______    _ _____         _                 ______ _                       
		| ___ \  | |_   _|       (_)                |  _  (_)                      
		| |_/ / _| |_| | _____  ___ _ __            | | | |_  __ _  __ _  ___ _ __ 
		| ___ \|__ __| |/ _ \ \/ / | '_ \           | | | | |/ _` |/ _` |/ _ \ '__|
		| |_/ /  | |_| | (_) >  <| | | | |  ______  | |/ /| | (_| | (_| |  __/ |   
		\____/   \___\_/\___/_/\_\_|_| |_| |______| |___/ |_|\__, |\__, |\___|_|   
								      __/ | __/ |          
								     |___/ |___/           

A web server of BtToxin_Digger can be found at http://bcam.hzau.edu.cn/BtToxin_Digger.

| Introduction | Installation | Database |Usage | Examples | Outputs | License | Feedback | Citation | FAQs | Updates |

• What is BtToxin_Digger?

  BtToxin_Digger is a high-throughput, automatic gene mining tool that can mine toxin genes, such as Cry, Cyt and Vip, etc, from Bacillus thuringiensis. The pipeline accepts multiple forms of input data including Reads, assembled genomes, CDSs, and protein sequences and can output rich and useful results. It is derived from the re-design of the tool BtToxin_scanner we developed previously. Compared with BtToxin_scanner, BtToxin_Digger has many improvements, as follows:

  • Can be run in batches, suitable for large-scale genome analysis.
  • Added genome assembly functions, including second-generation short-reads assembly, third-generation long-reads assembly, and hybrid assembly of short-reads and long-reads, to realize the full-automatic mining of genes from Reads to virulence factors. The previous three input files (assembled genomes, ORFs and protein sequences) are still supported, and genome assembly can be used independently.
  • Fixed a bug where BtToxin_Digger often reported errors when processing assembled genomes.
  • Added support for CDSs and not limited to ORFs.
  • The database has been updated, adding support for App, Gpp, Mcf, Mpf, Mpp, Mtx, Pra, Prb, Spp, Tpp, Cyt, Vip, Vpa, Vpb, Xpp (Crickmore, et al., 2020) and other virulence factors including Sip (Donovan, et al., 2006), Chitinase (Zhang, et al., 2014), InhA (Dalhammar and Steiner, 1984), Bmp1 (Luo, et al., 2013), Bel enhancin (Fang, et al., 2009), ZwA (He, et al., 1994).
  • BtToxin_Digger generates comprehensive and readable outputs including toxin list and sequence for each input; a matrix of all strains and the virulence factors it contains (behavior strain names, listed as virulence factor names), which can be used as virulence factors contained in the strain Database; and a file writes the information and sequences of all toxins (Table 1) to facilitate centralized processing and downstream analysis and experiment designs.
  • Added multi-thread support, greatly improving the running speed of the pipeline.

• Install with the source codes

  Not recommended, this method is complex and needs a lot of time to complete.

  • Before the installation, create a directory named "tools" in users home directory by executing the following command in the Linux terminal.

    # Create the directory
    mkdir ~/tools
    
    # Add the directory to Linux environment variable
    echo export PATH="$PATH:$HOME/tools" >> ~/.bashrc
    source ~/.bashrc
    
    # Enter the directory and download the dependency package to the tools directory
    cd ~/tools

  • Next, the required dependencies should be installed accoding to their official instructions.

    + As long as these software are added to the environment variables,
    + BtToxin_Digger can call them itself.

