Abstract:
Compositions and methods for isolating new variants of known gene sequences are provided. The methods find use in identifying variants, particularly homologs, in complex mixtures. Compositions comprise hybridization baits that hybridize to gene families of interest, particularly agricultural interest, in order to selectively enrich the polynucleotides of interest from complex mixtures. Bait sequences may be specific for a number of genes from distinct gene families of interest and may be designed to cover each gene of interest by at least 2-fold. Thus methods disclosed herein are drawn to an oligonucleotide hybridization gene capture approach for identification of new genes of interest from environmental samples. This approach bypasses the need for labor-intensive microbial strain isolation, permits simultaneous discovery of genes from multiple gene families of interest, and increases the potential to discover genes from low-abundance and unculturable organisms present in complex mixtures of environmental microbes.

Description:
FIELD OF THE INVENTION 
       [0001]    The invention is drawn to high throughput methods of gene discovery. 
       BACKGROUND OF THE INVENTION 
       [0002]    Given their diversity and abundance, microbial genomes represent an expansive untapped source for new gene discovery. Despite a relative lack of exploration, several gene families of agricultural and biomedical interest have been discovered in microbes and include genes that confer resistance to herbicides and pests in plants, as well as genes for antibiotic biosynthesis and antibiotic resistance. Current methods for new gene discovery from microbial genomes rely on screening isolated strains for activity in a bioassay and characterization of genes of interest by sequencing. However, complex samples containing mixed cultures of organisms often contain species that cannot be cultured or present other obstacles to performing traditional methods of gene discovery. Thus, a high throughput method of new gene identification where up to millions of culturable and non-culturable microbes can be queried simultaneously would be advantageous for identifying new genes or improved variants of known genes. 
       SUMMARY OF THE INVENTION 
       [0003]    Compositions and methods for isolating new variants of known gene sequences are provided. The methods find use in identifying variants, particularly homologs in complex mixtures. Compositions comprise hybridization baits that hybridize to gene families of interest, particularly agricultural interest, in order to selectively enrich the polynucleotides of interest from complex mixtures. Bait sequences may be specific for a number of genes from distinct gene families of interest and may be designed to cover each gene of interest by at least 2-fold. 
         [0004]    Thus methods disclosed herein are drawn to an oligonucleotide hybridization gene capture approach for identification of new genes of interest from environmental samples. This approach bypasses the need for labor-intensive microbial strain isolation, permits simultaneous discovery of genes from multiple gene families of interest, and increases the potential to discover genes from low-abundance and unculturable organisms present in complex mixtures of environmental microbes. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0005]    Methods for identifying variants of known gene sequences from complex mixtures are provided. The methods use labeled hybridization baits or bait sequences that correspond to a portion of known gene sequences to capture similar sequences from complex environmental samples. Once the DNA sequence is captured, subsequent sequencing and analysis can identify variants of the known gene sequences in a high throughput manner. 
         [0006]    The methods of the invention are capable of identifying and isolating gene sequences, and variants thereof, from a complex sample. By “complex sample” is intended any sample having DNA from more than one species of organism. In specific embodiments, the complex sample is an environmental sample, a biological sample, or a metagenomic sample. As used herein, the term “metagenome” or “metagenomic” refers to the collective genomes of all microorganisms present in a given habitat (Handelsman et al., (1998) Chem. Biol. 5: R245-R249; Microbial Metagenomics, Metatranscriptomics, and Metaproteomics. Methods in Enzymology vol. 531 DeLong, ed. (2013)). Environmental samples can be from soil, rivers, ponds, lakes, industrial wastewater, seawater, forests, agricultural lands on which crops are growing or have grown, samples of plants or animals or other organisms associated with microorganisms that may be present within or without the tissues of the plant or animal or other organism, or any other source having biodiversity. Complex samples also include colonies or cultures of microorganisms that are grown, collected in bulk, and pooled for storage and DNA preparation. For example, colonies can be grown on plates, in bottles, in other bulk containers and collected. In certain embodiments, complex samples are selected based on expected biodiversity that will allow for identification of gene sequences, and variants thereof. 
         [0007]    The method disclosed herein does not require purified samples of single organisms but rather is able to identify homologous sequences directly from uncharacterized mixes of populations of prokaryotic or eukaryotic organisms: from soil, from crude samples, and samples that are collected and/or mixed and not subjected to any purification. In this manner, the methods described herein can identify gene sequences, and variants thereof, from unculturable organisms, or those organisms that are difficult to culture. 
       I. Genes of Interest 
       [0008]    New gene sequences of interest, variants thereof, and variants of known gene sequences can be identified using the methods disclosed herein. As used herein, a “gene sequence of interest,” “target sequence,” or “target sequences” is intended to refer to a known gene sequence. Known genes of interest include cry genes (Hofte and Whiteley (1989) Microbiol. Rev. 53(2):242-255; U.S. Pat. Nos. 8,609,936, 8,609,937; cyt genes (or other hemolytic toxin or pest control genes, such as those listed in U.S. Pat. No. 8,067,671); mtx (or other mosquitocidal) genes; Binary toxins (such as those listed in U.S. Pat. No. 7,655,838); VIPs (or other vegetative insecticidal proteins, such as those listed in U.S. Pat. No. 8,344,307); SIPs (or other soluble insecticidal proteins); herbicide resistance genes such as EPSPS; HPPD; 16S rRNA sequences; and housekeeping genes. In particular embodiments, the gene of interest is of agricultural importance, such as genes that confer resistance to diseases and pests, and/or tolerance to herbicides in plants. Genes of interest can also be of biological, industrial, or medical interest such as genes as for antibiotic biosynthesis and antibiotic resistance, or biosynthesis of enzymes or other factors involved in bioremediation, bioconversion, industrial processes, detoxification, biofuel production, or compounds having cytotoxic, immune system priming or other therapeutic activity. Table 1 provides examples of gene sequences that can be used in the methods and compositions disclosed herein. The sequences and references provided herein are incorporated by reference. It is important to note that these sequences are provided merely as examples; any sequences can be used in the practice of the methods and compositions disclosed herein. 
         [0009]    The methods disclosed herein can identify variants of known sequences from multiple gene families of interest. As used herein, the term variants can refer to homologs, orthologs, and paralogs. While the activity of a variant may be altered compared to the gene of interest, the variant should retain the functionality of the gene of interest. For example, a variant may have increased activity, decreased activity, different spectrum of activity (e.g. for an insecticidal toxin gene) or any other alteration in activity when compared to the gene of interest. 
         [0010]    In general, “variants” is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a deletion and/or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a “native” or “wild type” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the native amino acid sequence of the gene of interest. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode the polypeptide of the gene of interest. Generally, variants of a particular polynucleotide disclosed herein will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide (e.g., a gene of interest) as determined by sequence alignment programs and parameters described elsewhere herein. 
         [0011]    Variants of a particular polynucleotide disclosed herein (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides disclosed herein is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. 
         [0012]    A. Sequence Analysis 
         [0013]    As used herein, “sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.). 
         [0014]    As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. 
         [0015]    Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. By “equivalent program” is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10. 
         [0016]    The use of the term “polynucleotide” is not intended to limit the present disclosure to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides can comprise ribonucleotides (RNA) and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides disclosed herein also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like. 
       II. Bait Sequences 
       [0017]    The methods and compositions described herein employ bait sequences to capture genes of interest, or variants thereof, from complex samples. As used herein a “bait sequence” or “bait” refers to a polynucleotide designed to hybridize to a gene of interest, or variant thereof. In specific embodiments bait sequences are single-stranded RNA sequences capable of hybridizing to a fragment of the gene of interest. For example, the RNA bait sequence can be complementary to the DNA sequence of a fragment of the gene sequence of interest. In some embodiments, the bait sequence is capable of hybridizing to a fragment of the gene of interest that is at least 50, at least 70, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 170, at least 200, at least 250, at least 400, at least 1000 contiguous nucleotides, and up to the full-length polynucleotide sequence of the gene of interest. The baits can be contiguous or sequential RNA or DNA sequences. In one embodiment, bait sequences are RNA sequences. RNA sequences cannot self-anneal and work to drive the hybridization. 
         [0018]    In specific embodiments, baits are at least 50, at least 70, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 170, at least 200, or at least 250 contiguous polynucleotides. For example, the bait sequence can be 50-200 nt, 70-150 nt, 100-140 nt, or 110-130 nt in length. The baits can be labeled with any detectable label in order to detect and/or capture the first hybridization complex comprised of a bait sequence hybridized to a fragment of the gene of interest, or variant thereof. In certain embodiments, the bait sequences are labeled with biotin, a hapten, or an affinity tag or the bait sequences are generated using biotinylated primers, e.g., where the baits are generated by nick-translation labeling of purified target organism DNA with biotinylated deoxynucleotides. In cases where the bait sequences are biotinylated, the target DNA can be captured using a binding partner, streptavidin molecule, attached to a solid phase. In specific embodiments, the baits are biotinylated RNA baits of about 120 nt in length. The baits may include adapter oligonucleotides suitable for PCR amplification, sequencing, or RNA transcription. The baits may include an RNA promoter or are RNA molecules prepared from DNA containing an RNA promoter (e.g., a T7 RNA promoter). Alternatively, antibodies specific for the RNA-DNA hybrid can be used (see, for example, WO2013164319 A1). In some embodiments, baits can be designed to 16S DNA sequences, or any other phylogenetically differential sequence, in order to capture sufficient portions of the 16S DNA to estimate the distribution of bacterial genera present in the sample. 
         [0019]    The bait sequences span substantially the entire sequence of the known gene. In some embodiments, the bait sequences are overlapping bait sequences. As used herein, “overlapping bait sequences” or “overlapping” refers to fragments of the gene of interest that are represented in more than one bait sequence. For example, any given 120 nt segment of a gene of interest can be represented by a bait sequence having a region complementary to nucleotides 1-60 of the fragment, another bait sequence having a region complementary to nucleotides 61-120 of the fragment, and a third bait sequence complementary to nucleotides 1-120. In some embodiments, at least 10, at least 30, at least 60, at least 90, or at least 120 nucleotides of each overlapping bait overlap with at least one other overlapping bait. In this manner, each nucleotide of a given gene of interest can be represented in at least 2 baits, which is referred to herein as being covered by at least 2× tiling. Accordingly the method described herein can use baits or labeled baits described herein that cover any gene of interest by at least 2× or at least 3× tiling. 
         [0020]    Baits for multiple genes can be used concurrently to hybridize with sample DNA prepared from a complex mixture. For example, if a given complex sample is to be screened for variants of multiple genes of interest, baits designed to each gene of interest can be combined in a bait pool prior to, or at the time of, mixing with prepared sample DNA. Accordingly, as used herein, a “bait pool” or “bait pools” refers to a mixture of baits designed to be specific for different fragments of an individual gene of interest and/or a mixture of baits designed to be specific for different genes of interest. “Distinct baits” refers to baits that are designed to be specific for different, or distinct, fragments of genes of interest. 
         [0021]    Accordingly, in some embodiments, a method for preparing an RNA bait pool for the identification of genes of interest is provided. A given RNA bait pool can be specific for at least 1, at least 2, at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 500, at least 750, at least 800, at least 900, at least 1,000, at least 1,500, at least 3,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 55,000, at least 60,000, or any other number of genes of interest. As used herein, a bait that is specific for a gene of interest is designed to hybridize to the gene of interest. A bait can be specific for more than one gene of interest or variants of a gene of interest. In specific embodiments, the sequences of the baits are designed to correspond to genes of interest using software tools such as Nimble Design (NimbleGen; Roche). 
       III. Methods of Isolating Genes of Interest, or Variants Thereof 
       [0022]    Methods of the invention include preparation of bait sequences, preparation of complex mixture libraries, hybridization selection, sequencing, and analysis. Such methods are set forth in the experimental section in more detail. Additionally, see NucleoSpin® Soil User Manual, Rev. 03, U.S. Publication No. 20130230857; Gnirke et al. (2009) Nature Biotechnology 27:182-189; SureSelect xT  Target Enrichment System for Illumina Paired-End Sequencing Library Protocol, Version 1.6; NimbleGen SeqCap EZ Library SR User&#39;s Guide, Version 4.3; and NimbleGen SeqCap EZ Library LR User&#39;s Guide, Version 2.0. All of which are herein incorporated by reference. 
         [0023]    Methods of preparing complex samples include fractionation and extraction of environmental samples comprising soil, rivers, ponds, lakes, industrial wastewater, seawater, forests, agricultural lands on which crops are growing or have grown, or any other source having biodiversity. Fractionation can include filtration and/or centrifugation to preferentially isolate microorganisms. In some embodiments, complex samples are selected based on expected biodiversity that will allow for identification of gene sequences, and variants thereof. Further methods of preparing complex samples include colonies or cultures of microorganisms that are grown, collected in bulk, and pooled for storage and DNA preparation. In certain embodiments, complex samples are subjected to heat treatment or pasteurization to enrich for microbial spores that are resistant to heating. In some embodiments, the colonies or cultures are grown in media that enrich for specific types of microbes or microbes having specific structural or functional properties, such as cell wall composition, resistance to an antibiotic or other compound, or ability to grow on a specific nutrient mix or specific compound as a source of an essential element, such as carbon, nitrogen, phosphorus, or potassium. 
         [0024]    In order to provide sample DNA for hybridization to baits as described elsewhere herein, the sample DNA must be prepared for hybridization. Preparing DNA from a complex sample for hybridization refers to any process wherein DNA from the sample is extracted and reduced in size sufficient for hybridization, herein referred to as fragmentation. For example, DNA can be extracted from any complex sample directly, or by isolating individual organisms from the complex sample prior to DNA isolation. In some embodiments, sample DNA is isolated from a pure culture or a mixed culture of microorganisms. DNA can be isolated by any method commonly known in the art for isolation of DNA from environmental or biological samples (see, e.g. Schneegurt et al. (2003) Current Issues in Molecular Biology 5:1-8; Zhou et al. (1996) Applied and Environmental Microbiology 62:316-322), including, but not limited to, the NucleoSpin Soil genomic DNA preparation kit (Macherey-Nagel GmbH &amp; Co., distributed in the US by Clontech). In one embodiment, extracted DNA can be enriched for any desired source of sample DNA. For example, extracted DNA can be enriched for prokaryotic DNA by amplification. As used herein, the term “enrich” or “enriched” refers to the process of increasing the concentration of a specific target DNA population. For example, DNA can be enriched by amplification, such as by PCR, such that the target DNA population is increased about 1.5-fold, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 15-fold, about 30-fold, about 50-fold, or about 100-fold. In certain embodiments, sample DNA is enriched by using 16S amplification. 
         [0025]    In some embodiments, after DNA is extracted from a complex sample, the extracted DNA is prepared for hybridization by fragmentation (e.g., by shearing) and/or end-labeling. End-labeling can use any end labels that are suitable for indexing, sequencing, or PCR amplification of the DNA. The fragmented sample DNA may be about 100-1000, 100-500, 125-400, 150-300, 200-2000, 100-3000, at least 100, at least 150, at least 200, at least 250, at least 300, or about 350 nucleotides in length. The detectable label may be, for example, biotin, a hapten, or an affinity tag. Thus, in certain embodiments, sample DNA is sheared and the ends of the sheared DNA fragments are repaired to yield blunt-ended fragments with 5′-phosphorylated ends. Sample DNA can further have a 3′-dA overhang prior to ligation to indexing-specific adaptors. Such ligated DNA can be purified and amplified using PCR in order to yield the prepared sample DNA for hybridization. In other embodiments, the sample DNA is prepared for hybridization by shearing, adaptor ligation, amplification, and purification. 
         [0026]    In some embodiments, RNA is prepared from complex samples. RNA isolated from complex samples contains genes expressed by the organisms or groups of organisms in a particular environment, which can have relevance to the physiological state of the organism(s) in that environment, and can provide information about what biochemical pathways are active in the particular environment (e.g. Booijink et al. 2010. Applied and Environmental Microbiology 76: 5533-5540). RNA so prepared can be reverse-transcribed into DNA for hybridization, amplification, and sequence analysis. 
         [0027]    Baits can be mixed with prepared sample DNA prior to hybridization by any means known in the art. The amount of baits added to the sample DNA should be sufficient to bind fragments of a gene of interest, or variant thereof. In some embodiments, a greater amount of baits is added to the mixture compared to the amount of sample DNA. The ratio of bait to sample DNA for hybridization can be about 1:4, about 1:3, about 1:2, about 1:1.8, about 1:1.6, about 1:1.4, about 1:1.2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 10:1, about 20:1, about 50:1, or about 100:1, and higher. 
         [0028]    While hybridization conditions may vary, hybridization of such bait sequences may be carried out under stringent conditions. By “stringent conditions” or “stringent hybridization conditions” is intended conditions under which the bait will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the bait can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). In specific embodiments, the prepared sample DNA is hybridized to the baits for 16-24 hours at about 45° C., about 50° C., about 55° C., about 60° C., or about 65° C. 
         [0029]    Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short baits (e.g., 10 to 50 nucleotides) and at least about 60° C. for long baits (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5×1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Other exemplary high-stringency conditions are those found in SureSelect XT  Target Enrichment System for Illumina Paired-End Sequencing Library Protocol, Version 1.6 and NimbleGen SeqCap EZ Library SR User&#39;s Guide, Version 4.3. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium. 
         [0030]    Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched bait. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with &gt;90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45° C. (aqueous solution) or 32° C. (formamide solution), it is optimal to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). 
         [0031]    As used herein, a hybridization complex refers to sample DNA fragments hybridizing to a bait. Following hybridization, the labeled baits can be separated based on the presence of the detectable label, and the unbound sequences are removed under appropriate wash conditions that remove the nonspecifically bound DNA and unbound DNA, but do not substantially remove the DNA that hybridizes specifically. The hybridization complex can be captured and purified from non-binding baits and sample DNA fragments. For example, the hybridization complex can be captured by using a streptavidin molecule attached to a solid phase, such as a bead or a magnetic bead. In such embodiments, the hybridization complex captured onto the streptavidin coated bead can be selected by magnetic bead selection. The captured sample DNA fragment can then be amplified and index tagged for multiplex sequencing. As used herein, “index tagging” refers to the addition of a known polynucleotide sequence in order to track the sequence or provide a template for PCR. Index tagging the captured sample DNA sequences can identify the DNA source in the case that multiple pools of captured and indexed DNA are sequenced together. As used herein, an “enrichment kit” or “enrichment kit for multiplex sequencing” refers to a kit designed with reagents and instructions for preparing DNA from a complex sample and hybridizing the prepared DNA with labeled baits. In certain embodiments, the enrichment kit further provides reagents and instructions for capture and purification of the hybridization complex and/or amplification of any captured fragments of the genes of interest. In specific embodiments, the enrichment kit is the SureSelect xT  Target Enrichment System for Illumina Paired-End Sequencing Library Protocol, Version 1.6. In other specific embodiments, the enrichment kit is as described in the NimbleGen SeqCap EZ Library SR User&#39;s Guide, Version 4.3 
         [0032]    Alternatively, the DNA from multiple complex samples can be indexed and amplified before hybridization. In such embodiments, the enrichment kit can be the SureSelect XT2  Target Enrichment System for Illumina Multiplexed Sequencing Protocol, Version D.0 
         [0033]    Following hybridization, the captured target organism DNA can be sequenced by any means known in the art. Sequencing of nucleic acids isolated by the methods described herein is, in certain embodiments, carried out using massively parallel short-read sequencing systems such as those provided by Illumina®, Inc, (HiSeq 1000, HiSeq 2000, HiSeq 2500, Genome Analyzers, MiSeq systems), Applied Biosystems™ Life Technologies (ABI PRISM® Sequence detection systems, SOLiD™ System, Ion PGM™ Sequencer, ion Proton™ Sequencer), because the read out generates more bases of sequence per sequencing unit than other sequencing methods that generate fewer but longer reads. Sequencing can also be carried out by methods generating longer reads, such as those provided by Oxford Nanopore Technologies® (GridiON, MiniON) or Pacific Biosciences (Pacbio RS II). Sequencing can also be carried out by standard Sanger dideoxy terminator sequencing methods and devices, or on other sequencing instruments, further as those described in, for example, United States patents and patent applications U.S. Pat. Nos. 5,888,737, 6,175,002, 5,695,934, 6,140,489, 5,863,722, 2007/007991, 2009/0247414, 2010/01 11768 and PCT application WO2007/123744 each of which is incorporated herein by reference in its entirety. 
         [0034]    Sequences can be assembled by any means known in the art. The sequences of individual fragments of genes of interest can be assembled to identify the full length sequence of the gene of interest, or variant thereof. In some embodiments, sequences are assembled using the CLC Bio suite of bioinformatics tools. Following assembly, sequences of genes of interest, or variants thereof, are searched (e.g., sequence similarity search) against a database of known sequences including those of the genes of interest in order to identify the gene of interest, or variant thereof. In this manner, new variants (i.e., homologs) of genes of interest can be identified from complex samples. 
       IV. Kits for Identification of a Gene of Interest, or Variant Thereof. 
       [0035]    Kits are provided for identifying genes of interest or variants thereof, by the methods disclosed herein. The kits include a bait pool or RNA bait pool, or reagents suitable for producing a bait pool specific for a gene of interest, along with other reagents, such as a solid phase containing a binding partner of any detectable label on the baits. In specific embodiments, the detectable label is biotin and the binding partner streptavidin or streptavidin adhered to magnetic beads. The kits may also include solutions for hybridization, washing, or eluting of the DNA/solid phase compositions described herein, or may include a concentrate of such solutions. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Exemplary Target Gene and Polynucleotides 
               
