Safety assessment of graphene-based materials (GBMs) including graphene oxide (GO) is essential for their safe use across many sectors of society. In particular, the link between specific material properties and biological effects needs to be further elucidated. Here, the effects of lateral dimensions of GO sheets in acute and chronic pulmonary responses after single intranasal instillation in mice are compared. Micrometer-sized GO induces stronger pulmonary inflammation than nanometer-sized GO, despite reduced translocation to the lungs. Genome-wide RNA sequencing also reveals distinct size-dependent effects of GO, in agreement with the histopathological results. Although large GO, but not the smallest GO, triggers the formation of granulomas that persists for up to 90 days, no pulmonary fibrosis is observed. These latter results can be partly explained by Raman imaging, which evidences the progressive biotransformation of GO into less graphitic structures. The findings demonstrate that lateral dimensions play a fundamental role in the pulmonary response to GO, and suggest that airborne exposure to micrometer-sized GO should be avoided in the production plant or applications, where aerosolized dispersions are likely to occur. These results are important toward the implementation of a safer-by-design approach for GBM products and applications, for the benefit of workers and end-users.

Ancient DNA has significantly improved our understanding of the evolution and population history of extinct megafauna. However, few studies have used complete ancient genomes to examine species responses to climate change prior to extinction. The woolly rhinoceros (Coelodonta antiquitatis) was a cold-adapted megaherbivore widely distributed across northern Eurasia during the Late Pleistocene and became extinct approximately 14 thousand years before present (ka BP). While humans and climate change have been proposed as potential causes of extinction [1-3], knowledge is limited on how the woolly rhinoceros was impacted by human arrival and climatic fluctuations [2]. Here, we use one complete nuclear genome and 14 mitogenomes to investigate the demographic history of woolly rhinoceros leading up to its extinction. Unlike other northern megafauna, the effective population size of woolly rhinoceros likely increased at 29.7 ka BP and subsequently remained stable until close to the species' extinction. Analysis of the nuclear genome from a ∼18.5-ka-old specimen did not indicate any increased inbreeding or reduced genetic diversity, suggesting that the population size remained steady for more than 13 ka following the arrival of humans [4]. The population contraction leading to extinction of the woolly rhinoceros may have thus been sudden and mostly driven by rapid warming in the Bølling-Allerød interstadial. Furthermore, we identify woolly rhinoceros-specific adaptations to arctic climate, similar to those of the woolly mammoth. This study highlights how species respond differently to climatic fluctuations and further illustrates the potential of palaeogenomics to study the evolutionary history of extinct species.

Recently developed high-throughput sequencing technology shows power to detect low-frequency disease-causing variants by deep sequencing of all known exons. We used exome sequencing to identify variants associated with morbid obesity. DNA from 100 morbidly obese adult subjects and 100 controls were pooled (n=10/pool), subjected to exome capture, and subsequent sequencing. At least 100 million sequencing reads were obtained from each pool. After several filtering steps and comparisons of observed frequencies of variants between obese and non-obese control pools, we systematically selected 144 obesity-enriched non-synonymous, splicing site or 5' upstream single-nucleotide variants for validation. We first genotyped 494 adult subjects with morbid obesity and 496 controls. Five obesity-associated variants (nominal P-value<0.05) were subsequently genotyped in 1425 morbidly obese and 782 controls. Out of the five variants, only rs62623713:A>G (NM_001040709:c.A296G:p.E99G) was confirmed. rs62623713 showed strong association with body mass index (beta=2.13 (1.09, 3.18), P=6.28 × 10(-5)) in a joint analysis of all 3197 genotyped subjects and had an odds ratio of 1.32 for obesity association. rs62623713 is a low-frequency (2.9% minor allele frequency) non-synonymous variant (E99G) in exon 4 of the synaptophysin-like 2 (SYPL2) gene. rs62623713 was not covered by Illumina or Affymetrix genotyping arrays used in previous genome-wide association studies. Mice lacking Sypl2 has been reported to display reduced body weight. In conclusion, using exome sequencing we identified a low-frequency coding variant in the SYPL2 gene that was associated with morbid obesity. This gene may be involved in the development of excess body fat.