    • BioPerl

      # Install cpanminus first
      curl -L https://cpanmin.us | perl - --sudo App::cpanminus
      
      # Install BioPerl with cpanm
      cpanm BioPerl

    • HMMER

      # Install HMMER3 with the tar package
      
      # Download the package
      wget http://eddylab.org/software/hmmer/hmmer.tar.gz
      
      # Decompress the package
      tar zxf hmmer.tar.gz
      
      # change to the package directory, notice the number behind the "hmmer-"
      cd hmmer-3.3.2
      
      # Configure
      ./configure --prefix ~/tools
      
      # Compiling the programs
      make
      
      # Just make a test
      make check
      
      # Install the tools
      make install

    • libsvm

      # Install LIBSVM with source codes
      
      # Clone the codes to tools directory
      git clone https://github.com/cjlin1/libsvm.git
      
      # Change directory to libsvm
      cd libsvm
      
      # Compiling the programs
      make
      
      # Add the directory to Linux environment variable
      echo export PATH="$PATH:$HOME/tools/libsvm" >> ~/.bashrc
      source ~/.bashrc

    • NCBI-blast+

      # Download the tool
      wget https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/ncbi-blast-2.11.0+-x64-linux.tar.gz
      
      # Decompress the file
      tar zxvf ncbi-blast-2.11.0+-x64-linux.tar.gz
      
      # Add the directory to Linux environment variable
      echo export PATH="$PATH:$HOME/tools/ncbi-blast-2.11.0+/bin" >> ~/.bashrc
      source ~/.bashrc

    • Perl

      Linux usually comes with Perl and does not need to be installed again.

    • PGCGAP >=version 1.0.24

      A light version of PGCGAP v1.0.29 was added in the GitHub repo of BtToxin_Digger now. So users just need to install a few dependencies of PGCGAP.

      • fastp
      • ABySS and its dependencies
      • Unicycler and its dependencies
      • canu
      • bwa
      • Racon
      • SPAdes

    • Perl modules

      • File::Tee
      • Pod::Usage
      • List::Util

        # Easy commands to install the three perl modules
        cpanm File::Tee
        cpanm Pod::Usage
        cpanm List::Util

  • Then, install BtToxin_Digger

    • Download the source codes

      git clone https://github.com/liaochenlanruo/BtToxin_Digger.git

    • Rename the main program and move affiliate scripts to the path of the main program

      # Rename BtToxin_Digger.pl to BtToxin_Digger
      mv BtToxin_Digger/BtToxin_Digger.pl BtToxin_Digger/BtToxin_Digger
      
      # Move the attachment scripts to the directory where the main program is located
      mv BtToxin_Digger/Scripts/*.pl BtToxin_Digger/
      
      mv BtToxin_Digger/pgcgap/* BtToxin_Digger/
      
      # Give the scripts executable permission
      chmod +x BtToxin_Digger/*

    • Add the path to the environment variable

      echo export PATH="$PATH:$HOME/tools/BtToxin_Digger" >> ~/.bashrc
      
      source ~/.bashrc

    • Test the installation

      Download the test data from the tests directory in the GitHub repo and change to the tests directory in the terminal. Run the following commands:

      BtToxin_Digger --SeqPath ./ --SequenceType nucl --Scaf_suffix .fna

      Users should get six files shown as in the Result_files directory.

• Install with Bioconda

  • First, If you don't have Miniconda in your system, please install it.

    # Assume Linux system
    wget https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh
    
    bash Miniconda3-latest-Linux-x86_64.sh
    
    source ~/.bashrc

  • Then, config the bioconda channels

    conda config --add channels defaults
    
    conda config --add channels bioconda
    
    conda config --add channels conda-forge

  • Now, install BtToxin_Digger with conda

    conda create -n toxin python=3
    
    conda activate toxin
    
    conda install bttoxin_digger

  • Test the installation

    Download the test data from the tests directory in the GitHub repo and change to the tests directory in the terminal. Run the following commands:

    conda activate toxin
    
    BtToxin_Digger --SeqPath ./ --SequenceType nucl --Scaf_suffix .fna

    Users should get six files shown as in the Result_files directory.