             
          
           
               
                   
                   
                 NCBI 
                 NCBI 
                   
                   
                   
               
               
                 Name 
                 Acc No. 
                 Protein 
                 Nuc 
                 Authors 
                 Year 
                 Source Strain 
               
               
                   
               
             
          
           
               
                 Cry1Aa1 
                 AAA22353 
                 142765 
                 142764 
                 Schnepf et al 
                 1985 
                 Bt kurstaki HD1 
               
               
                 Cry1Aa2 
                 AAA22552 
                 551713 
                 143100 
                 Shibano et al 
                 1985 
                 Bt sotto 
               
               
                 Cry1Aa3 
                 BAA00257 
                 216284 
                 216283 
                 Shimizu et al 
                 1988 
                 Bt aizawai IPL7 
               
               
                 Cry1Aa4 
                 CAA31886 
                 40267 
                 40266 
                 Masson et al 
                 1989 
                 Bt entomocidus 
               
               
                 Cry1Aa5 
                 BAA04468 
                 535781 
                 506190 
                 Udayasuriyan et al 
                 1994 
                 Bt Fu-2-7 
               
               
                 Cry1Aa6 
                 AAA86265 
                 1171233 
                 1171232 
                 Masson et al 
                 1994 
                 Bt kurstaki NRD-12 
               
               
                 Cry1Aa7 
                 AAD46139 
                 5669035 
                 5669034 
                 Osman et al 
                 1999 
                 Bt C12 
               
               
                 Cry1Aa8 
                 I26149 
                   
                   
                 Liu 
                 1996 
               
               
                 Cry1Aa9 
                 BAA77213 
                 4666284 
                 4666283 
                 Nagamatsu et al 
                 1999 
                 Bt dendrolimus T84A1 
               
               
                 Cry1Aa10 
                 AAD55382 
                 5901703 
                 5901702 
                 Hou and Chen 
                 1999 
                 Bt kurstaki HD-1-02 
               
               
                 Cry1Aa11 
                 CAA70856 
                 6687073 
                 6687072 
                 Tounsi et al 
                 1999 
                 Bt kurstaki 
               
               
                 Cry1Aa12 
                 AAP80146 
                 32344731 
                 32344730 
                 Yao et al 
                 2001 
                 Bt Ly30 
               
               
                 Cry1Aa13 
                 AAM44305 
                 21239436 
                 21239435 
                 Zhong et al 
                 2002 
                 Bt sotto 
               
               
                 Cry1Aa14 
                 AAP40639 
                 37781497 
                 37781496 
                 Ren et al 
                 2002 
                 unpublished 
               
               
                 Cry1Aa15 
                 AAY66993 
                 67089177 
                 67089176 
                 Sauka et al 
                 2005 
                 Bt INTA Mol-12 
               
               
                 Cry1Aa16 
                 HQ439776 
                   
                   
                 Liu et al 
                 2010 
                 Bt Ps9-E2 
               
               
                 Cry1Aa17 
                 HQ439788 
                   
                   
                 Liu et al 
                 2010 
                 Bt PS9-C12 
               
               
                 Cry1Aa18 
                 HQ439790 
                   
                   
                 Liu et al 
                 2010 
                 Bt PS9-D12 
               
               
                 Cry1Aa19 
                 HQ685121 
                 337732098 
                 337732097 
                 Li &amp; Luo 
                 2011 
                 Bt LS-R-21 
               
               
                 Cry1Aa20 
                 JF340156 
                   
                   
                 Kumari &amp; Kaur 
                 2011 
                 Bt SK-798 
               
               
                 Cry1Aa21 
                 JN651496 
                   
                   
                 Li Yuhong 
                 2011 
                 Bt LTS-209 
               
               
                 Cry1Aa22 
                 KC158223 
                   
                   
                 El Khoury et al 
                 2013 
                 Bt Lip 
               
               
                 Cry1Ab1 
                 AAA22330 
                 142720 
                 142719 
                 Wabiko et al 
                 1986 
                 Bt berliner 1715 
               
               
                 Cry1Ab2 
                 AAA22613 
                 143227 
                 143226 
                 Thorne et al 
                 1986 
                 Bt kurstaki 
               
               
                 Cry1Ab3 
                 AAA22561 
                 143124 
                 143123 
                 Geiser et al 
                 1986 
                 Bt kurstaki HD1 
               
               
                 Cry1Ab4 
                 BAA00071 
                 216280 
                 216279 
                 Kondo et al 
                 1987 
                 Bt kurstaki HD1 
               
               
                 Cry1Ab5 
                 CAA28405 
                 40255 
                 40254 
                 Hofte et al 
                 1986 
                 Bt berliner 1715 
               
               
                 Cry1Ab6 
                 AAA22420 
                 142886 
                 142885 
                 Hefford et al 
                 1987 
                 Bt kurstaki NRD-12 
               
               
                 Cry1Ab7 
                 CAA31620 
                 40278 
                 40277 
                 Haider &amp; Ellar 
                 1988 
                 Bt aizawai IC1 
               
               
                 Cry1Ab8 
                 AAA22551 
                 143099 
                 143098 
                 Oeda et al 
                 1987 
                 Bt aizawai IPL7 
               
               
                 Cry1Ab9 
                 CAA38701 
                 40273 
                 40272 
                 Chak &amp; Jen 
                 1993 
                 Bt aizawai HD133 
               
               
                 Cry1Ab10 
                 A29125 
                   
                   
                 Fischhoff et al 
                 1987 
                 Bt kurstaki HD1 
               
               
                 Cry1Ab11 
                 I12419 
                   
                   
                 Ely &amp; Tippett 
                 1995 
                 Bt A20 
               
               
                 Cry1Ab12 
                 AAC64003 
                 3746545 
                 3746544 
                 Silva-Werneck et al 
                 1998 
                 Bt kurstaki S93 
               
               
                 Cry1Ab13 
                 AAN76494 
                 25990352 
                 25990351 
                 Tan et al 
                 2002 
                 Bt c005 
               
               
                 Cry1Ab14 
                 AAG16877 
                 10440886 
                 10440885 
                 Meza-Basso &amp; Theoduloz 
                 2000 
                 Native Chilean Bt 
               
               
                 Cry1Ab15 
                 AAO13302 
                 27436100 
                 27436098 
                 Li et al 
                 2001 
                 Bt B-Hm-16 
               
               
                 Cry1Ab16 
                 AAK55546 
                 14190061 
                 14190060 
                 Yu et al 
                 2002 
                 Bt AC-11 
               
               
                 Cry1Ab17 
                 AAT46415 
                 48734426 
                 48734425 
                 Huang et al 
                 2004 
                 Bt WB9 
               
               
                 Cry1Ab18 
                 AAQ88259 
                 37048803 
                 37048802 
                 Stobdan et al 
                 2004 
                 Bt 
               
               
                 Cry1Ab19 
                 AAW31761 
                 56900936 
                 56900935 
                 Zhong et al 
                 2005 
                 Bt X-2 
               
               
                 Cry1Ab20 
                 ABB72460 
                 82395049 
                 82395048 
                 Liu et al 
                 2006 
                 BtC008 
               
               
                 Cry1Ab21 
                 ABS18384 
                 151655610 
                 151655609 
                 Swiecicka et al 
                 2007 
                 Bt IS5056 
               
               
                 Cry1Ab22 
                 ABW87320 
                 159024156 
                 159024155 
                 Wu and Feng 
                 2008 
                 BtS2491Ab 
               
               
                 Cry1Ab23 
                 HQ439777 
                   
                   
                 Liu et al 
                 2010 
                 Bt N32-2-2 
               
               
                 Cry1Ab24 
                 HQ439778 
                   
                   
                 Liu et al 
                 2010 
                 Bt HD12 
               
               
                 Cry1Ab25 
                 HQ685122 
                 337732100 
                 337732099 
                 Li &amp; Luo 
                 2011 
                 Bt LS-R-30 
               
               
                 Cry1Ab26 
                 HQ847729 
                 320090245 
                 320090244 
                 Prathap Reddy et al 
                 2011 
                 DOR BT-1 
               
               
                 Cry1Ab27 
                 JN135249 
                   
                   
                 Ammouneh et al 
                 2011 
               
               
                 Cry1Ab28 
                 JN135250 
                   
                   
                 Ammouneh et al 
                 2011 
               
               
                 Cry1Ab29 
                 JN135251 
                   
                   
                 Ammouneh et al 
                 2011 
               
               
                 Cry1Ab30 
                 JN135252 
                   
                   
                 Ammouneh et al 
                 2011 
               
               
                 Cry1Ab31 
                 JN135253 
                   
                   
                 Ammouneh et al 
                 2011 
               
               
                 Cry1Ab32 
                 JN135254 
                   
                   
                 Ammouneh et al 
                 2011 
               
               
                 Cry1Ab33 
                 AAS93798 
                   
                   
                 Li et al 
                 2012 
                 Bt kenyae K3 
               
               
                 Cry1Ab34 
                 KC156668 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry1Ab-like 
                 AAK14336 
                 13173238 
                 13173237 
                 Nagarathinam et al 
                 2001 
                 Bt kunthala RX24 
               
               
                 Cry1Ab-like 
                 AAK14337 
                 13173240 
                 13173239 
                 Nagarathinam et al 
                 2001 
                 Bt kunthala RX28 
               
               
                 Cry1Ab-like 
                 AAK14338 
                 13173242 
                 13173241 
                 Nagarathinam et al 
                 2001 
                 Bt kunthala RX27 
               
               
                 Cry1Ab-like 
                 ABG88858 
                 110734449 
                 110734448 
                 Lin et al 
                 2006 
                 Bt ly4a3 
               
               
                 Cry1Ac1 
                 AAA22331 
                   
                   
                 Adang et al 
                 1985 
                 Bt kurstaki HD73 
               
               
                 Cry1Ac2 
                 AAA22338 
                   
                   
                 Von Tersch et al 
                 1991 
                 Bt kenyae 
               
               
                 Cry1Ac3 
                 CAA38098 
                   
                   
                 Dardenne et al 
                 1990 
                 Bt BTS89A 
               
               
                 Cry1Ac4 
                 AAA73077 
                   
                   
                 Feitelson 
                 1991 
                 Bt kurstaki PS85A1 
               
               
                 Cry1Ac5 
                 AAA22339 
                   
                   
                 Feitelson 
                 1992 
                 Bt kurstaki PS81GG 
               
               
                 Cry1Ac6 
                 AAA86266 
                   
                   
                 Masson et al 
                 1994 
                 Bt kurstaki NRD-12 
               
               
                 Cry1Ac7 
                 AAB46989 
                   
                   
                 Herrera et al 
                 1994 
                 Bt kurstaki HD73 
               
               
                 Cry1Ac8 
                 AAC44841 
                   
                   
                 Omolo et al 
                 1997 
                 Bt kurstaki HD73 
               
               
                 Cry1Ac9 
                 AAB49768 
                   
                   
                 Gleave et al 
                 1992 
                 Bt DSIR732 
               
               
                 Cry1Ac10 
                 CAA05505 
                   
                   
                 Sun 
                 1997 
                 Bt kurstaki YBT-1520 
               
               
                 Cry1Ac11 
                 CAA10270 
                   
                   
                 Makhdoom &amp; Riazuddin 
                 1998 
               
               
                 Cry1Ac12 
                 I12418 
                   
                   
                 Ely &amp; Tippett 
                 1995 
                 Bt A20 
               
               
                 Cry1Ac13 
                 AAD38701 
                   
                   
                 Qiao et al 
                 1999 
                 Bt kurstaki HD1 
               
               
                 Cry1Ac14 
                 AAQ06607 
                   
                   
                 Yao et al 
                 2002 
                 Bt Ly30 
               
               
                 Cry1Ac15 
                 AAN07788 
                   
                   
                 Tzeng et al 
                 2001 
                 Bt from Taiwan 
               
               
                 Cry1Ac16 
                 AAU87037 
                   
                   
                 Zhao et al 
                 2005 
                 Bt H3 
               
               
                 Cry1Ac17 
                 AAX18704 
                   
                   
                 Hire et al 
                 2005 
                 Bt kenyae HD549 
               
               
                 Cry1Ac18 
                 AAY88347 
                   
                   
                 Kaur &amp; Allam 
                 2005 
                 Bt SK-729 
               
               
                 Cry1Ac19 
                 ABD37053 
                   
                   
                 Gao et al 
                 2005 
                 Bt C-33 
               
               
                 Cry1Ac20 
                 ABB89046 
                   
                   
                 Tan et al 
                 2005 
               
               
                 Cry1Ac21 
                 AAY66992 
                   
                   
                 Sauka et al 
                 2005 
                 INTA Mol-12 
               
               
                 Cry1Ac22 
                 ABZ01836 
                   
                   
                 Zhang &amp; Fang 
                 2008 
                 Bt W015-1 
               
               
                 Cry1Ac23 
                 CAQ30431 
                   
                   
                 Kashyap et al 
                 2008 
                 Bt 
               
               
                 Cry1Ac24 
                 ABL01535 
                   
                   
                 Arango et al 
                 2008 
                 Bt 146-158-01 
               
               
                 Cry1Ac25 
                 FJ513324 
                 237688242 
                 237688241 
                 Guan et al 
                 2011 
                 Bt Tm37-6 
               
               
                 Cry1Ac26 
                 FJ617446 
                 256003038 
                 256003037 
                 Guan et al 
                 2011 
                 Bt Tm41-4 
               
               
                 Cry1Ac27 
                 FJ617447 
                 256003040 
                 256003039 
                 Guan et al 
                 2011 
                 Bt Tm44-1B 
               
               
                 Cry1Ac28 
                 ACM90319 
                   
                   
                 Li et al 
                 2009 
                 Bt Q-12 
               
               
                 Cry1Ac29 
                 DQ438941 
                   
                   
                 Diego Sauka 
                 2009 
                 INTA TA24-6 
               
               
                 Cry1Ac30 
                 GQ227507 
                   
                   
                 Zhang et al 
                 2010 
                 Bt S1478-l 
               
               
                 Cry1Ac31 
                 GU446674 
                 319433505 
                   
                 Zhao et al 
                 2010 
                 Bt S3299-1 
               
               
                 Cry1Ac32 
                 HM061081 
                   
                   
                 Lu et al 
                 2010 
                 Bt ZQ-89 
               
               
                 Cry1Ac33 
                 GQ866913 
                 306977639 
                 306977638 
                 Kaur &amp; Meena 
                 2011 
                 Bt SK-711 
               
               
                 Cry1Ac34 
                 HQ230364 
                 314906994 
                   
                 Kaur &amp; Kumari 
                 2010 
                 Bt SK-783 
               
               
                 Cry1Ac35 
                 JF340157 
                   
                   
                 Kumari &amp; Kaur 
                 2011 
                 Bt SK-784 
               
               
                 Cry1Ac36 
                 JN387137 
                   
                   
                 Kumari &amp; Kaur 
                 2011 
                 Bt SK-958 
               
               
                 Cry1Ac37 
                 JQ317685 
                   
                   
                 Kumari &amp; Kaur 
                 2011 
                 Bt SK-793 
               
               
                 Cry1Ac38 
                 ACC86135 
                   
                   
                 Lin et al 
                 2008 
                 Bt LSZ9408 
               
               
                 Cry1Ad1 
                 AAA22340 
                   
                   
                 Feitelson 
                 1993 
                 Bt aizawai PS81I 
               
               
                 Cry1Ad2 
                 CAA01880 
                   
                   
                 Anonymous 
                 1995 
                 Bt PS81RR1 
               
               
                 Cry1Ae1 
                 AAA22410 
                   
                   
                 Lee &amp; Aronson 
                 1991 
                 Bt alesti 
               
               
                 Cry1Af1 
                 AAB82749 
                   
                   
                 Kang et al 
                 1997 
                 Bt NT0423 
               
               
                 Cry1Ag1 
                 AAD46137 
                   
                   
                 Mustafa 
                 1999 
               
               
                 Cry1Ah1 
                 AAQ14326 
                   
                   
                 Tan et al 
                 2000 
               
               
                 Cry1Ah2 
                 ABB76664 
                   
                   
                 Qi et al 
                 2005 
                 Bt alesti 
               
               
                 Cry1Ah3 
                 HQ439779 
                   
                   
                 Liu et al 
                 2010 
                 Bt S6 
               
               
                 Cry1Ai1 
                 AAO39719 
                   
                   
                 Wang et al 
                 2002 
               
               
                 Cry1Ai2 
                 HQ439780 
                   
                   
                 Liu et al 
                 2010 
                 Bt SC6H8 
               
               
                 Cry1A-like 
                 AAK14339 
                   
                   
                 Nagarathinam et al 
                 2001 
                 Bt kunthala nags3 
               
               
                 Cry1Ba1 
                 CAA29898 
                   
                   
                 Brizzard &amp; Whiteley 
                 1988 
                 Bt thuringiensis HD2 
               
