YACHT is a mathematically rigorous hypothesis test for the presence or absence of organisms in a metagenomic sample, based on average nucleotide identity (ANI).
The associated preprint can be found at: https://doi.org/10.1101/2023.04.18.537298. Please cite via:
Koslicki, D., White, S., Ma, C., & Novikov, A. (2023). YACHT: an ANI-based statistical test to detect microbial presence/absence in a metagenomic sample. bioRxiv, 2023-04.
We provide a demo to show how to use YACHT. Please follow the command lines below to try it out:
NUM_THREADS=64 # Adjust based on your machine's capabilities
cd demo # the 'demo' folder can be downloaded via command 'yacht download demo' if it doesn't exist
# build k-mer sketches for the query sample and ref genomes
yacht sketch sample --infile ./query_data/query_data.fq --kmer 31 --scaled 1000 --outfile sample.sig.zip
yacht sketch ref --infile ./ref_genomes --kmer 31 --scaled 1000 --outfile ref.sig.zip
# preprocess the reference genomes (training step)
yacht train --ref_file ref.sig.zip --ksize 31 --num_threads ${NUM_THREADS} --ani_thresh 0.95 --prefix 'demo_ani_thresh_0.95' --outdir ./ --force
# run YACHT algorithm to check the presence of reference genomes in the query sample (inference step)
yacht run --json demo_ani_thresh_0.95_config.json --sample_file sample.sig.zip --significance 0.99 --num_threads ${NUM_THREADS} --min_coverage_list 1 0.6 0.2 0.1 --out ./result.xlsx
# convert result to CAMI profile format (Optional)
yacht convert --yacht_output result.xlsx --sheet_name min_coverage0.2 --genome_to_taxid toy_genome_to_taxid.tsv --mode cami --sample_name 'MySample' --outfile_prefix cami_result --outdir ./There will be an output EXCEL file result.xlsx recoding the presence of reference genomes with different spreadsheets given the minimum coverage of 1 0.6 0.2 0.1.
- Installation
- Usage
Please note YACHT does not currently support MacOS. However, we are actively working on developing compatibility for this operating system and hope to have it available soon. During this time, we provide a docker container (see using docker section below) for those who need to run YACHT on MacOS.
YACHT is available on Conda can be installed via the steps below to install：
# create conda environment
conda create -n yacht_env
# activiate environment
conda activate yacht_env
# install YACHT
conda install -c conda-forge -c bioconda yachtYACHT requires Python 3.6 or higher and Conda. We recommend using a virtual environment to ensure a clean and isolated workspace. This can be accomplished using either Conda or Mamba (a faster alternative to Conda).
To create your Conda environment and install YACHT, follow these steps:
# Clone the YACHT repository
git clone https://github.com/KoslickiLab/YACHT.git
cd YACHT
# Create a new virtual environment named 'yacht_env'
conda env create -f env/yacht_env.yml
# Activate the newly created environment
conda activate yacht_env
# Install YACHT within the environment
pip install .If you prefer using Mamba instead of Conda, just simply repalce conda with mamba in the above commands.
If you prefer running YACHT on MacOS, you can choose to use docker with Act. To run YACHT on docker, simply execute "act" from the main YACHT folder, or "act --container-architecture linux/amd64" if you are on MacOS system.
YACHT can be run via the command line yacht <module>. Now it has three four main modules: download, sketch, train, run, and convert.
-
The
downloadmodule has three submodules:demo,default_ref_db, andpretrained_ref_db:democan automatically download the demo files to a specified folder:
# Example yacht download demo --outfolder ./demodefault_ref_dbcan automatically download pre-generated sketches of reference genomes from GTDB or GenBank as our input reference databases.
# Example for downloading the k31 sketches of representative genomes of GTDB rs214 version yacht download default_ref_db --database gtdb --db_version rs214 --gtdb_type reps --k 31 --outfolder ./Parameter Explanation database two options for default reference databases: 'genbank' or 'gtdb' db_version the version of database, options: "genbank-2022.03", "rs202", "rs207", "rs214" ncbi_organism the NCBI organism for the NCBI reference genome, options: "archaea", "bacteria", "fungi", "virus", "protozoa" gtdb_type for GTDB database, chooses "representative" genome version or "full" genome version k the length of k-mer outfolder the path to a folder where the downloaded file is expected to locate pretrained_ref_dbcan automatically download our pre-trained reference genome database that can be directly used as input foryacht trainmodule.
