The 1000 Genomes Project set out to provide a comprehensive description of common human genetic variation by applying whole-genome sequencing to a diverse set of individuals from multiple populations. Here we report completion of the project, having reconstructed the genomes of 2,504 individuals from 26 populations using a combination of low-coverage whole-genome sequencing, deep exome sequencing, and dense microarray genotyping. We characterized a broad spectrum of genetic variation, in total over 88 million variants (84.7 million single nucleotide polymorphisms (SNPs), 3.6 million short insertions/deletions (indels), and 60,000 structural variants), all phased onto high-quality haplotypes. This resource includes >99% of SNP variants with a frequency of >1% for a variety of ancestries. We describe the distribution of genetic variation across the global sample, and discuss the implications for common disease studies.

BackgroundCancer re-sequencing programs rely on DNA isolated from fresh snap frozen tissues, the preparation of which is combined with additional preservation efforts. Tissue samples at pathology departments are routinely stored as formalin-fixed and paraffin-embedded (FFPE) samples and their use would open up access to a variety of clinical trials. However, FFPE preparation is incompatible with many down-stream molecular biology techniques such as PCR based amplification methods and gene expression studies.Methodology/Principal FindingsHere we investigated the sample quality requirements of FFPE tissues for massively parallel short-read sequencing approaches. We evaluated key variables of pre-fixation, fixation related and post-fixation processes that occur in routine medical service (e.g. degree of autolysis, duration of fixation and of storage). We also investigated the influence of tissue storage time on sequencing quality by using material that was up to 18 years old. Finally, we analyzed normal and tumor breast tissues using the Sequencing by Synthesis technique (Illumina Genome Analyzer, Solexa) to simultaneously localize genome-wide copy number alterations and to detect genomic variations such as substitutions and point-deletions and/or insertions in FFPE tissue samples.Conclusions/SignificanceThe application of second generation sequencing techniques on small amounts of FFPE material opens up the possibility to analyze tissue samples which have been collected during routine clinical work as well as in the context of clinical trials. This is in particular important since FFPE samples are amply available from surgical tumor resections and histopathological diagnosis, and comprise tissue from precursor lesions, primary tumors, lymphogenic and/or hematogenic metastases. Large-scale studies using this tissue material will result in a better prediction of the prognosis of cancer patients and the early identification of patients which will respond to therapy.

Whole-genome transcriptome measurements are pivotal for characterizing molecular mechanisms of chemicals and predicting toxic classes, such as genotoxicity and carcinogenicity, from in vitro and in vivo assays. In recent years, deep sequencing technologies have been developed that hold the promise of measuring the transcriptome in a more complete and unbiased manner than DNA microarrays. Here, we applied this RNA-seq technology for the characterization of the transcriptomic responses in HepG2 cells upon exposure to benzo[a]pyrene (BaP), a well-known DNA damaging human carcinogen. Based on EnsEMBL genes, we demonstrate that RNA-seq detects ca 20% more genes than microarray-based technology but almost threefold more significantly differentially expressed genes. Functional enrichment analyses show that RNA-seq yields more insight into the biology and mechanisms related to the toxic effects caused by BaP, i.e., two- to fivefold more affected pathways and biological processes. Additionally, we demonstrate that RNA-seq allows detecting alternative isoform expression in many genes, including regulators of cell death and DNA repair such as TP53, BCL2 and XPA, which are relevant for genotoxic responses. Moreover, potentially novel isoforms were found, such as fragments of known transcripts, transcripts with additional exons, intron retention or exon-skipping events. The biological function(s) of these isoforms remain for the time being unknown. Finally, we demonstrate that RNA-seq enables the investigation of allele-specific gene expression, although no changes could be observed. Our results provide evidence that RNA-seq is a powerful tool for toxicology, which, compared with microarrays, is capable of generating novel and valuable information at the transcriptome level for characterizing deleterious effects caused by chemicals.

