Hostile

Hostile accurately removes host sequences from short and long read (meta)genomes, consuming paired or unpaired fastq[.gz] input. Batteries are included – a human reference genome is downloaded when run for the first time. Hostile is precise by default, removing an order of magnitude fewer microbial reads than existing approaches while removing >99.5% of real human reads from 1000 Genomes Project samples. For the best possible retention of microbial reads, use an existing index masked against bacterial and/or viral genomes, or make your own using the built-in masking utility. Read headers can be replaced with integers (using --rename) for privacy and smaller FASTQs. Heavy lifting is done with fast existing tools (Minimap2/Bowtie2 and Samtools). Bowtie2 is the default aligner for short (paired) reads while Minimap2 is default aligner for long reads. In benchmarks, bacterial Illumina reads were decontaminated at 32Mbp/s (210k reads/sec) and bacterial ONT reads at 22Mbp/s, using 8 alignment threads. Further information and benchmarks can be found in the paper and blog post. Please open an issue to report problems or otherwise reach out for help, advice etc.

Reference genomes (indexes)

The default index human-t2t-hla comprises T2T-CHM13v2.0 and IPD-IMGT/HLA v3.51, and is downloaded automatically when running Hostile unless another index is specified. Slightly higher microbial sequence retention is possible using a masked index, of which several are available. The index human-t2t-hla-argos985 is masked against 985 reference grade bacterial genomes including common human pathogens, while human-t2t-hla-argos985-mycob140 is further masked against mycobacterial genomes. To use a standard index, simply pass its name as the value of the --index argument which takes care of downloading and cacheing the relevant index. Automatic download can be disabled using the --offline flag, and --index can accept a path to a custom reference genome or Bowtie2 index. Object storage is provided by the ModMedMicro research group.

Performance of human-t2t-hla and human-t2t-hla-argos985-mycob140 was evaluated in the paper

Install

Installation with conda/mamba or Docker is recommended due to non-Python dependencies (Bowtie2, Minimap2, Samtools and Bedtools). Hostile is tested with Ubuntu Linux 22.04, MacOS 12, and under WSL for Windows.

Conda/mamba

conda create -y -n hostile -c conda-forge -c bioconda hostile
conda activate hostile

Docker

docker run quay.io/biocontainers/hostile:0.4.0--pyhdfd78af_0

# Build your own
wget https://raw.githubusercontent.com/bede/hostile/main/Dockerfile
docker build . --platform linux/amd64

Index installation (optional)

Hostile automatically downloads and caches the default index human-t2t-hla when run for the first time, meaning that there is no need to download an index in advance. Neverthless:

• To download and cache the default index (human-t2t-hla), run hostile fetch
• To list available indexes, run hostile fetch --list
• To download and cache another standard index, run e.g. hostile fetch --name human-t2t-hla-argos985
• To use a custom genome (made perhaps with hostile mask), run hostile clean --index path/to/genome.fa

Command line usage

$ hostile clean -h
usage: hostile clean [-h] --fastq1 FASTQ1 [--fastq2 FASTQ2] [--aligner {bowtie2,minimap2,auto}] [--index INDEX] [--invert] [--rename] [--reorder] [--out-dir OUT_DIR] [--threads THREADS] [--aligner-args ALIGNER_ARGS] [--force]
                     [--offline] [--debug]

Remove reads aligning to a target genome from fastq[.gz] input files.

options:
  -h, --help            show this help message and exit
  --fastq1 FASTQ1       path to forward fastq[.gz] file
  --fastq2 FASTQ2       optional path to reverse fastq[.gz] file
                        (default: None)
  --aligner {bowtie2,minimap2,auto}
                        alignment algorithm. Default is Bowtie2 (paired reads) & Minimap2 (unpaired reads)
                        (default: auto)
  --index INDEX         name of standard index or path to custom index
                        (default: human-t2t-hla)
  --invert              keep only reads aligning to the target genome (and their mates if applicable)
                        (default: False)
  --rename              replace read names with incrementing integers
                        (default: False)
  --reorder             ensure deterministic output order
                        (default: False)
  --out-dir OUT_DIR     path to output directory
                        (default: /Users/bede/Research/Git/hostile)
  --threads THREADS     number of alignment threads. A sensible default is chosen automatically
                        (default: 5)
  --aligner-args ALIGNER_ARGS
                        additional arguments for alignment
                        (default: )
  --force               overwrite existing output files
                        (default: False)
  --offline             disable automatic index download
                        (default: False)
  --debug               show debug messages
                        (default: False)

