Quick start

We assume here that the program is installed.

You can see all available options with:

integron_finder -h

For impatient

Go to the directory containing your input file(s), or specify the path to that file and call:

integron_finder mysequences.fst

or:

integron_finder path/to/mysequences.fst

It will perform a search, and outputs the results in a directory called Results_Integron_Finder_mysequences.

Input and Outputs

Inputs

integron_finder can take as an input:

  • a fasta file

  • a multi-fasta file

  • many (multi-)fasta files

  • several replicon in gembase format

Gembase format

Integron_Finder can use gembase formatted protein files instead of re-annotating genes with Prodigal. This feature enables the user to use Integron_Finder with its own annotations. The gembase format is the typical result of PanACoTA’s annotation step

Integron_Finder support gembase format v1 and v2

Gembase v1

It must contain, at least, a LSTINF and a Protein file per replicon. Folder structure must have the following architecture.

gembase/
├── Genes
│   ├── ACBA.0917.00019.gen       # genome in fasta format one seq/gene
│   └── ESCO001.C.00001.C001.gen
├── LSTINFO
│   ├── ACBA.0917.00019.lst       # information in space separated values
│   └── ESCO001.C.00001.C001.lst
├── Proteins
│   ├── ACBA.0917.00019.prt       # proteins in fasta format
│   └── ESCO001.C.00001.C001.prt
└── Replicons
    ├── ACBA.0917.00019.fna             # genome in fasta format one seq/replicon
    └── ESCO001.C.00001.C001.fst

5 first sequences headers in gembase/Genes/ACBA.0917.00019.gen file

>ACBA.0917.00019.b0001_00001 1215 tyrS | Tyrosine--tRNA ligase | 6.1.1.1 | similar to AA sequence:UniProtKB:P41256
>ACBA.0917.00019.i0001_00002 1128 anmK | Anhydro-N-acetylmuramic acid kinase | 2.7.1.170 | similar to AA sequence:UniProtKB:Q8EHB5
>ACBA.0917.00019.i0001_00003 846 ephA | Epoxide hydrolase A | 3.3.2.10 | similar to AA sequence:UniProtKB:I6YGS0
>ACBA.0917.00019.i0001_00004 336 erpA | Iron-sulfur cluster insertion protein ErpA | NA | similar to AA sequence:UniProtKB:P45344
>ACBA.0917.00019.i0001_00005 1005 NA | hypothetical protein | NA | NA
...

5 first sequences headers in gembase/Genes/ACBA.0917.00019.gen file

>ACBA.0917.00019.b0001_00001 1215 tyrS | Tyrosine--tRNA ligase | 6.1.1.1 | similar to AA sequence:UniProtKB:P41256
>ACBA.0917.00019.i0001_00002 1128 anmK | Anhydro-N-acetylmuramic acid kinase | 2.7.1.170 | similar to AA sequence:UniProtKB:Q8EHB5
>ACBA.0917.00019.i0001_00003 846 ephA | Epoxide hydrolase A | 3.3.2.10 | similar to AA sequence:UniProtKB:I6YGS0
>ACBA.0917.00019.i0001_00004 336 erpA | Iron-sulfur cluster insertion protein ErpA | NA | similar to AA sequence:UniProtKB:P45344
>ACBA.0917.00019.i0001_00005 1005 NA | hypothetical protein | NA | NA
...

5 first lines in gembase/LSTINFO/ACBA.0917.00019.lst

266     1480    C       CDS     ACBA.0917.00019.b0001_00001     tyrS    | Tyrosine--tRNA ligase | 6.1.1.1 | similar to AA sequence:UniProtKB:P41256
1560    2687    D       CDS     ACBA.0917.00019.i0001_00002     anmK    | Anhydro-N-acetylmuramic acid kinase | 2.7.1.170 | similar to AA sequence:UniProtKB:Q8EHB5
2815    3660    D       CDS     ACBA.0917.00019.i0001_00003     ephA    | Epoxide hydrolase A | 3.3.2.10 | similar to AA sequence:UniProtKB:I6YGS0
3716    4051    C       CDS     ACBA.0917.00019.i0001_00004     erpA    | Iron-sulfur cluster insertion protein ErpA | NA | similar to AA sequence:UniProtKB:P45344
4176    5180    C       CDS     ACBA.0917.00019.i0001_00005     NA      | hypothetical protein | NA | NA
...

all sequences headers in gembase/Replicons/ACBA.0917.00019.fna

>ACBA.0917.00019.0001
>ACBA.0917.00019.0002

Integron_finder will use LSTINF and Proteins folders. Hence if you want to analyze a replicon located in a gembase, the command line should look like

integron_finder –gembase data/Replicons/ACBA.0917.00019.fna

Gembase v2

In V2 format, there is one file per genome. If there are several chromosomes or chromosomes plus plasmids, phages, … all the sequences are in the same file.

