fARGene (Fragmented Antibiotic Resistance Gene iENntifiEr ) is a tool that takes either fragmented metagenomic data or longer sequences as input and predicts and delivers full-length antiobiotic resistance genes as output. The tool includes developed and optimized models for a number or resistance gene types, and the functionality to create and optimize models of your own choice of resistance genes.
The current version of the tool includes developed and optimized models for identification of the following resistance genes
- Class A beta-lactamases
- Subclass B1 and B2 beta-lactamases
- Subclass B3 beta-lactamases
- Class C beta-lactamases
- Class D beta-lactamases
- qnr
These instructions will get you a copy of the most up-to-date version of fARGene.
- Python 2.x
- EMBOSS transeq
- seqtk
- HMMER
- For short-read data:
- SPAdes 3.7.0 or later
- Trim Galore! (optional)
- ORFfinder (optional)
- For long-read data
- prodigal (optional)
Some of these requirements are optional but might affect the results. If you for example skip the installation of Trim Galore!, the option --no-quality-filtering must be used. If you skip the installation of ORFfinder/prodigal for short-read/long-read data, the option --no-orf-prediction must be used. fARGene expects these tools to be available in $PATH.
For the model creation package you additionally need the following packages:
- numpy
- matplotlib
- ClustalO
git clone https://github.com/fannyhb/fargene.git
cd fargene
python setup.py install
Note:
setup.py will look for and try to install numpy and matplotlib so make sure that you either:
- have these packages installed
- run setup.py as root or with sudo
- install the program in a conda environment.
Fragmented metagenomic input
fargene -i path/to/paired_end_fastqfiles/*.fastq --meta --hmm-model class_a -o output_dir -p num_of_processes
Note that the FASTQ files must be paired-end.
Genomes or longer contigs as nucleotide sequences as input
fargene -i path/to/fastafile(s)/*.fasta --hmm-model class_a -o output_dir
Genomes or longer contigs as protein sequences as input
fargene -i path/to/fastafile(s)/*.fasta --protein --hmm-model class_a -o output_dir
Where hmm-model can be any of the pre-defined models:
- Class A beta-lactamases
--hmm-model class_a - Subclass B1 and B2 beta-lactamases
--hmm-model class_b_1_2 - Subclass B3 beta-lactamases
--hmm-model class_b_3 - Class C beta-lactamases
--hmm-model class_c - Class D beta-lactamases
--hmm-model class_d_1--hmm-model class_d_2
- qnr
--hmm-model qnr
If you choose to use your own profile hidden Markov model you need to specify the score as follows:
--hmm-model /path/to/hmmfile.hmm --score full_length_threshold_score --meta-score fragmented_threshold_score_per_AA
Note that the meta score is given as score per aa.
If the input is not fragmented data then the --meta-score is not required.
It is also possible to change the scores for the predifened models, just add the option --score new_score and/or --meta-score new_meta_score.
Run fargene --help to get all the options of fargene.
usage: fargene [-h] --infiles INFILES [INFILES ...] --hmm-model HMM_MODEL
[--score LONG_SCORE] [--meta] [--meta-score META_SCORE]
[--output OUTDIR] [--force] [--tmp-dir TMP_DIR] [--protein]
[--processes PROCESSES] [--min-orf-length MIN_ORF_LENGTH]
[--retrieve-whole] [--no-orf-predict] [--no-quality-filtering]
[--no-assembly] [--orf-finder] [--store-peptides] [--rerun]
[--amino-dir AMINO_DIR] [--fasta-dir FASTA_DIR]
[--translation-format TRANS_FORMAT] [--loglevel {DEBUG,INFO}]
[--logfile LOGFILE]
Searches and retrieves new and previously known genes from fragmented
metagenomic data and genomes. Copyright (c) Fanny Berglund 2018.
optional arguments:
-h, --help show this help message and exit
--infiles INFILES [INFILES ...], -i INFILES [INFILES ...]
Input file(s) to be searched. Could either be in FASTA
or FASTQ format.
--hmm-model HMM_MODEL
The Hidden Markov Model that should be used to analyse
the data. Could either be one of the pre-defined
models or the path to a custom HMM.
--score LONG_SCORE, -sl LONG_SCORE
The threshold score for a sequence to be classified as
a (almost) complete gene (default: None).