Short reads, default index

$ hostile clean --fastq1 human_1_1.fastq.gz --fastq2 human_1_2.fastq.gz
INFO: Hostile version 1.0.0. Using Bowtie2 (paired reads)
INFO: Found cached standard index human-t2t-hla (Bowtie2)
INFO: Cleaning…
INFO: Cleaning complete
[
    {
        "version": "1.0.0",
        "aligner": "bowtie2",
        "index": "human-t2t-hla",
        "options": [],
        "fastq1_in_name": "human_1_1.fastq.gz",
        "fastq1_in_path": "/Users/bede/human_1_1.fastq.gz",
        "fastq1_out_name": "human_1_1.clean_1.fastq.gz",
        "fastq1_out_path": "/Users/bede/human_1_1.clean_1.fastq.gz",
        "reads_in": 2,
        "reads_out": 0,
        "reads_removed": 2,
        "reads_removed_proportion": 1.0,
        "fastq2_in_name": "human_1_2.fastq.gz",
        "fastq2_in_path": "/Users/bede/human_1_2.fastq.gz",
        "fastq2_out_name": "human_1_2.clean_2.fastq.gz",
        "fastq2_out_path": "/Users/bede/human_1_2.clean_2.fastq.gz"
    }
]

Short reads, masked index, save log

$ hostile clean --fastq1 human_1_1.fastq.gz --fastq2 human_1_2.fastq.gz --index human-t2t-hla-argos985 > log.json
INFO: Hostile version 1.0.0. Using Bowtie2 (paired reads)
INFO: Found cached standard index human-t2t-hla (Bowtie2)
INFO: Cleaning…
INFO: Cleaning complete

Long reads

$ hostile clean --fastq1 tests/data/tuberculosis_1_1.fastq.gz
INFO: Hostile version 1.0.0. Using Minimap2's long read preset
INFO: Found cached standard index human-t2t-hla (Minimap2)
INFO: Cleaning…
INFO: Cleaning complete
[
    {
        "version": "1.0.0",
        "aligner": "minimap2",
        "index": "human-t2t-hla",
        "options": [],
        "fastq1_in_name": "tuberculosis_1_1.fastq.gz",
        "fastq1_in_path": "/Users/bede/Research/Git/hostile/tests/data/tuberculosis_1_1.fastq.gz",
        "fastq1_out_name": "tuberculosis_1_1.clean.fastq.gz",
        "fastq1_out_path": "/Users/bede/Research/Git/hostile/tuberculosis_1_1.clean.fastq.gz",
        "reads_in": 1,
        "reads_out": 1,
        "reads_removed": 0,
        "reads_removed_proportion": 0.0
    }
]

Python usage

from pathlib import Path
from hostile.lib import clean_fastqs, clean_paired_fastqs

# Long reads, defaults
clean_fastqs(
    fastqs=[Path("reads.fastq.gz")],
)

# Paired short reads, various options, capture log
log = clean_paired_fastqs(
    fastqs=[(Path("reads_1.fastq.gz"), Path("reads_2.fastq.gz"))],
    index="human-t2t-hla-argos985",
    out_dir=Path("decontaminated-reads"),
  	rename=True,
    force=True,
    threads=4
)

print(log)

Masking reference genomes

The mask subcommand makes it easy to create custom-masked reference genomes and achieve maximum retention of specific target organisms:

hostile mask human.fasta lots-of-bacterial-genomes.fasta --threads 8

You may wish to use one of the existing reference genomes as a starting point. Masking uses Minimap2 to align 150mers of the supplied target genomes with the reference genome, and bedtools to mask all aligned regions with N. Both a masked genome (for Minimap2) and a masked Bowtie2 index is created.

Known issues

• Using more than 10 alignment threads may reduce performance, even on systems with enough CPU cores. A sensible default is therefore chosen automatically at runtime. To maximise performance on your system, some experimentation may be necessary.
• Minimap2 has an overhead of 30-90s for human genome indexing prior to starting decontamination. Surprisingly, loading a prebuilt index is not significantly faster. I hope to mitigate this in a future release.

Citation

Bede Constantinides, Martin Hunt, Derrick W Crook, Hostile: accurate decontamination of microbial host sequences, Bioinformatics, 2023; btad728, https://doi.org/10.1093/bioinformatics/btad728

@article{10.1093/bioinformatics/btad728,
    author = {Constantinides, Bede and Hunt, Martin and Crook, Derrick W},
    title = {Hostile: accurate decontamination of microbial host sequences},
    journal = {Bioinformatics},
    volume = {39},
    number = {12},
    pages = {btad728},
    year = {2023},
    month = {12},
    issn = {1367-4811},
    doi = {10.1093/bioinformatics/btad728},
    url = {https://doi.org/10.1093/bioinformatics/btad728},
    eprint = {https://academic.oup.com/bioinformatics/article-pdf/39/12/btad728/54850422/btad728.pdf},
}

Development install

git clone https://github.com/bede/hostile.git
cd hostile
conda env create -y -f environment.yml
conda activate hostile
pip install --editable '.[dev]'
pytest