               
                 Cry1Ba2 
                 CAA65003 
                   
                   
                 Soetaert 
                 1996 
                 Bt entomocidus HD110 
               
               
                 Cry1Ba3 
                 AAK63251 
                   
                   
                 Zhang et al 
                 2001 
               
               
                 Cry1Ba4 
                 AAK51084 
                   
                   
                 Nathan et al 
                 2001 
                 Bt entomocidus HD9 
               
               
                 Cry1Ba5 
                 ABO20894 
                   
                   
                 Song et al 
                 2007 
                 Bt sfw-12 
               
               
                 Cry1Ba6 
                 ABL60921 
                   
                   
                 Martins et al 
                 2006 
                 Bt S601 
               
               
                 Cry1Ba7 
                 HQ439781 
                   
                   
                 Liu et al 
                 2010 
                 Bt N17-37 
               
               
                 Cry1Bb1 
                 AAA22344 
                   
                   
                 Donovan et al 
                 1994 
                 Bt EG5847 
               
               
                 Cry1Bb2 
                 HQ439782 
                   
                   
                 Liu et al 
                 2010 
                 Bt WBT-2 
               
               
                 Cry1Bc1 
                 CAA86568 
                   
                   
                 Bishop et al 
                 1994 
                 Bt morrisoni 
               
               
                 Cry1Bd1 
                 AAD10292 
                   
                   
                 Kuo et al 
                 2000 
                 Bt wuhanensis HD525 
               
               
                 Cry1Bd2 
                 AAM93496 
                   
                   
                 Isakova et al 
                 2002 
                 Bt 834 
               
               
                 Cry1Be1 
                 AAC32850 
                   
                   
                 Payne et al 
                 1998 
                 Bt PS158C2 
               
               
                 Cry1Be2 
                 AAQ52387 
                   
                   
                 Baum et al 
                 2003 
               
               
                 Cry1Be3 
                 ACV96720 
                 259156864 
                   
                 Sun et al 
                 2010 
                 Bt g9 
               
               
                 Cry1Be4 
                 HM070026 
                   
                   
                 Shu et al 
                 2010 
               
               
                 Cry1Bf1 
                 CAC50778 
                   
                   
                 Arnaut et al 
                 2001 
               
               
                 Cry1Bf2 
                 AAQ52380 
                   
                   
                 Baum et al 
                 2003 
               
               
                 Cry1Bg1 
                 AAO39720 
                   
                   
                 Wang et al 
                 2002 
               
               
                 Cry1Bh1 
                 HQ589331 
                 315076091 
                   
                 Lira et al 
                 2010 
                 Bt PS46L 
               
               
                 Cry1Bi1 
                 KC156700 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry1Ca1 
                 CAA30396 
                   
                   
                 Honee et al 
                 1988 
                 Bt entomocidus 60.5 
               
               
                 Cry1Ca2 
                 CAA31951 
                   
                   
                 Sanchis et al 
                 1989 
                 Bt aizawai 7.29 
               
               
                 Cry1Ca3 
                 AAA22343 
                   
                   
                 Feitelson 
                 1993 
                 Bt aizawai PS81I 
               
               
                 Cry1Ca4 
                 CAA01886 
                   
                   
                 Van Mellaert et al 
                 1990 
                 Bt entomocidus HD110 
               
               
                 Cry1Ca5 
                 CAA65457 
                   
                   
                 Strizhov 
                 1996 
                 Bt aizawai 7.29 
               
               
                 Cry1Ca6 [1] 
                 AAF37224 
                   
                   
                 Yu et al 
                 2000 
                 Bt AF-2 
               
               
                 Cry1Ca7 
                 AAG50438 
                   
                   
                 Aixing et al 
                 2000 
                 Bt J8 
               
               
                 Cry1Ca8 
                 AAM00264 
                   
                   
                 Chen et al 
                 2001 
                 Bt c002 
               
               
                 Cry1Ca9 
                 AAL79362 
                   
                   
                 Kao et al 
                 2003 
                 Bt G10-01A 
               
               
                 Cry1Ca10 
                 AAN16462 
                   
                   
                 Lin et al 
                 2003 
                 Bt E05-20a 
               
               
                 Cry1Ca11 
                 AAX53094 
                   
                   
                 Cai et al 
                 2005 
                 Bt C-33 
               
               
                 Cry1Ca12 
                 HM070027 
                   
                   
                 Shu et al 
                 2010 
               
               
                 Cry1Ca13 
                 HQ412621 
                 312192962 
                   
                 Li &amp; Luo 
                 2010 
                 Bt LB-R-78 
               
               
                 Cry1Ca14 
                 JN651493 
                   
                   
                 Li Yuhong 
                 2011 
                 Bt LTS-38 
               
               
                 Cry1Cb1 
                 M97880 
                   
                   
                 Kalman et al 
                 1993 
                 Bt galleriae HD29 
               
               
                 Cry1Cb2 
                 AAG35409 
                   
                   
                 Song et al 
                 2000 
                 Bt c001 
               
               
                 Cry1Cb3 
                 ACD50894 
                   
                   
                 Huang et al 
                 2008 
                 Bt 087 
               
               
                 Cry1Cb-like 
                 AAX63901 
                   
                   
                 Thammasittirong et al 
                 2005 
                 Bt TA476-1 
               
               
                 Cry1Da1 
                 CAA38099 
                   
                   
                 Hofte et al 
                 1990 
                 Bt aizawai HD68 
               
               
                 Cry1Da2 
                 I76415 
                   
                   
                 Payne &amp; Sick 
                 1997 
               
               
                 Cry1Da3 
                 HQ439784 
                   
                   
                 Liu et al 
                 2010 
                 Bt HD12 
               
               
                 Cry1Db1 
                 CAA80234 
                   
                   
                 Lambert 
                 1993 
                 Bt BTS00349A 
               
               
                 Cry1Db2 
                 AAK48937 
                   
                   
                 Li et al 
                 2001 
                 Bt B-Pr-88 
               
               
                 Cry1Dc1 
                 ABK35074 
                   
                   
                 Lertwiriyawong et al 
                 2006 
                 Bt JC291 
               
               
                 Cry1Ea1 
                 CAA37933 
                   
                   
                 Visser et al 
                 1990 
                 Bt kenyae 4F1 
               
               
                 Cry1Ea2 
                 CAA39609 
                   
                   
                 Bosse et al 
                 1990 
                 Bt kenyae 
               
               
                 Cry1Ea3 
                 AAA22345 
                   
                   
                 Feitelson 
                 1991 
                 Bt kenyae PS81F 
               
               
                 Cry1Ea4 
                 AAD04732 
                   
                   
                 Barboza-Corona et al 
                 1998 
                 Bt kenyae LBIT-147 
               
               
                 Cry1Ea5 
                 A15535 
                   
                   
                 Botterman et al 
                 1994 
               
               
                 Cry1Ea6 
                 AAL50330 
                   
                   
                 Sun et al 
                 1999 
                 Bt YBT-032 
               
               
                 Cry1Ea7 
                 AAW72936 
                   
                   
                 Huehne et al 
                 2005 
                 Bt JC190 
               
               
                 Cry1Ea8 
                 ABX11258 
                   
                   
                 Huang et al 
                 2007 
                 Bt HZM2 
               
               
                 Cry1Ea9 
                 HQ439785 
                   
                   
                 Liu et al 
                 2010 
                 Bt S6 
               
               
                 Cry1Ea10 
                 ADR00398 
                   
                   
                 Goncalves et al 
                 2010 
                 Bt BR64 
               
               
                 Cry1Ea11 
                 JQ652456 
                   
                   
                 Lin Qunxin et al 
                 2012 
                 Bt 
               
               
                 Cry1Ea12 
                 KF601559 
                   
                   
                 Baonan He 
                 2013 
                 Bt strain V4 
               
               
                 Cry1Eb1 
                 AAA22346 
                   
                   
                 Feitelson 
                 1993 
                 Bt aizawai PS81A2 
               
               
                 Cry1Fa1 
                 AAA22348 
                   
                   
                 Chambers et al 
                 1991 
                 Bt aizawai EG6346 
               
               
                 Cry1Fa2 
                 AAA22347 
                   
                   
                 Feitelson 
                 1993 
                 Bt aizawai PS81I 
               
               
                 Cry1Fa3 
                 HM070028 
                   
                   
                 Shu et al 
                 2010 
               
               
                 Cry1Fa4 
                 HM439638 
                   
                   
                 Liu et al 
                 2010 
                 Bt mo3-D10 
               
               
                 Cry1Fb1 
                 CAA80235 
                   
                   
                 Lambert 
                 1993 
                 Bt BTS00349A 
               
               
                 Cry1Fb2 
                 BAA25298 
                   
                   
                 Masuda &amp; Asano 
                 1998 
                 Bt morrisoni INA67 
               
               
                 Cry1Fb3 
                 AAF21767 
                   
                   
                 Song et al 
                 1998 
                 Bt morrisoni 
               
               
                 Cry1Fb4 
                 AAC10641 
                   
                   
                 Payne et al 
                 1997 
               
               
                 Cry1Fb5 
                 AAO13295 
                   
                   
                 Li et al 
                 2001 
                 Bt B-Pr-88 
               
               
                 Cry1Fb6 
                 ACD50892 
                   
                   
                 Huang et al 
                 2008 
                 Bt 012 
               
               
                 Cry1Fb1 
                 ACD50893 
                   
                   
                 Huang et al 
                 2008 
                 Bt 087 
               
               
                 Cry1Ga1 
                 CAA80233 
                   
                   
                 Lambert 
                 1993 
                 Bt BTS0349A 
               
               
                 Cry1Ga2 
                 CAA70506 
                   
                   
                 Shevelev et al 
                 1997 
                 Bt wuhanensis 
               
               
                 Cry1Gb1 
                 AAD10291 
                   
                   
                 Kuo &amp; Chak 
                 1999 
                 Bt wuhanensis HD525 
               
               
                 Cry1Gb2 
                 AAO13756 
                   
                   
                 Li et al 
                 2000 
                 Bt B-Pr-88 
               
               
                 Cry1Gc1 
                 AAQ52381 
                   
                   
                 Baum et al 
                 2003 
               
               
                 Cry1Ha1 
                 CAA80236 
                   
                   
                 Lambert 
                 1993 
                 Bt BTS02069AA 
               
               
                 Cry1Hb1 
                 AAA79694 
                   
                   
                 Koo et al 
                 1995 
                 Bt morrisoni BF190 
               
               
                 Cry1Hb2 
                 HQ439786 
                   
                   
                 Liu et al 
                 2010 
                 Bt WBT-2 
               
               
                 Cry1H-like 
                 AAF01213 
                   
                   
                 Srifah et al 
                 1999 
                 Bt JC291 
               
               
                 Cry1Ia1 
                 CAA44633 
                   
                   
                 Tailor et al 
                 1992 
                 Bt kurstaki 
               
               
                 Cry1Ia2 
                 AAA22354 
                   
                   
                 Gleave et al 
                 1993 
                 Bt kurstaki 
               
               
                 Cry1Ia3 
                 AAC36999 
                   
                   
                 Shin et al 
                 1995 
                 Bt kurstaki HD1 
               
               
                 Cry1Ia4 
                 AAB00958 
                   
                   
                 Kostichka et al 
                 1996 
                 Bt AB88 
               
               
                 Cry1Ia5 
                 CAA70124 
                   
                   
                 Selvapandiyan 
                 1996 
                 Bt 61 
               
               
                 Cry1Ia6 
                 AAC26910 
                   
                   
                 Zhong et al 
                 1998 
                 Bt kurstaki S101 
               
               
                 Cry1Ia7 
                 AAM73516 
                   
                   
                 Porcar et al 
                 2000 
                 Bt 
               
               
                 Cry1Ia8 
                 AAK66742 
                   
                   
                 Song et al 
                 2001 
               
               
                 Cry1Ia9 
                 AAQ08616 
                   
                   
                 Yao et al 
                 2002 
                 Bt Ly30 
               
               
                 Cry1Ia10 
                 AAP86782 
                   
                   
                 Espindola et al 
                 2003 
                 Bt thuringiensis 
               
               
                 Cry1Ia11 
                 CAC85964 
                   
                   
                 Tounsi et al 
                 2003 
                 Bt kurstaki BNS3 
               
               
                 Cry1Ia12 
                 AAV53390 
                   
                   
                 Grossi de Sa et al 
                 2005 
                 Bt 
               
               
                 Cry1Ia13 
                 ABF83202 
                   
                   
                 Martins et al 
                 2006 
                 Bt 
               
               
                 Cry1Ia14 
                 ACG63871 
                   
                   
                 Liu &amp; Guo 
                 2008 
                 Bt11 
               
               
                 Cry1Ia15 
                 FJ617445 
                 256003036 
                 256003035 
                 Guan et al 
                 2011 
                 Bt E-1B 
               
               
                 Cry1Ia16 
                 FJ617448 
                 256003042 
                 256003041 
                 Guan et al 
                 2011 
                 Bt E-1A 
               
               
                 Cry1Ia17 
                 GU989199 
                   
                   
                 Li et al 
                 2010 
                 Bt MX2 
               
               
                 Cry1Ia18 
                 ADK23801 
                 300492624 
                   
                 Li et al 
                 2010 
                 Bt MX9 
               
               
                 Cry1Ia19 
                 HQ439787 
                   
                   
                 Liu et al 
                 2010 
                 Bt SC6H6 
               
               
                 Cry1Ia20 
                 JQ228426 
                   
                   
                 Zhao Can 
                 2011 
                 Bt wu1H-3 
               
               
                 Cry1Ia21 
                 JQ228424 
                   
                   
                 Zhao Can 
                 2011 
                 Bt you1D-9 
               
               
                 Cry1Ia22 
                 JQ228427 
                   
                   
                 Zhao Can 
                 2011 
                 Bt wu1E-3 
               
               
                 Cry1Ia23 
                 JQ228428 
                   
                   
                 Zhao Can 
                 2011 
                 Bt wu1E-4 
               
               
                 Cry1Ia24 
                 JQ228429 
                   
                   
                 Zhao Can 
                 2011 
                 Bt wu2B-6 
               
               
                 Cry1Ia25 
                 JQ228430 
                   
                   
                 Zhao Can 
                 2011 
                 Bt wu2G-11 
               
               
                 Cry1Ia26 
                 JQ228431 
                   
                   
                 Zhao Can 
                 2011 
                 Bt wu2G-12 
               
               
                 Cry1Ia27 
                 JQ228432 
                   
                   
                 Zhao Can 
                 2011 
                 Bt you2D-3 
               
               
                 Cry1Ia28 
                 JQ228433 
                   
                   
                 Zhao Can 
                 2011 
                 Bt you2E-3 
               
               
                 Cry1Ia29 
                 JQ228434 
                   
                   
                 Zhao Can 
                 2011 
                 Bt you2F-3 
               
               
                 Cry1Ia30 
                 JQ317686 
                   
                   
                 Kumari &amp; Kaur 
                 2011 
                 Bt 4J4 
               
               
                 Cry1Ia31 
                 JX944038 
                   
                   
                 Song et al 
                 2012 
                 Bt SC-7 
               
               
                 Cry1Ia32 
                 JX944039 
                   
                   
                 Song et al 
                 2012 
                 Bt SC-13 
               
               
                 Cry1Ia33 
                 JX944040 
                   
                   
                 Song et al 
                 2012 
                 Bt SC-51 
               
               
                 Cry1Ib1 
                 AAA82114 
                   
                   
                 Shin et al 
                 1995 
                 Bt entomocidus BP465 
               
               
                 Cry1Ib2 
                 ABW88019 
                   
                   
                 Guan et al 
                 2007 
                 Bt PP61 
               
               
                 Cry1Ib3 
                 ACD75515 
                   
                   
                 Liu &amp; Guo 
                 2008 
                 Bt GS8 
               
               
                 Cry1Ib4 
                 HM051227 
                 301641366 
                   
                 Zhao et al 
                 2010 
                 Bt BF-4 
               
               
                 Cry1Ib5 
                 HM070028 
                   
                   
                 Shu et al 
                 2010 
               
               
                 Cry1Ib6 
                 ADK38579 
                 300836937 
                   
                 Li et al 
                 2010 
                 Bt LB52 
               
               
                 Cry1Ib7 
                 JN571740 
                   
                   
                 Kumari &amp; Kaur 
                 2011 
                 Bt SK-935 
               
               
                 Cry1Ib8 
                 JN675714 
                   
                   
                 Swamy et al 
                 2011 
               
               
                 Cry1Ib9 
                 JN675715 
                   
                   
                 Swamy et al 
                 2011 
               
               
                 Cry1Ib10 
                 JN675716 
                   
                   
                 Swamy et al 
                 2011 
               
               
                 Cry1Ib11 
                 JQ228423 
                   
                   
                 Zhao Can 
                 2011 
                 Bt HD12 
               
               
                 Cry1Ic1 
                 AAC62933 
                   
                   
                 Osman et al 
                 1998 
                 Bt C18 
               
               
                 Cry1Ic2 
                 AAE71691 
                   
                   
                 Osman et al 
                 2001 
               
               
                 Cry1Id1 
                 AAD44366 
                   
                   
                 Choi 
                 2000 
               
               
                 Cry1Id2 
                 JQ228422 
                   
                   
                 Zhao Can 
                 2011 
                 Bt HD12 
               
               
                 Cry1Ie1 
                 AAG43526 
                   
                   
                 Song et al 
                 2000 
                 Bt BTC007 
               
               
                 Cry1Ie2 
                 HM439636 
                   
                   
                 Liu et al 
                 2010 
                 Bt T03B001 
               
               
                 Cry1Ie3 
                 KC156647 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry1Ie4 
                 KC156681 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry1If1 
                 AAQ52382 
                   
                   
                 Baum et al 
                 2003 
               
               
                 Cry1Ig1 
                 KC156701 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry1I-like 
                 AAC31094 
                   
