______ _ _____ _ ______ _
| ___ \ | |_ _| (_) | _ (_)
| |_/ / _| |_| | _____ ___ _ __ | | | |_ __ _ __ _ ___ _ __
| ___ \|__ __| |/ _ \ \/ / | '_ \ | | | | |/ _` |/ _` |/ _ \ '__|
| |_/ / | |_| | (_) > <| | | | | ______ | |/ /| | (_| | (_| | __/ |
\____/ \___\_/\___/_/\_\_|_| |_| |______| |___/ |_|\__, |\__, |\___|_|
__/ | __/ |
|___/ |___/
A web server of BtToxin_Digger can be found at https://bcam.hzau.edu.cn/BtToxin_Digger.
| Introduction | Installation | Database |Usage | Examples | Outputs | License | Feedback | Citation | FAQs | Updates |
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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.
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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.
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# Install cpanminus first curl -L https://cpanmin.us | perl - --sudo App::cpanminus # Install BioPerl with cpanm cpanm BioPerl
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# 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
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# 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
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# 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
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Linux usually comes with Perl and does not need to be installed again.
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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
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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
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- Then, install BtToxin_Digger
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Download the source codes
git clone https://github.com/liaochenlanruo/BtToxin_Digger.git
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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/*
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Add the path to the environment variable
echo export PATH="$PATH:$HOME/tools/BtToxin_Digger" >> ~/.bashrc source ~/.bashrc
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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.
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- Before the installation, create a directory named "tools" in users home directory by executing the following command in the Linux terminal.
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Install with Bioconda
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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
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Then, config the bioconda channels
conda config --add channels defaults conda config --add channels bioconda conda config --add channels conda-forge
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Now, install BtToxin_Digger with conda
conda create -n toxin python=3 conda activate toxin conda install bttoxin_digger
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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.
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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
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" )
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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>
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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>
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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>
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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>
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Processing assembled genomes
BtToxin_Digger --SeqPath <Assembled genome PATH> --SequenceType nucl --Scaf_suffix <suffix of genomes> --threads <INT>
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Processing protein sequences
