Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): Sequencing and profiling of functional transcripts constructed in expressed sequence tags (ESTs) of Coptotermes formosanus to fulfill the following objectives: a) Identifying and annotating of genes specifically associated with post-embryonic polyphenism (caste differentiation and development); b) Discovering of genes specifically responding to environmental cues and internal signals (pesticides, food sources, juvenile hormones, etc.); and c) Characterizing of genes uniquely involved in critical physiological pathways (digestion, molting, immunity, etc.). The fulfillment of these objectives would directly lead to achieving the following goals: a) providing biochemical/physiological/molecular bases for disruption of colony formation, development and survival; b) Discovering novel target site(s) that would be developed into new control strategies that could be incorporated into effective area-wide integrated management of Formosan subterranean termites. The overall objectives of this cooperative research are to determine genes that are unique to termites - specifically reproductive and development associated genes e.g. nymph or soldier formation mechanisms with specific emphasis on Coptotermes formosanus while also examining cellulosic material hydrolyzing enzymes for agriculture and industry. We will also concentrate on private and public database and website development strategies. Approach (from AD-416): A cDNA library representing expressed genes in each different developmental stage of Coptotermes formosanus has been constructed. To facilitate transcriptome analysis and rare gene discovery, repeated transcripts were proportionally removed from the cDNA library using the procedure of cDNA normalization. The cDNA library would contain approximately 400,000 independent clones, an estimate of at least 16X coverage of the entire expressed genes, provided that the protein-coding capacity of invertebrate genomes is in the range of 16,000 to 25,000 genes. The sequencing and gene data assembly of the cDNA library will be cooperatively conducted in JCVI. We will continue Sanger sequencing of EST clones and perform SoLID sequencing to determine differential gene expression. EST sequences will be compared against existing databases and annotated using the Basic Local Alignment Search Tool (BLAST). Batches of sequences will be sequentially released to GenBank for public access. Unique genes and singletons would be selected and gene expression analysis. Differentially-expressed genes or development-stage specific genes will be preferentially analyzed quantitatively (such as real-time PCR) or qualitatively (such as gene silencing by RNA interference). Last year, J. Craig Venter Institute (JCVI) completed whole genome sequencing and assembly of the nuclear genome of the Formosan Subterranean termite, Coptotermes formosanus, and also finished sequencing of Ribonucleic acid (RNA) samples from two developmental stages. The genomic Deoxyribonucleic acid (DNA) was used to prepare several fragmentary, Paired End and Mate Pair libraries and sequenced using Illumina GA-II and HiSeq platforms. This resulted in sequence coverage of over 50x after the reads were filtered based on quality. The draft assembly of the genome, generated by the ALLPATHS-LG assembler, has 8,478 scaffolds containing 72,457 contigs. The total scaffold length is 891,902,903 bp. The contig N50 is 29.2 kbp while the scaffold N50 is 1. 478 Mbp. This assembly is currently under annotation, while refinements to the assembly are ongoing. After the annotation is complete, we will compare it with that of the native subterranean termite Reticulitermes flavipes in order to further understand caste differentiation and development and provide information on cellulose digestion useful in cellulosic biofuel production. The RNA samples sequenced were from the Female Alates and the Worker Termites and the yields were 83,102,604 reads and 13,281,961 reads respectively. While the RNA samples from Nymphs and Male Alates were sequenced in the previous year and produced 22,493,290 reads and 21,234, 321 reads respectively, the RNA sample from the Pre-Soldier stage failed library preparation. The RNA-Seq data from the four stages will be analyzed and genes will be identified that may regulate processes associated with differentiation in order to determine possible biologically-based control measures for the termite.
