

Interpretation:

  • Ceftriaxone: Susceptible (no key penA mutations)
  • Azithromycin: Susceptible (no key 23S rRNA or mtrD mutations)
  • Ciprofloxacin: Susceptible (no key gyrA or parC mutations)
  • Treatment: Suitable for ceftriaxone + azithromycin dual therapy

Example: WHO_Q_SRR27944630 (XDR) 

ceftriaxone_NWT: penA.60.001,penA.A311V,penA.V316T
ceftriaxone_WT: penA.A501_WT
azithromycin_NWT: 23S.A2059G[99.9%]
azithromycin_WT: 23S.C2611_WT,mtrC.WT,mtrD.WT
ciprofloxacin_NWT: gyrA.D95_A,gyrA.S91F,parC.S87R
ciprofloxacin_WT: parC.D86_WT,parC.E91_WT

Interpretation:

  • Ceftriaxone: Resistant (mosaic penA with key resistance mutations)
  • Azithromycin: Resistant (23S rRNA mutation at position 2059)
  • Ciprofloxacin: Resistant (gyrA mutations at codons 91 and 95)
  • Treatment: XDR - requires alternative regimen or investigational drugs

Explore WHO_strains_sensitreat.html

Open the HTML file in your web browser:

open WHO_strains_sensitreat.html  # macOS
xdg-open WHO_strains_sensitreat.html  # Linux
start WHO_strains_sensitreat.html  # Windows

Interactive Features:

  • Click column headers to sort (▲/▼ indicators)
  • Search box for live filtering
  • Antibiotic filter (multi-select dropdown)
  • Color coding: Red=resistance (NWT), Blue=susceptible (WT)
  • Pagination (10/25/50/100/All entries per page)
  • WT columns to show or hide identified wildtype positions

Step 4: Assign Treatment Recommendations

The sensitreat module assigns evidence-based treatment regimens based on resistance profiles.

Command

python sensityper_v0.6.7.py sensitreat \
    --input_file WHO_strains_sensitype.tsv \
    --treatment_output WHO_strains_sensitreat.tsv

Arguments:

  • --input_file: Resistance TSV from Step 3
  • --treatment_output: Output file path for treatment recommendations
  • --available_antibiotics: (Optional) Limit to available antibiotics (default: all except azithromycin monotherapy)
  • --sensitreat_order: (Optional) Customize regimen priority order

Expected Runtime

  • ~2-5 seconds for 29 isolates

Expected Output Files

  1. alert_output.tsv - XDR/untreatable isolates flagged for review
  2. WHO_strains_sensitreat.tsv - Treatment recommendations per isolate
  3. WHO_strains_sensitreat.html - Combined tabbed HTML with both resistance and treatment tables

Pre-computed versions are available in examples/sensityper_output/.

Inspect WHO_strains_sensitreat.tsv

head -n 3 WHO_strains_sensitreat.tsv | cut -f1,4-6

Expected Output (truncated): 

isolate                   Predicted Profile                                    Recommended Treatment                              Comment
WHO_A_SRR27944640         ceftriaxone=YES, azithromycin=YES, ciprofloxacin=YES Ceftriaxone 1 g IM + Azithromycin 2 g orally      Acceptable combination therapy (RECOMMENDATION 2)
WHO_B_SRR27944628         ceftriaxone=YES, azithromycin=YES, ciprofloxacin=YES Ceftriaxone 1 g IM + Azithromycin 2 g orally      Acceptable combination therapy (RECOMMENDATION 2)

Inspect alert_output.tsv

cat alert_output.tsv

Expected Output (if XDR isolates present): 

Alert  isolate           treatment recommendation
XDR WHO_Q_SRR27944630   (UND) zoliflodacin

Alert Types:

  • XDR: Extensively Drug-Resistant (resistant to ceftriaxone, azithromycin, AND ciprofloxacin)
  • None: No suitable treatment available from specified antibiotics

Interpreting Treatment Recommendations

Example 1: WHO_A_SRR27944640 (Susceptible)

Predicted Profile:       ceftriaxone=YES, azithromycin=YES, ciprofloxacin=YES, spectinomycin=YES
Recommended Treatment:   Ceftriaxone 1 g IM + Azithromycin 2 g orally
Comment:                 Acceptable combination therapy (RECOMMENDATION 2)

