MHASS

Microbiome HiFi Amplicon Sequencing Simulator:
Preprint | Publication in Bioinformatics

A complete pipeline for simulating PacBio HiFi amplicon sequencing data for microbiome studies.

Installation

Note: Installation requires conda, samtools

git clone https://github.com/rhowardstone/MHASS.git
cd MHASS
bash install_dependencies.sh

After installation:

conda activate mhass

Usage

Note: For generating the ASV FASTA file required for MHASS ('amplicons.fa', below) from a collection of genomes, you may use our other tool: https://github.com/rhowardstone/AmpliconHunter

For an example script using AmpliconHunter in consort with MHASS, please refer to: https://github.com/rhowardstone/MHASS_evaluation

Simple test

mhass --amplicon-fasta Test-data_ATCC_16S_MSA-1003/amplicons.fa \
        --amplicon-genome-labels Test-data_ATCC_16S_MSA-1003/amplicon_labels.txt \
        --barcode-file Test-data_ATCC_16S_MSA-1003/barcodes.tsv --np-distribution-type lognormal \
        --output-dir testout 

This will produce a directory at ./testout, containing the output of the simulation:

• counts.tsv: Simulated ASV counts per sample
• counts_meta.tsv: Sample library sizes
• sample_barcode_map.tsv: Mapping of samples to barcodes
• sequence_file_mapping.tsv: Detailed template mappings
• combined_reads.fastq: Final output FASTQ file

Basic Command

mhass --amplicon-fasta amplicons.fa --amplicon-genome-labels labels.tsv --output-dir output/

Full Command with Options

usage: mhass [-h] --amplicon-fasta AMPLICON_FASTA --amplicon-genome-labels AMPLICON_GENOME_LABELS --output-dir OUTPUT_DIR
             [--num-samples NUM_SAMPLES] [--num-reads NUM_READS] [--var-intercept VAR_INTERCEPT] [--var-slope VAR_SLOPE]
             [--genome-distribution GENOME_DISTRIBUTION] [--barcode-file BARCODE_FILE] [--subread-accuracy SUBREAD_ACCURACY]
             [--difference-ratio DIFFERENCE_RATIO] [--np-distribution-type {empirical,lognormal}] [--lognormal-mu LOGNORMAL_MU] 
             [--lognormal-sigma LOGNORMAL_SIGMA] [--np-min NP_MIN] [--np-max NP_MAX] [--np-distribution NP_DISTRIBUTION] [--threads THREADS]

options:
  -h, --help            show this help message and exit
  --amplicon-fasta AMPLICON_FASTA
                        Input FASTA file of amplicons (default: None)
  --amplicon-genome-labels AMPLICON_GENOME_LABELS
                        TSV file mapping amplicons to genomes (asvid<TAB>genomeid) (default: None)
  --output-dir OUTPUT_DIR
                        Output directory for all files (default: None)
  --num-samples NUM_SAMPLES
                        Number of samples to simulate (default: 10)
  --num-reads NUM_READS
                        Number of reads per sample (default: 10000)
  --var-intercept VAR_INTERCEPT
                        Intercept for ASV variability model (controls baseline variation between samples)
                            (default: 1.47565981333483)
  --var-slope VAR_SLOPE
                        Slope for ASV variability model (how variation changes with abundance)
                            (default: -0.909890963463704)
  --genome-distribution GENOME_DISTRIBUTION
                        Distribution for genome abundances (uniform, lognormal, powerlaw, or empirical:<file>)
                            (default: uniform)
  --barcode-file BARCODE_FILE
                        TSV file with barcodes (id, forward, reverse) (default: None)
  --subread-accuracy SUBREAD_ACCURACY
                        Mean subread accuracy used in PBSIM (default: 0.65) (default: 0.65)
  --difference-ratio DIFFERENCE_RATIO
                        Difference (error) ratio for PBSIM (substitution:insertion:deletion).
                            Default 6:55:39 is for PacBio RS II. Use 22:45:33 for PacBio Sequel, 39:24:36 for ONT (default: 6:55:39)
  --np-distribution-type {empirical,lognormal}
                        Type of np distribution: empirical (from file) or lognormal (default: empirical)
  --lognormal-mu LOGNORMAL_MU
                        mu parameter for lognormal distribution of Number of Passes (default from empirical fit) (default: 3.88)
  --lognormal-sigma LOGNORMAL_SIGMA
                        sigma parameter for lognormal distribution of Num Passes (default from empirical fit) (default: 1.22)
  --np-min NP_MIN       Minimum np value when using lognormal distribution (default: 2)
  --np-max NP_MAX       Maximum np value when using lognormal distribution (default: 59)
  --np-distribution NP_DISTRIBUTION
                        TSV file with empirical num-passes distribution (default: None)
  --threads THREADS     Number of threads to use for parallel processing (default: 190)

Parameters

Required Parameters

• --amplicon-fasta: Input FASTA file of amplicon reference sequences.
• --amplicon-genome-labels: TSV file mapping each ASV to a genome ID. Format: asvid<TAB>genomeid.
• --output-dir: Path to the directory where all output files will be written.