Impacts
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) Sequencing and profiling of functional transcripts constructed in expressed sequence tags (ESTs) of Coptotermes formosanus to fulfill the following objectives: a) Identifying and annotating of genes specifically associated with post-embryonic polyphenism (caste differentiation and development); b) Discovering of genes specifically responding to environmental cues and internal signals (pesticides, food sources, juvenile hormones, etc.); and c) Characterizing of genes uniquely involved in critical physiological pathways (digestion, molting, immunity, etc.). The fulfillment of these objectives would directly lead to achieving the following goals: a) providing biochemical/physiological/molecular bases for disruption of colony formation, development and survival; b) Discovering novel target site(s) that would be developed into new control strategies that could be incorporated into effective area-wide integrated management of Formosan subterranean termites. The overall objectives of this cooperative research are to determine genes that are unique to termites - specifically reproductive and development associated genes e.g. nymph or soldier formation mechanisms with specific emphasis on Coptotermes formosanus while also examining cellulosic material hydrolyzing enzymes for agriculture and industry. We will also concentrate on private and public database and website development strategies. Approach (from AD-416) A cDNA library representing expressed genes in each different developmental stage of Coptotermes formosanus has been constructed. To facilitate transcriptome analysis and rare gene discovery, repeated transcripts were proportionally removed from the cDNA library using the procedure of cDNA normalization. The cDNA library would contain approximately 400,000 independent clones, an estimate of at least 16X coverage of the entire expressed genes, provided that the protein-coding capacity of invertebrate genomes is in the range of 16,000 to 25,000 genes. The sequencing and gene data assembly of the cDNA library will be cooperatively conducted in JCVI. We will continue Sanger sequencing of EST clones and perform SoLID sequencing to determine differential gene expression. EST sequences will be compared against existing databases and annotated using the Basic Local Alignment Search Tool (BLAST). Batches of sequences will be sequentially released to GenBank for public access. Unique genes and singletons would be selected and gene expression analysis. Differentially-expressed genes or development-stage specific genes will be preferentially analyzed quantitatively (such as real-time PCR) or qualitatively (such as gene silencing by RNA interference). In 2010, J. Craig Venter Institute (JCVI) completed determining the sequence of a portion of the genetic material from the Formosan Subterranean Termite (FST), Coptotermes formosanus. The 132,000 cDNA (complementary deoxyribonucleic acid) sequencing reads were determined to represent 25,000 unique sequences corresponding to individual genes from the termites and microorganisms living in the termite gut. Furthermore, JCVI initiated next generation sequencing of the genome of the FST. Thus far, 18-fold coverage has been thus far obtained and 80-fold coverage of the genome is expected by the end of July 2011. We expect to annotate the termite genome and compare it with that of the native subterranean termite Reticulitermes flavipes in order to further understand caste differentiation and development and provide information on cellulose digestion useful in cellulosic biofuel production. Our planned strategy is to characterize all individual stages of the termite to perform analysis of 7 individual termite stages and identify genes that may regulate processes associated with differentiation in order to determine possible biologically-based control measures for the termite. Progress in this project is monitored through Annual FST Technical Committee Meetings, reports, regular meetings with cooperators, routine phone calls, and e-mail correspondence.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) Sequencing and profiling of functional transcripts constructed in expressed sequence tags (ESTs) of Coptotermes formosanus to fulfill the following objectives: a) Identifying and annotating of genes specifically associated with post-embryonic polyphenism (caste differentiation and development); b) Discovering of genes specifically responding to environmental cues and internal signals (pesticides, food sources, juvenile hormones, etc.); and c) Characterizing of genes uniquely involved in critical physiological pathways (digestion, molting, immunity, etc.). The fulfillment of these objectives would directly lead to achieving the following goals: a) providing biochemical/physiological/molecular bases for disruption of colony formation, development and survival; b) Discovering novel target site(s) that would be developed into new control strategies that could be incorporated into effective area-wide integrated management of Formosan subterranean termites. The overall objectives of this cooperative research are to determine genes that are unique to termites - specifically reproductive and development associated genes e.g. nymph or soldier formation mechanisms with specific emphasis on Coptotermes formosanus while also examining cellulosic material hydrolyzing enzymes for agriculture and industry. We will also concentrate on private and public database and website development strategies. Approach (from AD-416) A cDNA library representing expressed genes in each different developmental stage of Coptotermes formosanus has been constructed. To facilitate transcriptome analysis and rare gene discovery, repeated transcripts were proportionally removed from the cDNA library using the procedure of cDNA normalization. The cDNA library would contain approximately 400,000 independent clones, an estimate of at least 16X coverage of the entire expressed genes, provided that the protein-coding capacity of invertebrate genomes is in the range of 16,000 to 25,000 