NGI offers spatial transcriptomics through the 10X Genomics Visium method, a technique recently nominated as Method of the Year by Nature Methods. The technology combines histology with unbiased transcriptomics in a spatial context.
The Illumina NovaSeq 6000 system is the largest of the Illumina sequencing instruments, able to run two flow cells independently of each other and generate massive sequencing depth at competitive prices.
A novel unstable duplication upstream of HAS2 predisposes to a breed-defining skin phenotype and a periodic fever syndrome in Chinese Shar-Pei dogs.
M Olsson, JRS Meadows, K Truvé, G Rosengren Pielberg, F Puppo, E Mauceli, J Quilez, N Tonomura, G Zanna, MJ Docampo, A Bassols, AC Avery, EK Karlsson, A Thomas, DL Kastner, E Bongcam-Rudloff, MT Webster, A Sanchez, A Hedhammar, EF Remmers, L Andersson, L Ferrer, L Tintle, K Lindblad-Toh
PLoS Genet., 7 (3) 1553-7404 (2011)
Hereditary periodic fever syndromes are characterized by recurrent episodes of fever and inflammation with no known pathogenic or autoimmune cause. In humans, several genes have been implicated in this group of diseases, but the majority of cases remain unexplained. A similar periodic fever syndrome is relatively frequent in the Chinese Shar-Pei breed of dogs. In the western world, Shar-Pei have been strongly selected for a distinctive thick and heavily folded skin. In this study, a mutation affecting both these traits was identified. Using genome-wide SNP analysis of Shar-Pei and other breeds, the strongest signal of a breed-specific selective sweep was located on chromosome 13. The same region also harbored the strongest genome-wide association (GWA) signal for susceptibility to the periodic fever syndrome (p(raw) = 2.3 × 10⁻⁶, p(genome) = 0.01). Dense targeted resequencing revealed two partially overlapping duplications, 14.3 Kb and 16.1 Kb in size, unique to Shar-Pei and upstream of the Hyaluronic Acid Synthase 2 (HAS2) gene. HAS2 encodes the rate-limiting enzyme synthesizing hyaluronan (HA), a major component of the skin. HA is up-regulated and accumulates in the thickened skin of Shar-Pei. A high copy number of the 16.1 Kb duplication was associated with an increased expression of HAS2 as well as the periodic fever syndrome (p < 0.0001). When fragmented, HA can act as a trigger of the innate immune system and stimulate sterile fever and inflammation. The strong selection for the skin phenotype therefore appears to enrich for a pleiotropic mutation predisposing these dogs to a periodic fever syndrome. The identification of HA as a major risk factor for this canine disease raises the potential of this glycosaminoglycan as a risk factor for human periodic fevers and as an important driver of chronic inflammation.
Labile Dissolved Organic Matter Compound Characteristics Select for Divergence in Marine Bacterial Activity and Transcription.
B Pontiller, S Martínez-García, D Lundin, J Pinhassi
Front Microbiol, 11 1664-302X (2020)
Bacteria play a key role in the planetary carbon cycle partly because they rapidly assimilate labile dissolved organic matter (DOM) in the ocean. However, knowledge of the molecular mechanisms at work when bacterioplankton metabolize distinct components of the DOM pool is still limited. We, therefore, conducted seawater culture enrichment experiments with ecologically relevant DOM, combining both polymer and monomer model compounds for distinct compound classes. This included carbohydrates (polysaccharides vs. monosaccharides), proteins (polypeptides vs. amino acids), and nucleic acids (DNA vs. nucleotides). We noted pronounced changes in bacterial growth, activity, and transcription related to DOM characteristics. Transcriptional responses differed between compound classes, with distinct gene sets ("core genes") distinguishing carbohydrates, proteins, and nucleic acids. Moreover, we found a strong divergence in functional transcription at the level of particular monomers and polymers (i.e., the condensation state), primarily in the carbohydrates and protein compound classes. These specific responses included a variety of cellular and metabolic processes that were mediated by distinct bacterial taxa, suggesting pronounced functional partitioning of organic matter. Collectively, our findings show that two important facets of DOM, compound class and condensation state, shape bacterial gene expression, and ultimately select for distinct bacterial (functional) groups. This emphasizes the interdependency of marine bacteria and labile carbon compounds for regulating the transformation of DOM in surface waters.