• Install with the docker container

  docker pull quay.io/biocontainers/bttoxin_digger:<tag>

  (See bttoxin_digger/tags for valid values for <tag>)

BtToxin_Digger --update-db

• 2021.08.02: Added Vip1Ea1, Vip1Ea2, Vip2Ah1, Vip2Ah2, Vip3Cb1, Cry1Ia37, Cry8Ba2, and Mpp82Aa1
• 2021.10.11: Added Cry1Ac40, Cry1Be6, Vpb1Ea1, Vpb1Ea2, Vpb4Aa2
• 2021.12.27: Added Cry1Ca16, Cry1Ia44, Cry3Aa13, Tpp80Ab1
• 2022.01.28: Added Cry1Da6, Cry3Aa14
• 2022.06.09: Added Cry1Ca17, Cyt8Aa1, Mcf1Ac1, Mpp75Ab1, Mpp75Ab2, Xpp84Aa1, Xpp85Aa1
• 2022.12.08: Added Cry1Bk1, Cry1Ia42, Cry1Ia43, Cry2Ab41, Cry9Ea12, Mpp51Aa4, Mpp83Aa1, Vip3Aa86, Vpb1Ac2, Xpp86Aa1

BtToxin_Digger [Options]

Options:

[--help]                      Print the help message and exit

[--version]                   Show version number of BtToxin_Digger and exit

[--threads (INT)]             Number of threads to be used ( Default 4 )

[--update-db]		      Update the toxin database

[--SeqPath (PATH)]            [Required] The path of input sequences ( Default "the current directory" )

[--SequenceType (STRING)]     [Required] Sequence type for inputs. "reads", "nucl", "orfs", and "prot" avaliable ( Default nucl )

[--platform (STRING)]         [Required] Sequencing Platform, "illumina", "pacbio", "oxford" and "hybrid" available ( Default illumina )

[--assemble_only (STRING)]    Only perform genome assembly without predicting toxins.

[--reads1 (STRING)]           [Required by "reads"] The suffix name of reads 1 ( for example: if the name of reads 1 is "YBT-1520_L1_I050.R1.clean.fastq.gz", "YBT-1520" is the strain same, so the suffix name should be ".R1.clean.fastq.gz" )

[--reads2 (STRING)]           [Required by "reads"] The suffix name of reads 2( not required by "oxford" and "pacbio". For example: if the name of reads 2 is "YBT-1520_2.fq", the suffix name should be _2.fq" )

[--suffix_len (INT)]          [Required by "reads"] (Strongly recommended) The suffix length of the reads file, that is the length of the reads name minus the length of the strain name. For example the --suffix_len of "YBT-1520_L1_I050.R1.clean.fastq.gz" is 26 ( "YBT-1520" is the strain name ) ( Default 0 )

[--short1 (STRING)]           [Required] FASTQ file of first short reads in each pair. Needed by hybrid assembly ( Default Unset )

[--short2 (STRING)]           [Required] FASTQ file of second short reads in each pair. Needed by hybrid assembly ( Default Unset )

[--long (STRING)]             [Required] FASTQ or FASTA file of long reads. Needed by hybrid assembly ( Default Unset )

[--hout (STRING)]             [Required] Output directory for hybrid assembly ( Default "../../Results/Assembles/Hybrid" )

[--genomeSize (STRING)]       [Required] An estimate of the size of the genome. Common suffixes are allowed, for example, 3.7m or 2.8g. Needed by PacBio data and Oxford data ( Default 6.07m )

[--Scaf_suffix (STRING)]      The suffix of scaffolds or genomes ( Default ".filtered.fas" )

[--orfs_suffix (STRING)]      The suffix of orfs files ( Default ".ffn" )

[--prot_suffix (STRING)]      The suffix of protein files ( Default ".faa" )

• Processing Illumina paired-end Reads

  BtToxin_Digger --SeqPath <Illumina Reads PATH> --SequenceType reads --platform illumina --reads1 <suffix name of reads 1> -reads2 <suffix name of reads 2> --threads <INT> --suffix_len <INT>

• Processing PacBio long Reads

  BtToxin_Digger --SeqPath <PacBio Reads PATH> --SequenceType reads --platform pacbio --reads1 <suffix name of PacBio reads> --threads <INT> --suffix_len <INT>