genes. The sequencing and gene data assembly of the cDNA library will be cooperatively conducted in JCVI. We will continue Sanger sequencing of EST clones and perform SoLID sequencing to determine differential gene expression. EST sequences will be compared against existing databases and annotated using the Basic Local Alignment Search Tool (BLAST). Batches of sequences will be sequentially released to GenBank for public access. Unique genes and singletons would be selected and gene expression analysis. Differentially-expressed genes or development-stage specific genes will be preferentially analyzed quantitatively (such as real-time PCR) or qualitatively (such as gene silencing by RNA interference). Last year, the JCVI continued analysis of sequences of pooled gene samples from all life stages of the Formosan subterranean termite, Coptotermes formosanus, obtained using the Sanger sequencing platform. The 132,000 gene sequencing reads were assembled into 25,000 unique sequences corresponding to individual transcripts (unigenes). Almost half of the transcripts showed similarity to known protein, while the other half did not, suggesting that they represent non-protein coding ribonucleic acids (RNAs). Among the former unigenes, over 50% unigenes (25% of all unigenes) shared similarity with other insect genomes such as the fly, bee, or wasp genomes. After insects, the second most represented class was Trichomonada, consistent with the presence of similar microrganisms in the termite gut. Many unigenes (940) in this group showed significant similarity to Trichomonas vaginalis genes. The search for carbohydrate-active enzymes, which may be involved in digestion of cellulose, identified five superfamilies: glycosidase (124 unigenes), polysaccharide lyase (2), carbohydrate-binding (32), carbohydrate esterase (11), and glycosyltransferase (80) superfamilies. The most abundant glycosidase families included GH5 (15 unigenes) and GH7 (14 unigenes). One unigene, corresponding to a cellulose digesting enzyme gene from the termite, was experimentally characterized as cellobiohydrolase (which digests cellulose from the end). The presence of GH7 enzymes was unexpected as these cellulases are usually found only in fungi. This suggests the presence of disease causing fungi, which have been identified before as potential biological pest control agents against termites. Another well represented glycosidase family was GH22 (13 unigenes) that contain lysozymes involved in defense against pathogens. The presence of a unigene classified as a member of a starch degrading enzyme family (GH14) typically found in plants and just a few bacteria was unusual. Another unexpected finding was the abundance of the carbohydrate-binding module family CBM13 (16 unigenes), which typically bind plant polysaccharides. In addition, unigenes from the chemical binding families were identified. Further computational analysis performed by the JCVI team demonstrated that additional sequencing is required to get sufficient genetic coverage of the termite and its symbionts. We have begun sequencing of RNA from all individual castes of the termite by using the novel RNA-Seq approach using the Illumina short-read sequencing technology. We have obtained over 40,000,000 reads from two castes (male alates and nymphs) and are continuing sequencing of the remaining castes. About 65% of reads aligned to the previously identified unigenes. We are currently assembling the reads and beginning determination and identification of the genes expressed in the various castes. Progress is monitored through regular e-mail exchanges and via teleconference calls.
Impacts
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) Sequencing and profiling of functional transcripts constructed in expressed sequence tags (ESTs) of Coptotermes formosanus to fulfill the following objectives: a) Identifying and annotating of genes specifically associated with post-embryonic polyphenism (caste differentiation and development); b) Discovering of genes specifically responding to environmental cues and internal signals (pesticides, food sources, juvenile hormones, etc.); and c) Characterizing of genes uniquely involved in critical physiological pathways (digestion, molting, immunity, etc.). The fulfillment of these objectives would directly lead to achieving the following goals: a) providing biochemical/physiological/molecular bases for disruption of colony formation, development and survival; b) Discovering novel target site(s) that would be developed into new control strategies that could be incorporated into effective area-wide integrated management of Formosan subterranean termites. The overall objectives of this cooperative research are to determine genes that are unique to termites - specifically reproductive and development associated genes e.g. nymph or soldier formation mechanisms with specific emphasis on Coptotermes formosanus while also examining cellulosic material hydrolyzing enzymes for agriculture and industry. We will also concentrate on private and public database and website development strategies. Approach (from AD-416) A cDNA library representing expressed genes in each different developmental stage of Coptotermes formosanus has been constructed. To facilitate transcriptome analysis and rare gene discovery, repeated transcripts were proportionally removed from the cDNA library using the procedure of cDNA normalization. The cDNA library would contain approximately 400,000 independent clones, an estimate of at least 16X coverage of the entire expressed genes, provided that the protein-coding capacity of invertebrate genomes is in the range of 16,000 to 25,000 genes. The sequencing and gene data assembly of the cDNA library will be cooperatively conducted in JCVI. We will continue Sanger sequencing of EST clones and perform SoLID sequencing to determine differential gene expression. EST sequences will be compared against existing databases and annotated using the Basic Local Alignment Search Tool (BLAST). Batches of sequences will be sequentially released to GenBank for public access. Unique