                   
                 Payne et al 
                 1998 
               
               
                 Cry1I-like 
                 ABG88859 
                   
                   
                 Lin &amp; Fang 
                 2006 
                 Bt ly4a3 
               
               
                 Cry1Ja1 
                 AAA22341 
                   
                   
                 Donovan 
                 1994 
                 Bt EG5847 
               
               
                 Cry1Ja2 
                 HM070030 
                   
                   
                 Shu et al 
                 2010 
               
               
                 Cry1Ja3 
                 JQ228425 
                   
                   
                 Zhao Shiyuan 
                 2011 
                 Bt FH21 
               
               
                 Cry1Jb1 
                 AAA98959 
                   
                   
                 Von Tersch &amp; Gonzalez 
                 1994 
                 Bt EG5092 
               
               
                 Cry1Jc1 
                 AAC31092 
                   
                   
                 Payne et al 
                 1998 
               
               
                 Cry1Jc2 
                 AAQ52372 
                   
                   
                 Baum et al 
                 2003 
               
               
                 Cry1Jd1 
                 CAC50779 
                   
                   
                 Arnaut et al 
                 2001 
                 Bt 
               
               
                 Cry1Ka1 
                 AAB00376 
                   
                   
                 Koo et al 
                 1995 
                 Bt morrisoni BF190 
               
               
                 Cry1Ka2 
                 HQ439783 
                   
                   
                 Liu et al 
                 2010 
                 Bt WBT-2 
               
               
                 Cry1La1 
                 AAS60191 
                   
                   
                 Je et al 
                 2004 
                 Bt kurstaki K1 
               
               
                 Cry1La2 
                 HM070031 
                   
                   
                 Shu et al 
                 2010 
               
               
                 Cry1Ma1 
                 FJ884067 
                   
                   
                 Noguera &amp; Ibarra 
                 2010 
                 LBIT 1189 
               
               
                 Cry1Ma2 
                 KC156659 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry1Na1 
                 KC156648 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry1Nb1 
                 KC156678 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry1-like 
                 AAC31091 
                   
                   
                 Payne et al 
                 1998 
               
               
                 Cry2Aa1 
                 AAA22335 
                   
                   
                 Donovan et al 
                 1989 
                 Bt kurstaki 
               
               
                 Cry2Aa2 
                 AAA83516 
                   
                   
                 Widner &amp; Whiteley 
                 1989 
                 Bt kurstaki HD1 
               
               
                 Cry2Aa3 
                 D86064 
                   
                   
                 Sasaki et al 
                 1997 
                 Bt sotto 
               
               
                 Cry2Aa4 
                 AAC04867 
                   
                   
                 Misra et al 
                 1998 
                 Bt kenyae HD549 
               
               
                 Cry2Aa5 
                 CAA10671 
                   
                   
                 Yu &amp; Pang 
                 1999 
                 Bt SL39 
               
               
                 Cry2Aa6 
                 CAA10672 
                   
                   
                 Yu &amp; Pang 
                 1999 
                 Bt YZ71 
               
               
                 Cry2Aa7 
                 CAA10670 
                   
                   
                 Yu &amp; Pang 
                 1999 
                 Bt CY29 
               
               
                 Cry2Aa8 
                 AAO13734 
                   
                   
                 Wei et al 
                 2000 
                 Bt Dongbei 66 
               
               
                 Cry2Aa9 
                 AAO13750 
                   
                   
                 Zhang et al 
                 2000 
               
               
                 Cry2Aa10 
                 AAQ04263 
                   
                   
                 Yao et al 
                 2001 
               
               
                 Cry2Aa11 
                 AAQ52384 
                   
                   
                 Baum et al 
                 2003 
               
               
                 Cry2Aa12 
                 ABI83671 
                   
                   
                 Tan et al 
                 2006 
                 Bt Rpp39 
               
               
                 Cry2Aa13 
                 ABL01536 
                   
                   
                 Arango et al 
                 2008 
                 Bt 146-158-01 
               
               
                 Cry2Aa14 
                 ACF04939 
                   
                   
                 Hire et al 
                 2008 
                 Bt HD-550 
               
               
                 Cry2Aa15 
                 JN426947 
                   
                   
                 Ammouneh et al 
                 2011 
                 Bt SSy77 
               
               
                 Cry2Aa16 
                 KF667522 
                   
                   
                 Baonan He 
                 2013 
                 Bt V4 
               
               
                 Cry2Aa17 
                 KF860848 
                   
                   
                 Guihua Chen et al 
                 2013 
               
               
                 Cry2Ab1 
                 AAA22342 
                   
                   
                 Widner &amp; Whiteley 
                 1989 
                 Bt kurstaki HD1 
               
               
                 Cry2Ab2 
                 CAA39075 
                   
                   
                 Dankocsik et al 
                 1990 
                 Bt kurstaki HD1 
               
               
                 Cry2Ab3 
                 AAG36762 
                   
                   
                 Chen et al 
                 1999 
                 Bt BTC002 
               
               
                 Cry2Ab4 
                 AAO13296 
                   
                   
                 Li et al 
                 2001 
                 Bt B-Pr-88 
               
               
                 Cry2Ab5 
                 AAQ04609 
                   
                   
                 Yao et al 
                 2001 
                 Bt ly30 
               
               
                 Cry2Ab6 
                 AAP59457 
                   
                   
                 Wang et al 
                 2003 
                 Bt WZ-7 
               
               
                 Cry2Ab7 
                 AAZ66347 
                   
                   
                 Udayasuriyan et al 
                 2005 
                 Bt 14-1 
               
               
                 Cry2Ab8 
                 ABC95996 
                   
                   
                 Huang et al 
                 2006 
                 Bt WB2 
               
               
                 Cry2Ab9 
                 ABC74968 
                   
                   
                 Zhang et al 
                 2005 
                 Bt LLB6 
               
               
                 Cry2Ab10 
                 ABM21766 
                   
                   
                 Lin et al 
                 2006 
                 Bt LyL 
               
               
                 Cry2Ab11 
                 CAM84575 
                   
                   
                 Saleem et al 
                 2007 
                 Bt CMBL-BT1 
               
               
                 Cry2Ab12 
                 ABM21764 
                   
                   
                 Lin et al 
                 2007 
                 Bt LyD 
               
               
                 Cry2Ab13 
                 ACG76120 
                   
                   
                 Zhu et al 
                 2008 
                 Bt ywc5-4 
               
               
                 Cry2Ab14 
                 ACG76121 
                   
                   
                 Zhu et al 
                 2008 
                 Bt Bts 
               
               
                 Cry2Ab15 
                 HM037126 
                 302634222 
                 302634221 
                 Zhao et al 
                 2011 
                 Bt BF-4 
               
               
                 Cry2Ab16 
                 GQ866914 
                 306977641 
                 306977640 
                 Katara &amp; Kaur 
                 2011 
                 SK-793 
               
               
                 Cry2Ab17 
                 HQ439789 
                   
                   
                 Liu et al 
                 2010 
                 Bt PS9-C12 
               
               
                 Cry2Ab18 
                 JN135255 
                   
                   
                 Ammouneh et al 
                 2011 
               
               
                 Cry2Ab19 
                 JN135256 
                   
                   
                 Ammouneh et al 
                 2011 
               
               
                 Cry2Ab20 
                 JN135257 
                   
                   
                 Ammouneh et al 
                 2011 
               
               
                 Cry2Ab21 
                 JN135258 
                   
                   
                 Ammouneh et al 
                 2011 
               
               
                 Cry2Ab22 
                 JN135259 
                   
                   
                 Ammouneh et al 
                 2011 
               
               
                 Cry2Ab23 
                 JN135260 
                   
                   
                 Ammouneh et al 
                 2011 
               
               
                 Cry2Ab24 
                 JN135261 
                   
                   
                 Ammouneh et al 
                 2011 
               
               
                 Cry2Ab25 
                 JN415485 
                   
                   
                 Sevim et al 
                 2013 
                 Btk MnD 
               
               
                 Cry2Ab26 
                 JN426946 
                   
                   
                 Ammouneh et al 
                 2011 
                 Bt SSy77 
               
               
                 Cry2Ab27 
                 JN415764 
                 344055822 
                 344055821 
                 Chankhamhaengdecha et al 
                 2011 
               
               
                 Cry2Ab28 
                 JN651494 
                   
                   
                 Li Yuhong 
                 2011 
                 Bt LTS-7 
               
               
                 Cry2Ab29 
                 KF860847 
                   
                   
                 Guihua Chen et al 
                 2013 
               
               
                 Cry2Ab30 
                 EU623976 
                   
                   
                 Lian Xu et al 
                 2013 
               
               
                 Cry2Ac1 
                 CAA40536 
                   
                   
                 Aronson 
                 1991 
                 Bt shanghai S1 
               
               
                 Cry2Ac2 
                 AAG35410 
                   
                   
                 Song et al 
                 2000 
               
               
                 Cry2Ac3 
                 AAQ52385 
                   
                   
                 Baum et al 
                 2003 
               
               
                 Cry2Ac4 
                 ABC95997 
                   
                   
                 Huang et al 
                 2006 
                 Bt WB9 
               
               
                 Cry2Ac5 
                 ABC74969 
                   
                   
                 Zhang et al 
                 2005 
               
               
                 Cry2Ac6 
                 ABC74793 
                   
                   
                 Xia et al 
                 2006 
                 Bt wuhanensis 
               
               
                 Cry2Ac7 
                 CAL18690 
                   
                   
                 Saleem et al 
                 2008 
                 Bt SBSBT-1 
               
               
                 Cry2Ac8 
                 CAM09325 
                   
                   
                 Saleem et al 
                 2007 
                 Bt CMBL-BT1 
               
               
                 Cry2Ac9 
                 CAM09326 
                   
                   
                 Saleem et al 
                 2007 
                 Bt CMBL-BT2 
               
               
                 Cry2Ac10 
                 ABN15104 
                   
                   
                 Bai et al 
                 2007 
                 Bt QCL-1 
               
               
                 Cry2Ac11 
                 CAM83895 
                   
                   
                 Saleem et al 
                 2007 
                 Bt HD29 
               
               
                 Cry2Ac12 
                 CAM83896 
                   
                   
                 Saleem et al 
                 2007 
                 Bt CMBL-BT3 
               
               
                 Cry2Ad1 
                 AAF09583 
                   
                   
                 Choi et al 
                 1999 
                 Bt BR30 
               
               
                 Cry2Ad2 
                 ABC86927 
                   
                   
                 Huang et al 
                 2006 
                 Bt WB10 
               
               
                 Cry2Ad3 
                 CAK29504 
                   
                   
                 Saleem et al 
                 2006 
                 Bt 5_2AcT(1) 
               
               
                 Cry2Ad4 
                 CAM32331 
                   
                   
                 Saleem et al 
                 2007 
                 Bt CMBL-BT2 
               
               
                 Cry2Ad5 
                 CAO78739 
                   
                   
                 Saleem et al 
                 2007 
                 Bt HD29 
               
               
                 Cry2Ae1 
                 AAQ52362 
                   
                   
                 Baum et al 
                 2003 
               
               
                 Cry2Af1 
                 ABO30519 
                   
                   
                 Beard et al 
                 2007 
                 Bt C81 
               
               
                 Cry2Af2 
                 GQ866915 
                 306977643 
                 306977642 
                 Katara &amp; Kaur 
                 2011 
                 SK-758 
               
               
                 Cry2Ag1 
                 ACH91610 
                   
                   
                 Zhu et al 
                 2008 
                 Bt JF19-2 
               
               
                 Cry2Ah1 
                 EU939453 
                 218963751 
                 218963750 
                 Zhang et al 
                 2011 
                 Bt SC6H8 
               
               
                 Cry2Ah2 
                 ACL80665 
                   
                   
                 Zhang et al 
                 2009 
                 Bt BRC-ZQL3 
               
               
                 Cry2Ah3 
                 GU073380 
                 309274394 
                 309274393 
                 Lixin Du 
                 2012 
                 HYW-8 
               
               
                 Cry2Ah4 
                 KC156702 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry2Ai1 
                 FJ788388 
                   
                 259166843 
                 Udayasuriyan et al 
                 2009 
                 Bt 
               
               
                 Cry2Aj1 
                   
                   
                   
                 Zhicheng Shen 
                 2009 
               
               
                 Cry2Ak1 
                 KC156660 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry2Ba1 
                 KC156658 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry2Ba2 
                 KF014123 
                   
                   
                 Guihua Chen et al 
                 2013 
               
               
                 Cry3Aa1 
                 AAA22336 
                   
                   
                 Herrnstadt et al 
                 1987 
                 Bt san diego 
               
               
                 Cry3Aa2 
                 AAA22541 
                   
                   
                 Sekar et al 
                 1987 
                 Bt tenebrionis 
               
               
                 Cry3Aa3 
                 CAA68482 
                   
                   
                 Hofte et al 
                 1987 
               
               
                 Cry3Aa4 
                 AAA22542 
                   
                   
                 McPherson et al 
                 1988 
                 Bt tenebrionis 
               
               
                 Cry3Aa5 
                 AAA50255 
                   
                   
                 Donovan et al 
                 1988 
                 Bt morrisoni EG2158 
               
               
                 Cry3Aa6 
                 AAC43266 
                   
                   
                 Adams et al 
                 1994 
                 Bt tenebrionis 
               
               
                 Cry3Aa7 
                 CAB41411 
                   
                   
                 Zhang et al 
                 1999 
                 Bt 22 
               
               
                 Cry3Aa8 
                 AAS79487 
                   
                   
                 Gao and Cai 
                 2004 
                 Bt YM-03 
               
               
                 Cry3Aa9 
                 AAW05659 
                   
                   
                 Bulla and Candas 
                 2004 
                 Bt UTD-001 
               
               
                 Cry3Aa10 
                 AAU29411 
                   
                   
                 Chen et al 
                 2004 
                 Bt 886 
               
               
                 Cry3Aa11 
                 AAW82872 
                   
                   
                 Kurt et al 
                 2005 
                 Bt tenebrionis Mm2 
               
               
                 Cry3Aa12 
                 ABY49136 
                   
                   
                 Sezen et al 
                 2008 
                 Bt tenebrionis 
               
               
                 Cry3Ba1 
                 CAA34983 
                   
                   
                 Sick et al 
                 1990 
                 Bt tolworthi 43F 
               
               
                 Cry3Ba2 
                 CAA00645 
                   
                   
                 Peferoen et al 
                 1990 
                 Bt PGSI208 
               
               
                 Cry3Ba3 
                 JQ397327 
                   
                   
                 Palma et al 
                 2011 
                 Bt 
               
               
                 Cry3Bb1 
                 AAA22334 
                   
                   
                 Donovan et al 
                 1992 
                 Bt EG4961 
               
               
                 Cry3Bb2 
                 AAA74198 
                   
                   
                 Donovan et al 
                 1995 
                 Bt EG5144 
               
               
                 Cry3Bb3 
                 I15475 
                   
                   
                 Peferoen et al 
                 1995 
               
               
                 Cry3Ca1 
                 CAA42469 
                   
                   
                 Lambert et al 
                 1992 
                 Bt kurstaki BtI109P 
               
               
                 Cry4Aa1 
                 CAA68485 
                   
                   
                 Ward &amp; Ellar 
                 1987 
                 Bt israelensis 
               
               
                 Cry4Aa2 
                 BAA00179 
                   
                   
                 Sen et al 
                 1988 
                 Bt israelensis HD522 
               
               
                 Cry4Aa3 
                 CAD30148 
                   
                   
                 Berry et al 
                 2002 
                 Bt israelensis 
               
               
                 Cry4Aa4 
                 AFB18317 
                 376008213 
                   
                 Li et al 
                 2012 
                 Bti BRC-LLP29 
               
               
                 Cry4A-like 
                 AAY96321 
                   
                   
                 Mahalakshmi et al 
                 2005 
                 Bt LDC-9 
               
               
                 Cry4Ba1 
                 CAA30312 
                   
                   
                 Chungjatpornchai et al 
                 1988 
                 Bt israelensis 4Q2-72 
               
               
                 Cry4Ba2 
                 CAA30114 
                   
                   
                 Tungpradubkul et al 
                 1988 
                 Bt israelensis 
               
               
                 Cry4Ba3 
                 AAA22337 
                   
                   
                 Yamamoto et al 
                 1988 
                 Bt israelensis 
               
               
                 Cry4Ba4 
                 BAA00178 
                   
                   
                 Sen et al 
                 1988 
                 Bt israelensis HD522 
               
               
                 Cry4Ba5 
                 CAD30095 
                   
                   
                 Berry et al 
                 2002 
                 Bt israelensis 
               
               
                 Cry4Ba-like 
                 ABC47686 
                   
                   
                 Mahalakshmi et al 
                 2005 
                 Bt LDC-9 
               
               
                 Cry4Ca1 
                 EU646202 
                 194396263 
                 194396262 
                 Shu et al 
                 2011 
                 Bt Y41 
               
               
                 Cry4Cb1 
                 FJ403208 
                 234203282 
                 234203281 
                 Zhu et al 
                 2010 
                 Bt HS18-l 
               
               
                 Cry4Cb2 
                 FJ597622 
                 256033943 
                 256033942 
                 Zhu et al 
                 2011 
                 Bt Ywc2-8 
               
               
                 Cry4Cc1 
                 FJ403207 
                 234203244 
                 234203243 
                 Zhu et al 
                 2011 
                 Bt MC28 
               
               
                 Cry5Aa1 
                 AAA67694 
                   
                   
                 Narva et al 
                 1994 
                 Bt darmstadiensis PS17 
               
               
                 Cry5Ab1 
                 AAA67693 
                   
                   
                 Narva et al 
                 1991 
                 Bt darmstadiensis PS17 
               
               
                 Cry5Ac1 
                 I34543 
                   
                   
                 Payne et al 
                 1997 
               
               
                 Cry5Ad1 
                 ABQ82087 
                   
                   
                 Lenane et al 
                 2007 
                 Bt L366 
               
               
                 Cry5Ba1 
                 AAA68598 
                   
                   
                 Foncerrada &amp; Narva 
                 1997 
                 Bt PS86Q3 
               
               
                 Cry5Ba2 
                 ABW88931 
                   
                   
                 Guo et al 
                 2008 
                 YBT 1518 
               
               
                 Cry5Ba3 
                 AFJ04417 
                 386277681 
                 386277680 
                 Wang et al 
                 2012 
                 Bt zjfc85 
               