• Processing Oxford long Reads

  BtToxin_Digger --SeqPath <Oxford Reads PATH> --SequenceType reads --platform oxford --reads1 <suffix name of Oxford reads> --threads <INT> --suffix_len <INT>

• Processing Hybrid Reads (Long reads + illumina short reads)

  BtToxin_Digger --SeqPath <Reads PATH> --SequenceType reads --platform hybrid --short1 <short reads 1> --short2 <short reads 2> --long <long reads> --threads <INT>

• Processing assembled genomes

  BtToxin_Digger --SeqPath <Assembled genome PATH> --SequenceType nucl --Scaf_suffix <suffix of genomes> --threads <INT>

• Processing protein sequences

  BtToxin_Digger --SeqPath <Protein file PATH> --SequenceType prot --prot_suffix <suffix of protein files> --threads <INT>

• Processing orfs sequences

  BtToxin_Digger --SeqPath <orfs file PATH> --SequenceType orfs --orfs_suffix <suffix of orfs files> --threads <INT>

• Results/Assembles/*: Genome assembly results;
• Results/Toxins/*.list: Toxin list of each strain;
• Results/Toxins/*.gbk: Toxin sequences in Genbank format of each strain;
• Results/Toxins/Bt_all_genes.table: A matrix describes Strains vs. Toxins. See Table 3 for details;
• Results/Toxins/All_Toxins.txt: A table containing all information and sequences of all toxin genes. See Table 1 and Table 2 for details.

Contents of '*.list':

Overview of prediction 	 Sequence type: nucl
Name	Cry	Cyt	Vip	Others	Summary
Rank1	1	0	0	0	1
Rank2	0	0	0	0	0
Rank3	0	0	0	0	0
Rank4	10	0	1	1	12
HMM_SVM	0	0	0	0
Summary	11	0	1	1	13
//


Toxin type: Cry protein
ID	Protein_ID		Protein_description	Length	Rank	BLAST	Best_hit	Hit length	Coverage	Identity	SVM	HMM
1	NHPK02000085.1_00018	+2 19034-20206 len=391	391	Rank1	YES	Cry78Aa1	359		97.21		32.66		NO	NO
2	NHPK02000099.1_00001	-1 5374-7407 len=678	678	Rank4	YES	Cry2Ab35	633		100.00		100.00		NO	YES
3	NHPK02000115.1_00002	+2 2-1342 len=447	447	Rank4	YES	Cry1Da2		1165		38.37		100.00		NO	YES
4	NHPK02000115.1_00006	+3 4275-7898 len=1208	1208	Rank4	YES	Cry1Ca15	1189		100.00		100.00		NO	YES
5	NHPK02000153.1_00001	-1 4176-6365 len=730	730	Rank4	YES	Cry1Ia40	719		100.00		100.00		YES	YES
6	NHPK02000168.1_00002	-3 1673-5146 len=1158	1158	Rank4	YES	Cry9Ea9		1150		100.00		100.00		YES	YES
7	NHPK02000196.1_00001	+3 723-2942 len=740	740	Rank4	YES	Cry1Da2		1165		63.43		100.00		NO	YES
8	NHPK02000240.1_00001	+2 2-1813 len=604	604	Rank4	YES	Cry1Ac16	1178		51.27		100.00		NO	YES
9	NHPK02000243.1_00001	+2 2-1738 len=579	579	Rank4	YES	Cry1Aa18	1225		47.27		100.00		NO	YES
10	NHPK02000256.1_00001	+2 2-1294 len=431	431	Rank4	YES	Cry1Ac30	1178		36.59		100.00		NO	YES
11	NHPK02000386.1_00001	-2 3-530 len=176	176	Rank4	YES	Cry1Ab11	695		25.32		99.43		NO	YES
//


Toxin type: Cyt protein
ID	Protein_ID	Protein_description	Length	Rank	BLAST	Best_hit	Hit length	Coverage	Identity	SVM	HMM
//


