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The human genome harbors numerous structural variants (SVs) which, due to their repetitive nature, are currently underexplored in short-read whole-genome sequencing approaches. Using single-molecule, real-time (SMRT) long-read sequencing technology in combination with FALCON-Unzip, we generated a de novo assembly of the diploid genome of a 115-year-old Dutch cognitively healthy woman. We combined this assembly with two previously published haploid assemblies (CHM1 and CHM13) and the GRCh38 reference genome to create a compendium of SVs that occur across five independent human haplotypes using the graph-based multi-genome aligner REVEAL. Across these five haplotypes, we detected 31,680 euchromatic SVs (>50 bp). Of these, ~62% were comprised of repetitive sequences with 'variable number tandem repeats' (VNTRs), ~10% were mobile elements (Alu, L1, and SVA), while the remaining variants were inversions and indels. We observed that VNTRs with GC-content >60% and repeat patterns longer than 15 bp were 21-fold enriched in the subtelomeric regions (within 5 Mb of the ends of chromosome arms). VNTR lengths can expand to exceed a critical length which is associated with impaired gene transcription. The genes that contained most VNTRs, of which PTPRN2 and DLGAP2 are the most prominent examples, were found to be predominantly expressed in the brain and associated with a wide variety of neurological disorders. Repeat-induced variation represents a sizeable fraction of the genetic variation in human genomes and should be included in investigations of genetic factors associated with phenotypic traits, specifically those associated with neurological disorders. We make available the long and short-read sequence data of the supercentenarian genome, and a compendium of SVs as identified across 5 human haplotypes. ispartof: TRANSLATIONAL PSYCHIATRY vol:10 issue:1 ispartof: location:United States status: published

ispartof: Journal of Allergy and Clinical Immunology vol:141 issue:2 pages:768-770 ispartof: location:United States status: published

In addition to Sanger sequencing, next-generation sequencing of gene panels and exomes has emerged as a standard diagnostic tool in many laboratories. However, these captures can miss regions, have poor efficiency, or capture pseudogenes which hamper proper diagnoses. One such example is the primary immunodeficiency-associated gene IKBKG. Its pseudogene IKBKGP1 makes traditional capture methods aspecific. We therefore developed a long-range PCR method to efficiently target IKBKG, as well as two associated genes (IRAK4 and MYD88), whilst bypassing the IKBKGP1 pseudogene. Sequencing accuracy was evaluated using both conventional short read technology and a newer long read single molecule sequencer. Different mapping and variant calling options were evaluated in their capability to bypass the pseudogene using both sequencing platforms. Based on these evaluations, we determined a robust diagnostic application for unambiguous sequencing and variant calling in IKBKG, IRAK4, and MYD88. This method allows rapid identification of selected primary immunodeficiency diseases in patients suffering from life-threatening invasive pyogenic bacterial infections. ispartof: The Journal of Molecular Diagnostics vol:20 issue:2 pages:195-202 ispartof: location:United States status: published

Fermented foods and beverages have been consumed by umans for over 8000 years. These fermentations were at first spontaneous process, without any control or knowledge of the icrobial driving force. This led to irregular and often inferior end roducts. It was not until the late 19th century that it was proposed o use a well-defined microbial starter culture for wine production, onsisting of one pure yeast strain. Although this greatly increased he reproducibility of the fermentations, the main difficulty was o select a strain with all beneficial characteristics necessary for n efficient and high-quality fermentation. Until recently, the ppropriate tools and knowledge where lacking to make a wellonsidered and scientifically-founded choice about which strain to ick. This explains why, even today, most beer yeasts are used based n historical rather than scientific reasons. However, the current nowledge and technologies now allow selection of superior yeasts trains that are optimally suited to perform their industrial tasks. In ur research team, for example, we are using state-of-the-art techologies to select and generate yeast strains with improved ethanol olerance, fermentation speed, attenuation and flavour production.

The positions of nucleosomes across the genome influence several cellular processes, including gene transcription. However, our understanding of the factors dictating where nucleosomes are located and how this affects gene regulation is still limited. Here, we perform an extensive in vivo study to investigate the influence of the neighboring chromatin structure on local nucleosome positioning and gene expression. Using truncated versions of the Saccharomyces cerevisiae URA3 gene, we show that nucleosome positions in the URA3 promoter are at least partly determined by the local DNA sequence, with so-called ‘antinucleosomal elements’ like poly(dA:dT) tracts being key determinants of nucleosome positions. In addition, we show that changes in the nucleosome positions in the URA3 promoter strongly affect the promoter activity. Most interestingly, in addition to demonstrating the effect of the local DNA sequence, our study provides novel in vivo evidence that nucleosome positions are also affected by the position of neighboring nucleosomes. Nucleosome structure may therefore be an important selective force for conservation of gene order on a chromosome, because relocating a gene to another genomic position (where the positions of neighboring nucleosomes are different from the original locus) can have dramatic consequences for the gene's nucleosome structure and thus its expression., Human Frontier Science Program (Strasbourg, France) (Grant no.RGY79/2007), European Research Council. Young Investigator Grant (241426), National Institutes of Health (U.S.) (Grant no. GM040266), National Institutes of Health (U.S.) (Grant no. GM035010)

Tolerance to high levels of ethanol is an ecologically and industrially relevant phenotype of microbes, but the molecular mechanisms underlying this complex trait remain largely unknown. Here, we use long-term experimental evolution of isogenic yeast populations of different initial ploidy to study adaptation to increasing levels of ethanol. Whole-genome sequencing of more than 30 evolved populations and over 100 adapted clones isolated throughout this two-year evolution experiment revealed how a complex interplay of de novo single nucleotide mutations, copy number variation, ploidy changes, mutator phenotypes, and clonal interference led to a significant increase in ethanol tolerance. Although the specific mutations differ between different evolved lineages, application of a novel computational pipeline, PheNetic, revealed that many mutations target functional modules involved in stress response, cell cycle regulation, DNA repair and respiration. Measuring the fitness effects of selected mutations introduced in non-evolved ethanol-sensitive cells revealed several adaptive mutations that had previously not been implicated in ethanol tolerance, including mutations in PRT1, VPS70 and MEX67. Interestingly, variation in VPS70 was recently identified as a QTL for ethanol tolerance in an industrial bio-ethanol strain. Taken together, our results show how, in contrast to adaptation to some other stresses, adaptation to a continuous complex and severe stress involves interplay of different evolutionary mechanisms. In addition, our study reveals functional modules involved in ethanol resistance and identifies several mutations that could help to improve the ethanol tolerance of industrial yeasts., Author Summary Organisms can evolve resistance to specific stress factors, which allows them to thrive in environments where non-adapted organisms fail to grow. However, the molecular mechanisms that underlie adaptation to complex stress factors that interfere with basic cellular processes are poorly understood. In this study, we reveal how yeast populations adapt to high ethanol concentrations, an ecologically and industrially relevant stress that is still poorly understood. We exposed six independent populations of genetically identical yeast cells to gradually increasing ethanol levels, and we monitored the changes in their DNA sequence over a two-year period. Together with novel computational analyses, we could identify the mutational dynamics and molecular mechanisms underlying increased ethanol resistance. Our results show how adaptation to high ethanol is complex and can be reached through different mutational pathways. Together, our study offers a detailed picture of how populations adapt to a complex continuous stress and identifies several mutations that increase ethanol resistance, which opens new routes to obtain superior biofuel yeast strains.