               
                 Cry5Ca1 
                 HM461869 
                   
                 328833584 
                 Sun et al 
                 2010 
                 Sbt003 
               
               
                 Cry5Ca2 
                 ZP_04123426 
                 228961871 
                   
                 Read et al 
                 2010 
                 Bt T13001 
               
               
                 Cry5Da1 
                 HM461870 
                   
                 328833586 
                 Sun et al 
                 2010 
                 Sbt003 
               
               
                 Cry5Da2 
                 ZP_04123980 
                 228962686 
                   
                 Read et al 
                 2010 
                 Bt T13001 
               
               
                 Cry5Ea1 
                 HM485580 
                   
                 339186758 
                 Sun et al 
                 2010 
                 Sbt003 
               
               
                 Cry5Ea2 
                 ZP_04124038 
                 228962776 
                   
                 Read et al 
                 2010 
                 Bt T13001 
               
               
                 Cry6Aa1 
                 AAA22357 
                   
                   
                 Narva et al 
                 1993 
                 Bt PS52A1 
               
               
                 Cry6Aa2 
                 AAM46849 
                   
                   
                 Bai et al 
                 2001 
                 YBT 1518 
               
               
                 Cry6Aa3 
                 ABH03377 
                   
                   
                 Jia et al 
                 2006 
                 Bt 96418 
               
               
                 Cry6Ba1 
                 AAA22358 
                   
                   
                 Narva et al 
                 1991 
                 Bt PS69D1 
               
               
                 Cry7Aa1 
                 AAA22351 
                   
                   
                 Lambert et al 
                 1992 
                 Bt galleriae PGSI245 
               
               
                 Cry7Ab1 
                 AAA21120 
                   
                   
                 Narva &amp; Fu 
                 1994 
                 Bt dakota HD511 
               
               
                 Cry7Ab2 
                 AAA21121 
                   
                   
                 Narva &amp; Fu 
                 1994 
                 Bt kumamotoensis 867 
               
               
                 Cry7Ab3 
                 ABX24522 
                   
                   
                 Song et al 
                 2008 
                 Bt WZ-9 
               
               
                 Cry7Ab4 
                 EU380678 
                 170877973 
                   
                 Deng et al 
                 2011 
                 Bt HQ122 
               
               
                 Cry7Ab5 
                 ABX79555 
                   
                   
                 Aguirre-Arzola et al 
                 2008 
                 Bt monterrey GM-33 
               
               
                 Cry7Ab6 
                 ACI44005 
                   
                   
                 Deng et al 
                 2008 
                 Bt HQ122 
               
               
                 Cry7Ab7 
                 ADB89216 
                   
                   
                 Wang et al 
                 2010 
                 Bt GW6 
               
               
                 Cry7Ab8 
                 GU145299 
                   
                   
                 Feng &amp; Guo 
                 2009 
               
               
                 Cry7Ab9 
                 ADD92572 
                   
                   
                 Li et al 
                 2010 
                 Bt QG-121 
               
               
                 Cry7Ba1 
                 ABB70817 
                   
                   
                 Zhang et al 
                 2006 
                 Bt huazhongensis 
               
               
                 Cry7Bb1 
                 KC156653 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry7Ca1 
                 ABR67863 
                   
                   
                 Gao et al 
                 2007 
                 Bt BTH-13 
               
               
                 Cry7Cb1 
                 KC156698 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry7Da1 
                 ACQ99547 
                   
                   
                 Yi et al 
                 2009 
                 Bt LH-2 
               
               
                 Cry7Da2 
                 HM572236 
                   
                 328751616 
                 Shu et al 
                 2010 
               
               
                 Cry7Da3 
                 KC156679 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry7Ea1 
                 HM035086 
                   
                 327505546 
                 Ming Sun et al 
                 2010 
                 Sbt009 
               
               
                 Cry7Ea2 
                 HM132124 
                   
                 327359579 
                 Shu et al 
                 2010 
               
               
                 Cry7Ea3 
                 EEM19403 
                   
                   
                 Read et al 
                 2010 
                 BGSC 4Y1 
               
               
                 Cry7Fa1 
                 HM035088 
                   
                 327505550 
                 Ming Sun et al 
                 2010 
                 SBt009 
               
               
                 Cry7Fa2 
                 EEM19090 
                   
                   
                 Read et al 
                 2010 
                 BGSC 4Y1 
               
               
                 Cry7Fb1 
                 HM572235 
                   
                 328751614 
                 Shu et al 
                 2010 
                 Bt 
               
               
                 Cry7Fb2 
                 KC156682 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry7Ga1 
                 HM572237 
                   
                 328751618 
                 Shu et al 
                 2010 
                 Bt 
               
               
                 Cry7Ga2 
                 KC156669 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry7Gb1 
                 KC156650 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry7Gc1 
                 KC156654 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry7Gd1 
                 KC156697 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry7Ha1 
                 KC156651 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry7Ia1 
                 KC156665 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry7Ja1 
                 KC156671 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry7Ka1 
                 KC156680 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry7Kb1 
                 BAM99306 
                   
                   
                 Takebe &amp; Azuma 
                 2013 
                 Bt dakota 
               
               
                 Cry7La1 
                 BAM99307 
                   
                   
                 Takebe &amp; Azuma 
                 2013 
                 Bt dakota 
               
               
                 Cry8Aa1 
                 AAA21117 
                   
                   
                 Narva &amp; Fu 
                 1992 
                 Bt kumamotoensis 
               
               
                 Cry8Ab1 
                 EU044830 
                   
                   
                 Cheng et al 
                 2007 
                 Bt B-JJX 
               
               
                 Cry8Ac1 
                 KC156662 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry8Ad1 
                 KC156684 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry8Ba1 
                 AAA21118 
                   
                   
                 Narva &amp; Fu 
                 1993 
                 Bt kumamotoensis 
               
               
                 Cry8Bb1 
                 CAD57542 
                   
                   
                 Abad et al 
                 2002 
               
               
                 Cry8Bc1 
                 CAD57543 
                   
                   
                 Abad et al 
                 2002 
               
               
                 Cry8Ca1 
                 AAA21119 
                   
                   
                 Sato et al. 
                 1995 
                 Bt japonensis Buibui 
               
               
                 Cry8Ca2 
                 AAR98783 
                   
                   
                 Shu et al 
                 2004 
                 Bt HBF-1 
               
               
                 Cry8Ca3 
                 EU625349 
                 194272339 
                 194272338 
                 Du et al 
                 2011 
                 Bt FTL-23 
               
               
                 Cry8Ca4 
                 ADB54826 
                   
                   
                 Li et al 
                 2010 
                 Bt S185 
               
               
                 Cry8Da1 
                 BAC07226 
                   
                   
                 Asano et al 
                 2002 
                 Bt galleriae 
               
               
                 Cry8Da2 
                 BD133574 
                   
                   
                 Asano et al 
                 2002 
                 Bt 
               
               
                 Cry8Da3 
                 BD133575 
                   
                   
                 Asano et al 
                 2002 
                 Bt 
               
               
                 Cry8Db1 
                 BAF93483 
                   
                   
                 Yamaguchi et al 
                 2007 
                 Bt BBT2-5 
               
               
                 Cry8Ea1 
                 AAQ73470 
                   
                   
                 Fuping et al 
                 2003 
                 Bt 185 
               
               
                 Cry8Ea2 
                 EU047597 
                   
                   
                 Liu et al 
                 2007 
                 Bt B-DLL 
               
               
                 Cry8Ea3 
                 KC855216 
                   
                   
                 Wei Wang 
                 2013 
               
               
                 Cry8Fa1 
                 AAT48690 
                   
                   
                 Shu et al 
                 2004 
                 Bt 185 
               
               
                 Cry8Fa2 
                 HQ174208 
                 307697880 
                   
                 Zang et al 
                 2010 
                 Bt DLL 
               
               
                 Cry8Fa3 
                 AFH78109 
                   
                   
                 Su et al 
                 2012 
                 Bt L-27 
               
               
                 Cry8Ga1 
                 AAT46073 
                   
                   
                 Shu et al 
                 2004 
                 Bt HBF-18 
               
               
                 Cry8Ga2 
                 ABC42043 
                   
                   
                 Yan et al 
                 2008 
                 Bt 145 
               
               
                 Cry8Ga3 
                 FJ198072 
                   
                   
                 Sun et al 
                 2010 
                 Bt FCD114 
               
               
                 Cry8Ha1 
                 AAW81032 
                   
                   
                 Fuping et al 
                 2011 
                 Bt 185 
               
               
                 Cry8Ia1 
                 EU381044 
                 170317962 
                 170317961 
                 Yan et al 
                 2008 
                 Bt su4 
               
               
                 Cry8Ia2 
                 GU073381 
                   
                 309274395 
                 Lixin Du et al 
                 2012 
                 Bt HW-11 
               
               
                 Cry8Ia3 
                 HM044664 
                   
                 328833556 
                 Ming Sun 
                 2010 
               
               
                 Cry8Ia4 
                 KC156674 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry8Ib1 
                 GU325772 
                   
                 314998609 
                 Ming Sun 
                 2012 
                 Bt F4 
               
               
                 Cry8Ib2 
                 KC156677 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry8Ja1 
                 EU625348 
                 194272337 
                 194272336 
                 Du et al 
                 2011 
                 Bt FPT-2 
               
               
                 Cry8Ka1 
                 FJ422558 
                 237506871 
                 237506870 
                 Oliveira et al 
                 2011 
               
               
                 Cry8Ka2 
                 ACN87262 
                   
                   
                 Noguera &amp; Ibarra 
                 2009 
                 Bt kenyae 
               
               
                 Cry8Kb1 
                 HM123758 
                   
                 310616446 
                 Jun Zhu et al 
                 2010 
                 ST8 
               
               
                 Cry8Kb2 
                 KC156675 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry8La1 
                 GU325771 
                 314998608 
                 314998607 
                 Ming Sun et al 
                 2012 
                 Bt F4 
               
               
                 Cry8Ma1 
                 HM044665 
                   
                 328833558 
                 Ming Sun et al 
                 2010 
                 Sbt016 
               
               
                 Cry8Ma2 
                 EEM86551 
                   
                   
                 Read et al 
                 2010 
                 BGSC 4CC1 
               
               
                 Cry8Ma3 
                 HM210574 
                   
                 305430488 
                 Jieyu Mao 
                 2010 
               
               
                 Cry8Na1 
                 HM640939 
                 302141260 
                 302141259 
                 Li et al 
                 2011 
                 BtQ52-7 
               
               
                 Cry8Pa1 
                 HQ388415 
                   
                 319769150 
                 Qiao Li 
                 2010 
                 Bt ST8 
               
               
                 Cry8Qa1 
                 HQ441166 
                   
                 321266472 
                 Hongxia Liang 
                 2010 
                 Bt ST8 
               
               
                 Cry8Qa2 
                 KC152468 
                   
                   
                 Amadio et al 
                 2012 
                 Bt INTA Fr7-4 
               
               
                 Cry8Ra1 
                 AFP87548 
                 400653691 
                   
                 Ben-Dov et al 
                 2012 
                 Bt R36 
               
               
                 Cry8Sa1 
                 JQ740599 
                   
                   
                 Singaravelu et al 
                 2012 
                 Bt Strain 62 
               
               
                 Cry8Ta1 
                 KC156673 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry8-like 
                 FJ770571 
                   
                   
                 Noguera &amp; Ibarra 
                 2009 
                 Bt canadensis 
               
               
                 Cry8-like 
                 ABS53003 
                   
                   
                 Mangena et al 
                 2007 
                 Bt 
               
               
                 Cry9Aa1 
                 CAA41122 
                   
                   
                 Shevelev et al 
                 1991 
                 Bt galleriae 
               
               
                 Cry9Aa2 
                 CAA41425 
                   
                   
                 Gleave et al 
                 1992 
                 Bt DSIR517 
               
               
                 Cry9Aa3 
                 GQ249293 
                   
                 293652149 
                 Su et al 
                 2012 
                 Bt SC5(D2) 
               
               
                 Cry9Aa4 
                 GQ249294 
                   
                 293652151 
                 Su et al 
                 2012 
                 Bt T03C001 
               
               
                 Cry9Aa5 
                 JX174110 
                   
                   
                 Naimov et al 
                 2012 
               
               
                 Cry9Aa like 
                 AAQ52376 
                   
                   
                 Baum et al 
                 2003 
               
               
                 Cry9Ba1 
                 CAA52927 
                   
                   
                 Shevelev et al 
                 1993 
                 Bt galleriae 
               
               
                 Cry9Ba2 
                 GU299522 
                   
                   
                 Zhao et al 
                 2010 
                 Bt B-SC5 
               
               
                 Cry9Bb1 
                 AAV28716 
                   
                   
                 Silva-Werneck et al 
                 2004 
                 Bt japonensis 
               
               
                 Cry9Ca1 
                 CAA85764 
                   
                   
                 Lambert et al 
                 1996 
                 Bt tolworthi 
               
               
                 Cry9Ca2 
                 AAQ52375 
                   
                   
                 Baum et al 
                 2003 
               
               
                 Cry9Da1 
                 BAA19948 
                   
                   
                 Asano 
                 1997 
                 Bt japonensis N141 
               
               
                 Cry9Da2 
                 AAB97923 
                   
                   
                 Wasano &amp; Ohba 
                 1998 
                 Bt japonensis 
               
               
                 Cry9Da3 
                 GQ249293 
                   
                 293652153 
                 Su et al 
                 2012 
                 Bt SC5 (D2) 
               
               
                 Cry9Da4 
                 GQ249297 
                   
                 293652157 
                 Su et al 
                 2012 
                 Bt T03B001 
               
               
                 Cry9Db1 
                 AAX78439 
                   
                   
                 Flannagan &amp; Abad 
                 2005 
                 Bt kurstaki DP1019 
               
               
                 Cry9Dc1 
                 KC156683 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry9Ea1 
                 BAA34908 
                   
                   
                 Midoh &amp; Oyama 
                 1998 
                 Bt aizawai SSK-10 
               
               
                 Cry9Ea2 
                 AAO12908 
                   
                   
                 Li et al 
                 2001 
                 Bt B-Hm-16 
               
               
                 Cry9Ea3 
                 ABM21765 
                   
                   
                 Lin et al 
                 2006 
                 Bt lyA 
               
               
                 Cry9Ea4 
                 ACE88267 
                   
                   
                 Zhu et al 
                 2008 
                 Bt ywc5-4 
               
               
                 Cry9Ea5 
                 ACF04743 
                   
                   
                 Zhu et al 
                 2008 
                 Bts 
               
               
                 Cry9Ea6 
                 ACG63872 
                   
                   
                 Liu &amp; Guo 
                 2008 
                 Bt 11 
               
               
                 Cry9Ea7 
                 FJ380927 
                   
                   
                 Sun et al 
                 2009 
                 Bt 4 
               
               
                 Cry9Ea8 
                 GQ249292 
                   
                 293652147 
                 Su et al 
                 2012 
                 Bt SC5(E8) 
               
               
                 Cry9Ea9 
                 JN651495 
                   
                   
                 Li Yuhong 
                 2011 
                 Bt LTS-7 
               
               
                 Cry9Eb1 
                 CAC50780 
                   
                   
                 Arnaut et al 
                 2001 
               
               
                 Cry9Eb2 
                 GQ249298 
                   
                 293652159 
                 Su et al 
                 2012 
                 Bt T23001 
               
               
                 Cry9Eb3 
                 KC156646 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry9Ec1 
                 AAC63366 
                   
                   
                 Wasano et al 
                 2003 
                 Bt galleriae 
               
               
                 Cry9Ed1 
                 AAX78440 
                   
                   
                 Flannagan &amp; Abad 
                 2005 
                 Bt kurstaki DP1019 
               
               
                 Cry9Ee1 
                 GQ249296 
                   
                 293652155 
                 Su et al 
                 2009 
                 Bt T03B001 
               
               
                 Cry9Ee2 
                 KC156664 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry9Fa1 
                 KC156692 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry9Ga1 
                 KC156699 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry9-like 
                 AAC63366 
                   
                   
                 Wasano et al 
                 1998 
                 Bt galleriae 
               
               
                 Cry10Aa1 
                 AAA22614 
                   
                   
                 Thorne et al 
                 1986 
                 Bt israelensis 
               
               
                 Cry10Aa2 
                 E00614 
                   
                   
                 Aran &amp; Toomasu 
                 1996 
                 Bt israelensis ONR-60A 
               
               
                 Cry10Aa3 
                 CAD30098 
                   
                   
                 Berry et al 
                 2002 
                 Bt israelensis 
               
               
                 Cry10Aa4 
                 AFB18318 
                   
                   
                 Li et al 
                 2012 
                 Bti BRC-LLP29 
               
               
                 Cry10A-like 
                 DQ167578 
                   
                   
                 Mahalakshmi et al 
                 2006 
                 Bt LDC-9 
               
               
                 Cry11Aa1 
                 AAA22352 
                   
                   
                 Donovan et al 
                 1988 
                 Bt israelensis 
               
               
                 Cry11Aa2 
                 AAA22611 
                   
                   
                 Adams et al 
                 1989 
                 Bt israelensis 
               
               
                 Cry11Aa3 
                 CAD30081 
                   
                   
                 Berry et al 
                 2002 
                 Bt israelensis 
               
               
                 Cry11Aa4 
                 AFB18319 
                   
                   
                 Li et al 
                 2012 
                 Bti BRC-LLP29 
               
               
                 Cry11Aa-like 
                 DQ166531 
                   
                   
                 Mahalakshmi et al 
                 2007 
                 Bt LDC-9 
               
               
                 Cry11Ba1 
                 CAA60504 
                   
                   
                 Delecluse et al 
                 1995 
                 Bt jegathesan 367 
               
               
                 Cry11Bb1 
                 AAC97162 
                   
                   
                 Orduz et al 
                 1998 
                 Bt medellin 
               
               
                 Cry11Bb2 
                 HM068615 
                   
                   
                 Melnikov et al 
                 2010 
                 Bt K34 
               
               
                 Cry12Aa1 
                 AAA22355 
                   
                   
                 Narva et al 
                 1991 
                 Bt PS33F2 
               
               
                 Cry13Aa1 
                 AAA22356 
                   
                   
                 Narva et al 
                 1992 
                 Bt PS63B 
               
               
                 Cry14Aa1 
                 AAA21516 
                   
                   
                 Narva et al 
                 1994 
                 Bt sotto PS80JJ1 
               
               
                 Cry14Ab1 
                 KC156652 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry15Aa1 
                 AAA22333 
                   
                   
                 Brown &amp; Whiteley 
                 1992 
                 Bt thompsoni 
               
               
                 Cry16Aa1 
                 CAA63860 
                   
                   
                 Barloy et al 
                 1996 
                 Cb malaysia CH18 
               
               
                 Cry17Aa1 
                 CAA67841 
                   
                   
                 Barloy et al 
                 1998 
                 Cb malaysia CH18 
               
               
                 Cry18Aa1 
                 CAA67506 
                   
                   
                 Zhang et al 
                 1997 
                 
                   Paenibacillus 
                 
               
               
                   
                   
                   
                   
                   
                   
                 
                   popilliae 
                 
               
               
                 Cry18Ba1 
                 AAF89667 
                   
                   
                 Patel et al 
                 1999 
                 
                   Paenibacillus 
                 
               
               