Several reports have recently demonstrated a detrimental role of Toll-like receptors (TLR) in cerebral ischemia, while there is little information about the endogenous ligands which activate TLR-signaling. The myeloid related proteins-8 and-14 (Mrp8/S100A8; Mrp14/S100A9) have recently been characterized as endogenous TLR4-agonists, and thus may mediate TLR-activation in cerebral ischemia. Interestingly, not only TLR-mRNAs, but also Mrp8 and Mrp14 mRNA were found to be induced in mouse brain between 3 and 48 h after transient 1 h focal cerebral ischemia/reperfusion. Mrp-protein was expressed in the ischemic hemisphere, and co-labeled with CD11b-positive cells. To test the hypothesis that Mrp-signaling contributes to the postischemic brain damage, we subjected Mrp14-deficient mice, which also lack Mrp8 protein expression, to focal cerebral ischemia. Mrp14-deficient mice had significantly smaller lesion volumes when compared to wild-type littermates (130+/-16 mm(3) vs. 105+/-28 mm(3)) at 2 days after transient focal cerebral ischemia (1 h), less brain swelling, and a reduced macrophage/microglia cell count in the ischemic hemisphere. We conclude that upregulation and signaling of Mrp-8 and-14 contribute to neuroinflammation and the progression of ischemic damage.

BackgroundSeveral organisms display dormancy and developmental arrest at embryonic stages. Long-term survival in the dormant form is usually associated with desiccation, orthodox plant seeds and Artemia cysts being well documented examples. Several aquatic invertebrates display dormancy during embryonic development and survive for tens or even hundreds of years in a hydrated form, raising the question of whether survival in the non-desiccated form of embryonic development depends on pathways similar to those occurring in desiccation tolerant forms.Methodology/Principal FindingsTo address this question, Illumina short read sequencing was used to generate transcription profiles from the resting and amictic eggs of an aquatic invertebrate, the rotifer, Brachionus plicatilis. These two types of egg have very different life histories, with the dormant or diapausing resting eggs, the result of the sexual cycle and amictic eggs, the non-dormant products of the asexual cycle. Significant transcriptional differences were found between the two types of egg, with amictic eggs rich in genes involved in the morphological development into a juvenile rotifer. In contrast, representatives of classical “stress” proteins: a small heat shock protein, ferritin and Late Embryogenesis Abundant (LEA) proteins were identified in resting eggs. More importantly however, was the identification of transcripts for messenger ribonucleoprotein particles which stabilise RNA. These inhibit translation and provide a valuable source of useful RNAs which can be rapidly activated on the exit from dormancy. Apoptotic genes were also present. Although apoptosis is inconsistent with maintenance of prolonged dormancy, an altered apoptotic pathway has been proposed for Artemia, and this may be the case with the rotifer.ConclusionsThese data represent the first transcriptional profiling of molecular processes associated with dormancy in a non-desiccated form and indicate important similarities in the molecular pathways activated in resting eggs compared with desiccated dormant forms, specifically plant seeds and Artemia.

Rotifer resting eggs retain their viability for several decades in a non-desiccated form and are of interest in discerning the processes associated with dormancy, since in most organisms this phenomenon is linked with desiccation. The expression pattern of candidate genes with biological functions associated with dormancy in several other organisms was examined in rotifers. High-throughput transcriptome profiling revealed three patterns of gene expression in resting eggs: (1) relatively highly expressed genes coding for LEA proteins and putative paralogs of the small heat shock protein family (shsp); (2) genes coding for ferritin (ferr), glutathione-6-transferase (gts) and HSP70, where some of the putative gene paralogs of these families show relatively high expression levels and other putative paralogs show relatively low expression levels in resting eggs; and (3) genes with relatively low expression levels in resting eggs, for trehalose-6-phosphate synthase (tps), fatty-acid binding proteins (fab) and of lipoprotein lipase (lpl) and the aquaporins gene family (aqp). Changes in the expression pattern of some members of putative gene families occurred during the obligatory dormant period of resting eggs. A transition was observed from an expression pattern of diapausing embryos to an expression pattern of amictic females, during hatching. Differences were also found in the expression pattern in the different types of females, especially in those carrying resting eggs, and in males compared with females. These results suggest putative functional significance to genes associated with dormancy in non-desiccated resting eggs. It could also be proposed that their occurrence in resting eggs is developmentally programmed to facilitate survival in case of desiccation.

A recombinant plasmid harboring both genomic termini of tupaia herpesvirus (THV) DNA was characterized by restriction enzyme analysis and by determination of the nucleotide sequence. A unique NotI cleavage site was found that is located approximately 19 base pairs upstream of the THV terminal junction. THV DNA fragments from virion DNA were analyzed by using the same restriction enzymes as for the recombinant plasmid. The comparative fine mapping of virion THV DNA revealed heterogeneous molecules of variable lengths with the NotI cleavage site conserved. A number of short direct and inverted repeats and palindromes were found surrounding the THV terminal joint. The THV repetitive sequences were compared with the repeats reported for the DNA termini of herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus and are discussed in respect to signals for a site-specific endonuclease required for packaging.