Daily Archives: September 20, 2012

CAMBRIDGE, Mass.--(BUSINESS WIRE)--

GnuBIO http://www.gnubio.com announced today that it has been awarded a $4.5 million Phase II SBIR grant over the next three years through the National Human Genome Research Institutes (NHGRI) Advanced DNA Sequencing Technology Program. After a rigorous process, the grant committee at the NIH chose six projects to fund for this competitive awards program aimed at further advancing DNA sequencing technologies. Out of the total of $19 million, the NHGRI awarded GnuBIO the largest annual amount.

The purpose of the NHGRI grant program is to significantly reduce the cost of genome sequencing while improving both accuracy and speed, while significantly reducing the cost of sequencing a genome. GnuBIO, whose platform is based on Microfluidic DNA Sequencing, will use this funding to further develop its core technology towards rapid and accurate whole genome sequencing for less than $1000.

GnuBIO is commercializing a platform technology that integrates target enrichment, PCR, sequencing and informatics into a single system. The instrument has demonstrated sequencing of genomic DNA amplicons with read lengths up to 1000bp and accuracy higher than 99.9% per base. Unlike other currently available commercial systems, the GnuBIO http://www.gnubio.com platform encompasses all of the steps required for DNA sequencing into a single cartridge, thereby obviating any sample preparation that is required for all other commercial sequencing platforms.

Sequencing reactions on the GnuBIO http://www.gnubio.com platform take place inside droplets using minute reagent volumes. The NHGRI grant will be used to further reduce the size of these droplets and increase parallel processing of sequencing reactions to enable extremely low cost and rapid sequencing of large targets such as exomes and genomes, said Tal Raz, Vice President of Molecular Biology at GnuBIO. Currently, GnuBIO is in the process of preparing for the launch of its beta system, a platform that is capable of inline selection of sequencing targets, inline PCR, inline sequencing, and real-time informatics. The commercial system has a targeted price of $50K USD.

The grant award comes at an ideal time for us as it complements our strategy to launch an affordable sequencing platform that is practical for everyday patient care, said John Boyce, President and CEO of GnuBIO. This grant will enable us to expand our R&D capacity and enhance our core technology by increasing the throughput capability and enabling whole-genome sequencing.

About GnuBIO: GnuBIO is a privately-held company developing next-generation desktop DNA sequencing technology that will compartmentalize the entire DNA sequencing process, combining all of the steps required for sequencing in a single system, and providing the only fully integrated next-generation sequencing workflow.The GnuBIO sequencingtechnology is based on an emulsion based microfluidic technology which also provides a scalable sequencing solution that allows for interrogation of single genes, gene panels or whole genomes. The user of his GnuBIO system simply injects the patient sample into the GnuBIO http://www.gnubio.com cartridge, the appropriate panel is run inclusive of gene capture, PCR, sequencing, and informatics analysis and the results are ready within hours. Unlike any other DNA sequencing system, the entire process is all on the chip, simplifying the complex sample preparation process and breaking the barrier of an obstacle that has prevented the widespread adoption of DNA sequencing.

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GnuBIO Awarded $4.5 Million in Funding from the National Human Genome Research Institute to Develop Lower Cost Genome ...

Public release date: 19-Sep-2012 [ | E-mail | Share ]

Contact: Ellen de Graffenreid edegraff@med.unc.edu 919-962-3405 University of North Carolina Health Care

Researchers have long known that individual diseases are associated with genes in specific locations of the genome. Genetics researchers at the University of North Carolina at Chapel Hill now have shown definitively that a small number of places in the human genome are associated with a large number and variety of diseases. In particular, several diseases of aging are associated with a locus which is more famous for its role in preventing cancer.

For this analysis, researchers at UNC Lineberger Comprehensive Cancer Center catalogued results from several hundred human Genome-Wide Association Studies (GWAS) from the National Human Genome Research Institute. These results provided an unbiased means to determine if varied different diseases mapped to common 'hotspot' regions of the human genome. This analysis showed that two different genomic locations are associated with two major subcategories of human disease.

"Our team is interested in understanding genetic susceptibility to diseases associated with aging, including cancer," said PhD student William Jeck, who was first author on the study, published in the journal Aging Cell.

The team examined the large NHGRI dataset and first eliminated hereditable traits such as eye or hair color and other non-disease traits like drug metabolism. The group then focused on variants identified from GWAS that contributed to actual diseases. Combining results from all of these studies, there was enough data to arrive at statistically valid conclusions. The team then mapped the disease associations to the appropriate locations of the genome, counting the number of unique diseases mapping to specific genomic regions, in order to see if disparate diseases mapped randomly throughout the genome, or clustered in hotspots.