                   
                   
                   
                   
                   
                   
                 
                   popilliae 
                 
               
               
                 Cry18Ca1 
                 AAF89668 
                   
                   
                 Patel et al 
                 1999 
                 
                   Paenibacillus 
                 
               
               
                   
                   
                   
                   
                   
                   
                 
                   popilliae 
                 
               
               
                 Cry19Aa1 
                 CAA68875 
                   
                   
                 Rosso &amp; Delecluse 
                 1996 
                 Bt jegathesan 367 
               
               
                 Cry19Ba1 
                 BAA32397 
                   
                   
                 Hwang et al 
                 1998 
                 Bt higo 
               
               
                 Cry19Ca1 
                 AFM37572 
                   
                   
                 Soufiane &amp; Cote 
                 2012 
                 BGSC 4CE1 
               
               
                 Cry20Aa1 
                 AAB93476 
                   
                   
                 Lee &amp; Gill 
                 1997 
                 Bt fukuokaensis 
               
               
                 Cry20Ba1 
                 ACS93601 
                   
                   
                 Noguera &amp; Ibarra 
                 2009 
                 Bt higo LBIT-976 
               
               
                 Cry20Ba2 
                 KC156694 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry20-like 
                 GQ144333 
                   
                   
                 Yi et al 
                 2009 
                 Bt Y-5 
               
               
                 Cry21Aa1 
                 I32932 
                   
                   
                 Payne et al 
                 1996 
               
               
                 Cry21Aa2 
                 I66477 
                   
                   
                 Feitelson 
                 1997 
               
               
                 Cry21Ba1 
                 BAC06484 
                   
                   
                 Sato &amp; Asano 
                 2002 
                 Bt roskildiensis 
               
               
                 Cry21Ca1 
                 JF521577 
                   
                   
                 Liu et al 
                 2013 
               
               
                 Cry21Ca2 
                 KC156687 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry21Da1 
                 JF521578 
                   
                   
                 Liu et al 
                 2011 
                 Sbt072 
               
               
                 Cry21Ea1 
                 KC865049 
                   
                   
                 Ming Sun 
                 2013 
               
               
                 Cry21Fa1 
                 KF701307 
                   
                   
                 Iatsenko et al 
                 2013 
               
               
                 Cry21Ga1 
                 KF771885 
                   
                   
                 Iatsenko et al 
                 2013 
               
               
                 Cry21Ha1 
                 KF771886 
                   
                   
                 Iatsenko et al 
                 2013 
               
               
                 Cry22Aa1 
                 I34547 
                   
                   
                 Payne et al 
                 1997 
               
               
                 Cry22Aa2 
                 CAD43579 
                   
                   
                 Isaac et al 
                 2002 
                 Bt 
               
               
                 Cry22Aa3 
                 ACD93211 
                   
                   
                 Du et al 
                 2008 
                 Bt FZ-4 
               
               
                 Cry22Ab1 
                 AAK50456 
                   
                   
                 Baum et al 
                 2000 
                 Bt EG4140 
               
               
                 Cry22Ab2 
                 CAD43577 
                   
                   
                 Isaac et al 
                 2002 
                 Bt 
               
               
                 Cry22Ba1 
                 CAD43578 
                   
                   
                 Isaac et al 
                 2002 
                 Bt 
               
               
                 Cry22Bb1 
                 KC156672 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry23Aa1 
                 AAF76375 
                   
                   
                 Donovan et al 
                 2000 
                 Bt 
               
               
                 Cry24Aa1 
                 AAC61891 
                   
                   
                 Kawalek and Gill 
                 1998 
                 Bt jegathesan 
               
               
                 Cry24Ba1 
                 BAD32657 
                   
                   
                 Ohgushi et al 
                 2004 
                 Bt sotto 
               
               
                 Cry24Ca1 
                 CAJ43600 
                   
                   
                 Beron &amp; Salerno 
                 2005 
                 Bt FCC-41 
               
               
                 Cry25Aa1 
                 AAC61892 
                   
                   
                 Kawalek and Gill 
                 1998 
                 Bt jegathesan 
               
               
                 Cry26Aa1 
                 AAD25075 
                   
                   
                 Wojciechowska et al 
                 1999 
                 Bt finitimus B-1166 
               
               
                 Cry27Aa1 
                 BAA82796 
                   
                   
                 Saitoh 
                 1999 
                 Bt higo 
               
               
                 Cry28Aa1 
                 AAD24189 
                   
                   
                 Wojciechowska et al 
                 1999 
                 Bt finitimus B-1161 
               
               
                 Cry28Aa2 
                 AAG00235 
                   
                   
                 Moore and Debro 
                 2000 
                 Bt finitimus 
               
               
                 Cry29Aa1 
                 CAC80985 
                   
                   
                 Delecluse et al 
                 2000 
                 Bt medellin 
               
               
                 Cry29Ba1 
                 KC865046 
                   
                   
                 Ming Sun 
                 2013 
               
               
                 Cry30Aa1 
                 CAC80986 
                   
                   
                 Delecluse et al 
                 2000 
                 Bt medellin 
               
               
                 Cry30Ba1 
                 BAD00052 
                   
                   
                 Ito et al 
                 2003 
                 Bt entomocidus 
               
               
                 Cry30Ca1 
                 BAD67157 
                   
                   
                 Ohgushi et al 
                 2004 
                 Bt sotto 
               
               
                 Cry30Ca2 
                 ACU24781 
                   
                   
                 Sun and Park 
                 2009 
                 Bt jegathesan 367 
               
               
                 Cry30Da1 
                 EF095955 
                   
                   
                 Shu et al 
                 2006 
                 Bt Y41 
               
               
                 Cry30Db1 
                 BAE80088 
                   
                   
                 Kishida et al 
                 2006 
                 Bt aizawai BUN1-14 
               
               
                 Cry30Ea1 
                 ACC95445 
                   
                   
                 Fang et al 
                 2007 
                 Bt S2160-1 
               
               
                 Cry30Ea2 
                 FJ499389 
                 237688240 
                 237688239 
                 Zhu et al 
                 2011 
                 Bt Ywc2-8 
               
               
                 Cry30Fa1 
                 ACI22625 
                   
                   
                 Tan et al 
                 2008 
                 Bt MC28 
               
               
                 Cry30Ga1 
                 ACG60020 
                   
                   
                 Zhu et al 
                 2008 
                 Bt HS18-1 
               
               
                 Cry30Ga2 
                 HQ638217 
                 320383831 
                 320383830 
                 Tian et al 
                 2010 
                 Bt S2160-1 
               
               
                 Cry31Aa1 
                 BAB11757 
                   
                   
                 Saitoh &amp; Mizuki 
                 2000 
                 Bt 84-HS-1-11 
               
               
                 Cry31Aa2 
                 AAL87458 
                   
                   
                 Jung and Cote 
                 2000 
                 Bt M15 
               
               
                 Cry31Aa3 
                 BAE79808 
                   
                   
                 Uemori et al 
                 2006 
                 Bt B0195 
               
               
                 Cry31Aa4 
                 BAF32571 
                   
                   
                 Yasutake et al 
                 2006 
                 Bt 79-25 
               
               
                 Cry31Aa5 
                 BAF32572 
                   
                   
                 Yasutake et al 
                 2006 
                 Bt 92-10 
               
               
                 Cry31Aa6 
                 BAI44026 
                   
                   
                 Nagamatsu et al 
                 2010 
                 M019 
               
               
                 Cry31Ab1 
                 BAE79809 
                   
                   
                 Uemori et al 
                 2006 
                 Bt B0195 
               
               
                 Cry31Ab2 
                 BAF32570 
                   
                   
                 Yasutake et al 
                 2006 
                 Bt 31-5 
               
               
                 Cry31Ac1 
                 BAF34368 
                   
                   
                 Yasutake et al 
                 2006 
                 Bt 87-29 
               
               
                 Cry31Ac2 
                 AB731600 
                   
                   
                 Hayakawa et al 
                 2012 
                 Bt B0462 
               
               
                 Cry31Ad1 
                 BAI44022 
                   
                   
                 Nagamatsu et al 
                 2010 
                 Bt MO19 
               
               
                 Cry32Aa1 
                 AAG36711 
                   
                   
                 Balasubramanian et al 
                 2001 
                 Bt yunnanensis 
               
               
                 Cry32Aa2 
                 GU063849 
                   
                 308445182 
                 Lixin Du et al 
                 2012 
                 Bt FBG-1 
               
               
                 Cry32Ab1 
                 GU063850 
                   
                 308445184 
                 Lixin Du et al 
                 2012 
                 Bt FZ-2 
               
               
                 Cry32Ba1 
                 BAB78601 
                   
                   
                 Takebe et al 
                 2001 
                 Bt 
               
               
                 Cry32Ca1 
                 BAB78602 
                   
                   
                 Takebe et al 
                 2001 
                 Bt 
               
               
                 Cry32Cb1 
                 KC156708 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32Da1 
                 BAB78603 
                   
                   
                 Takebe et al 
                 2001 
                 Bt 
               
               
                 Cry32Ea1 
                 GU324274 
                   
                 301299156 
                 Lixin Du 
                 2010 
                 Bt 
               
               
                 Cry32Ea2 
                 KC156686 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32Eb1 
                 KC156663 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32Fa1 
                 KC156656 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32Ga1 
                 KC156657 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32Ha1 
                 KC156661 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32Hb1 
                 KC156666 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32Ia1 
                 KC156667 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32Ja1 
                 KC156685 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32Ka1 
                 KC156688 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32La1 
                 KC156689 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32Ma1 
                 KC156690 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32Mb1 
                 KC156704 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32Na1 
                 KC156691 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32Oa1 
                 KC156703 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32Pa1 
                 KC156705 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32Qa1 
                 KC156706 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32Ra1 
                 KC156707 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32Sa1 
                 KC156709 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32Ta1 
                 KC156710 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry32Ua1 
                 KC156655 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry33Aa1 
                 AAL26871 
                   
                   
                 Kim et al 
                 2001 
                 Bt dakota 
               
               
                 Cry34Aa1 
                 AAG50341 
                   
                   
                 Ellis et al 
                 2001 
                 Bt PS80JJ1 
               
               
                 Cry34Aa2 
                 AAK64560 
                   
                   
                 Rupar et al 
                 2001 
                 Bt EG5899 
               
               
                 Cry34Aa3 
                 AAT29032 
                   
                   
                 Schnepf et al 
                 2004 
                 Bt PS69Q 
               
               
                 Cry34Aa4 
                 AAT29030 
                   
                   
                 Schnepf et al 
                 2004 
                 Bt PS185GG 
               
               
                 Cry34Ab1 
                 AAG41671 
                   
                   
                 Moellenbeck et al 
                 2001 
                 Bt PS149B1 
               
               
                 Cry34Ac1 
                 AAG50118 
                   
                   
                 Ellis et al 
                 2001 
                 Bt PS167H2 
               
               
                 Cry34Ac2 
                 AAK64562 
                   
                   
                 Rupar et al 
                 2001 
                 Bt EG9444 
               
               
                 Cry34Ac3 
                 AAT29029 
                   
                   
                 Schnepf et al 
                 2004 
                 Bt KR1369 
               
               
                 Cry34Ba1 
                 AAK64565 
                   
                   
                 Rupar et al 
                 2001 
                 Bt EG4851 
               
               
                 Cry34Ba2 
                 AAT29033 
                   
                   
                 Schnepf et al 
                 2004 
                 Bt PS201L3 
               
               
                 Cry34Ba3 
                 AAT29031 
                   
                   
                 Schnepf et al 
                 2004 
                 Bt PS201HH2 
               
               
                 Cry35Aa1 
                 AAG50342 
                   
                   
                 Ellis et al 
                 2001 
                 Bt PS80JJ1 
               
               
                 Cry35Aa2 
                 AAK64561 
                   
                   
                 Rupar et al 
                 2001 
                 Bt EG5899 
               
               
                 Cry35Aa3 
                 AAT29028 
                   
                   
                 Schnepf et al 
                 2004 
                 Bt PS69Q 
               
               
                 Cry35Aa4 
                 AAT29025 
                   
                   
                 Schnepf et al 
                 2004 
                 Bt PS185GG 
               
               
                 Cry35Ab1 
                 AAG41672 
                   
                   
                 Moellenbeck et al 
                 2001 
                 Bt PS149B1 
               
               
                 Cry35Ab2 
                 AAK64563 
                   
                   
                 Rupar et al 
                 2001 
                 Bt EG9444 
               
               
                 Cry35Ab3 
                 AY536891 
                   
                   
                 AAT29024 
                 2004 
                 Bt KR1369 
               
               
                 Cry35Ac1 
                 AAG50117 
                   
                   
                 Ellis et al 
                 2001 
                 Bt PS167H2 
               
               
                 Cry35Ba1 
                 AAK64566 
                   
                   
                 Rupar et al 
                 2001 
                 Bt EG4851 
               
               
                 Cry35Ba2 
                 AAT29027 
                   
                   
                 Schnepf et al 
                 2004 
                 Bt PS201L3 
               
               
                 Cry35Ba3 
                 AAT29026 
                   
                   
                 Schnepf et al 
                 2004 
                 Bt PS201HH2 
               
               
                 Cry36Aa1 
                 AAK64558 
                   
                   
                 Rupar et al 
                 2001 
                 Bt 
               
               
                 Cry37Aa1 
                 AAF76376 
                   
                   
                 Donovan et al 
                 2000 
                 Bt 
               
               
                 Cry38Aa1 
                 AAK64559 
                   
                   
                 Rupar et al 
                 2000 
                 Bt 
               
               
                 Cry39Aa1 
                 BAB72016 
                   
                   
                 Ito et al 
                 2001 
                 Bt aizawai 
               
               
                 Cry40Aa1 
                 BAB72018 
                   
                   
                 Ito et al 
                 2001 
                 Bt aizawai 
               
               
                 Cry40Ba1 
                 BAC77648 
                   
                   
                 Ito et al 
                 2003 
                 Bun1-14 
               
               
                 Cry40Ca1 
                 EU381045 
                 170317964 
                 170317963 
                 Shu et al 
                 2011 
                 Bt Y41 
               
               
                 Cry40Da1 
                 ACF15199 
                   
                   
                 Zhang et al 
                 2008 
                 Bt S2096-2 
               
               
                 Cry41Aa1 
                 BAD35157 
                   
                   
                 Yamashita et al 
                 2003 
                 Bt A1462 
               
               
                 Cry41Ab1 
                 BAD35163 
                   
                   
                 Yamashita et al 
                 2003 
                 Bt A1462 
               
               
                 Cry41Ba1 
                 HM461871 
                   
                 328833588 
                 Sun et al 
                 2010 
                 Sbt021 
               
               
                 Cry41Ba2 
                 ZP_04099652 
                 228936898 
                   
                 Read et al 
                 2010 
                 BGSC 4AW1 
               
               
                 Cry42Aa1 
                 BAD35166 
                   
                   
                 Yamashita et al 
                 2003 
                 Bt A1462 
               
               
                 Cry43Aa1 
                 BAD15301 
                   
                   
                 Yokoyama and Tanaka 
                 2003 
                 
                   P. lentimorbus 
                 
               
               
                   
                   
                   
                   
                   
                   
                 
                   semadara 
                 
               
               
                 Cry43Aa2 
                 BAD95474 
                   
                   
                 Nozawa 
                 2004 
                 
                   P. popilliae popilliae 
                 
               
               
                 Cry43Ba1 
                 BAD15303 
                   
                   
                 Yokoyama and Tanaka 
                 2003 
                 
                   P. lentimorbus 
                 
               
               
                   
                   
                   
                   
                   
                   
                 
                   semadara 
                 
               
               
                 Cry43Ca1 
                 KC156676 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry43Cb1 
                 KC156695 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry43Cc1 
                 KC156696 
                   
                   
                 Sampson et al 
                 2012 
               
               
                 Cry43-like 
                 BAD15305 
                   
                   
                 Yokoyama and Tanaka 
                 2003 
                 
                   P. lentimorbus 
                 
               
               
                   
                   
                   
                   
                   
                   
                 
                   semadara 
                 
               
               
                 Cry44Aa 
                 BAD08532 
                   
                   
                 Ito et al 
                 2004 
                 Bt entomocidus INA288 
               
               
                 Cry45Aa 
                 BAD22577 
                   
                   
                 Okumura et al 
                 2004 
                 Bt 89-T-34-22 
               
               
                 Cry46Aa 
                 BAC79010 
                   
                   
                 Ito et al 
                 2004 
                 Bt dakota 
               
               
                 Cry46Aa2 
                 BAG68906 
                   
                   
                 Ishikawa et al 
                 2008 
                 Bt A1470 
               
               
                 Cry46Ab 
                 BAD35170 
                   
                   
                 Yamagiwa et al 
                 2004 
                 Bt 
               
               
                 Cry47Aa 
                 AAY24695 
                   
                   
                 Kongsuwan et al 
                 2005 
                 Bt CAA890 
               
               
                 Cry48Aa 
                 CAJ18351 
                   
                   
                 Jones and Berry 
                 2005 
                 Bs IAB59 
               
               
                 Cry48Aa2 
                 CAJ86545 
                   
                   
                 Jones and Berry 
                 2006 
                 Bs 47-6B 
               
               
                 Cry48Aa3 
                 CAJ86546 
                   
                   
                 Jones and Berry 
                 2006 
                 Bs NHA15b 
               
               
                 Cry48Ab 
                 CAJ86548 
                   
                   
                 Jones and Berry 
                 2006 
                 Bs LP1G 
               
               
                 Cry48Ab2 
                 CAJ86549 
                   
                   
                 Jones and Berry 
                 2006 
                 Bs 2173 
               
               
                 Cry49Aa 
                 CAH56541 
                   
                   
                 Jones and Berry 
                 2005 
                 Bs IAB59 
               
               
                 Cry49Aa2 
                 CAJ86541 
                   
                   
                 Jones and Berry 
                 2006 
                 Bs 47-6B 
               
               
                 Cry49Aa3 
                 CAJ86543 
                   
                   
                 Jones and Berry 
                 2006 
                 BsNHA15b 
               
               
                 Cry49Aa4 
                 CAJ86544 
                   
                   
                 Jones and Berry 
                 2006 
                 Bs 2173 
               
               
                 Cry49Ab1 
                 CAJ86542 
                   
                   
                 Jones and Berry 
                 2006 
                 Bs LP1G 
               
               
                 Cry50Aa1 
                 BAE86999 
                 89885725 
                 89885724 
                 Ohgushi et al 
                 2006 
                 Bt sotto 
               
