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BtToxin_Digger: High-throughput Bacillus thuringiensis toxin mining pipeline




Introduction

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_Scanner often reported errors when processing assembled genomes.
  • Added support for CDSs and not limited to ORFs.
  • The database has been updated, adding support for Cyt, Vip and other virulence factors.
  • 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.

Installation

  • Required dependencies

  • Source codes

    The BtToxin_Digger codes can be downloaded Here or from GitHub.

  • Install with Bioconda - OSX/Linux/WSL

    conda create -n toxin python=3
    conda activate toxin
    conda install bttoxin_digger
  • Install with the docker container 
    docker pull quay.io/biocontainers/bttoxin_digger:<tag>

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

Usage

    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 )
    [--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" )

Examples

  • 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 Coding sequences 
    BtToxin_Digger --SeqPath <CDSs file PATH> --SequenceType orfs --orfs_suffix <suffix of orfs files> --threads <INT>

Outputs

  • 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/Btallgenes.table: A matrix describes Strains vs. Toxins;
  • Results/Toxins/All_Toxins.txt: A table containing all information and sequences of all toxin genes. See table 1 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


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.

License

BtToxin_Digger is free software, licensed under GPLv3.

Feedback/Issues

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

Citation

  • If you use this software please cite: Hualin Liu, Jinshui Zheng, Weixing Ye, Donghai Peng, Ming Sun. BtToxin_Digger: a comprehensive and high-throughput pipeline for mining toxin protein genes from Bacillus thuringiensis. 2020, available at https://github.com/BMBGenomics/BtToxin_Digger.

  • 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/v3.

FAQs

Updates

  • 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.


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