genes and singletons would be selected and gene expression analysis. Differentially-expressed genes or development-stage specific genes will be preferentially analyzed quantitatively (such as real-time PCR) or qualitatively (such as gene silencing by RNA interference). Significant Activities that Support Special Target Populations Last year, J. Craig Venter Institute (JCVI) completed determining the sequence of a portion of the genetic material obtained from samples from virtually all life stages of the Formosan subterranean termite, Coptotermes formosanus. The 132,000 cDNA (complementary deoxyribonucleic acid) sequencing reads were determined to represent 25,000 unique sequences corresponding to individual genes from the termites and microorganisms living in the termite gut. Over 25% of these unique transcripts showed similarity to known genes from C. formosanus, other termite species, other insect species, or protozoan symbionts (mutually beneficially microrganisms known to inhabit the gut of C. formosanus). These lists of characterized genes included lipopolysaccharide binding protein, trypsin binding protein, chitin binding protein, an actin binding protein, and a zinc binding dehydrogenase, as well as cellulases, glucosidases, hexamerins, and several odorant binding proteins. Unexpectedly, many sequencing reads showed no similarity to any known protein suggesting that they represent non-protein coding regions of the isolated genetic material. Further computational analysis performed by the JCVI team demonstrated that the additional analysis is required to determine all of the genes expressed during the development of the termite and its symbionts. Our planned strategy is to characterize all individual stages of the termite by using an instrument that can generate 75,000,000 genetic material sequence reads, while only 10,000,000 reads are believed to be sufficient to determine all of the genes that should be expressed. We will use this approach to perform analysis of 7 individual termite stages and identify genes that may regulate processes associated with differentiation in order to determine possible biologically-based control measures for the termite. This project was monitored through telephone calls, emails and meetings.
Impacts
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) Sequencing and profiling of functional transcripts constructed in expressed sequence tags (ESTs) of Coptotermes formosanus to fulfill the following objectives: a) Identifying and annotating of genes specifically associated with post-embryonic polyphenism (caste differentiation and development); b) Discovering of genes specifically responding to environmental cues and internal signals (pesticides, food sources, juvenile hormones, etc.); and c) Characterizing of genes uniquely involved in critical physiological pathways (digestion, molting, immunity, etc.). The fulfillment of these objectives would directly lead to achieving the following goals: a) providing biochemical/physiological/molecular bases for disruption of colony formation, development and survival; b) Discovering novel target site(s) that would be developed into new control strategies that could be incorporated into effective area-wide integrated management of Formosan subterranean termites. Approach (from AD-416) A cDNA library representing expressed genes in each different developmental stage of Coptotermes formosanus will be constructed. To facilitate transcriptome analysis and rare gene discovery, repeated transcripts will be proportionally removed from the cDNA library using the procedure of cDNA normalization. The cDNA library would contain approximately 400,000 independent clones, an estimate of at least 16X coverage of the entire expressed genes, provided that the protein-coding capacity of invertebrate genomes is in the range of 16,000 to 25,000 genes. The sequencing and gene data assembly of the cDNA library will be cooperatively conducted in JCVI. Eighty thousand clones will be initially sequenced at both 5�- and 3�-ends. The resulting EST sequences will be compared against existing databases and annotated using the Basic Local Alignment Search Tool (BLAST). Batches of sequences will be sequentially released to GenBank for public access. Unique genes and singletons would be selected for array and gene expression analysis. Differentially-expressed genes or development-stage specific genes will be preferentially analyzed quantitatively (such as real-time PCR) or qualitatively (such as gene silencing by RNA interference). Significant Activities that Support Special Target Populations J. C. Venter Institute has completed cDNA sequencing on approximately 150, 000 clones obtained using template Ribonucleic Acid (RNA) from virtually all life stages of the Formosan Subterranean termite, Coptotermes formosanus. Eighty-eight percent of the sequence reads were judged to be of good quality with an average read length of 555 base pairs. Sequence identities were found to include genes from C.formosanus, other termite species, other insect species and protozoan symbionts known from C. formosanus. Diribonucleic acid (DNA) sequence similarity was found to include genes from C. formosanus, other termite and insect species, and protozoan microorganisms known to be found in the gut of C. formosanus. Some genes of interest identified through sequence homology are lipopolysaccharide binding protein, trypsin binding protein, chitin binding protein, an actin binding protein, a zinc binding dehydrogenase among others. Further analysis is being completed to determine other sequence homologies and to determine possible gene function. Plans are underway for further sequencing to provide complete genome coverage of the termite and its gut symbiotic protozoans. We will then perform analysis to determine termite caste and colony growth and differentiation associated genes in order to determine possible biologically-based control measures for the termite. Progress of this project is monitored by phone calls, e-mails, teleconferences and regular mail.
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