Neutralizing antibodies have become an important tool in treating infectious diseases. Recently, two separate approaches yielded successful antibody treatments for Ebola—one from genetically humanized mice and the other from a human survivor. Here, we describe parallel efforts using both humanized mice and convalescent patients to generate antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, which yielded a large collection of fully human antibodies that were characterized for binding, neutralization, and three-dimensional structure. On the basis of these criteria, we selected pairs of highly potent individual antibodies that simultaneously bind the receptor binding domain of the spike protein, thereby providing ideal partners for a therapeutic antibody cocktail that aims to decrease the potential for virus escape mutants that might arise in response to selective pressure from a single-antibody treatment.

Bacillus thuringiensis (Bt) crystal (Cry) proteins are effective against a select number of insect pests, but improvements are needed to increase efficacy and decrease time to mortality for coleopteran pests. To gain insight into the Bt intoxication process in Coleoptera, we performed RNA-Seq on cDNA generated from the guts of Tenebrio molitor larvae that consumed either a control diet or a diet containing Cry3Aa protoxin. Approximately 134,090 and 124,287 sequence reads from the control and Cry3Aa-treated groups were assembled into 1,318 and 1,140 contigs, respectively. Enrichment analyses indicated that functions associated with mitochondrial respiration, signalling, maintenance of cell structure, membrane integrity, protein recycling/synthesis, and glycosyl hydrolases were significantly increased in Cry3Aa-treated larvae, whereas functions associated with many metabolic processes were reduced, especially glycolysis, tricarboxylic acid cycle, and fatty acid synthesis. Microarray analysis was used to evaluate temporal changes in gene expression after 6, 12 or 24 h of Cry3Aa exposure. Overall, microarray analysis indicated that transcripts related to allergens, chitin-binding proteins, glycosyl hydrolases, and tubulins were induced, and those related to immunity and metabolism were repressed in Cry3Aa-intoxicated larvae. The 24 h microarray data validated most of the RNA-Seq data. Of the three intoxication intervals, larvae demonstrated more differential expression of transcripts after 12 h exposure to Cry3Aa. Gene expression examined by three different methods in control vs. Cry3Aa-treated larvae at the 24 h time point indicated that transcripts encoding proteins with chitin-binding domain 3 were the most differentially expressed in Cry3Aa-intoxicated larvae. Overall, the data suggest that T. molitor larvae mount a complex response to Cry3Aa during the initial 24 h of intoxication. Data from this study represent the largest genetic sequence dataset for T. molitor to date. Furthermore, the methods in this study are useful for comparative analyses in organisms lacking a sequenced genome.

Leaf-cutter ants are one of the most important herbivorous insects in the Neotropics, harvesting vast quantities of fresh leaf material. The ants use leaves to cultivate a fungus that serves as the colony's primary food source. This obligate ant-fungus mutualism is one of the few occurrences of farming by non-humans and likely facilitated the formation of their massive colonies. Mature leaf-cutter ant colonies contain millions of workers ranging in size from small garden tenders to large soldiers, resulting in one of the most complex polymorphic caste systems within ants. To begin uncovering the genomic underpinnings of this system, we sequenced the genome of Atta cephalotes using 454 pyrosequencing. One prediction from this ant's lifestyle is that it has undergone genetic modifications that reflect its obligate dependence on the fungus for nutrients. Analysis of this genome sequence is consistent with this hypothesis, as we find evidence for reductions in genes related to nutrient acquisition. These include extensive reductions in serine proteases (which are likely unnecessary because proteolysis is not a primary mechanism used to process nutrients obtained from the fungus), a loss of genes involved in arginine biosynthesis (suggesting that this amino acid is obtained from the fungus), and the absence of a hexamerin (which sequesters amino acids during larval development in other insects). Following recent reports of genome sequences from other insects that engage in symbioses with beneficial microbes, the A. cephalotes genome provides new insights into the symbiotic lifestyle of this ant and advances our understanding of host-microbe symbioses.