Example of Gembase v2 architecture:

Gembase_v2_extraction/
    ├── Genes
    │   ├── VICH001.0523.00090.gen
    │   ├── VICH001.0523.00091.gen
    │   ├── ...
    ├── LST
    │   ├── VICH001.0523.00090.lst
    │   ├── VICH001.0523.00091.lst
    │   ├── ...
    ├── Microbial0523-genomes.info
    ├── Microbial0523-replicons.info
    ├── Proteins
    │   ├── VICH001.0523.00090.prt
    │   ├── VICH001.0523.00091.prt
    │   ├── ...
    ├── RNA
    │   ├── VICH001.0523.00090.rna
    │   ├── VICH001.0523.00091.rna
    │   ├── ...
    ├── Replicons
    │   ├── VICH001.0523.00090.fna
    │   ├── VICH001.0523.00091.fna
    │   ├── ...
    └── gff
        ├── VICH001.0523.00090.gff3
        ├── VICH001.0523.00091.gff3
        ├── ...

5 first sequences headers in gembase/Genes/VICH001.0523.00090.gen file

>VICH001.0523.00090.001C_00001  C       ATG     TAA     1       2961008 1128    mltC    FKV26_RS00005   WP_001230176.1  membrane-bound_lytic_murein_transglycosylase_MltC
ATGAGAAAGTTAGTGTTATGTATCACCGCTCTATTATTGAGTGGCTGTAGTCGTGAATTTATCGAGAAAATTTACGATGTTGATTACGAGCCCACTAACCGTTTCGCCAAGAACTTAGCCGAATTACCCGGACAGTTTCAGAAAGACACCGCCGCACTGGATGCATTAATCAACAGCTTCTCCGGCAATATCGAAAAACGTTGGGGGCGCCGCGAGCTA
>VICH001.0523.00090.001C_00002  C       ATG     TAG     913     1185    273     NA      FKV26_RS00010   WP_000124739.1  oxidative_damage_protection_protein
ATGGCTCGCACCGTATTTTGTACCCGATTGCAGAAAGAAGCCGATGGCTTGGATTTCCAACTCTATCCCGGAGAACTGGGCAAACGCATTTTCGACAACATCTGCAAAGAAGCTTGGGCACAATGGCAGACCAAACAGACCATGCTGATCAATGAAAAAAAACTCAACATGATGGATCCTGAGCACCGCAAATTGCTGGAGCAAGAAATGGTGAGCTTC
>VICH001.0523.00090.001C_00003  C       GTG     TAA     1199    2260    1062    mutY    FKV26_RS00015   WP_001995048.1  A/G-specific_adenine_glycosylase
...

VICH001.0523.00090 contains 2 chromosomes 001C and 002C

5 first lines of chromosome 1 and 2 in gembase/LST/VICH001.0523.00090.lst

1   2961008 C       CDS     VICH001.0523.00090.001C_00001   FKV26_RS00005   mltC    WP_001230176.1  membrane-bound_lytic_murein_transglycosylase_MltC       4.2.2.- COORDINATES:_similar_to_AA_sequence:RefSeq:WP_001230183.1       GO:0008933_-_lytic_transglycosylase_activity_[Evidence_IEA]     Derived_by_automated_computational_analysis_using_gene_prediction_method:_Protein_Homology.
913 1185    C       CDS     VICH001.0523.00090.001C_00002   FKV26_RS00010   NA      WP_000124739.1  oxidative_damage_protection_protein     NA      COORDINATES:_similar_to_AA_sequence:RefSeq:WP_005482430.1       GO:0005506_-_iron_ion_binding_[Evidence_IEA]    Derived_by_automated_computational_analysis_using_gene_prediction_method:_Protein_Homology.
1199        2260    C       CDS     VICH001.0523.00090.001C_00003   FKV26_RS00015   mutY    WP_001995048.1  A/G-specific_adenine_glycosylase        3.2.2.31        COORDINATES:_similar_to_AA_sequence:RefSeq:WP_014505785.1       GO:0019104_-_DNA_N-glycosylase_activity_[Evidence_IEA]  Derived_by_automated_computational_analysis_using_gene_prediction_method:_Protein_Homology.
2500        3219    D       CDS     VICH001.0523.00090.001C_00004   FKV26_RS00020   trmB    WP_000005581.1  tRNA_(guanosine(46)-N7)-methyltransferase_TrmB  2.1.1.33        COORDINATES:_similar_to_AA_sequence:RefSeq:WP_001898008.1       GO:0008176_-_tRNA_(guanine-N7-)-methyltransferase_activity_[Evidence_IEA]       Derived_by_automated_computational_analysis_using_gene_prediction_method:_Protein_Homology.
3330        4250    D       CDS VICH001.0523.00090.001C_00005       FKV26_RS00025   glsB    WP_000805054.1  glutaminase_B   3.5.1.2 COORDINATES:_similar_to_AA_sequence:RefSeq:WP_002046360.1       GO:0004359_-_glutaminase_activity_[Evidence_IEA]        Derived_by_automated_computational_analysis_using_gene_prediction_method:_Protein_Homology.
...
15      1373    D       CDS     VICH001.0523.00090.002C_02705   FKV26_RS13475   glpT    WP_000462380.1  glycerol-3-phosphate_transporter        NA      COORDINATES:_similar_to_AA_sequence:RefSeq:WP_019829515.1       GO:0015169_-_glycerol-3-phosphate_transmembrane_transporter_activity_[Evidence_IEA]     Derived_by_automated_computational_analysis_using_gene_prediction_method:_Protein_Homology.
1527    2594    D       CDS     VICH001.0523.00090.002C_02706   FKV26_RS13480   glpQ    WP_000917229.1  glycerophosphodiester_phosphodiesterase 3.1.4.46        COORDINATES:_similar_to_AA_sequence:RefSeq:WP_005524061.1       GO:0008081_-_phosphoric_diester_hydrolase_activity_[Evidence_IEA]       Derived_by_automated_computational_analysis_using_gene_prediction_method:_Protein_Homology.
2647    3327    C       CDS     VICH001.0523.00090.002C_02707   FKV26_RS13485   NA      WP_001177500.1  Crp/Fnr_family_transcriptional_regulator        NA      COORDINATES:_similar_to_AA_sequence:RefSeq:WP_001887744.1       NA      Derived_by_automated_computational_analysis_using_gene_prediction_method:_Protein_Homology.
3467    4198    C       CDS     VICH001.0523.00090.002C_02708   FKV26_RS13490   NA      WP_000027384.1  nucleoside_phosphorylase        NA      COORDINATES:_similar_to_AA_sequence:RefSeq:WP_001897923.1       NA      Derived_by_automated_computational_analysis_using_gene_prediction_method:_Protein_Homology.
4195    4920    C       CDS     VICH001.0523.00090.002C_02709   FKV26_RS13495   pnuC    WP_001995272.1  nicotinamide_riboside_transporter_PnuC  NA      COORDINATES:_similar_to_AA_sequence:RefSeq:WP_001933049.1       GO:0015663_-_nicotinamide_mononucleotide_transmembrane_transporter_activity_[Evidence_IEA]      Derived_by_automated_computational_analysis_using_gene_prediction_method:_Protein_Homology.

5 sequences of chromosome 1 and 2 in gembase/Proteins/VICH001.0523.00090.prt

>VICH001.0523.00090.001C_00001  C       ATG     TAA     1       2961008 375     mltC    FKV26_RS00005   WP_001230176.1  membrane-bound_lytic_murein_transglycosylase_MltC
MRKLVLCITALLLSGCSREFIEKIYDVDYEPTNRFAKNLAELPGQFQKDTAALDALINSFSGNIEKRWGRRELKIAGKNNYVKYIDNYLSRSEVNFTEGRIIVETVSPIDPKAHLRNAIITTLLTPDDPAHVDLFSSKDIELKGQPFLYQQVLDQDGQPIQWSWRANRYADY
>VICH001.0523.00090.001C_00002  C       ATG     TAG     913     1185    90      NA      FKV26_RS00010   WP_000124739.1  oxidative_damage_protection_protein
MARTVFCTRLQKEADGLDFQLYPGELGKRIFDNICKEAWAQWQTKQTMLINEKKLNMMDPEHRKLLEQEMVSFLFEGKEVHIEGYTPPAK
...
>VICH001.0523.00090.002C_02705  D       ATG     TAA     15      1373    452     glpT    FKV26_RS13475   WP_000462380.1  glycerol-3-phosphate_transporter
MFELFKPTAHTQRLPSDKVDSVYSRLRWQLFIGIFVGYAGYYLVRKNFSLAMPYLIEQGFSRGDLGVALGAVSIAYGLSKFLMGNVSDRSNPRYFLSAGLLLSALVMFCFGFMPWATGSITAMFILLFLNGWFQGMGWPACGRTMVHWWSRKER
>VICH001.0523.00090.002C_02706  D       ATG     TAA     1527    2594    355     glpQ    FKV26_RS13480   WP_000917229.1  glycerophosphodiester_phosphodiesterase
MLKPFSLSLLALACSTSLFSSIVSAEPIVIAHRGASGYLPEHTLEAKTLAYAMKPDYIEQDVVMTKDDQLVVLHDHYLDRVTDVAERFPNRARADGRYYAIDFTLAEIKTLRVTEGFNIDDQGKKVAGFPDRFPIWKGDFTVPTLAEEIELIQGLNKTLG

all sequences headers in gembase/Replicons/VICH001.0523.00090.fna

>VICH001.0523.00090.001C        2023-05-16      2961008 bp      Vibrio cholerae O1 strain AAS91 chromosome 1, complete sequence
>VICH001.0523.00090.002C        2023-05-16      1124701 bp      Vibrio cholerae O1 strain AAS91 chromosome 2, complete sequence