Toxin type: Vip protein
ID	Protein_ID		Protein_description	Length	Rank	BLAST	Best_hit	Hit length	Coverage	Identity	SVM	HMM
1	NHPK02000099.1_00004	-3 10013-12397 len=795	795	Rank4	YES	Vip3Aa12	789		100.00		100.00		NO	YES
//


Toxin type: Other toxins
ID	Protein_ID		Protein_description	Length	Rank	BLAST	Best_hit	Hit length	Coverage	Identity	SVM	HMM
1	NHPK02000017.1_00027	-3 15272-16258 len=329	329	Rank4	YES	Zwa5B-other	322		100.00		99.07		NO	NA
//

Contents of '*.gbk' (Partial):

LOCUS       NHPK02000017.1_00027        77664 bp    dna     linear   UNK
ACCESSION   NHPK02000017.1_00027
FEATURES             Location/Qualifiers
     Segment         complement(15272..16258)
                     /ETX_MTX2="NO"
                     /Endotoxin_C="NO"
                     /Endotoxin_M="NO"
                     /Endotoxin_N="NO"
                     /Endotoxin_mid="NO"
                     /Rank="Rank4"
                     /Segment_name="NHPK02000017.1_00027"
                     /Toxin_10="NO"
                     /blast_detail="Query_desc:-3 15272-16258 len=329
                     Query_Length:329	Query_Start-End:8-329	Hit_id:Zwa5B-other
                     Hit_desc:AAZ67352.1	Hit_length:322	Hit_Start-End:1-322
                     Aln_length:322	Percent_identity:99.0683229813665
                     E-value:0.0"
                     /blast_prediction="YES"
                     /hmm_detail=""
                     /hmm_prediction="NA"
                     /locus_tag="NHPK02000017.1_00027"
                     /protein_desc="-3 15272-16258 len=329"
                     /protein_id="NHPK02000017.1_00027"
                     /protein_len="329"
                     /svm_prediction="NO"
                     /translation="YRNEEAIMMYLNTKHINEMGVNWEETINVISKAVQSLDAEDFSQ
                     PIKPYLRFDDPANRIIAMPAYIGGEFKVSGIKWIASFPKNIEKGIQRAHSVTILNDAM
                     TGKPFATLNTAMVSVIRTASVTGLMIREFAKLRDLNNVKVGIIGFGPIGQMHLKMVTA
                     LLGDKIESVYLYDINGIKDELIPEDIYSKTQKVNAYEEAYNDADIFITCTVSAEGYID
                     KKPKDGALLLNVSLRDFKPDILEYTKSLVVDNWEEVCREKTDVERMHLERGLQKEDTV
                     SIADVVIRGALQNFPYDKAILFNPMGMAIFDVAIAAYYYQRAREKEIGVLLED"
ORIGIN      
        1 cttctaaccg caaggactgc ggggttagcc taatcaatta gagttcgata gaactcttta
       61 cttaggaatc cctcacttct aaacgaagtg aaagtggggt tagttcaaaa ctattaacta
      121 taatataccc tttcaagaaa ttttaaaaag gttgaagtag ccaaaaaata ttttccggaa
      181 taattagatt aatttctctt ttttgtatat ttatgttaaa ttattgttat cactacaaat
      241 ttattgaata attttaatac tctccccttt atactatggt aatatgtttt tcacaataca
      301 tattaccact ataattgcaa acatatataa acccatattt agaattttta agattctttc
      361 atagcattaa gatatttttt accttttagt ttgtttattc ttaattttta aactaaaata
      421 atatatattg gtaataatta aataaaattc caataattat aggaaggaga aaattatgaa

Table 1: Description of 'All_Toxins.txt'

Table 2. The contents of 'All_Toxins.txt' (Partial)

Footnote: BtToxin_Digger simultaneously uses BLAST, HMMER, and LIBSVM to predict insecticidal proteins. These three methods complement each other to discover insecticidal proteins as much as possible. However, due to the differences in algorithms, not all of them can identify a protein sequence as insecticidal protein at the same time. Columns 6, 7, and 8 of Table 2 show which algorithm or algorithms the insecticidal protein was identified by. YES indicates that the insecticidal protein was identified by the algorithm, while NO and NA indicate that the insecticidal protein was not identified by the algorithm. It should be noted that the insecticidal protein predicted by any method should be effective.