# Example for downloading the pretrained reference database that was trained from GTDB rs214 representative genomes with k=31 and ani_threshold=0.9995 yacht download pretrained_ref_db --database gtdb --db_version rs214 --k 31 --ani_thresh 0.9995 --outfolder ./Parameter Explanation database two options for default reference databases: 'genbank' or 'gtdb' db_version the version of database, options: "genbank-2022.03", "rs214" ncbi_organism the NCBI organism for the NCBI reference genome, options: "archaea", "bacteria", "fungi", "virus", "protozoa" ani_thresh the cutoff by which two organisms are considered indistinguishable (default: 0.95) k the length of k-mer outfolder the path to a folder where the downloaded file is expected to locate -
The
sketchmodule (note that it is a simple wrapper tosourmash) has two submodules:refandsample:refis used to sketch fasta files and make them as a reference database
# Example for sketching multiple fasta files as reference genomes in a given folder yacht sketch ref --infile ./demo/ref_genomes --kmer 31 --scaled 1000 --outfile ref.sig.zipParameter Explanation infile the path to a input FASTQ file or a folder containing multiple FASTQ files kmer the length of k-mer scaled the scaled factor outfile the path to a output file sampleis used to sketch the single-end or paired-end fasta file(s) and make it/them as a query sample.
# Example for sketching a FASTA/Q file as a metagenomic example yacht sketch sample --infile ./query_data/query_data.fq --kmer 31 --scaled 1000 --outfile sample.sig.zipParameter Explanation infile the input FASTA/Q file(s). For paired-end reads, provide two files kmer the length of k-mer scaled the scaled factor outfile the path to a output file -
The
trainmodule pre-reprocesses the given sketches of reference genomes (the.zipfile) to identify and merge the "identical' genomes based on the given ANI threshold (e.g., --ani_threshold 0.95). For an example, please refer to theyacht traincommand in the "Quick start" section. -
The
runmodule runs the YACHT algorithm to detect the presence of reference genomes in a given sample. For an example, please refer to theyacht runcommand in the "Quick start" section. -
The
convertmodule can covert YACHT result to other popular output formats (e.g., CAMI profiling format, BIOM format, GraphPlAn). For an example, please refer to theyacht convertcommand in the "Quick start" section.
This section simply introduces the analysis workflow for YACHT:
- Create Sketches of Your Reference Database Genomes and Your Sample:
- This step involves generating compact representations (sketches) of genomic data for efficient comparison and analysis.
- Preprocess the Reference Genomes:
- This is the training step of YACHT, aiming to identify and merge the "identical" genomes based on Average Nucleotide Identity (
ANI) using theani_threshparameter.
- This is the training step of YACHT, aiming to identify and merge the "identical" genomes based on Average Nucleotide Identity (
- Run YACHT algorithm:
- This step involves running the YACHT algorithm to detect the presence of reference genomes in your sample.
- Convert YACHT result to other output formats
- This step is optional if you prefer other output formats (e.g., CAMI profiling format, BIOM format) for the downstream analysis.
For each step of this workflow, please see more detailed description in the sections below.