Integron_finder will use LST (or LSTINF(O) for Gembase v1) and Proteins folders. Hence if you want to analyze a replicon located in a gembase, the command line should look like

integron_finder --gembase  data/My_base/Replicons/ACBA.0917.00019.fna

Note

If the replicon you want to analyze is not in the gembase directory, but you still want to take advantage of the gembase annotation, then you have to specify the –gembase-path option to indicate where to find it. A typical command line could be:

integron_finder --gembase --gembase-path data/gembase/  My_replicons/ACBA.0917.00019.fna

Note

The gembase format implies different default topology (Topology)

Custom protein file

Integron_Finder allow the user to provide it’s own protein file with it’s own annotations instead of using prodigal (default). In this case you have to specified the protein file to use with the option –prot-file and the path to the parser to extract information from this file –annot-parser. A typical command line could be:

integron_finder –prot-file <path/to/custom/protein /file> –annot-parser <my_annot_paser.py> <replicon_path>

The annotation parser must be a python file (with the .py extension’) with one function called description_parser This function must take one argument which is the header of a fasta sequence header (without the first character >) and must return a tuple with 4 elements:

  • sequence id: the id of the sequence (string)

  • start: the beginning position of the protein on the genome (positive int)

  • stop: the end position of the protein on the genome (positive int)

  • strand: the strand 1 if the protein os coded on the direct strand or -1 on the reverse

Below a description_parser to parse annotation in prodigal format:

from typing import Tuple


def description_parser(seq_header: str) -> Tuple[int, int, int, int]:
    """
    Extract description features from protein sequence fasta header

    :param str seq_header: the fasta header of a sequence (without '>' char)
    :return: features
                - sequence id (string)
                - start the beginning of the protein (positive integer)
                - stop the end position of the protein (positive integer)
                - strand 1 if prot is coded on direct strand or -1 if it's on reverse
    :rtypes: tuple (str, int, int, 1/-1)
    """
    id_, start, stop, strand, *_ = seq_header.split(" # ")
    start = int(start)
    stop = int(stop)
    strand = int(strand)
    return id_, start, stop, strand

Outputs

By default, integron_finder will output 3 files under Results_Integron_Finder_mysequences:

  • mysequences.integrons : A file with all integrons and their elements detected in all sequences in the input file.

  • mysequences.summary : A summary file with the number and type of integrons per sequence.

  • integron_finder.out : A copy standard output. The stdout can be silenced with the argument --mute

The amount of log in the standard output can be controlled with --verbose for more or --quiet for less, and both are cumulative arguments, eg. -vv or -qq.

Other files can be created on demand:

  • --gbk: Creates a Genbank files with all the annotations found (present in the .integrons file)

  • --pdf: Creates a simple pdf graphic with complete integrons

  • --split-results: Creates a .integrons a .summary file per replicon if the input is a multifasta file.

  • --keep-tmp: Keep temporary files. See Keep intermediate files for more.

For everyone

Note

The different options will be shown separately, but they can be used altogether unless otherwise stated.

Thorough local detection

This option allows a much more sensitive search of attC sites. It will be slower if integrons are found, but will be as fast if nothing is detected.

integron_finder mysequences.fst --local-max

CALIN detection

By default CALIN are reported if they are composed of at least 2 attC sites, in order to avoid false positives. This value was chosen as CALIN with 2 attC sites were unlikely to be false positive. The probability of a false CALIN with at least 2 attC sites within 4kb was estimated between 4.10^-6 and 7.10^-9. However, one can modify this value with the option –calin-threshold and use a lower or higher value depending on the risk one is willing to take:

integron_finder mysequences.fst --calin-threshold 1

Note

If --local-max is called, it will run around CALINs with single attC sites, even if --calin-threshold is 2. The filtering step is done after the search with local max in that case.

Functional annotation

This option allows to annotate cassettes given HMM profiles. As AMRFinderPlus database is distributed, to annotate antibiotic resistance genes, just use:

integron_finder mysequences.fst --func-annot

IntegronFinder will look in the directory Integron_Finder-x.x/data/Functional_annotation and use all .hmm files available to annotate. By default, there is only NCBIfam-AMRFinder.hmm, but one can add any other HMM file here. Alternatively, if one wants to use a database which is present elsewhere on the user’s computer without copying it into that directory, one can specify the following option

integron_finder mysequences.fst --path_func_annot bank_hmm

where bank_hmm is a file containing one absolute path to a hmm file per line, and you can comment out a line

~/Downloads/Integron_Finder-x.x/data/Functional_annotation/NCBIfam-AMRFinder.hmm
~/Documents/Data/Pfam-A.hmm
# ~/Documents/Data/Pfam-B.hmm

Here, annotation will be made using Pfam-A et NCBIfam-AMRFinder, but not Pfam-B. If a protein is hit by 2 different profiles, the one with the best e-value will be kept.