A New Look at Old Data Although the full genome of Caenorhabditis elegans has been available for over 10 years, only a portion of the full-length messenger RNAs (mRNAs) and their expression through development has been analyzed. By combining multiple methods, involving a careful reannotation of existing data sets, sequencing of poly-A captured RNAs from multiple developmental stages, as well as cloning and deep sequencing of a subset of genes, Mangone et al. (p. 432 , published online 3 June) defined ∼26,000 distinct mRNA three-prime untranslated regions (3′UTRs) for ∼85% of the protein-coding genes. Most C. elegans genes have more than one 3′UTR, arising through different mechanisms including trans-splicing–coupled cleavage and polyadenylation. The work highlights the importance of the 3'UTRs in the regulation of mRNA stability and translation.

The genetic structure of the indigenous hunter-gatherer peoples of southern Africa, the oldest known lineage of modern human, is important for understanding human diversity. Studies based on mitochondrial and small sets of nuclear markers have shown that these hunter-gatherers, known as Khoisan, San, or Bushmen, are genetically divergent from other humans. However, until now, fully sequenced human genomes have been limited to recently diverged populations. Here we present the complete genome sequences of an indigenous hunter-gatherer from the Kalahari Desert and a Bantu from southern Africa, as well as protein-coding regions from an additional three hunter-gatherers from disparate regions of the Kalahari. We characterize the extent of whole-genome and exome diversity among the five men, reporting 1.3 million novel DNA differences genome-wide, including 13,146 novel amino acid variants. In terms of nucleotide substitutions, the Bushmen seem to be, on average, more different from each other than, for example, a European and an Asian. Observed genomic differences between the hunter-gatherers and others may help to pinpoint genetic adaptations to an agricultural lifestyle. Adding the described variants to current databases will facilitate inclusion of southern Africans in medical research efforts, particularly when family and medical histories can be correlated with genome-wide data.

Tuberous sclerosis complex (TSC) is an autosomal dominant neurocutaneous syndrome caused by mutations in TSC1 and TSC2. However, 10-15% TSC patients have no mutation identified with conventional molecular diagnostic studies. We used the ultra-deep pyrosequencing technique of 454 Sequencing to search for mosaicism in 38 TSC patients who had no TSC1 or TSC2 mutation identified by conventional methods. Two TSC2 mutations were identified, each at 5.3% read frequency in different patients, consistent with mosaicism. Both mosaic mutations were confirmed by several methods. Five of 38 samples were found to have heterozygous non-mosaic mutations, which had been missed in earlier analyses. Several other possible low-frequency mosaic mutations were identified by deep sequencing, but were discarded as artifacts by secondary studies. The low frequency of detection of mosaic mutations, two (6%) of 33, suggests that the majority of TSC patients who have no mutation identified are not due to mosaicism, but rather other causes, which remain to be determined. These findings indicate the ability of deep sequencing, coupled with secondary confirmatory analyses, to detect low-frequency mosaic mutations.

A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (∼1.1 Gb) includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest.

Herbivores can gain indirect access to recalcitrant carbon present in plant cell walls through symbiotic associations with lignocellulolytic microbes. A paradigmatic example is the leaf-cutter ant (Tribe: Attini), which uses fresh leaves to cultivate a fungus for food in specialized gardens. Using a combination of sugar composition analyses, metagenomics, and whole-genome sequencing, we reveal that the fungus garden microbiome of leaf-cutter ants is composed of a diverse community of bacteria with high plant biomass-degrading capacity. Comparison of this microbiome?s predicted carbohydrate-degrading enzyme profile with other metagenomes shows closest similarity to the bovine rumen, indicating evolutionary convergence of plant biomass degrading potential between two important herbivorous animals. Genomic and physiological characterization of two dominant bacteria in the fungus garden microbiome provides evidence of their capacity to degrade cellulose. Given the recent interest in cellulosic biofuels, understanding how large-scale and rapid plant biomass degradation occurs in a highly evolved insect herbivore is of particular relevance for bioenergy.