--meta If the input data is paired end metagenomic data
(default: False).
--meta-score META_SCORE, -sm META_SCORE
The threshold score for a fragment to be classified as
a positive. Expressed as score per amino acid
(default: None).
--output OUTDIR, -o OUTDIR
The output directory for the whole run (default:
./fargene_output).
--force, -f Overwrite output directory if it exists (default:
False).
--tmp-dir TMP_DIR Directory for (sometimes large) intermediate files.
(default: OUT_DIR/tmpdir)
--protein If the input sequence(s) is amino acids (default:
False).
--processes PROCESSES, -p PROCESSES
Number of processes to be used when processing
metagenomic data (default: 1).
--min-orf-length MIN_ORF_LENGTH
The minimal length for a retrieved predicted ORF (nt).
(default: 90% of the length of the chosen hmm.)
--retrieve-whole Use this flag if the whole sequence where a hit is
detected should be retrieved (default: False).
--no-orf-predict Do not perform ORF prediction.
--no-quality-filtering
Use if no quality control should be performed on the
metagenomic data (default: False).
--no-assembly Use if you want to skip the assembly and retrieval of
contigs for metagenomic data (default: False).
--orf-finder Use NCBI ORFfinder instead of prodigal for ORF
prediction of genomes/contigs (default: False).
--store-peptides, -sp
Store the translated sequences. Useful if you plan to
redo the analysis using a different model and want to
skip the preprocessing steps (default: False).
--rerun Use of you want to redo the analysis or do the
analysis using a different model and have kept either
the nucletide or amino acid sequences. Please note
that this only works if the input data is the same for
both runs (default: False).
--amino-dir AMINO_DIR
Where the amino acid sequences generated by the method
are located. Only to be used in combination with
--rerun
--fasta-dir FASTA_DIR
Where the nucleotide sequences in FASTA generated by
previous runs of the method are located. Only to be
used in combination with --rerun
--translation-format TRANS_FORMAT
The translation format that transeq should use.
(default: pearson)
--loglevel {DEBUG,INFO}
Set logging level (default: INFO).
--logfile LOGFILE Logfile (default: fargene_analysis.log).
The two most important output files are predicted-orfs.fasta and predicted-orfs-amino.fasta which are located in output_dir/predictedGenes/.
This directory also contains retrieved-contigs.fasta and retrieved-contigs-peptides.fasta. Where the first file contains the (complete) contigs that passed the final full-length classification, and the second file contains the parts of the contigs that passed the final classification step that aligned with the HMM, as amino acid sequences. Note that the second file is a prediction of where the genes are located on the contig and does usually not include the start and/or the stop of the genes.
A summary of the analysis can be found in output_dir/results_summary.txt and the logfile is found in output_dir/novelGeneFinder.log.
Below is a summary of the remaining output:
| File/Directory | Description |
|---|---|
| hmmsearchresults/ | All the output files from hmmsearch. |
| retrievedFragments/ | Each fragment that were classified as positive, together with its read-pair. |
| retrievedFragments/all_retrieved_[12].fastq | All quality controlled retrieved fragments gathered in two files. |
| retrievedFragments/trimmedReads/ | The quality controlled retrieved fragments from each input file. |
| tmpdir/ | Various files that can be deleted if you don't want to redo the analysis. |
| tmpdir/* positives.out | List of ids for sequences/genes that were classified as positives. |
| tmpdir/infile(s).fasta | The from FASTQ to FASTA converted input files. |
| tmpdir/infiles(s)-amino.fasta | The translated input sequences. Are only saved if option --store-peptides is used. |
| spades_assembly/ | The output from the SPAdes assembly. |
The output is basically the same as for the metagenomic input. The most important difference is the file
input_file-hmm_model_name-filtered.fasta located in output_dir/predictedGenes. This file contains the sequences that passed the final classification step, but only the parts that where predicted by the HMM to be part of the gene. Since this prediction is rather conservative, the start/stop of the genes are usually not included here. The file input_file-hmm_model_name-filtered-peptides.fasta is the above file translated in the same frame as the gene is predicted to be located.