Search for promoter and attI sites

By default integron_finder look for attC sites and site-specific integron integrase,, If you want to search for known promoters (integrase, Pc-int1 and Pc-int3) and AttI sites in integrons elements you need to add the --promoter-attI option on the command line.

Keep intermediate results

Integrons finder needs some intermediate results to run completely. It includes notably the protein file in fasta (mysequences.prt), but also the outputs from hmmer and infernal. A folder containing these outputs is generated for each replicon and have name tmp_<replicon_id> This directory is removed at the end. You can keep this directory to analyse further each integron_finder steps with the option --keep-tmp. Using this argument allows you to rerun integron_finder on the same sequences without redetecting proteins and attC sites. It is useful if one wants to change clustering parameters, evalues of attC sites, or size of them. Note that it won’t search for new attC sites so it is better to start with relaxed parameters and then rerun integron_finder with more strict parameters. See the section for integron diggers for more informations

For each tmp file, there are:

  • <replicon_id>.fst: a single fasta file with the replicon_name

  • <replicon_id>.prt: a multifasta file with the sequences of the detected proteins.

  • <replicon_id>_intI_table.res: hmm result for the intI hmm profile in tabular format

  • <replicon_id>_intI.res: hmm result for the intI hmm profile

  • <replicon_id>_phage_int_table.res: hmm result for the tyrosine recombinase hmm profile in tabular format

  • <replicon_id>_phage_int.res: hmm result for the tyrosine recombinase hmm profile in tabular format

  • <replicon_id>_attc_table.res: cmsearch result for the attC sites covariance model in tabular format

  • <replicon_id>_attc.res: significant (according to evalue-attc) attC sites aligned in stockholm format

  • integron_max.pickle: pickle file so integron_finder reuse this instead of re-running the local_max part

Topology

For regular sequence file format

By default, IntegronFinder assumes that

  • your replicon is considered as circular if there is only one replicon in the input file.

  • your replicons are considered as linear if there are several replicons in the input file.

For sequence in GemBase format

By default, IntegronFinder assumes that

  • Your replicon is considered as circular if it’s a Complete Relicon and it’s a Chromosome or Plasmid. Even there are several replicons in the same file.

  • If your replicons are a Phage, Other or a Draft. They will be considered by default as linear.

Whatever The sequence format

However, you can change this default behavior and specify the default topology with options --circ or --lin on the command line:

integron_finder --lin mylinearsequence.fst
integron_finder --circ mycircularsequence.fst

If you have multiple replicon in the input file with different topologies you can specify a topology for each replicon by providing a topology file. The syntax for the topology file is simple:

  • one topology by line

  • one line start by the seqid followed by ‘circ’ or ‘lin’ for circular or linear topologies.

example:

seq_id_1 circ
seq_id_2 lin

You can also mix the options --circ or --lin with option --topology-file:

integron_finder --circ --topology-file path/to/topofile mysequencess.fst

In the example above the default topology is set to circular. The replicons specified in topofile supersede the default topology.

Warning

However, if the replicon is smaller than 4 x dt (where dt is the distance threshold, so 4kb by default), the replicon is considered linear to avoid clustering problem. The topology used to searching integron is report in the *.integrons file

For big data people

Parallelization

The time limiting part are HMMER (search integrase) and INFERNAL (search attC sites). So if you have to analyze one or few replicons the user can set the number of CPU used by HMMER and INFERNAL:

integron_finder mysequences.fst --cpu 4

Default is 1.

Warning

Increasing too much (usually above 4) the number of CPUs used may lower the performance of the software. Please refer to the documentation of HMMER and INFERNAL for more details.

If you want to deal with a fasta file with a lot of replicons (from 10 to more than thousand) we provide a workflow to parallelize the execution of the data. This mean that we cut the data input into chunks (by default of one replicon) then execute IntegronFinder in parallel on each replicon (the number of parallel tasks can be limited) then aggregate the results in one global summary. The workflow use the nextflow framework and can be run on a single machine or a cluster.

First, you have to install nextflow first, and integron_finder. Then we provide 2 files (you need to download them from the IntegronFinder github repo.)

  • parallel_integron_finder.nf which is the workflow itself in nextflow syntax

  • nextflow.config which is a configuration file to execute the workflow.

The workflow file should not be modified. Whereas the profile must be adapted to the local architecture.