               
                 Cry50Ba1 
                 GU446675 
                   
                   
                 Zhang &amp; Fang 
                 2011 
                 Bt S2160-1 
               
               
                 Cry50Ba2 
                 GU446676 
                   
                   
                 Zhang et al 
                 2011 
                 Bt S3161-3 
               
               
                 Cry51Aa1 
                 ABI14444 
                 112253719 
                 112253718 
                 Meng et al 
                 2006 
                 Bt F14-1 
               
               
                 Cry51Aa2 
                 GU570697 
                   
                   
                 Baum et al 
                 2011 
                 EG2934 
               
               
                 Cry52Aa1 
                 EF613489 
                   
                   
                 Shu et al 
                 2010 
                 Bt Y41 
               
               
                 Cry52Ba1 
                 FJ361760 
                 227976386 
                 227976385 
                 Zhu et al 
                 2010 
                 Bt BM59-2 
               
               
                 Cry53Aa1 
                 EF633476 
                   
                   
                 Shu et al 
                 2010 
                 Bt Y41 
               
               
                 Cry53Ab1 
                 FJ361759 
                 227976384 
                 227976383 
                 Zhu et al 
                 2011 
                 Bt MC28 
               
               
                 Cry54Aa1 
                 ACA52194 
                 169261091 
                 169261090 
                 Tan et al 
                 2009 
                 Bt MC28 
               
               
                 Cry54Aa2 
                 GQ140349 
                   
                 291010566 
                 Lixin Du et al 
                 2012 
                 Bt FBG25 
               
               
                 Cry54Ab1 
                 JQ916908 
                   
                   
                 Guan Peng 
                 2012 
                 Bt MC28 
               
               
                 Cry54Ba1 
                 GU446677 
                   
                   
                 Zhang &amp; Fang 
                 2010 
                 Bt S2160-1 
               
               
                 Cry55Aa1 
                 ABW88932 
                   
                   
                 Guo et al 
                 2008 
                 YBT 1518 
               
               
                 Cry55Aa2 
                 AAE33526 
                 10056620 
                   
                 Bradfisch et al 
                 2000 
                 Bt Y41 
               
               
                 Cry55Aa3 
                 HG764207 
                   
                   
                 Balasubramani et al 
                 2013 
                 Bt T44 
               
               
                 Cry56Aa1 
                 ACU57499 
                 256033941 
                 256033940 
                 Zhu et al 
                 2010 
                 Bt Ywc2-8 
               
               
                 Cry56Aa2 
                 GQ483512 
                 300837105 
                 300837104 
                 Guan et al 
                 2009 
                 Bt G7-1 
               
               
                 Cry56Aa3 
                 JX025567 
                   
                   
                 Qiao Li et al 
                 2012 
                 Bt HS18-1 
               
               
                 Cry57Aa1 
                 ANC87261 
                 225348555 
                 225348554 
                 Noguera &amp; Ibarra 
                 2009 
                 Bt kim 
               
               
                 Cry57Ab1 
                 KF638650 
                   
                   
                 Guowang Zhou 
                 2013 
               
               
                 Cry58Aa1 
                 ANC87260 
                 225348553 
                 225348552 
                 Noguera &amp; Ibarra 
                 2009 
                 Bt entomocidus 
               
               
                 Cry59Ba1 
                 JN790647 
                   
                   
                 Qiao Li et al 
                 2012 
                 Bt Bm59-2 
               
               
                 Cry59Aa1 
                 ACR43758 
                 239638225 
                 239638224 
                 Noguera &amp; Ibarra 
                 2009 
                 Bt kim LBIT-980 
               
               
                 Cry60Aa1 
                 ACU24782 
                 255653180 
                 255653179 
                 Sun and Park 
                 2009 
                 Bt jegathesan 
               
               
                 Cry60Aa2 
                 EAO57254 
                 74494162 
                 74494143 
                 Anderson et al 
                 2005 
                 Bt israelensis 
               
               
                 Cry60Aa3 
                 EEM99278 
                 228854669 
                 228854666 
                 Read et al 
                 2009 
                 Bt IBL 4222 
               
               
                 Cry60Ba1 
                 GU810818 
                 292398077 
                 292398076 
                 Sun and Park 
                 2009 
                 Bt malayensis 
               
               
                 Cry60Ba2 
                 EAO57253 
                   
                   
                 Anderson et al 
                 2005 
                 Bt israelensis 
               
               
                 Cry60Ba3 
                 EEM99279 
                   
                   
                 Read et al 
                 2009 
                 Bt IBL 4222 
               
               
                 Cry61Aa1 
                 HM035087 
                   
                 327505548 
                 Ming Sun et al 
                 2010 
                 Sbt009 
               
               
                 Cry61Aa2 
                 HM132125 
                   
                 327359581 
                 Shu et al 
                 2010 
               
               
                 Cry61Aa3 
                 EEM19308 
                 228770790 
                 228770789 
                 Read et al 
                 2010 
                 BGSC 4Y1 
               
               
                 Cry62Aa1 
                 HM054509 
                   
                 302753235 
                 Jun Zhu et al 
                 2010 
                 ST7 
               
               
                 Cry63Aa1 
                 BAI44028 
                 260268375 
                   
                 Nagamatsu et al 
                 2010 
                 MO19 
               
               
                 Cry64Aa1 
                 BAJ05397 
                 294661779 
                   
                 Ekino et al 
                 2010 
                 Bt tohokuensis 
               
               
                 Cry65Aa1 
                 HM461868 
                   
                 328833581 
                 Sun et al 
                 2010 
                 SBt 003 
               
               
                 Cry65Aa2 
                 ZP_04123838 
                 228962456 
                   
                 Read et al 
                 2010 
                 T13001 
               
               
                 Cry66Aa1 
                 HM485581 
                   
                 339186760 
                 Sun et al 
                 2010 
                 SBt 021 
               
               
                 Cry66Aa2 
                 ZP_04099945 
                 228937265 
                   
                 Read et al 
                 2010 
                 BGSC 4AW1 
               
               
                 Cry67Aa1 
                 HM485582 
                   
                 339186762 
                 Sun et al 
                 2010 
                 SBt 009 
               
               
                 Cry67Aa2 
                 ZP_04148882 
                 228988817 
                   
                 Read et al 
                 2010 
                 BGSC 4Y1 
               
               
                 Cry68Aa1 
                 HQ113114 
                   
                 327466752 
                 Peng Guan et al 
                 2012 
                 Bt MC28 
               
               
                 Cry69Aa1 
                 HQ401006 
                   
                 332139130 
                 Peng Guan 
                 2011 
                 Bt MC28 
               
               
                 Cry69Aa2 
                 JQ821388 
                   
                   
                 Peng Guan 
                 2012 
                 Bt MC28 
               
               
                 Cry69Ab1 
                 JN209957 
                   
                   
                 Yujie Tang 
                 2011 
                 Bt hs18-1 
               
               
                 Cry70Aa1 
                 JN646781 
                   
                   
                 Qiao Li 
                 2011 
                 Bt hs18-1 
               
               
                 Cry70Ba1 
                 ADO51070 
                 308756031 
                   
                 Guan et al 
                 2011 
                 Bt MC28 
               
               
                 Cry70Bb1 
                 EEL67276 
                 228715456 
                   
                 Read et al 
                 2009 
                 Bc AH603 
               
               
                 Cry71Aa1 
                 JX025568 
                   
                   
                 Qiao Li et al 
                 2012 
                 Bt Hs18-1 
               
               
                 Cry72Aa1 
                 JX025569 
                   
                   
                 Qiao Li et al 
                 2012 
                 Bt Hs18-1 
               
               
                 Cyt1Aa1 
                 X03182 
                   
                   
                 Waalwijk et al 
                 1985 
                 Bt israelensis 
               
               
                 Cyt1Aa2 
                 X04338 
                   
                   
                 Ward &amp; Ellar 
                 1986 
                 Bt israelensis 
               
               
                 Cyt1Aa3 
                 Y00135 
                   
                   
                 Earp &amp; Ellar 
                 1987 
                 Bt morrisoni PG14 
               
               
                 Cyt1Aa4 
                 M35968 
                   
                   
                 Galjart et al 
                 1987 
                 Bt morrisoni PG14 
               
               
                 Cyt1Aa5 
                 AL731825 
                   
                   
                 Berry et al 
                 2002 
                 Bt israelensis 
               
               
                 Cyt1Aa6 
                 ABC17640 
                   
                   
                 Zhang et al 
                 2005 
                 Bt LLP29 
               
               
                 Cyt1Aa7 
                 KF152888 
                   
                   
                 Qinyang Hong 
                 2013 
                 Bt BRC-HQY1 
               
               
                 Cyt1Aa-like 
                 ABB01172 
                   
                   
                 Mahalakshmi 
                 2007 
                 Bt LDC-9 
               
               
                 Cyt1Ab1 
                 X98793 
                   
                   
                 Thiery et al 
                 1997 
                 Bt medellin 
               
               
                 Cyt1Ba1 
                 U37196 
                   
                   
                 Payne et al 
                 1995 
                 Bt neoleoensis 
               
               
                 Cyt1Ca1 
                 AL731825 
                   
                   
                 Berry et al 
                 2002 
                 Bt israelensis 
               
               
                 Cyt1Da1 
                 HQ113115 
                   
                 317575156 
                 Peng Guan 
                 2012 
                 Bt MC28 
               
               
                 Cyt1Da2 
                 JN226105 
                   
                 354551244 
                 Yujie Tang 
                 2011 
                 hs18-1 
               
               
                 Cyt2Aa1 
                 Z14147 
                   
                   
                 Koni &amp; Ellar 
                 1993 
                 Bt kyushuensis 
               
               
                 Cyt2Aa2 
                 AF472606 
                   
                   
                 Promdonkoy &amp; Panyim 
                 2001 
                 Bt darmstadiensis73E10 
               
               
                 Cyt2Aa3 
                 EU835185 
                   
                   
                 Zhu et al 
                 2008 
                 Bt MC28 
               
               
                 Cyt2Aa4 
                 AEG19547 
                   
                   
                 Guo et al 
                 2011 
                 Bt WFS-97 
               
               
                 Cyt2Ba1 
                 U52043 
                   
                   
                 Guerchicoff et al 
                 1997 
                 Bt israelensis 4Q2 
               
               
                 Cyt2Ba2 
                 AF020789 
                   
                   
                 Guerchicoff et al 
                 1997 
                 Bt israelensis PG14 
               
               
                 Cyt2Ba3 
                 AF022884 
                   
                   
                 Guerchicoff et al 
                 1997 
                 Bt fuokukaensis 
               
               
                 Cyt2Ba4 
                 AF022885 
                   
                   
                 Guerchicoff et al 
                 1997 
                 Bt morrisoni HD12 
               
               
                 Cyt2Ba5 
                 AF022886 
                   
                   
                 Guerchicoff et al 
                 1997 
                 Bt morrisoni HD518 
               
               
                 Cyt2Ba6 
                 AF034926 
                   
                   
                 Guerchicoff et al 
                 1997 
                 Bt tenebrionis 
               
               
                 Cyt2Ba7 
                 AF215645 
                   
                   
                 Yu &amp; Pang 
                 2000 
                 Bt T301 
               
               
                 Cyt2Ba8 
                 AF215646 
                   
                   
                 Yu &amp; Pang 
                 2000 
                 Bt T36 
               
               
                 Cyt2Ba9 
                 AL731825 
                   
                   
                 Berry et al 
                 2002 
                 Bt israelensis 
               
               
                 Cyt2Ba10 
                 ACX54358 
                   
                   
                 Sauka &amp; Benintende 
                 2009 
                 Bti HD 567 
               
               
                 Cyt2Ba11 
                 ACX54359 
                   
                   
                 Sauka &amp; Benintende 
                 2009 
                 Bti HD 522 
               
               
                 Cyt2Ba12 
                 ACX54360 
                   
                   
                 Sauka &amp; Benintende 
                 2009 
                 Bti INTA H41-1 
               
               
                 Cyt2Ba13 
                 FJ205865 
                 209168617 
                   
                 Sauka &amp; Benintende 
                 2009 
                 INTA 160-2 
               
               
                 Cyt2Ba14 
                 FJ205866 
                 209168619 
                   
                 Sauka &amp; Benintende 
                 2009 
                 Bti IPS82 
               
               
                 Cyt2Ba15 
                 JF283552 
                 342360662 
                 342360661 
                 Zhang et al 
                 2011 
                 Bt LLP29 
               
               
                 Cyt2Ba-like 
                 ABE99695 
                   
                   
                 Mahalakshmi et al 
                 2007 
                 Bt LDC-9 
               
               
                 Cyt2Bb1 
                 U82519 
                   
                   
                 Cheong &amp; Gill 
                 1997 
                 Bt jegathesan 
               
               
                 Cyt2Bc1 
                 CAC80987 
                   
                   
                 Delecluse et al 
                 1999 
                 Bt medellin 
               
               
                 Cyt2B-like 
                 DQ341380 
                   
                   
                 Zhang et al 
                 2005 
               
               
                 Cyt2Ca1 
                 AAK50455 
                   
                   
                 Baum et al 
                 2001 
                 Bt 
               
               
                 Cyt3Aa1 
                 HM596591 
                   
                 305433345 
                 Zhu Jun 
                 2010 
                 Bt TD516 
               
               
                   
               
             
          
         
       
     
         [0036]    The article “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one or more element. 
         [0037]    All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. 
         [0038]    Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. 
         [0039]    The following examples are offered by way of illustration and not by way of limitation. 
       EXPERIMENTAL 
     Example 1 
       [0040]    Sampling and DNA preparation: Soil samples were collected from diverse environmental niches on private property in NC. Genomic DNA was prepared from 400 mg of each sample with the NucleoSpin Soil preparation kit from Clontech. In an alternative method, genomic DNA was prepared with the PowerMax Soil DNA Isolation Kit from Mo Bio Laboratories. Prior to DNA extraction, intact samples were preserved as glycerol stocks for future identification of the organism bearing genes of interest and for retrieval of complete gene sequences. Yields of DNA from soil samples ranged from 0.36 to 9.1 micrograms with A260/A280 ratios ranging from 1.50 to 1.89 (Table 2). Because soil DNA preparations have been reported to inhibit PCR reactions, which could hinder the gene enrichment protocol, DNA samples were used as template for PCR with primers designed against the microbial 16S rRNA. Samples 1-4 yielded a PCR product (Table 2), and those 4 samples were used for gene enrichment experiments. Additional DNA samples were prepared from pools of cultured environmental microbes containing up to 25,000 colonies. 
         [0041]    To enrich these microbial pools for organisms likely to contain genes of interest, samples collected from about 920 diverse environmental sources were either (1) pasteurized to select for spore formers before plating on 0.1× LB medium, or (2) plated on media that selects for certain bacteria. Selection for certain species could include growth of environmental samples on defined carbon sources (for example, starch, mannitol, succinate or acetate), antibiotics (for example, cephalothin, vancomycin, polymyxin, kanamycin, neomycin, doxycycline, ampicillin, trimethoprim or sulfonamides), chromogenic substrates (for example, enzyme substrates such as phospholipase substrates, lecithinase substrates, cofactor metabolism substrates, nucleosidase substrates, glucosidase substrates, metalloprotease substrates and the like). Carbon or nitrogen sources could also be, for example, herbicides or other substrates that might select for organisms with herbicide detoxification or metabolism genes. Soil DNA preparations were spiked with genomic DNA from 4 organisms known to contain genes of interest at various ratios to serve as positive controls for the process (Table 2). In an alternative method, DNA from positive control strains was not included. 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Environmental Sample 
                 DNA Yield 
                 A260/ 
                 A260/ 
                   
               
               
                   
                 Description 
                 (μg) 
                 A280 
                 A230 
                 PCR 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 Pond (Center) 
                 2.9 
                 1.79 
                 1.76 
                 Yes 
               
               
                 2 
                 Forest 
                 6.5 
                 1.81 
                 1.59 
                 Yes 
               
               
                 3 
                 Pond (Edge) 
                 0.36 
                 1.50 
                 1.28 
                 Yes 
               
               
                 4 
                 Garden 
                 6.9 
                 1.86 
                 1.62 
                 Yes 
               
               
                 5  
                 Peach orchid 
                 9.1 
                 1.89 
                 2.05 
                 No 
               
               
                 6  
                 Front yard 
                 9.1 
                 1.64 
                 1.04 
                 No 
               
               
                 7  
                 Broom sedge 
                 8.7 
                 1.74 
                 1.57 
                 No 
               
               
                 8  
                   100 pooled colonies 
                   
                   
                   
                 Yes 
               
               
                 9  
                   1000 pooled colonies 
                   
                   
                   
                 Yes 
               
               
                 10 
                 10,000 pooled colonies 
                   
                   
                   
                 Yes 
               
               
                 11 
                 25,000 pooled colonies 
                   
                   
                   
                 Yes 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Environmental sources for DNA preparations with yields and spectrophotometric 
               
               
                 quality assessments. 
               
             
          
           
               
                   
                 # Microbes 
                   
                   
                 Approx. copy 
               
               
                   
                 screened 
                 Microbial DNA source 
                 BT spike 
                 #/gene 
               
               
                   
               
             
          
           
               
                 Positive control 
               
             
          
           
               
                 1 
                 25 
                 BT only 
                 25 strains 
                 10,000,000 
               
             
          
           
               
                 Soil DNA spiked with BT DNA 
               
             
          
           
               
                 2 
                 ND 
                 Soil 1-4 
                 1/50,000,000 (60 fg) 
                 5 
               
               
                 3 
                 ND 
                 Soil 1-4 
                 1/1,000,000 (3 pg) 
                 250 
               
               
                 4 
                 ND 
                 Soil 1-4 
                 1/50,000 (60 pg) 
                 5,000 
               
               
                 5 
                 ND 
                 Soil 1-4 
                 1/1000 (3 ng) 
                 250,000 
               
             
          
           
               
                 Colonies from pasteurized collections spiked with BT colonies before DNA preparation 
               
             
          
           
               
                 6 
                 100 
                 Pasteurized collections 
                 1 colony each x4 (1/25) 
                 2,500,00 
               
               
                 7 
                 1000 
                 Pasteurized collections 
                 1 colony each x4 (1/250) 
                 250,000 
               
               
                 8 
                 10,000 
                 Pasteurized collections 
                 1 colony each x4 (1/2500) 
                 25,000 
               
               
                 9 
                 25,000 
                 Pasteurized collections 
                 1 colony each x4 (1/6250) 
                 10,000 
               
               
                 10 
                 10,000 
                 Pasteurized collections 
                 10 colonies each x4 (1/250) 
                 250,000 
               
               
                 11 
                 10,000 
                 Pasteurized collections 
                 100 colonies each x4 (1/25)  
                 2,500,000 
               
               
                   
               
             
          
         
       
     
         [0042]    Table 3: Experimental design for gene enrichment experiments: DNA inputs for capture reactions including the environmental sample (described in Table 2), genes used as positive controls and the representation of genomic DNA from the positive control strains as a ratio to total DNA input. 
         [0043]    Oligonucleotide baits: Baits for gene capture consisted of approximately 30,000 biotinylated 120 base RNA oligonucleotides that were designed against approximately 900 genes and represent 9 distinct gene families of agricultural interest (Table 4). The process is used iteratively such that each subsequent round of hybridization includes baits designed to genes discovered in a previous round of gene discovery. In addition to genes of interest, additional sequences were included as positive controls (housekeeping genes) and for microbe species identification (16S rRNA). Starting points for baits were staggered at 60 bases to confer 2× coverage for each gene. Baits were synthesized at Agilent with the SureSelect technology. However, additional products for similar use are available from Agilent and other vendors including NimbleGen (SeqCap EZ), Mycroarray (MYbaits), Integrated DNA Technologies (XGen), and LC Sciences (OligoMix). 
         [0000]    
       
         
               
             
               
               
             
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Gene families queried in capture reactions with the number of genes  
               
               
                 queried for each family. 
               