You will need a reference database in the form of sourmash sketches of a collection of microbial genomes. There are a variety of pre-created databases available at: https://sourmash.readthedocs.io/en/latest/databases.html. Our code uses the "Zipfile collection" format, and we suggest using the GTDB genomic representatives database:
yacht download default_ref_db --database gtdb --db_version rs214 --gtdb_type reps --k 31 --outfolder ./wget https://farm.cse.ucdavis.edu/~ctbrown/sourmash-db/gtdb-rs214/gtdb-rs214-reps.k31.zipIf you want to use a custom database, you will need to create a Sourmash sketch Zipfile collection from the FASTA/FASTQ files of your reference database genomes (see Sourmash documentation for details). In brief, this can be accomplished via the following commands:
If you have a single FASTA file with one genome per record:
# the command below is equivalent to: sourmash sketch dna -f -p k=31,scaled=1000,abund --singleton <path to your multi-FASTA file> -o training_database.sig.zip
yacht sketch ref --infile <path to your multi-FASTA file> --kmer 31 --scaled 1000 --outfile training_database.sig.zipIf you have a directory of FASTA files, one per genome:
# the command below is equivalent to: find <path of foler containg FASTA/FASTQ files> > dataset.csv; sourmash sketch fromfile dataset.csv -p dna,k=31,scaled=1000,abund -o training_database.sig.zip
yacht sketch ref --infile <path of foler containg FASTA/FASTQ files> --kmer 31 --scaled 1000 --outfile training_database.sig.zipCreating a sketch of your sample metagenome is an essential step in the YACHT workflow. This process involves using the same k-mer size and scale factor that were used for the reference database. You can use the following commands to implement this step:
# For a single-end FASTA/Q file
# the command below is equivalent to: sourmash sketch dna -f -p k=31,scaled=1000,abund -o sample.sig.zip <input FASTA/Q file>
yacht sketch sample --infile <input FASTA/Q file> --kmer 31 --scaled 1000 --outfile sample.sig.zip
# For pair-end FASTA/Q files, you need to separately specify two FASTA/Q files
# the command below is equivalent to: cat <FASTA/Q file 1> <FASTA/Q file 2> > combine.fastq (or combine.fasta); sourmash sketch dna -f -p k=31,scaled=1000,abund -o sample.sig.zip combine.fastq (or combine.fasta)
yacht sketch sample --infile <FASTA/Q file 1> <FASTA/Q file 2> --kmer 31 --scaled 1000 --outfile sample.sig.zipNote: Sourmash database offers three available k values (21, 31, and 51), allowing you to select the one that best suits your particular analytical needs. The scale factor serves as an indicator of data compression, and if your dataset is small, you might consider using a smaller value (corresponding to a higher portion of genomes retained in the sketch).
Warning: the training process is time-consuming on large database
In our benchmark with GTDB representive genomes, it takes 15 minutes using 16 threads, 50GB of MEM on a system equipped with a 3.5GHz AMD EPYC 7763 64-Core Processor. You can use the pre-trained database (see here) to skip this step. The processing time can be significant when executed on GTDB all genomes OR with limited resources. If only part of genomes are needed, one may use sourmash sig command to extract signatures of interests only.
The command yacht train extracts the sketches from the Zipfile-format reference database, and then turns them into a form usable by YACHT. In particular, it removes one of any two organisms that have ANI greater than the user-specified threshold as these two organisms are too close to be "distinguishable".
yacht train --ref_file gtdb-rs214-reps.k31.zip --ksize 31 --num_threads 32 --ani_thresh 0.95 --prefix 'gtdb_ani_thresh_0.95' --outdir ./The most important parameter of this script is --ani_thresh: this is average nucleotide identity (ANI) value equal to or below which two organisms are considered distinct. For example, if --ani_thresh is set to 0.95, then two organisms with ANI > 0.95 will be considered indistinguishable. For the organisms with ANI > 0.95, only the one with the largest number of unique kmers will be kept. If there is a tie in the number of unique kmers, one organism will be randomly selected. The default value of --ani_thresh is 0.95. The --ani_thresh value chosen here must match the one chosen for the YACHT algorithm (see below).
| Parameter | Explanation |
|---|---|
| --ref_file | the path to the sourmash signature database zip file |
| --ksize | the length of k-mer, must match the k size used in previous sketching steps (default: 31) |
| --num_threads | the number of threads to use for parallelization (default: 16) |
| --ani_thresh | the cutoff by which two organisms are considered indistinguishable (default: 0.95) |
| --prefix | the prefix for output folders and files (see details below) |
| --outdir | the path to output directory where the results and intermediate files will be genreated |
| File (names starting with prefix) | Content |
|---|---|
| _config.json | A JSON file stores the required information needed to run the next YACHT algorithm |
| _manifest.tsv | A TSV file contains organisms and their relevant info after removing the similar ones |
| _removed_orgs_to_corr_orgas_mapping.tsv | A TSV file with two columns: removed organism names ('removed_org') and their similar genomes ('corr_orgs') |
For convenience, we have provided some pre-trained reference database for the GenBank and GTDB genomes on Zenodo. If any of them is suitable for your study, you can simply run the following command to download it and skip the training step below. Note: download of pre-trained data is provided in the yacht download feature, please see here for more details about yacht download.