In-depth transcriptome analysis reveals novel TARs and prevalent antisense transcription in human cell lines.
D Klevebring, M Bjursell, O Emanuelsson, J Lundeberg
PLoS ONE, 5 (3) 1932-6203 (2010)
Several recent studies have indicated that transcription is pervasive in regions outside of protein coding genes and that short antisense transcripts can originate from the promoter and terminator regions of genes. Here we investigate transcription of fragments longer than 200 nucleotides, focusing on antisense transcription for known protein coding genes and intergenic transcription. We find that roughly 12% to 16% of all reads that originate from promoter and terminator regions, respectively, map antisense to the gene in question. Furthermore, we detect a high number of novel transcriptionally active regions (TARs) that are generally expressed at a lower level than protein coding genes. We find that the correlation between RNA-seq data and microarray data is dependent on the gene length, with longer genes showing a better correlation. We detect high antisense transcriptional activity from promoter, terminator and intron regions of protein-coding genes and identify a vast number of previously unidentified TARs, including putative novel EGFR transcripts. This shows that in-depth analysis of the transcriptome using RNA-seq is a valuable tool for understanding complex transcriptional events. Furthermore, the development of new algorithms for estimation of gene expression from RNA-seq data is necessary to minimize length bias.
Development of gut microbiota during the first 2 years of life.
M Wernroth, S Peura, AM Hedman, S Hetty, S Vicenzi, B Kennedy, K Fall, B Svennblad, E Andolf, G Pershagen, J Theorell-Haglöw, D Nguyen, S Sayols-Baixeras, KF Dekkers, S Bertilsson, C Almqvist, J Dicksved, T Fall
Sci Rep, 12 (1) 2045-2322 (2022)
Although development of microbiota in childhood has been linked to chronic immune-related conditions, early childhood determinants of microbiota development have not been fully elucidated. We used 16S rRNA sequencing to analyse faecal and saliva samples from 83 children at four time-points during their first 2 years of life and from their mothers. Our findings confirm that gut microbiota in infants have low diversity and highlight that some properties are shared with the oral microbiota, although inter-individual differences are present. A considerable convergence in gut microbiota composition was noted across the first 2 years of life, towards a more diverse adult-like microbiota. Mode of delivery accounted for some of the inter-individual variation in early childhood, but with a pronounced attenuation over time. Our study extends previous research with further characterization of the major shift in gut microbiota composition during the first 2 years of life.
The surface waters at the ultramafic ophiolitic outcrop in Chimaera, Turkey, are characterized by high pH values and high metal levels due to the percolation of fluids through areas of active serpentinization. We describe the influence of the liquid chemistry, mineralogy, and H2 and CH4 levels on the bacterial community structure in a semidry, exposed, ultramafic environment. The bacterial and archaeal community structures were monitored using Illumina sequencing targeting the 16S rRNA gene. At all sampling points, four phyla, Proteobacteria, Actinobacteria, Chloroflexi, and Acidobacteria, accounted for the majority of taxa. Members of the Chloroflexi phylum dominated low-diversity sites, whereas Proteobacteria dominated high-diversity sites. Methane, nitrogen, iron, and hydrogen oxidizers were detected as well as archaea and metal-resistant bacteria.IMPORTANCE Our study is a comprehensive microbial investigation of the Chimaera ophiolite. DNA has been extracted from 16 sites in the area and has been studied from microbial and geochemical points of view. We describe a microbial community structure that is dependent on terrestrial, serpentinization-driven abiotic H2, which is poorly studied due to the rarity of these environments on Earth.
Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high-throughput (22286-37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (∼1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full-length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost-effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput.
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