Table 3. The contents of 'Bt_all_genes.table'

Footnote: The float number represent the identity of blast search, and the integer number represent the number of toxins predicted by HMM and SVM. Genes with a blast coverage less than 50% are not shown in this table.

BtToxin_Digger is free software, licensed under GPLv3.

Please report any issues about usage of the software to the issues page.

• If you use this software please cite: Liu H, Zheng J, Yu Y, Ye W, Peng D, Sun M. BtToxin_Digger: a comprehensive and high-throughput pipeline for mining toxin protein genes from Bacillus thuringiensis. Bioinformatics, 2021. DOI: 10.1093/bioinformatics/btab506.

• If you used the genome assembly function, please also cite: Liu H, Xin B, Zheng J, Zhong H, Yu Y, Peng D, Sun M. Build a bioinformatics analysis platform and apply it to routine analysis of microbial genomics and comparative genomics. Protocol exchange, 2020. DOI: 10.21203/rs.2.21224/v5.

• v1.0.2

  • Fixed a "Can not find path" error.

• v1.0.3

  • Fixed a bug of "get_all_info_nucl.pl", which can not get the gene location and strand information of some toxins.

• v1.0.4

  • Updated the database and models to support the latest clasiffication of Bt toxins.

• v1.0.5

  • The name of strains with no toxin found will be outputed into the file "Strains_without_toxins_found.txt".

• v1.0.6

  • Update to adapt to PGCGAP v1.0.23, and to delete the intermediate files.

• v1.0.7

  • To delete the intermediate files.

• v1.0.8

  • To delete the intermediate files.

• v1.0.9

  • Update the database to keep it consistent with the BPPRC (to 2021-06-07).
  • More HMM models are added.
  • The filter length of amino acid sequence was reduced from 115 aa to 75 aa.
  • Solved a problem: BioPerl reported an error when the length of the sequence in the input file was inconsistent.
  • A light version of PGCGAP was added to the BtToxin_Digger repo to simplify software installation.

• v1.0.10

  • Users can use the command "BtToxin_Digger --update-db" to update their local database.
  • The toxin database was updated to 2021.08.02.

• Crickmore, N., et al. A structure-based nomenclature for Bacillus thuringiensis and other bacteria-derived pesticidal proteins. Journal of Invertebrate Pathology 2020:107438.

• Donovan, W.P., et al. Discovery and characterization of Sip1A: A novel secreted protein from Bacillus thuringiensis with activity against coleopteran larvae. Appl. Microbiol. Biotechnol. 2006;72(4):713-719.

• Zhang, L.L., et al. Biological Activity of Bacillus thuringiensis (Bacillales: Bacillaceae) Chitinase Against Caenorhabditis elegans (Rhabditida: Rhabditidae). J. Econ. Entomol. 2014;107(2):551-558.

• Dalhammar, G. and Steiner, H. Characterization of inhibitor A, a protease from Bacillus thuringiensis which degrades attacins and cecropins, two classes of antibacterial proteins in insects. Eur. J. Biochem. 1984;139(2):247-252.

• Luo, X., et al. Bacillus thuringiensis Metalloproteinase Bmp1 Functions as a Nematicidal Virulence Factor. Appl. Environ. Microbiol. 2013;79(2):460.

• Fang, S., et al. Bacillus thuringiensis bel protein enhances the toxicity of Cry1Ac protein to Helicoverpa armigera larvae by degrading insect intestinal mucin. Appl. Environ. Microbiol. 2009;75(16):5237-5243.

• He, H., et al. Zwittermicin A, an antifungal and plant protection agent from Bacillus cereus. Tetrahedron Lett. 1994;35(16):2499-2502.