             
          
           
               
                 Gene Family 
                 # genes 
               
               
                   
               
             
          
           
               
                 Cry 
                 640 
               
               
                 Cyt 
                 7 
               
               
                 Mtx 
                 25 
               
               
                 Binary 
                 33 
               
               
                 Vip 
                 104 
               
               
                 Sip 
                 2 
               
               
                 Misc. toxins 
                 25 
               
               
                 EPSPS 
                 14 
               
               
                 HPPD 
                 22 
               
               
                 16S 
                 373 
               
               
                 Housekeeping 
                 8 
               
               
                 TOTAL 
                 1253 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Example baits designed against Cry1Aa1. 
               
             
          
           
               
                 Base 
                   
                   
               
               
                 pair 
                 SEQ 
                   
               
               
                 range 
                 ID 
                 Sequence 
               
               
                   
               
             
          
           
               
                    1 . . . 120 
                 1 
                 ATGGATAACAATCCGAACATCAATGAATGCATTCCTTATAATTGTTTAAGTAACCCTGAAGTAGAAGTATT 
               
               
                   
                   
                 AGGTGGAGAAAGAATAGAAACTGGTTACACCCCAATCGATATTTCCTTG 
               
               
                   
               
               
                   61 . . . 180 
                 2 
                 GTAGAAGTATTAGGTGGAGAAAGAATAGAAACTGGTTACACCCCAATCGATATTTCCTTGTCGCTAACGC 
               
               
                   
                   
                 AATTTCTTTTGAGTGAATTTGTTCCCGGTGCTGGATTTGTGTTAGGACTA 
               
               
                   
               
               
                  121 . . . 240 
                 3 
                 TCGCTAACGCAATTTCTTTTGAGTGAATTTGTTCCCGGTGCTGGATTTGTGTTAGGACTAGTTGATATAATA 
               
               
                   
                   
                 TGGGGAATTTTTGGTCCCTCTCAATGGGACGCATTTCCTGTACAAATT 
               
               
                   
               
               
                  181 . . . 300 
                 4 
                 GTTGATATAATATGGGGAATTTTTGGTCCCTCTCAATGGGACGCATTTCCTGTACAAATTGAACAGTTAAT 
               
               
                   
                   
                 TAACCAAAGAATAGAAGAATTCGCTAGGAACCAAGCCATTTCTAGATTA 
               
               
                   
               
               
                  241 . . . 360 
                 5 
                 GAACAGTTAATTAACCAAAGAATAGAAGAATTCGCTAGGAACCAAGCCATTTCTAGATTAGAAGGACTAA 
               
               
                   
                   
                 GCAATCTTTATCAAATTTACGCAGAATCTTTTAGAGAGTGGGAAGCAGAT 
               
               
                   
               
               
                  301 . . . 420 
                 6 
                 GAAGGACTAAGCAATCTTTATCAAATTTACGCAGAATCTTTTAGAGAGTGGGAAGCAGATCCTACTAATCC 
               
               
                   
                   
                 AGCATTAAGAGAAGAGATGCGTATTCAATTCAATGACATGAACAGTGCC 
               
               
                   
               
               
                  361 . . . 480 
                 7 
                 CCTACTAATCCAGCATTAAGAGAAGAGATGCGTATTCAATTCAATGACATGAACAGTGCCCTTACAACCGC 
               
               
                   
                   
                 TATTCCTCTTTTGGCAGTTCAAAATTATCAAGTTCCTCTTTTATCAGTA 
               
               
                   
               
               
                  421 . . . 540 
                 8 
                 CTTACAACCGCTATTCCTCTTTTGGCAGTTCAAAATTATCAAGTTCCTCTTTTATCAGTATATGTTCAAGCTG 
               
               
                   
                   
                 CAAATTTACATTTATCAGTTTTGAGAGATGTTTCAGTGTTTGGACAA 
               
               
                   
               
               
                  481 . . . 600 
                 9 
                 TATGTTCAAGCTGCAAATTTACATTTATCAGTTTTGAGAGATGTTTCAGTGTTTGGACAAAGGTGGGGATT 
               
               
                   
                   
                 TGATGCCGCGACTATCAATAGTCGTTATAATGATTTAACTAGGCTTATT 
               
               
                   
               
               
                  541 . . . 660 
                 10 
                 AGGTGGGGATTTGATGCCGCGACTATCAATAGTCGTTATAATGATTTAACTAGGCTTATTGGCAACTATAC 
               
               
                   
                   
                 AGATTATGCTGTGCGCTGGTACAATACGGGATTAGAGCGTGTATGGGGA 
               
               
                   
               
               
                  601 . . . 720 
                 11 
                 GGCAACTATACAGATTATGCTGTGCGCTGGTACAATACGGGATTAGAGCGTGTATGGGGACCGGATTCTA 
               
               
                   
                   
                 GAGATTGGGTAAGGTATAATCAATTTAGAAGAGAGCTAACACTTACTGTA 
               
               
                   
               
               
                  661 . . . 780 
                 12 
                 CCGGATTCTAGAGATTGGGTAAGGTATAATCAATTTAGAAGAGAGCTAACACTTACTGTATTAGATATCGT 
               
               
                   
                   
                 TGCTCTATTCTCAAATTATGATAGTCGAAGGTATCCAATTCGAACAGTT 
               
               
                   
               
               
                  721 . . . 840 
                 13 
                 TTAGATATCGTTGCTCTATTCTCAAATTATGATAGTCGAAGGTATCCAATTCGAACAGTTTCCCAATTAACA 
               
               
                   
                   
                 AGAGAAATTTATACGAACCCAGTATTAGAAAATTTTGATGGTAGTTTT 
               
               
                   
               
               
                  781 . . . 900 
                 14 
                 TCCCAATTAACAAGAGAAATTTATACGAACCCAGTATTAGAAAATTTTGATGGTAGTTTTCGTGGAATGGC 
               
               
                   
                   
                 TCAGAGAATAGAACAGAATATTAGGCAACCACATCTTATGGATATCCTT 
               
               
                   
               
               
                  841 . . . 960 
                 15 
                 CGTGGAATGGCTCAGAGAATAGAACAGAATATTAGGCAACCACATCTTATGGATATCCTTAATAGTATAA 
               
               
                   
                   
                 CCATTTATACTGATGTGCATAGAGGCTTTAATTATTGGTCAGGGCATCAA 
               
               
                   
               
               
                  901 . . . 1020 
                 16 
                 AATAGTATAACCATTTATACTGATGTGCATAGAGGCTTTAATTATTGGTCAGGGCATCAAATAACAGCTTC 
               
               
                   
                   
                 TCCTGTAGGGTTTTCAGGACCAGAATTCGCATTCCCTTTATTTGGGAAT 
               
               
                   
               
               
                  961 . . . 1080 
                 17 
                 ATAACAGCTTCTCCTGTAGGGTTTTCAGGACCAGAATTCGCATTCCCTTTATTTGGGAATGCGGGGAATGC 
               
               
                   
                   
                 AGCTCCACCCGTACTTGTCTCATTAACTGGTTTGGGGATTTTTAGAACA 
               
               
                   
               
               
                 1021 . . . 1140 
                 18 
                 GCGGGGAATGCAGCTCCACCCGTACTTGTCTCATTAACTGGTTTGGGGATTTTTAGAACATTATCTTCACC 
               
               
                   
                   
                 TTTATATAGAAGAATTATACTTGGTTCAGGCCCAAATAATCAGGAACTG 
               
               
                   
               
               
                 1081 . . . 1200 
                 19 
                 TTATCTTCACCTTTATATAGAAGAATTATACTTGGTTCAGGCCCAAATAATCAGGAACTGTTTGTCCTTGAT 
               
               
                   
                   
                 GGAACGGAGTTTTCTTTTGCCTCCCTAACGACCAACTTGCCTTCCACT 
               
               
                   
               
               
                 1141 . . . 1260 
                 20 
                 TTTGTCCTTGATGGAACGGAGTTTTCTTTTGCCTCCCTAACGACCAACTTGCCTTCCACTATATATAGACAA 
               
               
                   
                   
                 AGGGGTACAGTCGATTCACTAGATGTAATACCGCCACAGGATAATAGT 
               
               
                   
               
               
                 1201 . . . 1320 
                 21 
                 ATATATAGACAAAGGGGTACAGTCGATTCACTAGATGTAATACCGCCACAGGATAATAGTGTACCACCTC 
               
               
                   
                   
                 GTGCGGGATTTAGCCATCGATTGAGTCATGTTACAATGCTGAGCCAAGCA 
               
               
                   
               
               
                 1261 . . . 1380 
                 22 
                 GTACCACCTCGTGCGGGATTTAGCCATCGATTGAGTCATGTTACAATGCTGAGCCAAGCAGCTGGAGCAG 
               
               
                   
                   
                 TTTACACCTTGAGAGCTCCAACGTTTTCTTGGCAGCATCGCAGTGCTGAA 
               
               
                   
               
             
          
         
       
     
         [0044]    Gene capture reactions: 3 μg of DNA was used as starting material for the procedure. DNA shearing, capture, post-capture washing and gene amplification are performed in accordance with Agilent SureSelect specifications. Throughout the procedure, DNA is purified with the Agencourt AMPure XP beads, and DNA quality was evaluated with the Agilent TapeStation. Briefly, DNA is sheared to an approximate length of 800 bp using a Covaris Focused-ultrasonicator. In an alternative method, DNA is sheared to lengths from about 400 to about 2000 bp, including about 500 bp, about 600 bp, about 700 bp, about 900 bp, about 1000 bp, about 1200 bp, about 1400 bp, about 1600 bp, about 1800 bp. The Agilent SureSelect Library Prep Kit was used to repair ends, add A bases, ligate the paired-end adaptor and amplify the adaptor-ligated fragments. Prepped DNA samples were lyophilized to contain 750 ng in 3.4 0_, and mixed with Agilent SureSelect Hybridization buffers, Capture Library Mix and Block Mix. Hybridization was performed for at least 16 hours at 65° C. In an alternative method, hybridization is performed at a lower temperature (55° C.). DNAs hybridized to biotinylated baits were precipitated with Dynabeads MyOne Streptavidin T1 magnetic beads and washed with SureSelect Binding and Wash Buffers. Captured DNAs were PCR-amplified to add index tags and pooled for multiplexed sequencing. 
         [0045]    Genomic DNA libraries were generated by adding a predetermined amount of sample DNA to, for example, the Paired End Sample prep kit PE-1021001 Inc) following manufacturer&#39;s protocol. Briefly, DNA fragments were generated by random shearing and conjugated to a pair of oligonucleotides in a forked adaptor configuration. The ligated products are amplified using two oligonucleotide primers, resulting in double-stranded blunt-ended products having a different adaptor sequence on either end. The libraries once generated are applied to a flow cell for cluster generation. 
         [0046]    Clusters were formed prior to sequencing using the TruSeq PE v3 cluster kit (ILLUMINA, Inc.) following manufacturer&#39;s instructions. Briefly, products from a DNA library preparation were denatured and single strands annealed to complementary oligonucleotides on the flow cell surface. A new strand was copied from the original strand in an extension reaction and the original strand was removed by denaturation. The adaptor sequence of the copied strand was annealed to a surface-bound complementary oligonucleotide, forming a bridge and generating a new site for synthesis of a second strand. Multiple cycles of annealing, extension and denaturation in isothermal conditions resulted in growth of clusters, each approximately 1 μm in physical diameter. 
         [0047]    The DNA, in each cluster was linearized by cleavage within one adaptor sequence and denatured, generating single-stranded template for sequencing by synthesis (SBS) to obtain a sequence read. To perform paired-read sequencing, the products of read I can be removed by denaturation, the template was used to generate a bridge, the second strand was re-synthesized and the opposite strand was cleaved to provide the template for the second read. Sequencing was performed using the ILLUMINA, Inc. V4 SBS kit with 100 base paired-end reads on the HiSeq 2000. Briefly, DNA templates were sequenced by repeated cycles of polymerase-directed single base extension. To ensure base-by-base nucleotide incorporation in a stepwise manner, a set of four reversible terminators, A, C, G, and T, each labeled with a different removable fluorophore, was used. The use of modified nucleotides allowed incorporation to be driven essentially to completion without risk of over-incorporation. It also enabled addition of all four nucleotides simultaneously minimizing risk of misincorporation. After each cycle of incorporation, the identity of the inserted base was determined by laser-induced excitation of the fluorophores and fluorescence imaging was recorded. The fluorescent dye and linker were removed to regenerate an available group ready for the next cycle of nucleotide addition. The HiSeq sequencing instrument is designed to perform multiple cycles of sequencing chemistry and imaging to collect sequence data automatically from each cluster on the surface of each lane of an eight ane flow cell. 
         [0048]    Bioinformatics: Sequences were assembled using the CLC Bio suite of bioinformatics tools. The presence of genes of interest (Table 4) was determined by BLAST query against a database of those genes of interest. Diversity of organisms present in the sample can be evaluated from 16S identifications. Process QC was evaluated based on retrieval of positive control sequences that are included in the reactions. To assess the capacity of this approach for new gene discovery, assembled genes, as well as individual sequencing reads, were BLASTed against baits and gene sequences published in public databases including NCBI and PatentLens. The lowest % identity read to a bait was 66%, while the lowest % identity to a gene was 77%. Example genes that were captured and sequenced with this method are shown in Table 6. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Examples of homologs to targeted genes captured and sequenced with the method. 
               
             
          
           
               
                 Sequence 
                 % Identity 
                 Closest Homolog 
                 Hit Length (bp) 
               
               
                   
               
             
          
           
               
                 contig_5548_ORF_complement(905 . . . 4639)  
                 100 
                 Cry5Ba2_DNA 
                 3735 
               
               
                 contig_1122_ORF_585 . . . 4268 
                 100 
                 Cry1Ba5_DNA 
                 3699 
               
               
                 contig_3847_ORF_1348 . . . 4338 
                 100 
                 Cry9Aa1_DNA 
                 2991 
               
               
                 contig_2073_ORF_442 . . . 3243 
                 100 
                 Cry9Ea2_DNA 
                 2808 
               
               
                 contig_594_ORF_complement(3 . . . 2144) 
                 100 
                 Cry1Aa12_DNA 
                 2142 
               
               
                 contig_6343_ORF_120 . . . 2279 
                 100 
                 Cry1Cb2_DNA 
                 2094 
               
               
                 contig_681_ORF_2 . . . 1471 
                 100 
                 Cry1Aa11_DNA 
                 1470 
               
               
                 contig_1643_ORF_complement(321 . . . 1277)  
                 100 
                 Cry1Db1_DNA 
                 972 
               
               
                 contig_5152_ORF_664 . . . 1554 
                 100 
                 Cry22Ba1_DNA 
                 891 
               
               
                 contig_680_ORF_2284 . . . 5106 
                 100 
                 Cry1Db2_DNA 
                 2823 
               
               
                 contig_239_ORF_complement(936 . . . 2756) 
                 100 
                 Cry32Aa1_DNA 
                 1821 
               
               
                 contig_529_ORF_complement(2060 . . . 5443)  
                 100 
                 Cry28Aa2_DNA 
                 3384 
               
               
                 contig_50404_ORF_complement(696 . . . 4109) 
                 100 
                 Cry7Ab6_DNA 
                 3431 
               
               
                 contig_656_ORF_469 . . . 3327 
                 100 
                 Cry1Fb3_DNA 
                 2862 
               
               
                 contig_6166_ORF_3 . . . 1049 
                 100 
                 Cry9Eb1_DNA 
                 1050 
               
               
                 contig_1758_ORF_complement(120 . . . 2522)  
                 99 
                 Cry1Ka1_DNA 
                 2392 
               
               
                 contig_657_ORF_382 . . . 1284 
                 98.23 
                 Cry1Ab17_DNA 
                 895 
               
               
                 contig_1904_ORF_complement(3 . . . 893) 
                 98.2 
                 Cry22Ba1_DNA 
                 891 
               
               
                 contig_6742_ORF_complement(2 . . . 3454) 
                 98 
                 Cry5Ad1_DNA 
                 1810 
               
               
                 contig_7322_ORF_670 . . . 4182 
                 95 
                 Cry7Ba1_DNA 
                 1563 
               
               
                 contig_1907_ORF_complement(2 . . . 907) 
                 94 
                 Cry22Ba1_DNA 
                 906 
               
               
                 contig_5549_ORF_complement(2 . . . 1201) 
                 94 
                 Cry21Ba1_DNA 
                 1195 
               
               
                 contig_1906_ORF_complement(2 . . . 1666) 
                 94 
                 Cry22Ab1_DNA 
                 1665 
               
               
                 contig_595_ORF_complement(2214 . . . 5711)  
                 93 
                 Cry1Hb1_DNA 
                 3506 
               
               
                 contig_238_ORF_complement(63 . . . 3749) 
                 92 
                 Cry32Ca1_DNA 
                 1787 
               
               
                 contig_5912_ORF_322 . . . 3852 
                 91 
                 Cry21Ba1_DNA 
                 3545 
               
               
                 contig_1905_ORF_complement(2 . . . 1795) 
                 90 
                 Cry22Ba1_DNA 
                 1327 
               
               
                 contig_228_ORF_complement(3 . . . 3560) 
                 89 
                 Cry32Da1_DNA 
                 3282 
               
               
                 contig_50688_ORF_1 . . . 1122 
                 80 
                 Cry8Da1_DNA 
                 1125 
               
               
                 contig_50689_ORF_complement(662 . . . 1507) 
                 77 
                 Cry28Aa2_DNA 
                 842 
               
               
                   
               
             
          
         
       
     
         [0049]    Novel Gene Confirmation: To confirm actual physical presence of genes predicted from sample enrichment, capture, and sequencing of captured DNA, oligonucleotide primers were designed to amplify sequences from DNA samples to confirm that the actual sequence matched the predicted sequence. Genes were called “novel homologs” if they contained domain characteristics of a targeted known gene but had less than 95% identity to a known gene.