Tuberous sclerosis complex (TSC) is an often severe neurocutaneous syndrome. Cortical tubers are the predominant neuropathological finding in TSC, and their number and location has been shown to correlate roughly with the severity of neurologic features in TSC. Past studies have shown that genomic deletion events in TSC1 or TSC2 are very rare in tubers, and suggested the potential involvement of the MAPK pathway in their pathogenesis. We used deep sequencing to assess all coding exons of TSC1 and TSC2, and the activating mutation hot spots within KRAS in 46 tubers from TSC patients. Germline heterozygous mutations were identified in 81% of tubers. The same secondary mutation in TSC2 was identified in six tuber samples from one individual. Further study showed that this second hit mutation was widely distributed in the cortex from one cerebral hemisphere of this individual at frequencies up to 10%. No other secondary mutations were found in the other 40 tubers analyzed. These data indicate that small second hit mutations in any of these three genes are very rare in TSC tubers. However, in one TSC individual, a second hit TSC2 point mutation occurred early during brain development, and likely contributed to tuber formation.

Killer whales (Orcinus orca) currently comprise a single, cosmopolitan species with a diverse diet. However, studies over the last 30 yr have revealed populations of sympatric "ecotypes" with discrete prey preferences, morphology, and behaviors. Although these ecotypes avoid social interactions and are not known to interbreed, genetic studies to date have found extremely low levels of diversity in the mitochondrial control region, and few clear phylogeographic patterns worldwide. This low level of diversity is likely due to low mitochondrial mutation rates that are common to cetaceans. Using killer whales as a case study, we have developed a method to readily sequence, assemble, and analyze complete mitochondrial genomes from large numbers of samples to more accurately assess phylogeography and estimate divergence times. This represents an important tool for wildlife management, not only for killer whales but for many marine taxa. We used high-throughput sequencing to survey whole mitochondrial genome variation of 139 samples from the North Pacific, North Atlantic, and southern oceans. Phylogenetic analysis indicated that each of the known ecotypes represents a strongly supported clade with divergence times ranging from approximately 150,000 to 700,000 yr ago. We recommend that three named ecotypes be elevated to full species, and that the remaining types be recognized as subspecies pending additional data. Establishing appropriate taxonomic designations will greatly aid in understanding the ecological impacts and conservation needs of these important marine predators. We predict that phylogeographic mitogenomics will become an important tool for improved statistical phylogeography and more precise estimates of divergence times.

Whole‐genome association studies of complex traits in higher eukaryotes require a high density of single nucleotide polymorphism (SNP) markers at genome‐wide coverage. To design high‐throughput, multiplexed SNP genotyping assays, researchers must first discover large numbers of SNPs by extensively resequencing multiple individuals or lines. For SNP discovery approaches using short read‐lengths that next‐generation DNA sequencing technologies offer, the highly repetitive and duplicated nature of large plant genomes presents additional challenges. Here, we describe a genomic library construction procedure that facilitates pyrosequencing of genic and low‐copy regions in plant genomes, and a customized computational pipeline to analyze and assemble short reads (100–200 bp), identify allelic reference sequence comparisons, and call SNPs with a high degree of accuracy. With maize (Zea mays L.) as the test organism in a pilot experiment, the implementation of these methods resulted in the identification of 126,683 putative SNPs between two maize inbred lines at an estimated false discovery rate (FDR) of 15.1%. We estimated rates of false SNP discovery using an internal control, and we validated these FDR rates with an external SNP dataset that was generated using locus‐specific PCR amplification and Sanger sequencing. These results show that this approach has wide applicability for efficiently and accurately detecting gene‐enriched SNPs in large, complex plant genomes.

