# Patent

## Title
High throughput discovery of new genes from complex mixtures of environmental microbes

## 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.

## Background
FIELD OF THE INVENTION
The invention is drawn to high throughput methods of gene discovery.
BACKGROUND OF THE INVENTION
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
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.


DETAILED DESCRIPTION OF THE INVENTION
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.
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.
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
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.
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.
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.
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.
A. Sequence Analysis
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.).
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.
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.
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
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.
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.
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.
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.
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 NimbleDesign® (NimbleGen®; Roche).
III. Methods of Isolating Genes of Interest, or Variants Thereof
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; SureSelectXT® Target Enrichment System for Illumina Inc. Paired-End Sequencing Library Protocol, Version 1.6; NimbleGen® SeqCap® EZ Library SR User's Guide, Version 4.3; and NimbleGen SeqCap EZ Library LR User's Guide, Version 2.0. All of which are herein incorporated by reference.
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.
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 & 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.
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.
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.
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.
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.
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 SureSelectXT® Target Enrichment System for Illumina Inc. Paired-End Sequencing Library Protocol, Version 1.6 and NimbleGen® SeqCap® EZ Library SR User'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.
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 >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.).
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 SureSelectXT® Target Enrichment System for Illumina Inc. 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's Guide, Version 4.3.
Alternatively, the DNA from multiple complex samples can be indexed and amplified before hybridization. In such embodiments, the enrichment kit can be the SureSelectXT2 Target Enrichment System for Illumina Multiplexed Sequencing Protocol, Version D.0
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 BiosystemsTM Life Technologies (ABI PRISM® Sequence detection systems, SOLiDTM System, Ion PGMTM Sequencer, ion ProtonTM 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, and 5,863,722, U.S. Pat. Appl. Nos. 2007/007991, 2009/0247414, and 2010/0111768, and PCT application WO2007/123744, each of which is incorporated herein by reference in its entirety.
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.
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.







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 & Luo
2011
Bt LS-R-21


Cry1Aa20
JF340156


Kumari & 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 & Ellar
1988
Bt aizawai IC1


Cry1Ab8
AAA22551
143099
143098
Oeda et al
1987
Bt aizawai IPL7


Cry1Ab9
CAA38701
40273
40272
Chak & Jen
1993
Bt aizawai HD133


Cry1Ab10
A29125


Fischhoff et al
1987
Bt kurstaki HD1


Cry1Ab11
I12419


Ely & 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 & 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 & 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 & Riazuddin
1998


Cry1Ac12
I12418


Ely & 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 & 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 & 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 & Meena
2011
Bt SK-711


Cry1Ac34
HQ230364
314906994

Kaur & Kumari
2010
Bt SK-783


Cry1Ac35
JF340157


Kumari & Kaur
2011
Bt SK-784


Cry1Ac36
JN387137


Kumari & Kaur
2011
Bt SK-958


Cry1Ac37
JQ317685


Kumari & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & Pang
1999
Bt SL39


Cry2Aa6
CAA10672


Yu & Pang
1999
Bt YZ71


Cry2Aa7
CAA10670


Yu & 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 & 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 & 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 & 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 & 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 & 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 & Fu
1994
Bt dakota HD511


Cry7Ab2
AAA21121


Narva & 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 & 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 & Azuma
2013
Bt dakota


Cry7La1
BAM99307


Takebe & Azuma
2013
Bt dakota


Cry8Aa1
AAA21117


Narva & 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 & 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 & 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 & 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 & 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 & Abad
2005
Bt kurstaki DP1019


Cry9Dc1
KC156683


Sampson et al
2012


Cry9Ea1
BAA34908


Midoh & 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 & 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 & 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 & 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 & 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 & Delecluse
1996
Bt jegathesan 367


Cry19Ba1
BAA32397


Hwang et al
1998
Bt higo


Cry19Ca1
AFM37572


Soufiane & Cote
2012
BGSC 4CE1


Cry20Aa1
AAB93476


Lee & Gill
1997
Bt fukuokaensis


Cry20Ba1
ACS93601


Noguera & 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 & 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 & 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 & 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 & 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 & 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 & Ibarra
2009
Bt kim


Cry57Ab1
KF638650


Guowang Zhou
2013


Cry58Aa1
ANC87260
225348553
225348552
Noguera & Ibarra
2009
Bt entomocidus


Cry59Ba1
JN790647


Qiao Li et al
2012
Bt Bm59-2


Cry59Aa1
ACR43758
239638225
239638224
Noguera & 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 & Ellar
1986
Bt israelensis


Cyt1Aa3
Y00135


Earp & 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 & Ellar
1993
Bt kyushuensis


Cyt2Aa2
AF472606


Promdonkoy & 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 & Pang
2000
Bt T301