"What we ended up with is a very interesting distribution of disease risk across the genome. More than 90 percent of the genome lacked any disease loci. Surprisingly, however, lots of diseases mapped to two specific loci, which soared above all of the others in terms of multi-disease risk. The first locus at chromosome 6p21, is where the major histocompatibility (MHC) locus resides. The MHC is critical for tissue typing for organ and bone marrow transplantation, and was known to be an important disease risk locus before genome-wide studies were available. Genes at this locus determine susceptibility to a wide variety of autoimmune diseases such as arthritis, celiac disease, Type I diabetes, asthma, psoriasis, and lupus," said Jeck.

"The second place where disease associations clustered is the INK4/ARF (or CDKN2a) tumor suppressor locus. This area, in particular, was the location for diseases associated with aging: atherosclerosis, heart attacks, stroke, Type II diabetes, glaucoma and various cancers." he added.

"The finding that INK4/ARF is associated with lots of cancer, and MHC is associated with lots of diseases of immunity is not surprisingthese associations were known. What is surprising is the diversity of diseases mapping to just two small places: 30 percent of all tested human diseases mapped to one of these two places. This means that genotypes at these loci determine a substantial fraction of a person's resistance or susceptibility to multiple independent diseases," said Ned Sharpless, MD, Wellcome Distinguished Professor of Cancer Research and Associate Director of Translational Research at UNC Lineberger.

Another interesting finding was the apparent role of two biological processes in multi-disease association. In addition to the MHC and INK4/ARF loci, five less significant hotspot loci were also identified. Of the seven total hotspot loci, however, all contained genes associated with either immunity or cellular senescence. Cellular senescence is a permanent form of cellular growth arrest, and it is an important means whereby normal cells are prevented from becoming cancerous. It has been long known that senescent cells accumulate with aging, and may cause aspects of aging. This new analysis provides evidence that genetic differences in an individual's ability to regulate the immune response and activate cellular senescence determine their susceptibility to many seemingly disparate diseases.

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Diseases of aging map to a few 'hotspots' on the human genome

Comparison with seven other sequenced genomes identified 8,654 oyster-specific genes (Supplementary Text E3.1) that are probably important in the evolution and adaptation of oysters and other molluscs. With oysters being the only representative, these genes could be shared by other molluscs. Among these genes, gene ontology terms related to protein binding, apoptosis, cytokine activity and inflammatory response are highly enriched (P<0.0001; Supplementary Text E2 and Supplementary Table 17), indicating over-representation of some host-defence genes against biotic and abiotic stress. Manual examination shows that several gene families related to defence pathways, including protein folding, oxidation and anti-oxidation, apoptosis and immune responses, are expanded in C. gigas (Fig. 3a and Supplementary Table 18). The oyster genome contains 88 heat shock protein 70 (HSP70) genes, which have crucial roles in protecting cells against heat and other stresses, compared with ~17 in humans and 39 in sea urchins. Phylogenetic analysis finds clustering of 71 oyster HSP70 genes to themselves, suggesting that the expansion is specific to the oyster (Supplementary Fig. 19). Also expanded are cytochrome P450 (Supplementary Fig. 20) and multi-copper oxidase gene families, which are important in the biotransformation of endobiotic and xenobiotic chemicals26, and extracellular superoxide dismutases, which are important in defence against oxidative stress. The oyster genome has 48 genes coding for inhibitor of apoptosis proteins (IAPs), compared with 8 in humans and 7 in sea urchins, indicating a powerful anti-apoptosis system in oysters. Genes encoding lectin-like proteins, including C-type lectin, fibrinogen-related proteins and C1q domain-containing proteins (C1QDCs), are highly over-represented in the oyster genome (P<0.0001; Supplementary Table 18); these genes have important roles in the innate immune response in invertebrates27, 28, 29. Interestingly, many immune-related genes, including genes coding for Gram-negative bacteria-binding proteins, peptidoglycan-recognition proteins, defensin, C-type-lectin-domain-containing proteins and C1QDCs, are highly expressed in the digestive gland (Supplementary Fig. 21), indicating that the digestive system of this filter feeder is an important first-line defence organ against pathogens.