Endogenous small interfering RNAs (endo-siRNAs) regulate diverse gene expression programs in eukaryotes by either binding and cleaving mRNA targets or mediating heterochromatin formation; however, the mechanisms of endo-siRNA biogenesis, sorting, and target regulation remain poorly understood. Here we report the identification and function of a specific class of germline-generated endo-siRNAs in Caenorhabditis elegans that are 26 nt in length and contain a guanine at the first nucleotide position (i.e., 26G RNAs). 26G RNAs regulate gene expression during spermatogenesis and zygotic development, and their biogenesis requires the ERI-1 exonuclease and the RRF-3 RNA-dependent RNA polymerase (RdRP). Remarkably, we identified two nonoverlapping subclasses of 26G RNAs that sort into specific RNA-induced silencing complexes (RISCs) and differentially regulate distinct mRNA targets. Class I 26G RNAs target genes are expressed during spermatogenesis, whereas class II 26G RNAs are maternally inherited and silence gene expression during zygotic development. These findings implicate a class of endo-siRNAs in the global regulation of transcriptional programs required for fertility and development.

During development, proliferation of cerebellar granule neuron precursors (CGNPs), candidate cells-of-origin for the pediatric brain tumor medulloblastoma, requires signaling by Sonic hedgehog (Shh) and insulin-like growth factor (IGF), whose pathways are also implicated in medulloblastoma. One of the consequences of IGF signaling is inactivation of the mTOR-suppressing Tuberous Sclerosis Complex (TSC), comprised of TSC1 and TSC2, leading to increased mRNA translation. We show that mice in which TSC function is impaired display increased mTOR pathway activation, enhanced CGNP proliferation, GSK-3 alpha/beta inactivation, and cytoplasmic localization of the cyclindependent kinase (cdk) inhibitor p27, which has been proposed to cause its inactivation or gain of oncogenic functions. We observed the same characteristics in wild-type primary cultures of CGNPs in which TSC1 and/or TSC2 were knocked down, and in mouse medulloblastomas induced by ectopic Shh pathway activation. Moreover, Shh-induced mouse medulloblastomas manifested Akt-mediated TSC2 inactivation, and the mutant TSC2 allele synergized with aberrant Shh signaling to increase medulloblastoma incidence in mice. Driving exogenous TSC2 expression in Shhinduced medulloblastoma cells corrected p27 localization and reduced proliferation. GSK-3 alpha/beta inactivation in the tumors in vivo and in primary CGNP cultures was mTOR-dependent, whereas p27 cytoplasmic localization was regulated upstream of mTOR, by TSC2. These results indicate that a balance between Shh mitogenic signaling and TSC function regulating new protein synthesis and cdk inhibition is essential for normal development and prevention of tumor formation or expansion. POLYCYSTIN-1 REGULATES MTORC1 IN AN ERKS-DEPENDENT, AKTINDEPENDENT MANNER IN VITRO AND IN VIVO Monika Pema, Gianfranco Distefano, Claas Wodarczyk and Alessandra Boletta , 1 Dulbecco Telethon Institute (DTI) at Dibit, San Raffaele Scientific Institute, Milan-ITALY Introduction Polycystin-1 (PC-1) the product of the gene most commonly mutated in Autosomal Dominant Polycystic Kidney Disease (ADPKD) regulates both proliferation and apoptosis, consistent with misregulation of both biological properties observed in ADPKD cystic epithelia. Several studies have recently linked the function of PC-1 to that of Tuberin. Furthermore, studies performed in several different animal models as well as on patients-derived tissues have reveled that the mTOR cascade is upregulated in Polycystic Kidney Diseases, although the precise mechanism of this disregulation was not investigated. We have recently shown using a series of loss and gain of function in vitro models that PC-1 controls cell growth (size) in addition to and independently of its action on proliferation. We have shown that PC-1 achieves this effect by inhibiting the mTORC1 pathway and its downstream effectors S6K and 4EBP1 in a Tsc2-dependent manner. Furthermore, PC-1 is able to regulate Tsc2 by modulating its ERKs-specific phosphorylation in S664 (Distefano et al, MCB, 2009). Notably, we also have found that PC-1 induces full activation of Akt, suggesting a role of PC-1 in regulating mTORC2 as well, but this does not result in Tsc2 phosphorylation in Akt-specific sites, suggesting that there is a disconnect between the PI3k/Akt pathway and mTORC1 in response to PC-1. Methods In order to further investigate the mechanism by which PC-1 regulates the mTORC1 and 2 in vivo, we have generated a bona fide ADPKD mouse model by crossing a conditional mouse model generated in our laboratory (Wodarczyk et al., submitted), in which the last two exons of Pkd1 are flanked by two loxP sites, with a mouse harbouring a kidneyspecific Cre recombinase (Ksp-Cre, kindly provided by Dr. P. Igarashi, Southewestern University, TX-USA). Pkd1 flox/-:Ksp-Cre mice undergo