The file nextflow.config provide for profiles:

  • a standard profile for local use

  • a cluster profile

  • a standard profile using apptainer container

  • a cluster profile using apptainer container

so now install nextflow. If you have capsule error like

CAPSULE EXCEPTION: Error resolving dependencies. while processing attribute Allow-Snapshots: false (for stack trace, run with -Dcapsule.log=verbose)
Unable to initialize nextflow environment

install nextflow (>=0.29.0) as follow (change the nextflow version with the last release)

wget -O nextflow http://www.nextflow.io/releases/v0.30.2/nextflow-0.30.2-all
chmod 777 nextflow

for more details see: https://github.com/nextflow-io/nextflow/issues/770#issuecomment-400384617

How to get parallel_integron_finder

The release contains the workflow parallel_integron_finder.nf and the nextflow.config at the top level of the archive But If you use pip to install Integron_Finder you have not easily access to them. But they can be downloaded or executed directly by using nextflow.

to download it

nextflow pull gem-pasteur/Integron_Finder

to get the latest version or use -r option to specify a version

nextflow pull -r release_2.0 gem-pasteur/Integron_Finder

to see what you download

nextflow see Integron_Finder

to execute it directly

nextflow run gem-pasteur/Integron_Finder -profile standard --replicons all_coli.fst --circ

or:

nextflow run -r release_2.0 gem-pasteur/Integron_Finder -profile standard --replicons all_coli.fst --circ

standard profile

This profile is used if you want to parallelize IntegronFinder on your machine. You can specify the number of tasks in parallel by setting the queueSize value

standard {
        executor {
            name = 'local'
            queueSize = 7
        }
        process{
            executor = 'local'
            $integron_finder{
                errorStrategy = 'ignore'
                cpu=params.cpu
            }
        }
 }

If you installed IntegronFinder with apptainer, just uncomment the container line in the script, and set the proper path to the container.

All options available in non parallel version are also available for the parallel one. except the --outdir which is not available and --replicons option which is specific to the parallelized version. --replicons allows to specify the path of a file containing the replicons.

A typical command line will be:

./parallel_integron_finder.nf -profile standard --replicons all_coli.fst --circ

Note

Joker as * or ? can be used in path to specify several files as input.

But do not forget to protect the wild card from the shell for instance by enclosing your glob pattern with simple quote.

nextflow run -profile standard parallel_integron_finder.nf --replicons 'replicons_dir/*.fst'

Two asterisks, i.e. **, works like * but crosses directory boundaries. Curly brackets specify a collection of sub-patterns.

nextflow run -profile standard parallel_integron_finder.nf --replicons 'data/**.fa'
nextflow run -profile standard parallel_integron_finder.nf --replicons 'data/**/*.fa'
nextflow run -profile standard parallel_integron_finder.nf --replicons 'data/file_{1,2}.fa'

The first line will match files ending with the suffix .fa in the data folder and recursively in all its sub-folders. While the second one only match the files which have the same suffix in any sub-folder in the data path. Finally the last example capture two files: data/file_1.fa, data/file_2.fa

More than one path or glob pattern can be specified in one time using comma. Do not insert spaces surrounding the comma

nextflow run -profile standard parallel_integron_finder --replicons 'some/path/*.fa,other/path/*.fst'

The command above will analyze all files ending by .fa in /some/path with .fst extension in other/path

For further details see: https://www.nextflow.io/docs/latest/channel.html#frompath

Note

The option –outdir is not allowed. Because you can specify several replicon files as input, So in this circumstances specify only one name for the output is a none sense.

Note

The options starting with one dash are for nextflow workflow engine, whereas the options starting by two dashes are for integron_finder workflow.

Note

Replicons will be considered linear by default (see above), here we use –circ to consider replicons circular.

Note

If you specify several input files, the split and merge steps will be parallelized.

If you execute this line, 2 kinds of directories will be created.

  • One named work containing lot of subdirectories this for all jobs launch by nextflow.

  • Directories named Results_Integron_Finder_XXX where XXX is the name of the replicon file. So, one directory per replicon file will be created. These directories contain the final results as in non parallel version.

cluster profile

The cluster profile is intended to work on a cluster managed by SLURM. If your cluster is managed by an other drm replace executor name by the right value (see nextflow supported cluster )

You can also manage

  • The number of tasks in parallel with the executor.queueSize parameter (here 500). If you remove this line, the system will send in parallel as many jobs as there are replicons in your data set.

  • The queue (or partition in SLURM terminology) with process.queue parameter (here common,dedicated)

  • and some options specific to your cluster management systems with process.clusterOptions parameter

    cluster {
    executor {
        name = 'slurm'
        queueSize = 500
    }

    process{
        executor = 'slurm'
        queue= 'common,dedicated'
        clusterOptions = '--qos=fast'
        $integron_finder{
            cpu=params.cpu
        }
    }
}

To run the parallel version on a cluster, for instance on a cluster managed by slurm, I can launch the main nextflow process in one slot. The parallelization and the submission on the other slots is made by nextflow itself. Below a command line to run parallel_integron_finder and use 2 cpus per integron_finder task, each integron_finder task can be executed on a different machine, each integron_finder task claim 2 cores (cpus in nextflow terminology) to speed up the attC sites or integrase search:

sbatch --qos fast -p common nextflow run  parallel_integron_finder.nf -profile cluster --replicons all_coli.fst --cpu 2 --local-max --gbk --circ

The results will be the same as described in local execution.