a, Expansion and expression of key genes in major stress-response pathways in C. gigas. Genes include HSPs and HSF in the heat-shock response; GRP78, CRT, CNX, GRP94, PERK, IRE1 and EIF2a in the endoplasmic reticulum unfolded-protein response (UPRER); IAPs, BCL2 like, BAG, BI1, caspases, FADD and TNFR in apoptotic pathways; CYP450 and MO in oxidation; and SOD, GPX, PRX and CAT in anti-oxidation. Boxes with bold black borders indicate gene families (HSPs, IAPs and SODs) expanded in C. gigas, and the filled colours correspond to their degree of upregulation in RPKMtreatment/RPKMcontrol by stress, found in 61 transcriptomes from oysters challenged with 9 types of stressors (Supplementary Text G2 and Supplementary Table 23). b, Venn diagram of common and unique genes expressed in response to temperature, salinity, air exposure and heavy-metal stress (zinc, cadmium, copper, lead and mercury), showing overlap of responses. c, Number of genes with and without detectable paralogues differentially expressed under stress and normal conditions, showing that genes responding to stress are more likely to have paralogues (P<11010; 2 test). Green sections of the pie chart represent 1,442, 809, 358, 550 and 7,938 paralogues for air exposure, metal, temperature, salinity and normal conditions, respectively.

To investigate genome-wide responses to stress, we sequenced 61 transcriptomes from C. gigas subjected to nine stressors, including temperature, salinity, air exposure and heavy metals (Supplementary Text G1 and Supplementary Tables 19 and 20). We found that 5,844 genes were differentially expressed under at least one stressor, and genes responding to different stressors showed significant overlap (Fig. 3b and Supplementary Fig. 23a). Air exposure induced a response from the largest number of genes (4,420), indicating that air exposure is a major stressor and that oysters have evolved an extensive gene set in defence. Genes differentially expressed in response to stress are more likely to have paralogues (Fig. 3c), suggesting that expansion and selective retention of duplicated defence-related genes are probably important to oyster adaptation. Under most stressors, genes coding for HSPs, histones, IAPs and protein biogenesis were upregulated, and those for protein degradation downregulated, pointing to concerted responses to maintain cellular homeostasis30 (Supplementary Text G3 and Supplementary Table 21). Genes involved in the unfolded protein response to cellular stress in the endoplasmic reticulum (coding for calreticulin, calnexin, 78- and 94-kDa glucose-regulated proteins) were upregulated, indicating that protein quality control is critical in cellular homeostasis under stress.

Air exposure induced up to 67-fold upregulation of five highly expressed IAPs (Supplementary Fig. 24a). Other inhibitors of apoptosis were also upregulated: BCL2 up to fourfold and BAG up to 12-fold (Supplementary Fig. 24b). These apoptosis inhibitors were also highly upregulated under heat and low salinity stress. These findings, along with the expansion of IAPs, suggest that a powerful anti-apoptosis system exists and may be critical for the amazing endurance of oysters to air exposure and other stresses. The existence of an intrinsic apoptosis pathway in invertebrates has been controversial, and parts of the pathways have only recently been demonstrated for two lophotrochozoans31, 32. The finding of key genes belonging to both intrinsic (BAX, BAK, BAG, BCL2, BI1 and procaspase) and extrinsic (TNFR and caspase 8) apoptosis pathways indicates that oysters have advanced apoptosis systems. Powerful inhibition of apoptosis as shown by genomic and transcriptomic analyses may be central to the ability of oysters to tolerate prolonged air exposure and other stresses.

Heat stress induced a ~2,000-fold increase in expression of five highly inducible HSP70 genes or a 13.9-fold increase in average expression of all HSP70 genes, amounting to 4.2% of all transcripts (Supplementary Figs 24c and 25). The genomic expansion and massive upregulation of HSP genes help to explain why C. gigas can tolerate temperatures as high as 49C when exposed to summer sun at low tide33. HSP genes were also upregulated under other stressors and may be central to the oyster defence against all stresses (Supplementary Fig. 25). HSP genes may also inhibit apoptosis by binding to effector caspases34.

Genes involved in signal transduction, including genes coding for G-protein-coupled receptors and Ras GTPase, were also activated by stressors (Supplementary Fig. 24f) and over-represented in the oyster genome (Supplementary Table 11). These regulators may have a role in orchestrating stress responses, which seem to be well coordinated (Fig. 3a and Supplementary Fig. 25). The expansion of key defence genes and the strong, complex transcriptomic response to stress highlight the sophisticated genomic adaptations of the oyster to sessile life in a highly stressful environment.

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The oyster genome reveals stress adaptation and complexity of shell formation

Public release date: 19-Sep-2012 [ | E-mail | Share ]

Contact: Jia Liu liujia@genomics.cn BGI Shenzhen

September 19, Shenzhen, China An international research team, led by Institute of Oceanology of Chinese Academy of Sciences and BGI, has completed the sequencing, assembly and analysis of Pacific oyster (Crassostrea gigas) genomethe first mollusk genome to be sequencedthat will help to fill a void in our understanding of the species-rich but poorly explored mollusc family. The study, published online today in Nature, reveals the unique adaptations of oysters to highly stressful environment and the complexity mechanism of shell formation.

"The accomplishment is a major breakthrough in the international Conchological research, with great advancement in the fields of Conchology and Marine Biology." said, Professor Fusui Zhang, Academician of Chinese Academy of Sciences, and a well-known Chinese Scientist of Conchology, "The study will provide valuable resources for studying the biology and genetic improvement of molluscs and other marine species. "

Oysters are a soft-bodied invertebrate with a double-hinged shell, which make up an essential part of many aquatic ecosystems. They have a global distribution and for many years they have much higher annual production than any other freshwater or marine organisms. In addition to its economic and ecological importance, the unique biological characteristics of oyster make it an important model for studying marine adaptations, inducing a great deal of biological and genomics research. The completed sequencing of oyster genome will provide a new horizon into understanding its natural mechanisms such as the adaptations to environmental stresses and shell formation, better exploration of marine gene resource, , among others.

Unlike many mammals and social insects, oyster as well as many other marine invertebrates is known to be highly polymorphism, which is a challenge for de novo assembling based on current strategies. In this study, researchers sequenced and assembled the Pacific oyster genome using a combination of short reads and a "Divide and Conquer" fosmid-pooling strategy. This is a novel approach developed by BGI, which can be used to study the genomes with high level of heterozygosity and/or repetitive sequences. After data process, the assembled oyster genome size is about 559 Mb, with a total of ~28,000 genes.

Based on the genomic and transcriptomic analysis results, researchers uncovered an extensive set of genes that allow oysters to adapt and cope with environmental stresses, such as temperature variation and changes in salinity, air exposure and heavy metals. For example, the expansion of heat shock protein 70 (HSP 70) may help explain why Pacific oyster can tolerate high temperatures as HSP family is expanded and highly expressed when in high temperature. The expansion of inhibitors of apoptosis proteins (IAPs), along with other findings, suggested that a powerful anti-apoptosis system exists and may be critical for oyster's amazing endurance to air exposure and other stresses. One notable finding on development is that the oyster Hox gene cluster was broken, and there are unusual gene losses and expansions of the TALE and PRD classes. Hox genes are essential and play critical important role in body plan, the Hox clusters are found to be more conserved in many organisms.

Researchers found paralogs might have the function to change the gene expression for better coping with the stresses. This result suggested that expansion and selective retention of duplicated defense-related genes are probably important to oyster's adaptation. Moreover, many immune-related genes were highly expressed in the digestive gland of the oyster, which indicated its digestive system was an important first-line defense organ against pathogens for the filter-feeder. The shell provides a strong protection against predation and desiccation in sessile marine animals such as oysters. At present, two models have been proposed for molluscan shell formation, but neither of them is accurate enough. In this study, by sequencing the peptides in the shell, researchers identified 259 shell proteins, and further analysis revealed that shell formation was a far more complex process than previously thought. They found many diverse proteins may play important roles in matrix construction and modification. The typical ECM proteins such as Laminin and some collagens were highly expressed in shells, suggesting that shell matrix has similarities to the ECM of animal connective tissues and basal lamina. Hemocytes may mediate fibronectin (FN)-like fibril formation in the shell matrix as they do in ECM. Furthermore, the functional diversity of proteins showed that the cells and exosome may participate in the shell formation.

Xiaodong Fang, Primary Investigator of this project at BGI, said, "The assembly approach of Oyster genome opens a new way for researchers to better crack the genomes with high-heterozygosity and high-polymorphism. The Oyster genome sheds insights into the comprehensive understanding of mollusc genomes or even lophotrochozoa genomes at the whole genome-wide level, with focuses on the studies of diversity, evolutionary adaptive mechanisms, developmental biology as well as genomics-assisted breeding. "

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Oyster genome uncover the stress adaptation and complexity of shell formation