kidney specific inactivation of the floxed allele and develop massive renal cysts after birth, ultimately leading to death by the second week of life Results Biochemical analysis of lysates from polycystic kidneys at P4 and P8 shows hyperphosphorylation of downstream effectors of mTORC1, such as p70S6K and S6Rp as compared to controls. Furthermore, MEK1/2 and ERKs, but not Akt, are strongly upregulated, further suggesting that the ERKs, but not Akt control mTORC1 upregulation in ADPKD. Notably, immunohistochemistry on P4 cystic kidneys shows that the majority of cysts stain positive for phospho S6Rp (>70%), but we also observe a high proportion of cysts that do not show hyperactivation of the mTORC1 cascade. Conclusions We propose a model by which PC-1 is able to regulate the mTORC1 cascade through an atypical mechanism that might be protective of cyst transformation, but this alone cannot justify renal cyst formation and expansion. Funding This work was supported by the italian Telethon Career Program (TCR05007) and by the European Community FP6 (MC-EXT2785). SINGLE-CELL GENETIC REMOVAL OF TSC1 IN CORTICAL RADIAL GLIA David Feliciano, Jean-Claude Platel and Angelique Bordey Yale University School of Medicine, New Haven, CT 06520-8082, USA In Tuberous Sclerosis Complex (TSC) patients, mutations in one of two tumor suppressor genes, TSC1 or TSC2, result in the formation of lesions. The mechanisms leading to TSC lesions and associated seizure generation during perinatal life remain unclear. The recent finding that radial glia act as neural progenitors led us to hypothesize that deletion of TSC genes in perinatal neural progenitor cells (i.e. radial glia) contributes to the generation of TSC lesions, and we propose to identify the mechanisms underlying that process. To test our hypothesis, we genetically removed Tsc1 in transgenic mice carrying conditional Tsc1 alleles (fl) from embryonic progenitor cells using in vivo electroporation of a Cre recombinase-containing plasmid. We found that removal of Tsc1 in subsets of cortical radial glia at embryonic day 16 (E16) did not disrupt the proper migration of cortical pyramidal neurons to layer II/III. However, Tsc1-null neurons displayed increased phosphorylation of S6 as expected following loss of functional Tsc1-Tsc2 complex and increased soma size. Next considering that TSC patients have a systemic loss of one Tsc1 allele, in future experiments we will use a double hit model in transgenic mice carrying fl and mutant (mut, non-functional) Tsc1 alleles. These data suggest that formation of TSC-like lesions such as cortical tubers requires using mice with a systemic loss of Tsc1 prior removing the second allele. Support: TSC Alliance MODULATION OF HYPOXIA INDUCIBLE FACTOR-1-ALPHA Kayleigh Dodd and Andrew Tee. Institute of Medical Genetics, Cardiff University, Heath Park, Cardiff, CF14 4XN, United Kingdom Hypoxia-Inducible Factor-1-alpha (HIF1-alpha) is a transcription factor essential for the maintenance of oxygen homeostasis. Under hypoxia, the HIF1-alpha subunit becomes stable enough to bind to the constitutively expressed beta subunit. It then translocates into the nucleus where it binds to HIF response elements located upstream of promoters on various target genes, thus triggering their gene expression. HIF1-alpha is known to regulate numerous targets which can in turn modulate cellular processes to increase O2 saturation, including angiogenesis, glucose transport and erythropoiesis. Previous work has demonstrated that HIF1-alpha is modulated via the mTOR pathway and is sensitive to rapamycin inhibition. Clinical research has also verified that TS sufferers show high secretion levels of the HIF1-alpha target VEGF. HIF dysregulation may, therefore, play a significant role in the pathogenesis of TS patients. Increased HIF1-alpha activity will facilitate the formation and development of harmatomas by modulating the blood supply to increase the delivery of oxygen and nutrients to the core of the tumour. This makes it a potentially appealing therapeutic target for treatment of TS. The mechanisms behind mTOR dependent HIF regulation have yet to be fully elucidated, therefore we have carried out a series of experiments to attempt to further clarify the pathway. The use of various techniques including a luciferase reporter construct, kinase assays and quantitative-pcr have allowed us to confirm HIF1-alpha as a target of mTOR that is responsive to insulin stimulation but subject to rapamycin inhibition. Furthermore we have been able to eliminate the mTOR target S6K1 as a regulator of HIF1-alpha. We found HIF-1-alpha is regulated by 4E-BP1, however further analysis also suggests a potential secondary mechanism for HIF1-alpha inhibition which is dependent upon TSC2 but independent of the mTORC1 complex. Functional analysis of various patient derived mutants of TSC2 has also revealed the importance of the calmodulin binding domain (also referred to as the transcriptional activation domain) of TSC2 in regulating HIF1-