apptainer (formely singularity) profiles

If you use the integron_finder image with the apptainer container executor, use the profile standard_apptainer. With the command line below nextflow will download parallel_integron_finder from github and download the integron_finder image from the docker hub and convert it to apptainer on the fly so you haven’t to install anything except nextflow and apptainer.

nextflow run gem-pasteur/Integron_Finder -profile standard_apptainer --replicons all_coli.fst --circ

You can also use the integron_finder apptainer image on a cluster, for this use the profile cluster_apptainer.

sbatch --qos fast -p common nextflow run  gem-pasteur/Integron_Finder:2.0 -profile cluster_apptainer --replicons all_coli.fst --cpu 2 --local-max --gbk --circ

In the case of your cluster cannot reach the world wide web. you have to download the apptainer image

apptainer pull --name Integron_Finder docker pull gempasteur/integron_finder:<tag>

the move the image on your cluster modify the nextflow.config to point on the location of the image, and adapt the cluster options (executor, queue, …) to your architecture

 cluster_apptainer {
        executor {
            name = 'slurm'
            queueSize = 500
        }

        process {
            container = /path/to/integron_finder/image
            queue = 'common,dedicated'
            clusterOptions = '--qos=fast'
            withName: integron_finder {
                cpus = params.cpu
            }
        }
        singularity {
            enabled = true
            runOptions = '-B /pasteur'
            autoMounts = false
       }
    }
}

then run it

sbatch --qos fast -p common nextflow run  ./parallel_integron_finder.nf -profile cluster_singualrity --replicons all_coli.fst --cpu 2 --local-max --gbk --circ

If you want to have more details about the jobs execution you can add some options to generate report:

Execution report

To enable the creation of this report add the -with-report command line option when launching the pipeline execution. For example:

nextflow run  ./parallel_integron_finder.nf -profile standard -with-report [file name] --replicons

It creates an HTML execution report: a single document which includes many useful metrics about a workflow execution. For further details see https://www.nextflow.io/docs/latest/tracing.html#execution-report

Trace report

In order to create the execution trace file add the -with-trace command line option when launching the pipeline execution. For example:

nextflow run  ./parallel_integron_finder.nf -profile standard -with-trace --replicons

It creates an HTML timeline for all processes executed in your pipeline. For further details see https://www.nextflow.io/docs/latest/tracing.html#timeline-report

Timeline report

To enable the creation of the timeline report add the -with-timeline command line option when launching the pipeline execution. For example:

nextflow run  ./parallel_integron_finder.nf -profile standard -with-timeline [file name] --replicons ...

It creates an execution tracing file that contains some useful information about each process executed in your pipeline script, including: submission time, start time, completion time, cpu and memory used. For further details see https://www.nextflow.io/docs/latest/tracing.html#trace-report

For integron diggers

Many options are set to prevent false positives. However, one may want higher sensitivity at the expense of having potentially false positives. Ultimately, only experimental experiments will tell whether a given attC sites or integrase is functional.

Also, note that because of how local_max works (ie. around already detected elements), true attC sites may be found thanks to false attC sites, because false attC sites may trigger local_max around them. Hence, one may want to use very relaxed parameters first with the --keep-tmp flag to rerun the analysis on the same data while restrincting the parameters.

Clustering of elements

attC sites are clustered together if they are on the same strand and if they are less than 4 kb apart (-dt 4000 by default). To cluster an array of attC sites and an integron integrase, they also must be less than 4 kb apart. This value has been empirically estimated and is consistent with previous observations showing that biggest gene cassettes are about 2 kb long. This value of 4 kb can be modified though:

integron_finder mysequences.fst --distance-thresh 10000

or, equivalently:

integron_finder mysequences.fst -dt 10000

This sets the threshold for clustering to 10 kb.

Note

The option --outdir allows you to chose the location of the Results folder (Results_Integron_Finder_mysequences). If this folder already exists, IntegronFinder will not re-run analyses already done, except functional annotation. It allows you to re-run rapidly IntegronFinder with a different --distance-thresh value. Functional annotation needs to re-run each time because depending on the aggregation parameters, the proteins associated with an integron might change.

Integrase

We use two HMM profiles for the detection of the integron integrase. One for tyrosine recombinase and one for a specific part of the integron integrase. To be specific we use the intersection of both hits, but one might want to use the union of both hits (and sees whether it exists cluster of attC sites nearby non integron-integrase…). To do so, use:

integron_finder mysequences.fst --union-integrases

attC evalue

The default evalue is 1. Sometimes, degenerated attC sites can have a evalue above 1 and one may want to increase this value to have a better sensitivity.

integron_finder mysequences.fst --evalue-attc 5

Here is a plot of how the sensitivity and false positive rate evolve as a function of the evalue:

attC evalue

Note

If one wants to have maximum sensitivity, use a high evalue (max is 10), and then integron_finder can be run again on the same data with a lower evalue. It won’t work the other way around (starting with low evalue), as attC sites are not searched again.