# remember to replace <zendo_id> and <file_name> for your case before running it
curl --cookie zenodo-cookies.txt "https://zenodo.org/records/<zendo_id>/files/<file_name>?download=1" --output <file_name>
# Example
# curl --cookie zenodo-cookies.txt "https://zenodo.org/records/10113534/files/genbank-2022.03-archaea-k31_0.80_pretrained.zip?download=1" --output genbank-2022.03-archaea-k31_0.80_pretrained.zipPlease note that if you plan to use these pre-trained reference databases, once you download and unzip it. You need to change the paths within the config json file (e.g., gtdb-rs214-reps.k31_0.9995_config.json) to the correct paths in your machine.
After this, you are ready to perform the hypothesis test via yacht run for each organism in your reference database. This can be accomplished with something like:
yacht run --json 'gtdb_ani_thresh_0.95_config.json' --sample_file 'sample.sig.zip' --num_threads 32 --keep_raw --significance 0.99 --min_coverage_list 1 0.5 0.1 0.05 0.01 --out ./result.xlsxThe --significance parameter is basically akin to your confidence level: how sure do you want to be that the organism is present? Higher leads to more false negatives, lower leads to more false positives.
The --min_coverage_list parameter dictates a list of min_coverage which indicates what percentage (value in [0,1]) of the distinct k-mers (think: whole genome) must have been sequenced and present in my sample to qualify as that organism as being "present." Setting this to 1 is usually safe, but if you have a very low coverage sample, you may want to lower this value. Setting it higher will lead to more false negatives, setting it lower will lead to more false positives (pretty rapidly).
| Parameter | Explanation |
|---|---|
| --json | the path to a json file generated by the make_training_data_from_sketches.py script (see above) |
| --significance | minimum probability of individual true negative (default: 0.99) |
| --num_threads | the number of threads to use for parallelization (default: 16) |
| --keep_raw | keep the raw result (i.e. min_coverage=1) no matter if the user specifies it |
| --show_all | Show all organisms (no matter if present) |
| --min_coverage_list | a list of min_coverage values, see more detailed description above (default: 1, 0.5, 0.1, 0.05, 0.01) |
| --out | path to output excel result (default: './result.xlsx') |
The output file will be an EXCEL file; column descriptions can be found here. The most important are the following:
organism_name: The name of the organismin_sample_est: A boolean value either False or True: if False, there was not enough evidence to claim this organism is present in the sample.p_vals: Probability of observing this or more extreme result at the given ANI threshold, assuming the null hypothesis.
Other interesting columns include:
num_exclusive_kmers_to_genome: How many k-mers were found in this organism and no othersnum_matches: How many k-mers were found in this organism and the sampleacceptance_threshold_*: How many k-mers must be found in this organism to be considered "present" at the given ANI threshold. Hence,in_sample_estis True ifnum_matches>=acceptance_threshold_*(adjusting by coverage if desired).alt_confidence_mut_rate_*: What the mutation rate (1-ANI) would need to be to get your false positive to match the false negative rate of 1-significance(adjusting by coverage if desired).
When we get the EXCEL result file from run_YACHT.py, you can run yacht convert to covert the YACHT result to other popular output formats (Currently, only cami, biom, graphplan are supported).
Note: Before you run yacht convert, you need to prepare a TSV file genome_to_taxid.tsv containing two columns: genome ID (genome_id) and its corresponding taxid (taxid). An example can be found here. You need to prepare it according to the reference database genomes you used.
Then you are ready to run yacht convert with something like:
yacht convert --yacht_output 'result.xlsx' --sheet_name 'min_coverage0.01' --genome_to_taxid 'genome_to_taxid.tsv' --mode 'cami' --sample_name 'MySample' --outfile_prefix 'cami_result' --outdir ./| Parameter | Explanation |
|---|---|
| --yacht_output | the path to the output excel file generated by run_YACHT.py |
| --sheet_name | specify which spreadsheet result you want to covert from |
| --genome_to_taxid | the path to the location of genome_to_taxid.tsv you prepared |
| --mode | specify to which output format you want to convert (e.g., 'cami', 'biom', 'graphplan') |
| --sample_name | A random name you would like to show in header of the cami file. Default: Sample1.' |
| --outfile_prefix | the prefix of the output file. Default: result |
| --outdir | the path to output directory where the results will be genreated |