Clinical Interpretation:

  • Regimen: Standard dual therapy (first-line)
  • Route: Ceftriaxone intramuscular (IM), Azithromycin oral
  • Rationale: Susceptible to both agents, combination prevents resistance development
  • Category: RECOMMENDATION 2 (preferred dual therapy)

Example 2: WHO_R_SRR27944629 (Ceftriaxone-Resistant)

Predicted Profile:       ceftriaxone=NO, azithromycin=YES, ciprofloxacin=NO, spectinomycin=YES
Recommended Treatment:   Spectinomycin 2 g IM + Azithromycin 2 g orally
Comment:                 Alternative regimen (RECOMMENDATION 3)

Clinical Interpretation:

  • Regimen: Alternative dual therapy (ceftriaxone resistance detected)
  • Route: Both intramuscular or oral
  • Rationale: Avoid ceftriaxone due to resistance, use spectinomycin + azithromycin
  • Category: RECOMMENDATION 3 (alternative regimen)
  • ⚠️ Important: Spectinomycin has lower cure rates for pharyngeal infections - avoid when possible

Example 3: WHO_Q_SRR27944630 (XDR)

Predicted Profile:       ceftriaxone=NO, azithromycin=NO, ciprofloxacin=NO, spectinomycin=YES
Recommended Treatment:   Spectinomycin 2 g IM
Comment:                 Lower cure rates in oropharyngeal infection; avoid for pharyngeal disease when possible (RECOMMENDATION 1)
Alert:                   XDR

Clinical Interpretation:

  • Regimen: Spectinomycin monotherapy (only option)
  • Alert: XDR isolate - flagged for clinical review
  • Action Required:
  • Confirm infection site (spectinomycin ineffective for pharyngeal)
  • Consider investigational drugs (zoliflodacin)
  • Consult infectious disease specialist
  • Report to public health authorities
  • Ensure test of cure after treatment

Treatment Recommendation Categories

RECOMMENDATION 1 - Guideline-based monotherapy:

  • Ceftriaxone 1 g IM
  • Used when: Azithromycin resistance detected OR azithromycin unavailable

RECOMMENDATION 2 - Acceptable combination therapy:

  • Ceftriaxone 1 g IM + Azithromycin 2 g orally
  • Used when: Both agents susceptible
  • Preferred regimen for uncomplicated gonorrhea

RECOMMENDATION 3 - Alternative regimen:

  • Spectinomycin 2 g IM + Azithromycin 2 g orally
  • Used when: Ceftriaxone resistance detected
  • ⚠️ Lower cure rates for pharyngeal infections

Investigational:

  • Zoliflodacin 3 g orally (single dose)
  • Phase 3 trial data: non-inferior to ceftriaxone+azithromycin for urogenital infection
  • Not yet widely available

Step 5: Explore Combined HTML Report

The combined HTML report is the primary output for clinical interpretation and surveillance.

Open WHO_strains_sensitreat.html

open WHO_strains_sensitreat.html  # macOS
xdg-open WHO_strains_sensitreat.html  # Linux
start WHO_strains_sensitreat.html  # Windows

A pre-computed version is available at examples/sensityper_output/WHO_strains_sensitreat.html.

HTML Structure

Alert Banner (if applicable): 

⚠️ Clinical Alerts
• Isolate WHO_Q_SRR27944630: XDR (Extensively Drug-Resistant) - Requires clinical review

Tab Navigation: - Tab 1: Treatment Recommendations (shown by default) - Tab 2: Resistance Profile (detailed mechanisms)

Tab 1: Treatment Recommendations

Columns (4 total):

  1. Isolate - Sample identifier
  2. Predicted Profile - Color-coded susceptibility badges
  3. Green badge = YES (susceptible)
  4. Gray badge = NO (resistant)
  5. Recommended Treatment - Full regimen with dosing instructions
  6. Comment - Clinical guidance and recommendation category

Interactive Features:

  • Sort: Click column headers
  • Search: Live filtering across all columns
  • Pagination: 10/25/50/100/All entries per page

Treatment Recommendations tab — colour-coded susceptibility badges and regimen assignments per isolate

Tab 2: Resistance Profile

Columns (varies by antibiotic):

  • isolate
  • treatment recommendation
  • For each antibiotic: {antibiotic}_NWT, {antibiotic}_WT