attC size

By default, attC sites’ size ranges from 40 to 200bp. This can be changed with the --min-attc-size or --max-attc-size parameters:

integron_finder mysequences.fst --min-attc-size 50 --max-attc-size 100

Palindromes

attC sites are more or less palindromic sequences, and sometimes, a single attC site can be detected on the 2 strands. By default, the one with the highest evalue is discarded, but you can choose to keep them with the following option:

integron_finder mysequences.fst --keep-palindromes

attC alignements

One can get the alignements of attC sites in the temporary files (use --keep-tmp) to have them. Under Results_Integron_Finder_mysequences/tmp_repliconA/repliconA_attc.res one can find alignements of attC sites from repliconA, in Stokholm format, where R and L core regions are aligned with each others:

# STOCKHOLM 1.0
#=GF AU Infernal 1.1.2

ACBA.0917.00019.0001/315102-315161         GUCUAACAAUUC---GUUCAAGCcgacgccgcu.................................................ucgcggcgcgGCUUAACUCAAGC----GUUAGAU
#=GR ACBA.0917.00019.0001/315102-315161 PP ************...******************.................................................***********************....*******
ACBA.0917.00019.0001/313260-313368         ACCUAACAAUUC---GUUCAAGCcgagaucgcuucgcggccgcggaguuguucggaaaaauugucacaacgccgcggccgcaaagcgcuccgGCUUAACUCAGGC----GUUGGGC
#=GR ACBA.0917.00019.0001/313260-313368 PP ************...******************************************************************************************....*******
ACBA.0917.00019.0001/313837-313906         GCCCAACAUGGC---GCUCAAGCcgaccggccagcccu.......................................gcgggcuguccgucgGCUUAGCUAGGGC----GUUAGAG
#=GR ACBA.0917.00019.0001/313837-313906 PP ************...***********************.......................................****************************....*******
#=GC SS_cons                               <<<<<<<--------<<<-<<<<.....................................................................>>>>>>>---------->>>>>>>
#=GC RF                                    [Rsec=]========[=Lsec=].....................................................................[Lprim]==========[Rprim]
//

Which you can manipulate easily with esl-alimanip tools provided by infernal (the following examples should work if your cmsearch is in your PATH). You can convert the same alignement in dna alphabet (cmsearch use RNA alphabet):

$ esl-alimanip --dna Results_Integron_Finder_mysequences/tmp_ACBA.0917.00019.0001/ACBA.0917.00019.0001_attc.res
# STOCKHOLM 1.0
#=GF AU Infernal 1.1.2

ACBA.0917.00019.0001/315102-315161         GTCTAACAATTC---GTTCAAGCCGACGCCGCT-------------------------------------------------TCGCGGCGCGGCTTAACTCAAGC----GTTAGAT
#=GR ACBA.0917.00019.0001/315102-315161 PP ************...******************.................................................***********************....*******
ACBA.0917.00019.0001/313260-313368         ACCTAACAATTC---GTTCAAGCCGAGATCGCTTCGCGGCCGCGGAGTTGTTCGGAAAAATTGTCACAACGCCGCGGCCGCAAAGCGCTCCGGCTTAACTCAGGC----GTTGGGC
#=GR ACBA.0917.00019.0001/313260-313368 PP ************...******************************************************************************************....*******
ACBA.0917.00019.0001/313837-313906         GCCCAACATGGC---GCTCAAGCCGACCGGCCAGCCCT---------------------------------------GCGGGCTGTCCGTCGGCTTAGCTAGGGC----GTTAGAG
#=GR ACBA.0917.00019.0001/313837-313906 PP ************...***********************.......................................****************************....*******
#=GC SS_cons                               <<<<<<<--------<<<-<<<<.....................................................................>>>>>>>---------->>>>>>>
#=GC RF                                    [Rsec=]========[=Lsec=].....................................................................[Lprim]==========[Rprim]
//

You can also convert it to fasta format:

$ esl-alimanip --dna --outformat afa Results_Integron_Finder_mysequences/tmp_ACBA.0917.00019.0001/ACBA.0917.00019.0001_attc.res
>ACBA.0917.00019.0001/315102-315161
GTCTAACAATTC---GTTCAAGCCGACGCCGCT---------------------------
----------------------TCGCGGCGCGGCTTAACTCAAGC----GTTAGAT
>ACBA.0917.00019.0001/313260-313368
ACCTAACAATTC---GTTCAAGCCGAGATCGCTTCGCGGCCGCGGAGTTGTTCGGAAAAA
TTGTCACAACGCCGCGGCCGCAAAGCGCTCCGGCTTAACTCAGGC----GTTGGGC
>ACBA.0917.00019.0001/313837-313906
GCCCAACATGGC---GCTCAAGCCGACCGGCCAGCCCT----------------------
-----------------GCGGGCTGTCCGTCGGCTTAGCTAGGGC----GTTAGAG

The possible outformat are:

  • stockholm

  • pfam

  • a2m

  • psiblast

  • afa