BACKGROUND:The next generation sequencing technologies provide new options to characterize the transcriptome and to develop affordable tools for functional genomics. We describe here an innovative approach for this purpose and demonstrate its potential also for non-model species.RESULTS:The method we developed is based on 454 sequencing of 3' cDNA fragments from a normalized library constructed from pooled RNAs to generate, through de novo reads assembly, a large catalog of unique transcripts in organisms for which a comprehensive collection of transcripts or the complete genome sequence, is not available. This "virtual transcriptome" provides extensive coverage depth, and can be used for the setting up of a comprehensive microarray based expression analysis. We evaluated the potential of this approach by monitoring gene expression during berry maturation in Vitis vinifera as if no other sequence information was available for this species. The microarray designed on the berries' transcriptome derived from half of a 454 run detected the expression of 19,609 genes, and proved to be more informative than one of the most comprehensive grape microarrays available to date, the GrapeArray 1.2 developed by the Italian-French Public Consortium for Grapevine Genome Characterization, which could detect the expression of 15,556 genes in the same samples.CONCLUSION:This approach provides a powerful method to rapidly build up an extensive catalog of unique transcripts that can be successfully used to develop a microarray for large scale analysis of gene expression in any species, without the need for prior sequence knowledge.

Major histocompatibility complex (MHC) genetics dictate adaptive cellular immune responses, making robust MHC genotyping methods essential for studies of infectious disease, vaccine development and transplantation. Nonhuman primates provide essential preclinical models for these areas of biomedical research. Unfortunately, given the unparalleled complexity of macaque MHCs, existing methodologies are inadequate for MHC typing of these key model animals. Here we use pyrosequencing of complementary DNA-PCR amplicons as a general approach to determine comprehensive MHC class I genotypes in nonhuman primates. More than 500 unique MHC class I sequences were resolved by sequence-based typing of rhesus, cynomolgus and pig-tailed macaques, nearly half of which have not been reported previously. The remarkable sensitivity of this approach in macaques demonstrates that pyrosequencing is viable for ultra-high-throughput MHC genotyping of primates, including humans.