Recently a number of high profile crime cases (e.g. the "Golden State Killer") have successfully been solved or given new leads with the use of genome wide DNA data in combination with pairwise matching from individuals present in genealogy DNA databases. Such databases will primarily involve distant relatives which in turn require a large amount of genetic information, in the range of several hundred thousand to millions of SNPs, to be genotyped. While it nowadays is fairly straightforward to obtain such as data from high quality and high quantity DNA, it is still a challenge for degraded DNA of low quantity such in the case of forensic samples. Here we present a successful effort in obtaining genome-wide genotype data from human remains. The goal was to get investigative leads in order to identify the remains of an unknown male ("the Ekeby man") that was found murdered in the south of Sweden in 2003. Whole-genome sequencing was performed on DNA originating from a bone sample. Three replicates of libraries were prepared using ThruPLEX DNA-seq Kit (Takara) which were sequenced on a HiSeq X instrument (Illumina). A mean coverage of 30X was obtained when the sequencing reads were mapped to a human reference genome. Following further bioinformatic processing, allele calling, quality checks and filtering to match the genealogy DNA database SNPs, genotypes for approximately one million SNPs were established. The resulting SNP genotypes were then used to search for relatives in the genealogy DNA database GEDmatch (www.gedmatch.com). A candidate list of relatives was obtained which was further processed using traditional genealogy methods in order to get leads about the identity of the unknown. In summary, this report shows how whole-genome sequencing successfully can be applied on forensic samples to create the SNP genotypes required for searches in genealogy DNA databases for the purpose of generating leads to identify missing or unknown persons, including perpetrators and victims.

Heterozygous pathogenic variants in the FN1 gene, encoding fibronectin (FN), have recently been shown to be associated with a skeletal disorder in some individuals affected by spondylometaphyseal dysplasia with "corner fractures" (SMD-CF). The most striking feature characterizing SMD-CF is irregularly shaped metaphyses giving the appearance of "corner fractures". An array of secondary features, including developmental coxa vara, ovoid vertebral bodies and severe scoliosis, may also be present. FN is an important extracellular matrix component for bone and cartilage development. Here we report five patients affected by this subtype of SMD-CF caused by five novel FN1 missense mutations: p.Cys123Tyr, p.Cys169Tyr, p.Cys213Tyr, p.Cys231Trp and p.Cys258Tyr. All individuals shared a substitution of a cysteine residue, disrupting disulfide bonds in the FN type-I assembly domains located in the N-terminal assembly region. The abnormal metaphyseal ossification and "corner fracture" appearances were the most remarkable clinical feature in these patients. In addition, generalized skeletal fragility with low-trauma bilateral femoral fractures was identified in one patient. Interestingly, the distal femoral changes in this patient healed with skeletal maturation. Our report expands the phenotypic and genetic spectrum of the FN1-related SMD-CF and emphasizes the importance of FN in bone formation and possibly also in the maintenance of bone strength.

Conifer-feeding bark beetles are important herbivores and decomposers in forest ecosystems. These species complete their life cycle in nutritionally poor substrates and some can kill enormous numbers of trees during population outbreaks. The Eurasian spruce bark beetle (Ips typographus) can destroy >100 million m3 of spruce in a single year. We report a 236.8 Mb I. typographus genome assembly using PacBio long-read sequencing. The final phased assembly has a contig N50 of 6.65 Mb in 272 contigs and is predicted to contain 23,923 protein-coding genes. We reveal expanded gene families associated with plant cell wall degradation, including pectinases, aspartyl proteases, and glycosyl hydrolases. This genome sequence from the genus Ips provides timely resources to address questions about the evolutionary biology of the true weevils (Curculionidae), one of the most species-rich animal families. In forests of today, increasingly stressed by global warming, this draft genome may assist in developing pest control strategies to mitigate outbreaks.

The aim of this project was to implement long-read sequencing for BCR-ABL1 TKI resistance mutation screening in a clinical setting for patients undergoing treatment for chronic myeloid leukemia. Processes were established for registering and transferring samples from the clinic to an academic sequencing facility for long-read sequencing. An automated analysis pipeline for detecting mutations was established, and an information system was implemented comprising features for data management, analysis and visualization. Clinical validation was performed by identifying BCR-ABL1 TKI resistance mutations by Sanger and long-read sequencing in parallel. The developed software is available as open source via GitHub at https://github.com/pharmbio/clamp. The information system enabled traceable transfer of samples from the clinic to the sequencing facility, robust and automated analysis of the long-read sequence data, and communication of results from sequence analysis in a reporting format that could be easily interpreted and acted upon by clinical experts. In a validation study, all 17 resistance mutations found by Sanger sequencing were also detected by long-read sequencing. An additional 16 mutations were found only by long-read sequencing, all of them with frequencies below the limit of detection for Sanger sequencing. The clonal distributions of co-existing mutations were automatically resolved through the long-read data analysis. After the implementation and validation, the clinical laboratory switched their routine protocol from using Sanger to long-read sequencing for this application. Long-read sequencing delivers results with higher sensitivity compared to Sanger sequencing and enables earlier detection of emerging TKI resistance mutations. The developed processes, analysis workflow, and software components lower barriers for adoption and could be extended to other applications.