The methylotrophic yeast Pichia pastoris is widely used for the production of proteins and as a model organism for studying peroxisomal biogenesis and methanol assimilation. P. pastoris strains capable of human-type N-glycosylation are now available, which increases the utility of this organism for biopharmaceutical production. Despite its biotechnological importance, relatively few genetic tools or engineered strains have been generated for P. pastoris. To facilitate progress in these areas, we present the 9.43 Mbp genomic sequence of the GS115 strain of P. pastoris. We also provide manually curated annotation for its 5,313 protein-coding genes.The methylotrophic yeast Pichia pastoris is by far the most commonly used yeast species in the production of recombinant proteins 1 and is employed in laboratories around the world to produce proteins for basic research and medical applications. It is also an important model organism for the investigation of peroxisomal proliferation and methanol assimilation. The P. pastoris expression technology has been commercially available for many years. P. pastoris grows to high cell density, provides tightly controlled methanol-inducible transgene expression and efficiently secretes heterologous proteins in defined media. Several P. pastoris-produced biopharmaceuticals that are either not glycosylated (such as human serum albumin 2 ) or for which glycosylation is needed only for proper folding (such as several vaccines 3 ) are already on the market. An important recent breakthrough has been the development of P. pastoris strains with humantype N-glycosylation [4][5][6] . Humanized glycosylation will further increase the importance of P. pastoris for biopharmaceutical production; indeed, proteins produced with this system are moving into clinical development 7 . Moreover, monoclonal antibodies can be made at gramper-liter scale in the humanized glycosylation-homogenous strains 8 .For further strain engineering, a better understanding of all aspects of the yeast's protein production machinery is needed, and a number of studies relating to P. pastoris's secretory system and engineered promoters have been forthcoming 9,10 . To facilitate the investigation of P. pastoris and other methylotrophic yeasts, we present the 9.43 Mbp genomic sequence of the GS115 strain of P. pastoris.

Monozygotic (MZ) twins are considered being genetically identical, therefore they cannot be differentiated using standard forensic DNA testing. Here we describe how identification of extremely rare mutations by ultra-deep next generation sequencing can solve such cases. We sequenced DNA from sperm samples of two twins and from a blood sample of the child of one twin. Bioinformatics analysis revealed five single nucleotide polymorphisms (SNPs) present in the twin father and the child, but not in the twin uncle. The SNPs were confirmed by classical Sanger sequencing. Our results give experimental evidence for the hypothesis that rare mutations will occur early after the human blastocyst has split into two, the origin of twins, and that such mutations will be carried on into somatic tissue and the germline. The method provides a solution to solve paternity and forensic cases involving monozygotic twins as alleged fathers or originators of DNA traces.

HighlightsDeep sequencing has potential as an improved adventitious virus screening method.15 laboratories sequenced a common reagent containing 25 target viruses.6 viruses were detected by all lab, the remainder were detected by 4–14 labs.A wide range of sample preparation and bioinformatics methods is currently used.A common reference material is essential to enable results to be compared.

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Identification of the potential donor(s) of human germline-derived cells is an issue in many criminal investigations and in paternity testing. The experimental and statistical methodology necessary to work up such cases is well established but may be more challenging if monozygotic (MZ) twins are involved. Then, elaborate genome-wide searches are required for the detection of early somatic mutations that distinguish the cell sample and its donor from the other twin, usually relying upon reference material other than semen (e.g. saliva). The first such cases, involving either criminal sexual offenses or paternity disputes, have been processed successfully by Eurofins Genomics and Forensics Campus. However, when presenting the experimental results in court, common forensic genetic practice requires that the residual uncertainty about donorship is quantified in the form of a likelihood ratio (LR). Hence, we developed a general mathematical framework for LR calculation, presented herein, which allows quantification of the evidence in favour of the true donor in the respective cases, based upon observed DNA sequencing read counts.

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