If you want to search the same dataset for different types of genes it is recommended to use the --rerun option in order to avoid repeating the time consuming preprocessing steps. This can be used on the converted FASTA files generated by the first analysis with fargene as shown below
First analysis
fargene -i path/to/paired_end_fastqfiles/*.fastq --meta --hmm-model class_a -o class_a_out -p num_of_processes
To repeat the analysis with another model
fargene -i path/to/paired_end_fastqfiles/*.fastq --meta --hmm-model class_c -o class_c_out -p num_of_processes
--rerun --fasta-dir class_a_out/tmpdir/
Depending on the size of your storage and the size of the dataset, the option --store-peptides can be used in the first analysis. This will significantly speed up the analysis for the following models, but be aware of the potential huge amounts of data that will be stored. The commands can then look as follows
First analysis
fargene -i path/to/paired_end_fastqfiles/*.fastq --meta --hmm-model class_a -o class_a_out -p num_of_processes
--store-peptides
To repeat the analysis with another model
fargene -i path/to/paired_end_fastqfiles/*.fastq --meta --hmm-model class_c -o class_c_out -p num_of_processes
--rerun --amino-dir class_a_out/tmpdir/
To create and optimize threshold score for a new model using the default values (full-length genes and 10 000 fragments of length 33 aa)
fargene_model_creation --reference-sequences file_with_reference_sequences.fasta
--negative-sequences file_with_negative_sequences.fasta --modelname name_of_my_new_model
--output output_dir
Where both the reference and negative sequences should be protein sequences.
Run fargene_model_creation --help to get all the options of the model creation and optimization
usage: fargene_model_creation [-h] --reference-sequences REFERENCE_SEQUENCES
[--negative-sequences NEGATIVE_SEQUENCES]
[--output OUTPUT_DIR] [--modelname MODELNAME]
[--fragment-lengths FRAGMENT_LENGTHS]
[--num-fragments NUM_FRAGMENTS] [--only-sens]
[--only-spec] [--only-full-length] [--only-fragments]
A program to create and optimize profile hidden Markov models
optional arguments:
-h, --help show this help message and exit
--reference-sequences REFERENCE_SEQUENCES, -rin REFERENCE_SEQUENCES
The sequences that the model should be built of.
--negative-sequences NEGATIVE_SEQUENCES, -nin NEGATIVE_SEQUENCES
The sequences that should be used as the negative
dataset. Should preferable be similar sequences but
without the desired phenotype.
--output OUTPUT_DIR, -o OUTPUT_DIR
The directory where the output should be saved.
--modelname MODELNAME
The name of the new model
--fragment-lengths FRAGMENT_LENGTHS, -l FRAGMENT_LENGTHS
The length (aa) of the fragments that should be used
to determine the threshold score for metagenomic
input. (default: 33 AA)
--num-fragments NUM_FRAGMENTS
The number of fragments that should be created from
each gene. (default: 10 000)
--only-sens Should be used if only sensitivity of the model should
be estimated.
--only-spec Should be used if only the specificity of the model
should be estimated.
--only-full-length Should be used if you only want to optimize the
threshold score for full length genes.
--only-fragments Should be used if you only want to optimize the
threshold score for full fragmented genes.
The created HMM is located in output_dir/hmmerModel and is called modelname.hmm.
The two most important files from the estimation of sensitivity and specificity are called resulting_sensitivity_specificity_modelname_full_length.txt and resulting_sensitivity_specificity_modelname_fragmentlength.txt and are located in output_dir/results/. These contain the estimated senstivity and specificity as a function of the domain score from the hmmsearch.
This results is also visualized in the figures resulting_sensitivity_specificity_modelname_full_length.png resulting_sensitivity_specificity_modelname_fragmentlength.png.
The combined results from the hmmsearch of full-length sequences are called modelname-hmmsearch-refrence-sequences-full-length.txtand modelname-hmmsearch-negative-sequences-full-length.txt and are useful to detect genes responsible for potenial outline scores.
For a tutorial of how to use fargene click here.
fARGene comes with a third tool called pick_long_reads which can extract sequences longer than a certain length.
Usage for nucleotide sequences:
pick_long_reads -i all_contigs.fasta --length 800 -o contigs_longer_than_800nt.fasta
Usage for protein sequences:
pick_long_reads -i all_contigs-amino.fasta --length 250 --cut-stars -o contigs_longer_than_250aa.fasta
This project is licensed under the MIT License - see the LICENSE.md file for details.