Cyt2Ba8
AF215646


Yu & Pang
2000
Bt T36


Cyt2Ba9
AL731825


Berry et al
2002
Bt israelensis


Cyt2Ba10
ACX54358


Sauka & Benintende
2009
Bti HD 567


Cyt2Ba11
ACX54359


Sauka & Benintende
2009
Bti HD 522


Cyt2Ba12
ACX54360


Sauka & Benintende
2009
Bti INTA H41-1


Cyt2Ba13
FJ205865
209168617

Sauka & Benintende
2009
INTA 160-2


Cyt2Ba14
FJ205866
209168619

Sauka & 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 & 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









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.
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.
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.
The following examples are offered by way of illustration and not by way of limitation.
EXPERIMENTAL
Example 1
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.
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.
















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
















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









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.

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®).







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
















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









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-UltrasonicatorTM. 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 μl, 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® MyOneTM 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.
Genomic DNA libraries were generated by adding a predetermined amount of sample DNA to, for example, the Paired End Sample prep kit PE-102-1001 (ILLUMINA, Inc.) following manufacturer'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.
Clusters were formed prior to sequencing using the TruSeq PE v3 cluster kit (ILLUMINA, Inc.) following manufacturer'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.
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. SBS kit v4 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 lane flow cell.
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.







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









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.

## Claims

1. A method for identifying a variant of a gene of interest in a complex sample comprising:
a) obtaining a complex sample comprising a variant of a gene of interest;
b) preparing DNA from said complex sample for hybridization thereby forming a prepared sample DNA, the prepared sample DNA comprising said variant of said gene of interest;
c) mixing said prepared sample DNA with a labeled bait pool comprising polynucleotide sequences complementary to said gene of interest;
d) hybridizing the prepared sample DNA to said labeled bait pool under conditions that allow for hybridization of a labeled bait in said labeled bait pool with said variant of said gene of interest to form one or more hybridization complexes,
wherein said variant of said gene of interest in the hybridization complexes comprises captured DNA;
e) sequencing said captured DNA;
f) identifying a full length gene sequence of said variant of said gene of interest by assembling sequences of said captured DNA; and
g) identifying said variant of said gene of interest from said full length gene sequence, by performing a sequence similarity search using the full length gene sequence against a database of known sequences, said variant of said gene of interest having less than 95% identity to said gene of interest.

2. The method of claim 1, wherein said complex sample is an environmental sample.

3. The method of claim 1, wherein said complex sample is a mixed culture of at least two organisms.

4. The method of claim 1, wherein said complex sample is a mixed culture of more than two organisms collected from a petri plate.

5. The method of claim 1, wherein said labeled bait pools comprise labeled baits specific for at least 500 genes of interest.

6. The method of claim 1, wherein said labeled bait pool comprises at least 50 distinct labeled baits that are mixed with said prepared sample DNA.

7. The method of claim 1, wherein said labeled bait pool comprises labeled baits that are 50-200 nt in length.

8. The method of claim 1, wherein said labeled baits are labeled with biotin, a hapten, or an affinity tag.

9. The method of claim 1, wherein said labeled baits comprise overlapping labeled baits, said overlapping labeled baits comprising at least two labeled baits that are complimentary to a portion of a gene of interest, wherein the at least two labeled baits comprise different DNA sequences that are partially overlapping.

10. The method of claim 9, wherein at least 10, at least 30, at least 60, at least 90, or at least 120 nucleotides of each overlapping labeled bait overlap with at least one other overlapping labeled bait.

11. The method of claim 9, wherein said labeled baits cover each gene of interest by at least 2×.

12. The method of claim 1, wherein said variant is a homolog of said gene of interest.

13. The method of claim 1, wherein said prepared sample DNA is enriched prior to mixing with said labeled baits.

14. The method of claim 1, wherein said labeled baits are designed to target 16S DNA.

15. The method of claim 1, wherein said hybridization complex is captured and purified from unbound prepared sample DNA.

16. The method of claim 15, wherein said hybridization complex is captured using a streptavidin molecule attached to a solid phase.

17. The method of claim 16, wherein said solid phase is a magnetic bead.

18. The method of claim 1, wherein steps b), c), and d) are performed using an enrichment kit for multiplex sequencing.

19. The method of claim 1, wherein said captured DNA from said hybridization complex is amplified and index tagged prior to said sequencing.

20. The method of claim 1, wherein said sequencing comprises multiplex sequencing with gene fragments from different environmental samples.

21. The method of claim 1, wherein said labeled bait pool comprises labeled baits that are 70-150 nt in length.

22. The method of claim 1, wherein said labeled bait pool comprises labeled baits that are 100-140 nt in length.

23. The method of claim 1, wherein said labeled bait pool comprises labeled baits that are 110-130 nt in length.