Interactive Features:

  • Sort: Click column headers
  • Search: Live filtering
  • Antibiotic Filter: Multi-select dropdown
  • Pagination: Same as Tab 1

Resistance Profile tab — NWT (red) and WT (blue) columns for all antibiotics

WT columns can also be hidden with the 'Show WT' switch: Resistance Profile tab — filtered view showing only NWT columns (WT columns hidden)

Complete Pipeline (Alternative Approach)

Instead of running modules separately, use the pipeline module for end-to-end automation. The pre-computed outputs in examples/ were generated with this single command:

Single Command

python sensityper_v0.6.7.py pipeline \
    --modules ariba,sensitype,sensitreat \
    --other_arguments '--input_dirs /path/to/PRJNA1067895_Illumina \
                       --output_dir PRJNA1067895_ARIBA \
                       --sensiscript_outfile WHO_strains_sensitype.tsv \
                       --treatment_output WHO_strains_sensitreat.tsv'

What Happens

  1. ARIBA module: Processes FASTQ files → ariba_output/ariba_summary.csv
  2. Sensitype module: Analyzes ARIBA output → WHO_strains_sensitype.tsv
  3. Sensitreat module: Assigns treatments → WHO_strains_sensitreat.tsv + WHO_strains_sensitreat.html

Benefits of the pipeline mode

  • Single command: Less manual intervention
  • Optimized HTML: Only one combined HTML generated (no duplicate standalone HTML)
  • Automatic chaining: Output of one module feeds into next

When to Use Separate Modules

  • Re-running analysis: Already have ARIBA results, only need to update treatment logic
  • Custom antibiotics: Want to analyze different antibiotic subsets
  • Debugging: Isolate specific module failures
  • External ARIBA: Using ARIBA results from another study

Next Steps

Using Your Own Data

  1. Prepare FASTQ files:
  2. Ensure paired-end reads (R1/R2)
  3. Follow naming convention

  4. Place in a single directory

  5. Run pipeline: 

    python sensityper_v0.6.7.py pipeline ariba,sensitype,sensitreat \
    --other_arguments "--input_dirs /path/to/your/fastqs \
                       --output_dir my_analysis \
                       --sensiscript_outfile my_resistance.tsv \
                       --treatment_output my_treatment.tsv \
                       --threads 16"

  6. Review outputs:

  7. Check alert_output.tsv for XDR isolates
  8. Open the generated HTML report for interactive results
  9. Share HTML with clinical team (self-contained, no external dependencies)

Customizing treatment recommendations

Limit to available antibiotics: 

python sensityper_v0.6.7.py sensitreat \
    --input_file my_resistance.tsv \
    --available_antibiotics ceftriaxone,azithromycin,spectinomycin \
    --treatment_output my_treatment.tsv

Change regimen priority: 

python sensityper_v0.6.7.py sensitreat \
    --input_file my_resistance.tsv \
    --sensitreat_order ceftriaxone,azithromycin+spectinomycin,spectinomycin \
    --treatment_output my_treatment.tsv

Batch processing multiple studies

# Process multiple sequencing runs
python sensityper_v0.6.7.py ariba \
    --input_dirs /data/run1,/data/run2,/data/run3 \
    --output_dir batch_ariba \
    --threads 32

# Continue pipeline
python sensityper_v0.6.7.py pipeline sensitype,sensitreat \
    --input_AMRtable batch_ariba/ariba_summary.csv \
    --sensiscript_outfile batch_resistance.tsv \
    --treatment_output batch_treatment.tsv

Clinical Reminder

This tool is for surveillance and research. Clinical decisions should consider: - Patient allergies and contraindications - Infection site (pharyngeal vs. urogenital) - Local resistance patterns - Current treatment guidelines - Treatment history and previous failures

Always consult infectious disease specialists for: - XDR isolates - Treatment failures - Severe infections (disseminated, PID) - Immunocompromised patients

Additional Resources

  • README.md - Installation and quick start
  • ARIBA documentation - https://github.com/sanger-pathogens/ariba
  • WHO gonorrhea guidelines - https://www.who.int/publications/i/item/9789241549691
  • CDC gonorrhea treatment - https://www.cdc.gov/std/treatment-guidelines/gonorrhea.htm

Questions or Issues?

  • Open an issue on GitHub!