Cancers arise by the gradual accumulation of mutations in multiple genes. We now use shotgun pyrosequencing to characterize RNA mutations and expression levels unique to malignant pleural mesotheliomas (MPMs) and not present in control tissues. On average, 266 Mb of cDNA were sequenced from each of four MPMs, from a control pulmonary adenocarcinoma (ADCA), and from normal lung tissue. Previously observed differences in MPM RNA expression levels were confirmed. Point mutations were identified by using criteria that require the presence of the mutation in at least four reads and in both cDNA strands and the absence of the mutation from sequence databases, normal adjacent tissues, and other controls. In the four MPMs, 15 nonsynonymous mutations were discovered: 7 were point mutations, 3 were deletions, 4 were exclusively expressed as a consequence of imputed epigenetic silencing, and 1 was putatively expressed as a consequence of RNA editing. Notably, each MPM had a different mutation profile, and no mutated gene was previously implicated in MPM. Of the seven point mutations, three were observed in at least one tumor from 49 other MPM patients. The mutations were in genes that could be causally related to cancer and included XRCC6, PDZK1IP1, ACTR1A, and AVEN.

Recently, genome-wide association studies have identified loci across a segment of chromosome 8q24 (128,100,000-128,700,000) associated with the risk of breast, colon and prostate cancers. At least three regions of 8q24 have been independently associated with prostate cancer risk; the most centromeric of which appears to be population specific. Haplotypes in two contiguous but independent loci, marked by rs6983267 and rs1447295, have been identified in the Cancer Genetic Markers of Susceptibility project ( http://cgems.cancer.gov ), which genotyped more than 5,000 prostate cancer cases and 5,000 controls of European origin. The rs6983267 locus is also strongly associated with colorectal cancer. To ascertain a comprehensive catalog of common single-nucleotide polymorphisms (SNPs) across the two regions, we conducted a resequence analysis of 136 kb (chr8: 128,473,000-128,609,802) using the Roche/454 next-generation sequencing technology in 39 prostate cancer cases and 40 controls of European origin. We have characterized a comprehensive catalog of common (MAF > 1%) SNPs within this region, including 442 novel SNPs and have determined the pattern of linkage disequilibrium across the region. Our study has generated a detailed map of genetic variation across the region, which should be useful for choosing SNPs for fine mapping of association signals in 8q24 and investigations of the functional consequences of select common variants.

Background With a whole genome duplication event and wealth of biological data, salmonids are excellent model organisms for studying evolutionary processes, fates of duplicated genes and genetic and physiological processes associated with complex behavioral phenotypes. It is surprising therefore, that no salmonid genome has been sequenced. Atlantic salmon ( Salmo salar ) is a good representative salmonid for sequencing given its importance in aquaculture and the genomic resources available. However, the size and complexity of the genome combined with the lack of a sequenced reference genome from a closely related fish makes assembly challenging. Given the cost and time limitations of Sanger sequencing as well as recent improvements to next generation sequencing technologies, we examined the feasibility of using the Genome Sequencer (GS) FLX pyrosequencing system to obtain the sequence of a salmonid genome. Eight pooled BACs belonging to a minimum tiling path covering ~1 Mb of the Atlantic salmon genome were sequenced by GS FLX shotgun and Long Paired End sequencing and compared with a ninth BAC sequenced by Sanger sequencing of a shotgun library. Results An initial assembly using only GS FLX shotgun sequences (average read length 248.5 bp) with ~30× coverage allowed gene identification, but was incomplete even when 126 Sanger-generated BAC-end sequences (~0.09× coverage) were incorporated. The addition of paired end sequencing reads (additional ~26× coverage) produced a final assembly comprising 175 contigs assembled into four scaffolds with 171 gaps. Sanger sequencing of the ninth BAC (~10.5× coverage) produced nine contigs and two scaffolds. The number of scaffolds produced by the GS FLX assembly was comparable to Sanger-generated sequencing; however, the number of gaps was much higher in the GS FLX assembly. Conclusion These results represent the first use of GS FLX paired end reads for de novo sequence assembly. Our data demonstrated that this improved the GS FLX assemblies; however, with respect to de novo sequencing of complex genomes, the GS FLX technology is limited to gene mining and establishing a set of ordered sequence contigs. Currently, for a salmonid reference sequence, it appears that a substantial portion of sequencing should be done using Sanger technology.