Optional Parameters

• --num-samples: Number of synthetic samples to generate (default: 10).
• --num-reads: Number of reads per sample (default: 10000).
• --var-intercept: Intercept of the abundance variability model across samples (default: 1.47565981333483).
• --var-slope: Slope of the abundance variability model as a function of ASV abundance (default: -0.909890963463704).
• --genome-distribution: Relative genome abundance distribution model. Options: uniform, lognormal, powerlaw, or empirical:<file.tsv> (default: uniform).
• --barcode-file: Optional TSV file of barcodes with columns: id, forward, reverse.
• --subread-accuracy: Mean subread accuracy used for PBSIM3 simulation (default: 0.65).
• --difference-ratio: Difference (error) ratio for PBSIM in format substitution:insertion:deletion. Each value must be 0-1000. Default 6:55:39 is for PacBio RS II. Use 22:45:33 for PacBio Sequel, 39:24:36 for ONT (default: 6:55:39).
• --np-distribution-type: Distribution type for number of passes. Options: empirical or lognormal (default: empirical).
• --lognormal-mu: mu parameter for lognormal number-of-passes distribution (default: 3.88).
• --lognormal-sigma: sigma parameter for lognormal number-of-passes distribution (default: 1.22).
• --np-min: Minimum number of passes for lognormal distribution (default: 2).
• --np-max: Maximum number of passes for lognormal distribution (default: 59).
• --np-distribution: TSV file specifying empirical number-of-passes distribution. Required if --np-distribution-type is empirical.
• --threads: Number of parallel threads to use for simulation (default: 190 or all CPUs if unspecified).

Input File Formats

1. Amplicon FASTA

>ASV001
ATCGATCGATCGATCG...

2. Amplicon-to-Genome Label TSV

asvid	genomeid
ASV001	Genome1
ASV002	Genome2

3. Barcode File (optional)

BarcodeID	ForwardBarcode	ReverseBarcode
BC01	CACATATCAGAGTGCG	TGGCGTGCATGATTCGA

4. Num-Passes Distribution (optional)

num_passes	count
2	19
3	23159
...

5. Empirical Genome Abundance (if used)

genomeid	proportion
Genome1	0.45
Genome2	0.30

Workflow

1. Count Simulation: Uses metaSPARSim to simulate ASV abundance across samples based on genome distribution.
2. Template Creation: Templates are created per ASV×?Sample with barcodes and sampled np values.
3. Read Simulation: PBSIM3 simulates PacBio subreads per template with user-defined --subread-accuracy.
4. CCS Processing: Subreads are collapsed into CCS reads using ccs.
5. Read Relabeling: All CCS reads are relabeled and merged into combined_reads.fastq.
6. Cleanup: Intermediate files are cleaned up, leaving only final outputs.

Examples

mhass --amplicon-fasta test/amplicons.fa \
      --amplicon-genome-labels test/labels.tsv \
      --output-dir output/ \
      --num-samples 5 \
      --num-reads 1000 \
      --threads 4 \
      --subread-accuracy 0.9

Example for PacBio Sequel

mhass --amplicon-fasta test/amplicons.fa \
      --amplicon-genome-labels test/labels.tsv \
      --output-dir output_sequel/ \
      --num-samples 5 \
      --num-reads 1000 \
      --difference-ratio 22:45:33 \
      --threads 4

Example for Oxford Nanopore

mhass --amplicon-fasta test/amplicons.fa \
      --amplicon-genome-labels test/labels.tsv \
      --output-dir output_ont/ \
      --num-samples 5 \
      --num-reads 1000 \
      --difference-ratio 39:24:36 \
      --threads 4

For more usage information, please see our separate evaluation repository: https://github.com/rhowardstone/MHASS_evaluation

Citation

If our work is useful to you, please cite as:

Rye Howard-Stone, Ion I Măndoiu, MHASS: Microbiome HiFi Amplicon Sequencing Simulator, Bioinformatics, 2025;, btaf656, https://doi.org/10.1093/bioinformatics/btaf656