BtToxin_Digger --SeqPath <Protein file PATH> --SequenceType prot --prot_suffix <suffix of protein files> --threads <INT>
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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'
| Header | Description |
|---|---|
| Strain | The name of your input |
| Protein_id | The protein ID |
| Protein_len | The length of protein sequence |
| Strand | Positive or negative strand where the gene comes from |
| Gene location on scaffold | Gene coordinates on the genome |
| SVM | Is the protein predicted by SVM |
| BLAST | Is the protein predicted by BLAST |
| HMM | Is the protein predicted by HMM |
| Hit_id | The subject sequence ID |
| Hit_length | The length of subject sequence |
| Aln_length | alignment length (sequence overlap) |
| Query start-end | Start and end of alignment in query |
| Hit stard-end | Start and end of alignment in subject |
| Identity | Percentage of identical matches |
| Evalue of blast | Expect value of BLAST |
| Hmm hit | The subject model ID |
| Hmm hit length | The length of subject model sequence |
| Evalue of Hmm | Expect value of HMM |
| Nomenclature | Bt nomenclature containing 4 Ranks |
| Endotoxin_N | Whether the Cry protein contain Endotoxin_N domain |
| Endotoxin_M | Whether the Cry protein contain Endotoxin_M domain |
| Endotoxin_C | Whether the Cry protein contain Endotoxin_C domain |
| Endotoxin_mid | Whether the Cry protein contain Endotoxin_mid domain |
| Toxin_10 | Whether the Cry protein contain Toxin_10 domain |
| ETX_MTX2 | Whether the Cry protein contain ETX_MTX2 domain |
| Gene sequence | The nucleotide sequence of the toxin |
| Protein sequence | Amino acid sequence of the toxin |
| Scaffold sequence | The scaffold sequence where the toxin gene is located |
Table 2. The contents of 'All_Toxins.txt' (Partial)
| Strain | Protein_id | Protein_len | Strand | Gene location on scaffold | SVM | BLAST | HMM | Hit_id | Hit_length | Aln_length | Query start-end | Hit stard-end | Identity | Evalue of blast | Hmm hit | Hmm hit length | Evalue of Hmm | Nomenclature | Endotoxin_N | Endotoxin_M | Endotoxin_C | Endotoxin_mid | Toxin_10 | ETX_MTX2 | Gene sequence | Protein sequence | Scaffold sequence |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1126_1 | NHPK02000017.1_00027 | 329 | -3 | 10001-10987 | NO | YES | NA | Zwa5B-other | 322 | 322 | 8-329 | 1-322 | 99.0683229813665 | 0.0 | NA | NA | NA | Rank4 | NO | NO | NO | NO | NO | NO | GTCTTCTAATAAAACACCTATTTCCTTTTCCCTAGCCCTTTGATAGTAATAAGCAGCAATGGCTACATCAAAAATGGCCATACCCATTGGATTAAATAATATTGCTTTGTCATAAGGGAAGTTTTGCAGTGCTCCTCGGATTACAACGTCAGCAATTGATACTGTATCTTCTTTTTGTAAACCTCTTTCAAGGTGCATCCTTTCAACATCAGTTTTTTCACGACAGACTTCTTCCCAATTATCAACAACTAGGGATTTTGTATATTCTAAGATATCAGGCTTAAAATCACGAAGAGAAACATTGAGTAGTAGCGCTCCATCTTTAGGTTTCTTATCAATATAACCTTCTGCAGAAACAGTACAAGTAATAAAAATATCAGCATCATTATAGGCTTCTTCATAAGCATTAACCTTTTGAGTTTTAGAATAAATATCCTCAGGAATTAATTCGTCTTTAATTCCATTGATATCATATAGGTAAACACTTTCGATTTTATCGCCTAATAGGGCAGTTACCATCTTTAAATGCATCTGACCAATAGGTCCAAATCCAATAATTCCAACTTTAACATTATTTAAATCTCTTAACTTGGCAAACTCTCGAATCATTAACCCTGTTACAGAAGCTGTTCGTATTACACTGACCATTGCTGTATTAAGCGTCGCAAATGGTTTTCCAGTCATAGCATCATTTAATATAGTAACTGAATGAGCACGTTGAATACCTTTCTCAATATTCTTCGGAAAACTAGCTATCCATTTGATTCCTGAAACCTTAAATTCTCCTCCAATATAAGCTGGCATAGCAATAATTCTATTAGCCGGATCATCAAAACGTAAGTATGGCTTAATTGGCTGAGAAAAATCTTCTGCATCAAGACTTTGTACTGCCTTTGAAATGACATTTATTGTTTCTTCCCAATTAACACCCATTTCATTGATATGTTTAGTATTTAAATACATCATAATTGCTTCCTCATTTCTATA | YRNEEAIMMYLNTKHINEMGVNWEETINVISKAVQSLDAEDFSQPIKPYLRFDDPANRIIAMPAYIGGEFKVSGIKWIASFPKNIEKGIQRAHSVTILNDAMTGKPFATLNTAMVSVIRTASVTGLMIREFAKLRDLNNVKVGIIGFGPIGQMHLKMVTALLGDKIESVYLYDINGIKDELIPEDIYSKTQKVNAYEEAYNDADIFITCTVSAEGYIDKKPKDGALLLNVSLRDFKPDILEYTKSLVVDNWEEVCREKTDVERMHLERGLQKEDTVSIADVVIRGALQNFPYDKAILFNPMGMAIFDVAIAAYYYQRAREKEIGVLLED | TCTCAATACGAACCAGTAAAAATGGTTCAGGGCGTTGAAGATACTGGACGTAATTCTTTTAGCCAAGCTAGTTTTTCAAACGTACTTCTAAGAACAGATACATCTGGAGCTACCTACAAAAGTTGGAACGGTTCAATTTCTTCCTCATTTTCTAAACACGGTACTTATGGTAACAGTTTCACAGTTTATTCTCAAGTGCCATTAAGCGCAAGTTTACGCTAAAATTGTTCTTGTATACGAATAATTTTGTGAAAAAGGTAGCTGTACTTTTGTACAGCTACCTTTTTCATATGGTGAATTTTTAATAGATATTGTATCAGACATAAGTTACATTCTTCATATTTTAACTTCTTTGAATTCAATAATACTTAATCAGCTTGTCTTATTACTTCACTTAACGTATCGAGATACAGCTTATAGCAACTACAACACGCTTTATTATTAATAATACCACTCTTCTTAATTTAACTTTAATTCCATTGCATTGATTTTTTAATAATAAATATACAACTTAGTAATGATAATAGATATACAATAAGAATAATATACAACTGTTTTGGTTCAATGCTATCGATATAAAGACTCATAATTGGATATGACCAAGGAAACAGTATCCAATAGTTTGTATGCGAGATAAAAAAGCTACCCAACCCGCCTATAACCCCTACTAATAATGAAAGTAACGGGATTTTATAAAATATTATATTTATTAAAAAATGTAAATTCACAATAGTTAAAGAGCTAATCACCATAAAAAGTATAGAAAAAATTAATCTTTGATAAACAGTCCCAAGATTATCAACCGTTAATACTATAGGTATACTAGTTATGACTAAAGCACATATTAAATAACCAAAAGTAATTATTAAGCAAAACAAAAACTTATTACTTAATATAGTTTTTAAAGTGATTCCGTGATAAATAGCAGACATATATCCTTTTCTTCTGAACTCGTTTACAAAAGAAAATGACACTAATAGACCTATTGCAATAGGTAAAAATAATCTACAAAAAAGACTTAGAATCGCTATATGCATGAAAACGCCATCTTTTGATTGAGATGATACAGTTAGAAAGATTTGAAAACTAATTGGTATCAACAGAAATACTAGTAACAGGATATACAATCCATTATGAAGACTTTTTTTATATTCACCTAATATACCCACTTATTTCACCCTTTATTATTCATATGTATAATATTTAAAACTCACAGCTAATAACATAATTCCTAAAATATAGATTGAAGAACTTGCAAGATATATTAAAATAGTTGTTTCTTTCATCATTACATCACTTACTATATTCAATGCATAAAATATTGGAAATGTCTTTGTGAATACTACAGGAAATAATCCATAAGTTACGCTAAAAAAAATCCAAATAACTGACAAGGACGCCGCAAGAATTCCCTTTTTTATAATCAAATGAAAAAGTAATTGAGAATATGGTATTGCTAAAGATAGAATTGTACAAAGCATCAACATTTTCAAAAAATAAAGTATAGATATTTCATATCCATTTATAAAAATATAGATACCCATAACTGAAAAATTTAAAATATTAAAGATTAAATAAAACAGTATAAACAATAAATTTTTAGCTACCAATAATTTAAATTTTGAAATTGGCTTTGTATAGATAATGGCCCAAGTACCATATTTATTTTCAGAAGAAATGATAAAATACCACATACAAGCAGAAAAAATGCTTAATGTTAGTGTATTAAAGTTTACTGTAATTGCTATAAGATTGGTAGATTGAAACATATCGGGATTATTCTTGTTAAAAATAAAATAACTTATACACAAAAAGCTTGATAATAATGAAAAAAACAAAGAAAATAACAATAATTTTCTCCCACCAATTTTCCTCATCTCACTATTCACACTTTTAAAGATAAATTGCATTATGATAGTAACTCCTTTTGTTTAGTTAAAGATAAAAATACTTCCTCTAATGTTAGCTTTTTAGGGCTAACTTCAAAAATATCTATATCATTTTCAATTAATATTCTGGTTAAATTAGGAACACTATTTTGATCTATCATTAGTTTGAATACCCCTTCTTTTTCTATGCTATAAGGTATATCCTTTGTATTTAAAATCCTTTCTGTGGCAGTAGTGTCATTAACATGAACAATATATTCTCTAGAAGTAATAATACTGTTTAAATTCCCTTCATAGAGTAAGTTACCCTTATGCAGTAATGCTATGTAATCTGCTATCATTTCGATTTCAGCTAAATTATGACTAGAAATAAATATTGTCTTCCCCTCATTTTTTACTAAATGAAGTAATAAGCGCCTTATCTCGTGTACTCCCTCTGGGTCTAATCCATTTGTCGGCTCATCAAGAATTAGTATTTTAGGATCATTAAGAATAGCAAAGGAAATACCTAGCCTTTGTTTCATTCCTAAAGAAAAATCTTTCACTTTTTTATCTTTTGCTTTATATATTCCTGTAATTTTTAATACTCTGTCTATTTCATCTAAAGGCTTATTAATCATTTTTTGTACATAGGCTAAATTTTCATATGCTGTCAAATTAGGATAGTAGGTAGGTGAATCAATAAAACAACCCACATTTTCAAATAATTCTTCTTTCCAATCTTTGATATCTTTATTATTAAATAATATCGTTCCTTTATCTAATGGAATTAATCCTAATAGCATTCGCATTGTTGTTGTTTTTCCTGCACCATTTGGTCCAATAAAAGCATATAAACTTCCTTCAGGAACAGATAAGTTTAAATCCCTTACAATGGATTGATTGTCAAAAGACTTTGTTAAATTTTTAGTTTGGATTGCTAATTTCATTTTGTTCCTCACTTTCTTATTCTCAAATAAAAGTAAGTATCTATTAAATTCTTGATCTATTAGATATTCTATTTACCCTTTACCTAATGTTTGCAATTTGGATTTAGAAAATATTTCTCGTTGTTTTAATTTTTTATCAGTAAGTTCATAAACTCTATCTACACGCTCTAAAATATTTTCATCATGAGTGATAATAATCTTAGTGGATGGTATGGCAAATATATTTTCAATAACAGCATTTGCCGTTTCCCTATCTAAATGGTTAGTTGGTTCATCCAAAATAAGAATCGGATATTCTTTCATGAACAATCGTAACATAGCCAACCTTTGTCTTTGGCCACCGGAGAAATTTGTTCCTCCCTCTGAGATTAAAATATCATTTATACTTTGAAATTGAATATAATTACTCAGATTCAATTTATCAATAGACTCCTGGATTTTTGAAGGATCATAATTTATATCAAAGTAAGATAAATTCTCAGATAAACTTCCTCTAAATAAAACTCCATCCTGAGTCATTAGTCCTACATTTTTTCTTAGTTCCTCACTGTTCCAATCTTTAATATCTCTATCGTCTATTAATATATTACCCTTATCGGGAGTATGCAATTTTAATAATAAGTTAGCTAAAGTGGACTTACCACTGCCACTTTCTCCAACAATAGCAATACTTTCGTTATTTTTAATGTTCAAACTTAAGTTTTCTATTATTTTTTTGTCATCAAAACTAAACGAAATATTATTAAATTGAATCGATCCTCTAAGAATGTTTACTTTAGTATCCGATCCATCTTTTTCTAGTTCCTCTTCCAAGATATCTAAAGTTCTTAATAAAAGTGGTTTAACTGAATTCCAATTTAAAATTGATCCTATAATTGTTGAAACCGGAGAAAATATAATACTAGATATAGCAATAAAGAAGAAAATATTACTTAAATCCCCTTCTCCTTTACTAAAATTATAAAATCCTACAGCAAGCATTATGATAATAAATATTGTTGATAGGGAACTATTAAATGATTCTAAAGCATTTTGTTTTAGCGCTCGTTTATGTACTGTATCTAAGTATTTATTATAATCTTTTTGCCATACAGATTTAATTCTAGAAAAAATCCCTGTAGCCTTTACATAATACATCGCTTTTAATGATTCATTAATTTTCCCTCTATGTTCTCCTAGGTAATACGTTTCGACTAAATTAGCATTATATATGAATTTCCCTAAGGAAATATTGATAAATAGGGAACTAGCAATAAAAAACATCAATATTAAAGTATAAAAAGTCTCAGTATATAATAGATACATCAATAATGGAATGATAACTACTATTGATATAATCATTTCAATTAGATCCTCTAAAATATATCCTCGTATTGATTCTAGGCTCATGATTCTTACATTTAAATCCCCTGCATGACGTGTAGTAATTTTTTCTATGGGGGTATTAAACATATGCTCAAAAACTTTTGTTGTACTTTTTCTATCAATTTCTTTCTGAAAGTTTACTTTTATAATAGAAGTTATAGATTTTATCCCTATAACAACGATTAAACTTCCCAAAATAATTAAAAGGTATCTTACATCCACTTCCTTTGAAGCATTCAAAATATCCATAAACTTTTTCAAAATAAAGGGGAAAGCAAGAATTGAGATTTGTGCTAACAATGCTAATAGTGCTAATAAAACAATCTGGCTAGGAGATACAAAATGATCAATATATCTAAGTAATATATGTTTTTCACGCCTTTTAGTATTTGGCTGTATATCTTTTTTGTTTTCTATTACTAGGATATAGTCACAGAACATTTCCTCAAAACTTCTTATATCAATTATCATCGGCCCTGCCTTAGGATCTACAATATAAAATTTATTTGCTTTAACCCTTTCAATAATTACAAAGTGATTAAAGCCCCAAAATGCTATCATCGGCTTTTGTATCCTTTGTAAATCAGTTATATTTTCTATCCTATATGCAGTTGCAGAAAGTCCATACGATTTACTAACTTTTTTAATATCCAACAAACTCCATGCCGATTTTTTATTGATAATTTTACTATTTCTAATTTCCGAAGCTTGTACTGCTGAACCATAATAATGCATAATCATTGCTAGACAACTAGGCCCACAATCAAAAGATGTCATTTGCGGTATAAATTTAATTTTCTTCCTTAACATATTTATCACCTATTCTAGGCATTAAAGTATAAAATGCTATTTAGTTTATTATCCTCTAACCTTTGTAAAAAATACAAAATACTTCCTAGCCCTGTAAATAATGCGATTTTATGAGGATTAAAATCTTCTATATCAAAATTATTCAAAAAGTTATTTGTTACTTTCATAACAATTTGTTTTTCATTGCGCTCGAGCATACCTCTATTTTCTAGCTCCAATAAAAAGTCTAGTTTCCCCATTAAACCATGACATAAACAATAATCACTATTTTCATGTAATGTACTTAAGATTTGTTTTTTAAAAAAAATTAAATCTGATTGTATATCCTCATTTTGATACGATTCTAAAATTTTAATTCTCGAGATTCCCATTCCACTAAATCCTTTACACCAGCTATCTGTATACTCATATTCACTCATACTCATTTTCTCACTTTTATGTATACCATGTATTACTCTATCGTACTTATTTGTTTTTAATATGCCTTTTAACTTATAAAATGCGAGAAGTTGTCCTGATAATCCATGAACAAACCCCTTTAATGAATCCTCTAAATTTAGAGTATCGTGAGCTTTCCAATATACAAATTCATTATGTGTTTGCATATTATTGACAATGTAGTCACCGAGATTTTCAGCCACTTTAAGCAATATTTTATAATTAGGATTAATTTGATAGAACTTAACTGCCGTTAAAATTAGACCAGCTGAACCCCCTAATATATCAAAATGCTTATCGCTATATTTACCTTTTTCAATATCAGAGTTGACATCCATAAATATTTGCAATATCAATCTTTCATCGATAGATGTTATATGTGGTAAATTTGTTAAATAGTAAGAAATAATTGCATTAACACCATTTACAAAACCATAATTATTTTTATTTTTGTTTAATATTTCTTGCAGGGCTTTTTGTTCAAGTTTATTTATAATTTCAATCTGATTAGGATCAGCATTATTTCTCAATAATGAACTTATTCCCAATAATCCATCATATATTCCACTATTCAAATGGTTTATCTCAAGCTCCCCTTGCCAATTTGTTTTTAATCCAATAAAATCTACAGCACCGTCTTTTGATATATAACTGCCATCAAGAATTTTCCCTGTAATTGCTTCCTTTATTTTATTTGTAGGTAAATTAGTTCGAAAATTAATTGTTTCCTTTACTGTATTCAGCTTATATAGATTGTCATTTTGATTAGAAAGGGATATTTCTATAATTTTTCTTTGTAATAAAAAGTCAGCATATGAAATTTTTTTAAATTTTGTTTCTAGATCTATTTCATTATTTGATAAACTTTCGCAAGACTCCCATTCATACTTATTAAAGAAATATGGAACATCATAGGATAATAGTGCCTCTATCTCTTTTTGAATAATATCTTTCCTAGTAGCCAAGTATATGTTTTTTTCAAGAATATTCCGCAAATATTTTATAGTCTTTAATTTATTCTCTAATAAATCCGGAACATTTAGTCTATCTAATAAAAGGGTATAGACTTTTGTATTTCGATATACTTTTCGATAATTCCATTTAGAAACACTATTTTTTAATATACACATATACTCCTCTTTGTTTTTTAAAACTGAGTTATATGCATTCTCAAAACCTTTAATAATATCATCTATATAATTATCATACTCGTATATATCATCGTTAAGAAACGGTAAATTCTGAACCGTATCTTCTACTATTGTCTCTTCTCTTATTTCAATTGGATTCGATGTAAATTCATTTTTTATTTTAATTGTTTTTACCAAAGACTTCATTACTCTCCCAATTGCACTGTAATCTCTTATTACCTCCCTACCTCCACTAAACCTTATAGGTAGCATTCTAGAATTTAATACTGAATTAGATAATATAAAACTACTGGTTTCTTCAGTAACACTTGGATTTAATGTACTTCCTAATGTCTCAAAATCAATTATGACTGGTGAGGATTGTTTAGCTATGATATTTTCATTATGTATGTCACTTCCATTAATCAAATACATTACCGAAAGCACTACACCTAAATTATAATAATAATCTTTAATTTGCTCATTAGATTCACATGATTCATGATTAATGTATTCCATATATCCATAATTTCTTTTATTAACTACTTGCGGAATAAAGATATCCGTATTTAAATCTTTATTCAGTTGATTAATTAAACTATGATATAGTATAGAATTTTTCAAATCGACTGGTTTTAAAATTACCTTATTATTAAATTTATCTTCTAAAAGAATTGTTCTCTTTCCATTATTATGGTAGTCACCTAGGTTAATAACTTTTTCTACATGCATAATATTAAAACCCGACATTTTATGAATTTCTTTTTGATCTTTTTCAAGAGCACCTTGCAACCAAATCATATAATTTATTTCATTTTCAATGATTGTAAAAATTTTTTCGTTTAATACGGGTAATATTTTAAAAAAATTCATATAAAATTTTTTAAAATGAAAGAAGTTCTTTGTAAAATACTTATATTCTTCATTGGTATCCATTCCTCTTAAAGCATTTTCTACCCTAAGTTTATTTATGTATACAATTAAGGATGGCCTTAATACTTGGTATAGTCTTTTTTCTAAATCAAACATTATCGATTCAGAAACCTTCTCAAAATTACCAAAAAAATCAATTTTATTTATTATCATATTTTTAAAGAATACTACATATGTTTGTATTATTTTTTGAAAGTTATAGTTTTCGTCATCCTTAATGACATCATTACATGAAAAACCCTCTAAAAAGATAACAAAATTTTTTTCTAGATATTCTTTTGTTACTAAAGAATGTGATCCATTATCTATTGTATTCAAAATTCCATGTGAATTATTTTTCATAATAACCTCTTCTTTTTATAGGTAGGAGAAACAGTTAGAAATTCATTCTCATTAAAGAGAGTAAGGCCTTTCGCCTTACTCTCTTTAACTATTAGTAGCATTTTTTACAAATTCTTTGTCCCATAATCGTGACACATGCTACAGGCACATTCCAAGGACAATCTTTAGTTAAAGTTTCAACCCAACCAGGTCCTGCTCCACCAACTAATTGTTCAATCTCTTCATCTTCTACGACTTGTAAATATTTTTCAGTTTCCATTTCAATACCTACCTTTTATTTTTTTGAAAATGATGTTATAAATTCAATAATTTACCACAACATTATCTAAAAACAATATACCATTAAATGTTAATAAAAAAAAGATATATTTATAGAAAAATAAAATAATAGGATATTTAACTATATTAATATAATTAAAGGAGACTCACTTACTATATCATTAAAATTTAAAGGCTATTTTCCAAGTTAATATTTTTAAAATTTTATAAATTTATTAATAATCAATGCTGTTTAATAATTTAAATAATTCTTCTAATACCTGAATAACGATTAAGAATATTAATTAAACTTGTAAAAAATAGATTTTCAGAAGATATAGCTCAAGAATATTTTACCATAAAATAGGATAATCAATTCTTTTTTTAATAGTATAATGCTTCGTCAGAAAATTTTAATCATGAATTGCAAAAAGCATGTTAAAAATAAAAAGATGAAATTGCAAGTATATTCACTTCACAATCCCATCTCTTCTAAAAATTGTATATCATCAACTCTCTTGATCCTCGCAATTCTACCAAATTAATAGCAACACATTATGTAGATATAGGTTTATACTATTCTACATTTAAATACATGTAATATTGCCTATACTTTGTTATTTTCATGTGAAACTAATTCACTATATATCTCTTTCTCTATATTTTTCAAGAGAATTTCCAACCTATTTTTTGTAGCTAGATTAAAATGTGGATTTAATTCTTTTATTATAAATTCAAGTATCTCAAAGCTTATGTAATCTCCTAATTCAAGTGAATATTGATCACCCAAGAAAGTTTTTAATTTTTTAGAGATCTCCACTTTATCTTTTTTGGTAATATTTACCCCATCCGATAACAAACCTCTCTTAGATTAGTCTTAAATACTAATTCAAAAAGTTTTTATAACATAATAAAAATATTTTGGAATTAATATAAATCATATAATGAAATTTTTACAAATAAATAAAAAATATACATCATAATCATTAAACAATATATTAAGCATCGTAAACATATATTGGCTCGTCATAAAGGCGTAAACATTTTGACCAATTTCCACATAACGTTTCCCATTCACCAGCAGGATATACTTCCGTACGCAATATATGAAGTGGTATACGTAAATTCAATAACCGACCACTAAGCGTCGTACGAGTAACTCCCGCTGGCATTAAAAACTTTGCTTCAATTACCTTTTTCAGTTCCATCAACGAGTATTCTGGATAACTCATCAATATTTCATTTGAATGAGGCCAGACCTCAGTATCATTTGAATGACGGCGTACCGTATACTCATGTTGATATAAGCTGACAATACGATGCCATATCTCTAAATGATTAAATAAATCGCTATTTAAATCCCTTGCATATAAAAAATGTACTTGTTTATTTGCTTCTGTAATTTTTGCAAGCAAACGTTTATTAGCAATAATCTTACCTGACCAAATTATTGTAGGTTCTTTCTTTAAATTATTTACCCATTCCCCCATTGGTAATAAATGATTCCAAGCACGAATTTTTATTTTTTCATCAGGTACTACCTGTACAGCAACGCGTTTACAACCTAATGCCATCATAGCCTTCATACGATGGGCACCATCAAGTAACAGATAATTACCATCACTAAGCTCAGATACTAAGGGAGGATTCCGTAAAACACCTTCGCTTTCCATTACACGACAAATTTGTTCCAATCTTTTGTGTTCATATGATTCATGAAAGCGAATTTGCTTGGGATGTAAGAGATCTAAGGCCGAAATAATTTCACTCATAAAAACAGCTCCTCAAATAATTACATAAATTCTAAGCTTATCAAATAACTAATTTTCTATAATGATTAGTCGTGTTTACCGATATTTAGCAGCTATTAGACAATTATTTCAAACTACGTTTAATAATTTTACTAGTCTTCTAATAAAACACCTATTTCCTTTTCCCTAGCCCTTTGATAGTAATAAGCAGCAATGGCTACATCAAAAATGGCCATACCCATTGGATTAAATAATATTGCTTTGTCATAAGGGAAGTTTTGCAGTGCTCCTCGGATTACAACGTCAGCAATTGATACTGTATCTTCTTTTTGTAAACCTCTTTCAAGGTGCATCCTTTCAACATCAGTTTTTTCACGACAGACTTCTTCCCAATTATCAACAACTAGGGATTTTGTATATTCTAAGATATCAGGCTTAAAATCACGAAGAGAAACATTGAGTAGTAGCGCTCCATCTTTAGGTTTCTTATCAATATAACCTTCTGCAGAAACAGTACAAGTAATAAAAATATCAGCATCATTATAGGCTTCTTCATAAGCATTAACCTTTTGAGTTTTAGAATAAATATCCTCAGGAATTAATTCGTCTTTAATTCCATTGATATCATATAGGTAAACACTTTCGATTTTATCGCCTAATAGGGCAGTTACCATCTTTAAATGCATCTGACCAATAGGTCCAAATCCAATAATTCCAACTTTAACATTATTTAAATCTCTTAACTTGGCAAACTCTCGAATCATTAACCCTGTTACAGAAGCTGTTCGTATTACACTGACCATTGCTGTATTAAGCGTCGCAAATGGTTTTCCAGTCATAGCATCATTTAATATAGTAACTGAATGAGCACGTTGAATACCTTTCTCAATATTCTTCGGAAAACTAGCTATCCATTTGATTCCTGAAACCTTAAATTCTCCTCCAATATAAGCTGGCATAGCAATAATTCTATTAGCCGGATCATCAAAACGTAAGTATGGCTTAATTGGCTGAGAAAAATCTTCTGCATCAAGACTTTGTACTGCCTTTGAAATGACATTTATTGTTTCTTCCCAATTAACACCCATTTCATTGATATGTTTAGTATTTAAATACATCATAATTGCTTCCTCATTTCTATATCATTTTTATAAAGAAACTAATTGGTCTTCTACTGATTTTTTTGTATTAAGCCATTCTACCCATTCTACGTTGTAAATAGTTGATGTGTAAGCTTGTCCATTATCAGGACACAAGAAAACAACATTAGGTGTATTTTGAACATCCCTATTTTCAAAATACTTTTGGATTGCATAATATGAAGTCCCTGATGATCCACCAGCAAATATTGCATGTTTATTAAATAGCTCATAACAACCTGCAACAGTATGGACCTCCGGTACAATCATTACATCATCAATCAATGCTTTTTTAACCATACCAGGTATCATACTGGCACCAATTCCTGGTATGTACCGTTTACGAGGCTTATCACCAAAAATAATGGACCCTTGACTATCTACCGCAATAATTTTAATATTAGGGAATTTTTCTTTTAATCGTGTAGATACCCCAGCGATAGTTCCTCCAGTACTCACTCCAATGAAGGCATAATCTAACTGTTTAAAATCATTAGATATTTCTTCACCAATTCCTTGATAATGGGCTTCAAAGTTATCAGCATTATTATATTGATTTGTCCAGTATGCATTAGGAATAGTATTTAAAAGTTCTTCCACCTTATTTAAACGCGTTAATAAATAGCCACCTGTTTCATCTCGTTCATCCACTTTAGCTACTTGATAGGAAGTTGCTCTCAAAAAATTCTCATAACTATCATTAATATTTGGATCAATAACTGGTATGAATTTCAGGCCGATATACCTACAAAGAGTAGCAAGAGCAACCGCAAAGTTACCAGATGAAGATTCGATAATTGTAGAATTTTCAGTCACTTCACCGCGACTTATTGCTGATTTTAAAATATGGTGAGCAGCACGCACTTTAACACTATTCATTAAATTGTGATATTCTAGTTTTGCATACAGATTAATCTTTTCATGCTCTAATTTAATCATAGGTGTGTTGCCGATTACCCTTTCTAAACTCTCTAATTTCTTGAGCATAATTTGCTTCCTTTCTTGGTTAAAATCTAAAAAAATAATTAAAGATAAATAGTTGTAAATGTTCTTTCGAATGTATTTCAAATAGAACTTATACCTGAAAGACAATAAATGCTAGATTCTTTAGTATCGATAAGCTAAGCACCATTCAAGTACTGCCATAGCACTATTTAGCTTGTTTTCGGCCTGGGTAAATACAATACTTTTCGGTCCATCAATAACTGTTGCTGCTACTTCTTCCCCCCGGTGTGCTGGGAGGTCATGCATGAAAGTAGCTTCCGGATATAAAGTCAAAATTCTCTCTGTCACTGCAAATGGATTAAAGGCATCTTTCCATAAAGGATCTATTTTGCTTGTCCCTGTTGTTTGCCACCTTGTTGTATATACAACATCCACTTCACTAGGTAACATTTTTATATCATGACATTCAATAACTTTAGCACCAGATAATCGTGCATATTCTTTTGATTTTTCAAGCACACTTGGAGACACTCCATAACCAGCAGGTGTAAAAAGATATAACTCCGTACCTGGAAAACGAGATAAAGAAAGAGCTAGAGCTGCAGCACTGTTATTACCTTCTCCCATATATAAAACCCGTAATCCAGCTATTTCACCAAATTTTTGTTTCATTGTTGTTAAGTCTGTTAGCGCTTGTGTTGGATGTTCTTCCGTACTCATTGCATTAATGACTGCCATACGACTTTGAGATGCCAGCTTTTCCATTTCCTTTTGACTATCTGCAGTTCTTGCTACTAGTGCATCCAGCATTCGCGAAAGTACTTGAGTAGTGTCTTCTATAGACTCCCCTGTATTTTCTTGCAAATCATTTGGACCATACGTTACAATTTGTGCACCCATCTTTAATGCTGCAACAGAAAATGCCGATCGAGTTCTAGTCGATGTTTTACGGAAGTAAATCCCAATTATATTTCCCGCTAATATTTGATTAGGTTGTGATTTACCTGTGGCAAACTCTACCCCACGAGTTACAATTTGGTTAATATCACTATCAGTAAGATCCTCAAGAGTAATTAAATGTTTTCGAATAGCCATTATTATATTACCTCCCTTTTGAATAATTGCATGTAACAAAAATGTATTTACATATGTATGTAAATAAAAACTACTAAACAGCTAAAGTAAAAATAATAGATTGGAAATATAATTCATGTGGATCCATTACCACACTTTTCTATAATTTTTTTTAAACTAATGTATAGCAGTTGATATATAGCCTATATGAACTCGAATACTATTTTTACTCAGAAATACTTACATATAATTCTATTGTTAGTCTATATTCCTATTTTTTATGATATAGCTCTGCTGAATATATTACACACTACAACACTAAAAATATACTCAAAATACAGAATTTTCAGCTTTCTCAATTCTTTCATCAATTTTCTGTCAATTCATACTCAAATTATCTTTTTTACTAATAATATACATACAATGTTGGCTGTTTCTTTTATAGCCCACTTCTAAAAAATCCACCCATTCAAAAATAACGTTTTCCGGATTGTCAGTTAAGATACAAATTACTGCTGCAGTCATTAAAGAGTCCTCTTTAAAAGAGATATACTCCTTCTACTGGCTCGCTATTTTTAGTTATGATAACCTCTTAATTGTTACAGCTATAACGCTAACAATTGTTATTGCAACCATTGATCTAAGTAAACAAACAATGAATGACATACTAATATATACACAAAGTAATCTATTAAGCGTAAAATGATTGCATCTCCAGCAAGTTTAATTACTTATTCAGATCTTTGCTTTTATAATTTTCAAAATTAAATTTCCGAAAATTTTCTTTCCTTAAGGAAATATCGATCATTTTAAACTATGTAAGAATGCTTATCTTCTATAAAGTAATTACACAAATCAACAAAGTCAATTTCAATTATTGATTCTGGAAAACTATCTAAAGAAGCACAACAAGAAAGCTTATAATCTTTACCAATAGTAAACTTTTTATAATAAACCTCATTAGATAGCATGTCACCATAAAATATTATATTTTTTTTAATAACAAAAGAGTTTAAAGGTATAGTTAGTCCTTTCCCTATCCATTTAACAAAGCTTTCTTTTATAGTCCATAATTCATAAAACACATCTAATTGTTGTTCTACATTTAATGAATTTAAATAATTAAATTCTTCCTCAGTAAATATATTTTTAATCATTGTAGTATCCATGGGCCGGACTTTTTCTACATCTATTCCTACTTCTTCCTCATGAATAGCTCCAACAACCCAACTTTCGGAATGCGATATATTAAAATGAAAATTATTTAACTCATCCACATAAGGCTTTCCGTATTCATTGTATTTATATTTAATATCTTTATTTTCTTTTGAGAAATTTGTAATAATTAAATATCTTATTATAATGTCTCCAATTAAGGCGCTATATTGGTCGGGCTTTCTTTTATATTTCTGTATTTTCATCTGCTTTTCTTTTGAGACCAAACTCATAAGTTTCTGCATAATGTTATGTTCTATATTTCCTGGCACACGTACCTTATATATATTCATTATTTCCTTCCCACCTAACTTCTTATATAAATATAAAAAACTCTCAATGAATAATTCCTATTTATTTTTGATTAGTAAAAAACATTTTCAGAGGATAATATTCTATTTTACATTCATATTTTCGACTTATTTTCTAACCTCTTTATGTCTATTATTGAATGGGCTCAAAGAAAAAAGTCTCATAATATGTTATACACGAATTTATGAGACTTTTAGAAAACTAATAGTGTACAATTTCAAATAATATTTCACCTATTTACATGTGCTAATAAGAGCATTTGTTTAAAACTCATTTTTATACTCTTTACCAACTCATGAATATGCGGTTTCTCTAACATACTCAGATGGGAACCTGGCACTTGCAAAGCGTATACTTCCCCTGATGTATATTCATTCCATCTGTTATAATCTACTAGTGGGTGAATGTCATTAATAGATGCATTAAATAGAAAGATATCTGCTTTTATTTTTTGTTTACAATTATATTTTAGGTATGCATATCTATTAGCTATCATTACTTTTAACTTATTCATCATTGGATCTTCAAAATTTTGCTGACAACTATTTTTATTCAATGTAAATTTTTTCAGTAAAGATTCTATTAATTGTTCTTCACTCATTTGCTCAAAACTAATTTTCTCAATCCCTAACTGATCATTGAATTTTTCCAATTCTTCAAAGGCATTCTTAATATTTAAACTAAGAATCTCCTTACCTTGTTCAATAGGATGAACATCAAGTAAACCTAGAAAACTAACTTTATCACCTAGTTCTTCTAATTTTCTAGCCATTTCAAATGCAACTATTCCTCCAAAAGACCATCCTAATAAGGCATATGGCCCTTCTTTTTTTACTTGTTTAATCTCTTCTATATATCTTACCGCCATTTCCTCTACAGATAAGTTTGAAAATCTATTATCATCATATCCTATAGATTGTAACCCATATACAGTCTTATCCTCTCCTAATTCTCTAGCCAAATCATAATAGTTTAATATGCCCCCTCCTTGTCCATGAACAATGAACCATTGACTATCCTTGTTTGTCCCATTTTGAATTGGAATTAAACACTCACTGTCTATTCCTTTATTCCTACTTATTACATCACTTAATTGTTCAATAGTAGCATTTTGAAATAATAAACTTAATGGCAGTTGCACATTAAACATTCTCTTGATATTTTCGAATAACTTTAATCCCTTAAGAGAATGACCACCTAATTCAAAGAAATTATCATTTATACCAATATTATTTACGCCTAATATACTACTCCAAATATCAATTAAACTACTATCTATGTCATTTCTTGGTGGTACATAATTACTATTGTCCAGTGTATTTAGCTTCGGTAACTTACTTCTATCTATTTTCCCATTTTGTGTTAATGGTATATTGTGAATTGGTATGAGTTGTTGAGGTATCATATAATGTGGTAACTTTGTTGCCAAATATGCTCTTACCTCTGGAATAGGAATATCTTTTTCTGTAACTACGTATGCACATAAATACTTTTCCCCAGCTTCATCTTCTTGATCTATTACTACGGCCGTTTTGATTGTCTCATATTTTAACAAACTCGCTTCTATCTCACCTAGTTCTATTCGATATCCCCTTATTTTTACTTGATGATCGACTCGGCCCAAGTATTCAATGTTACCATCAGGTAGCCACCTTGCTATATCCCCTGTTTTATATAGTTTCTCACCTCGTTCAAATGGATGATCAATAAATTTATCTGCTGTTAATTCTGTTCGATTGATGTATCCTCTAGCTAACCCTATGCCAGAGATACATATTTCACCTGGAACACCTATAGGTTGTATCCTATAAAATGAATCCAATATATAAATTTTAGTATTTAACAATGGTGAACCTATCGGTACGTTTTGTGTTGTAATTTCTTTATTACTCTCATATCGATAAGATGTACAACAAACTGTAGCCTCTGTAGGTCCGTATCCATTTAATATTTGGAGGTTCCCCCTAAATAGATGATCGTATTTTGCGAGTAATTCTGTTTTAATGGGTTCTACTCCCACAAGTAGCTTATTTAACACTATCTTCTGGTTATCTCTAACAAAATAATCATATATTTCATTTAATAAAGTAGGTGGAATATATGATAATGTAACCTGTTCTTCAAGAATAACTTGTACAAGTTTTGATACATCAAACTTCTCACCTTGATAAATAGTCATTCTTGCGCCATATATCAATGGGACAAATATCTCAAAAATAGTAACATCAAAAGAAATACTACTTGAGAATAGAACGTTATCAGTTATCCCTATATCTTGAGAGAAATCTTCATACATTGCACACAAAAAATTAGTCAAGGATCGATGTTCAATCATTACTCCTTTAGGTTGTCCTGTAGAACCTGATGTATAAATAACATACGCTAAGTTGTGAGGTTCTATCATCATTTGCATGTCTTCTCCTGGTTCTTCTTCAAAAGACATATCCATTAGATCTATTACATTACCTTGGAATTCTATCCCCTTTATAATAGAGTTTTGATGTACTAATACATGTGAACACCCACTGTCTGTCAGCATATATTCCACTCTTTGTTTTGGTAAGGCGGTATCAATTGGTAAATACGCCCCACCTGCTTTTAAAACACCTAATATACCTATAACCATCTCGATGGATCGTTCCATCATCACACCAACAATGGATTCTCTTTTAACTCCTTGGTCTAATAACCTTCTCGCCAACTGATTAGCTTTTATATTTAATTCATTATATGTAATTCCTTTTTCATTACATACAACGGCTATTTGATTAGGGTTCCGTTTTACTTGTTCTTCAAACATTTTATGCACTAATAAATGATTAGAATTTGAATTCTCTTTTTTGTTAAATTCATTCATAATACAATGTTCTTCTTCTATAGATAACATATTAATATTACGCAATCGTACTCTAGGGTTATTAGTTACTTCCTCAACTATATTTGTAAAATGTACCATTAACCTTTCTATTGTCTCCGCTTTAAATAACTTAGTACTATATTCTACTTTTAAATGAATGTTATTATCTATTTCCGTTGCTACTAATGACAAATCAAATTTTGAAACTGACTGCTTAAACGGATACGGTGTAAATTCTAATTCACCAATAGATATTGGATTCATATCCATGTTTTGAAAAACAAACATGGTATCAAATAATGGATTTCTACTTGTATCCCTATGCAAGTCTAAACCTTCTAATAGTTCTTCAAAAGGATAGTCTTGATTTTCATAAGCTTCTAATGTATTGAGTTTTAATCTACTCAAAAACTCAATAAACTCATCGTCATTTTCTAGATAATTTCTCATTACCAAAGTATTAATAAACATACCAATCATATGATTAGTATCAGAATGAGACCTTCCAGCAATAGGCGAACCTACAATAATGTCTTCTTGACCTGTGTATCTAGATAAAAGTATGTTATAAATGGCCAATAAAATCATATATGGCGTAGTACCAGTTTCAGTTGCTAGCTTATTTACTTTAAAAGTTAAATCTCTTCCCAAATTAAAAGAACAAACATTACCTTTAAAGCTTTGTATAGTCGGTCTTTGAAAATCGGTTGGAAAATTTAAAACCGGGAGTTCTCCTTTTAAAGTTGTTAACCAATAATTCTTTTGTTCACTAATTAGATTCTTATAGTAAGGTCCATTTTGCCACATCACATAGTCTTTGTACTGCACTCTCAATTTTGGAAGCTCATTTCCTTTATACAATTCTACAAACTCTTTTATTAATATCCCCATTGATAAACCATCAGATATTATATGATGCATATCTACTACAAGGATATGTCTTTCTTCTGCTATCCTTAAAAGCAACACCCTTAATAATGGAGGTTTTGATAAATCAAATGGACTTATAAACTCATGTATTAAATAATCCGCATCTTTTTCATTTACATGAACGTATTCAATATTGAAATCTACATTAGGCTCAATTTTCTGCACTAATTCCCCATCTAAAATTTGAAAAGAAGTTCTTAATATTTCATGTCTCTCAATTAAAGATTGAAATATATTTTCAAACTTATCTTTACAAATATCCCCTTCTACTTTAAGTATTGTGGGCATATTATAAGTTGTATTTGTACCATCTTCGAATTGATCTACTATAAACATTCTCTTTTGTGACGTAGAAGCTAAATAATACTCTTGTTGTTTTACAGGTTCTATGGAAATATAATTACTTTTTTCCATTTCTAATATACATTTTGAAAAATCAACCAAAATAGGAAACTTAAATAGGGATTTAATAGATAATTGCACATTAAATTCTTTATTAACGATAGAAATTAGCCTAGCAGCCTTTAATGAATGACCACCTATCTCAAAAAAATTATCCCGTATTCCTACCCTTTGTATTCCTAATACATCTTTCCAAATCTCAACTAATTTTCTTTCTGTAGAATTTGTCGGCTCTAGATGACTAGATTTCAAATTATTTATAGGTTGAGGTAATTTTTTTCTATCTATTTTTCCGTTTTGTGTTAACGGTATGTTTTGAATGGATATGATTTGTTGAGGTATCATATAATATGGTAACTTGGTTGCTAAATATGCTCTTACCTCTGGAATAGGAATATCTTTTTCGGTAACTACATACGCACATAAATACTTTTCTCCACTTTCATCTTCTCGCTGTATTACTACGGCGGTTTTGATTGTCTCATATTTTAACAAACTTGCTTCTATCTCACCTAGTTCTATTCGATATCCCCTTATTTTCACTTGATGATCAACTCGTCCCAAGTATTCTATGTTACCATCAGGTAGCCATCTCGCTATATCTCCTGTTTTATATAATTTCTCACCATGTTCAAATGGATGATCAATAAATTTATCTGCTGTTAATTCTTTTCGATTGATGTATCCCCTAGCTAACCCTATGCCAGAAATACATATTTCTCCTGGAACACCTATAGGTTGTAGCCTGTGAAATGAATCCAATATATAAATTTTAGTATTTAACAATGGCGAACCTATTGGTACGTTTTGTATTGTAATTTCTTTATCCCTCTCATATTGATAAGATGTACAACAGACTGTAGCCTCTGTAGGTCCGTATAAATTTAATATTTGGAGGTTCCCCCTAAATAGATGATCGTATTTTGCAAGTAATTCTGTTTTAATTGGTTCTACTCCCACGAAAAGTTTATTTAACGATATCTTCTGATTCGCCCTTACAAAATAATCATATATTTCATTTAATAAGGTAGGTGGAATATATGCTAACGTGACCTGTTCCTCAAGAATGACTTGTACAAGTTTTGGTACATCAAACTTCTCACCTTGATAAATAGTCATTCTTGCTCCATATACCAATGGGACAAATATTTCAAATATAGTAACATCAAAAGAAATACTACTTGAGAATAGTACATTATCACTTATCCCTATATCTTGAGAGAAGTCCTCATACATTGCACACAAAAAATTAGTTAAGGAACGATGTTCAATCATTACCCCTTTGGGCTGGCCTGTAGAACCTGATGTATAAATAACATATGCCAAATTTTGAGGTTCCATCGTTATCTGCAAATCTTCTACTTGTTCTTCTTCAAAAGGAATATCCATTAGATTTATTACACTACCTTGAAATGCTACTCCCTTTATAATAGAGTTTTGATATGTTAGTACATGTGAACACCCGCTGTCTGTCAGCATATATTCCACTCTTTGTTTTGGTAACTCGGTATCAATCGGTAAATACGCCCCACCCGCTTTTAAGATACCTAATATGCCTACAATCATCTCAATAGAGCGTTCCATCATCACACCAACAATGGATTCTCTTTTAACTCCCTGGTCTAATAACCTTCTCGCCAACTGATTAGCTTTTATATTTAATTGTTTATATGTAATTTCTTTTCCATTACATACAATCGCTATTTGATTAGGATTCTGTTTTACCTGCTCTTCAAATAATTGAGGAGCTGTTACAGTCTCACAAAGTAATGTTTTATGAACTCTAGTCGTATGATTGAAATCAAATAATATTTGATTCTTTTCTGTTTTAGGCATTACATCTAGATCCATTGCAGATTTATTTGGATCTTTCATTAATATATCTAAAATATTATATAAATGATTAACTATTCTACTAACTAATCCTTCACTATATAAAATAGAATTGTAATCCACTTGAACCTTTAACTGTTCTTCATTTTTCATAAACCTAATAACCATATCAGAGTTTATTTTATCTGTACTCTCATAACAATGAATATCATCTAACATTACTATTGTATTTAATAAGGGAAGATTATTACTCTCTCCATCTAGACTTAATAATTGAGTAAGTTTATTAAAAGGAAAATGACAATGTTCATTTGACTCTAAAATTGTTTCTTTAATTTTATATATTATTTCTTTAAAATTATCTTCTTGATTTATTTGAGTACGTAATAATAAAAAGTTGTTTTGAAAAACCGTCTCTTCTTGACCTTGTTTAAA 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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'
| - | Cry1Ac16 | Cry1Ca15 | Cry1Da2 | Cry1Ia40 | Cry2Aa10 | Cry2Ab35 | Cry78Aa1 | Cry9Ea9 | HMM_Cry_len_492 | HMM_Cyt_len_531 | HMM_Cyt_len_533 | HMM_Cyt_len_615 | Sip1A2-other | Vip1Aa2 | Vip3Aa12 | Zwa5B-other |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1126_1 | 100.00 | 100.00 | 100.00 | 100.00 | 100.00 | 32.66 | 100.00 | 100.00 | 99.07 | |||||||
| AFS094730 | 36.14 | 1 | 1 | 92.41 | 29.46 | |||||||||||
| AFS095482 | 1 | 1 | ||||||||||||||
| AK47 | 100.00 | 100.00 | 100.00 | 1 | 100.00 | 100.00 |
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.
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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.
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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.
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v1.0.2
- Fixed a "Can not find path" error.
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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.
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v1.0.4
- Updated the database and models to support the latest clasiffication of Bt toxins.
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v1.0.5
- The name of strains with no toxin found will be outputed into the file "Strains_without_toxins_found.txt".
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v1.0.6
- Update to adapt to PGCGAP v1.0.23, and to delete the intermediate files.
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v1.0.7
- To delete the intermediate files.
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v1.0.8
- To delete the intermediate files.
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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.
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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.
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Crickmore, N., et al. A structure-based nomenclature for Bacillus thuringiensis and other bacteria-derived pesticidal proteins. Journal of Invertebrate Pathology 2020:107438.
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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.
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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.
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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.
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Luo, X., et al. Bacillus thuringiensis Metalloproteinase Bmp1 Functions as a Nematicidal Virulence Factor. Appl. Environ. Microbiol. 2013;79(2):460.
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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.
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He, H., et al. Zwittermicin A, an antifungal and plant protection agent from Bacillus cereus. Tetrahedron Lett. 1994;35(16):2499-2502.