Transcriptomic landscape of Atlantic salmon (Salmo salar L.) skin.

In this study, we present the first spatial transcriptomic atlas of Atlantic salmon skin using the Visium Spatial Gene Expression protocol. We utilized frozen skin tissue from 4 distinct sites, namely the operculum, pectoral and caudal fins, and scaly skin at the flank of the fish close to the lateral line, obtained from 2 Atlantic salmon (150 g). High-quality frozen tissue sections were obtained by embedding tissue in optimal cutting temperature media prior to freezing and sectioning. Further, we generated libraries and spatial transcriptomic maps, achieving a minimum of 80 million reads per sample with mapping efficiencies ranging from 79.3 to 89.4%. Our analysis revealed the detection of over 80,000 transcripts and nearly 30,000 genes in each sample. Among the tissue types observed in the skin, the epithelial tissues exhibited the highest number of transcripts (unique molecular identifier counts), followed by muscle tissue, loose and fibrous connective tissue, and bone. Notably, the widest nodes in the transcriptome network were shared among the epithelial clusters, while dermal tissues showed less consistency, which is likely attributable to the presence of multiple cell types at different body locations. Additionally, we identified collagen type 1 as the most prominent gene family in the skin, while keratins were found to be abundant in the epithelial tissue. Furthermore, we successfully identified gene markers specific to epithelial tissue, bone, and mesenchyme. To validate their expression patterns, we conducted a meta-analysis of the microarray database, which confirmed high expression levels of these markers in mucosal organs, skin, gills, and the olfactory rosette.

Warmer temperature during asexual reproduction induce methylome, transcriptomic, and lasting phenotypic changes in Fragaria vesca ecotypes.

Plants must adapt with increasing speed to global warming to maintain their fitness. One rapid adaptation mechanism is epigenetic memory, which may provide organisms sufficient time to adapt to climate change. We studied how the perennial Fragaria vesca adapted to warmer temperatures (28°C vs. 18°C) over three asexual generations. Differences in flowering time, stolon number, and petiole length were induced by warmer temperature in one or more ecotypes after three asexual generations and persisted in a common garden environment. Induced methylome changes differed between the four ecotypes from Norway, Iceland, Italy, and Spain, but shared methylome responses were also identified. Most differentially methylated regions (DMRs) occurred in the CHG context, and most CHG and CHH DMRs were hypermethylated at the warmer temperature. In eight CHG DMR peaks, a highly similar methylation pattern could be observed between ecotypes. On average, 13% of the differentially methylated genes between ecotypes also showed a temperature-induced change in gene expression. We observed ecotype-specific methylation and expression patterns for genes related to gibberellin metabolism, flowering time, and epigenetic mechanisms. Furthermore, we observed a negative correlation with gene expression when repetitive elements were found near (±2 kb) or inside genes. In conclusion, lasting phenotypic changes indicative of an epigenetic memory were induced by warmer temperature and were accompanied by changes in DNA methylation patterns. Both shared methylation patterns and transcriptome differences between F. vesca accessions were observed, indicating that DNA methylation may be involved in both general and ecotype-specific phenotypic variation.

Epitype-inducing temperatures drive DNA methylation changes during somatic embryogenesis in the long-lived gymnosperm Norway spruce.

An epigenetic memory of the temperature sum experienced during embryogenesis is part of the climatic adaptation strategy of the long-lived gymnosperm Norway spruce. This memory has a lasting effect on the timing of bud phenology and frost tolerance in the resulting epitype trees. The epigenetic memory is well characterized phenotypically and at the transcriptome level, but to what extent DNA methylation changes are involved have not previously been determined. To address this, we analyzed somatic epitype embryos of Norway spruce clones produced at contrasting epitype-inducing conditions (18 and 28°C). We screened for differential DNA methylation in 2744 genes related mainly to the epigenetic machinery, circadian clock, and phenology. Of these genes, 68% displayed differential DNA methylation patterns between contrasting epitype embryos in at least one methylation context (CpG, CHG, CHH). Several genes related to the epigenetic machinery (e.g., DNA methyltransferases, ARGONAUTE) and the control of bud phenology (FTL genes) were differentially methylated. This indicates that the epitype-inducing temperature conditions induce an epigenetic memory involving specific DNA methylation changes in Norway spruce.

Intergenerational effects of early life-stage temperature modulation on gene expression and DNA methylation in Atlantic cod (Gadus morhua).

After suffering several collapses, the cod farming industry is now in the process of trying to re-establish itself. We have used material from Norway's National Cod Breeding Program to study how different early life-stage temperature regimes affect DNA methylation and gene expression. Long-term effects were detected by sampling fish several weeks after the end of differential treatments, and offspring from the different exposure groups was also sampled. Many overlapping genes were found between the different exposure groups and generations, coupled with genes associated with differential CpG methylation levels. Genes involved in muscle fibre development, general metabolic processes and formation of deformities were significantly affected, and genes relevant for intergenerational transfer of epigenetic marks were also detected. We believe the use of environmental cues can be a useful strategy for improving the production of Atlantic cod.

Major transcriptomic differences are induced by warmer temperature conditions experienced during asexual and sexual reproduction in Fragaria vesca ecotypes.

A major challenge for plants in a rapidly changing climate is to adapt to rising temperatures. Some plants adapt to temperature conditions by generating an epigenetic memory that can be transmitted both meiotically and mitotically. Such epigenetic memories may increase phenotypic variation to global warming and provide time for adaptation to occur through classical genetic selection. The goal of this study was to understand how warmer temperature conditions experienced during sexual and asexual reproduction affect the transcriptomes of different strawberry (Fragaria vesca) ecotypes. We let four European F. vesca ecotypes reproduce at two contrasting temperatures (18 and 28°C), either asexually through stolon formation for several generations, or sexually by seeds (achenes). We then analyzed the transcriptome of unfolding leaves, with emphasis on differential expression of genes belonging to the epigenetic machinery. For asexually reproduced plants we found a general transcriptomic response to temperature conditions but for sexually reproduced plants we found less significant responses. We predicted several splicing isoforms for important genes (e.g. a SOC1, LHY, and SVP homolog), and found significantly more differentially presented splicing event variants following asexual vs. sexual reproduction. This difference could be due to the stochastic character of recombination during meiosis or to differential creation or erasure of epigenetic marks during embryogenesis and seed development. Strikingly, very few differentially expressed genes were shared between ecotypes, perhaps because ecotypes differ greatly both genetically and epigenetically. Genes related to the epigenetic machinery were predominantly upregulated at 28°C during asexual reproduction but downregulated after sexual reproduction, indicating that temperature-induced change affects the epigenetic machinery differently during the two types of reproduction.

Dose rate dependent reduction in chromatin accessibility at transcriptional start sites long time after exposure to gamma radiation.

Ionizing radiation (IR) impact cellular and molecular processes that require chromatin remodelling relevant for cellular integrity. However, the cellular implications of ionizing radiation (IR) delivered per time unit (dose rate) are still debated. This study investigates whether the dose rate is relevant for inflicting changes to the epigenome, represented by chromatin accessibility, or whether it is the total dose that is decisive. CBA/CaOlaHsd mice were whole-body exposed to either chronic low dose rate (2.5 mGy/h for 54 d) or the higher dose rates (10 mGy/h for 14 d and 100 mGy/h for 30 h) of gamma radiation (60Co, total dose: 3 Gy). Chromatin accessibility was analysed in liver tissue samples using Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-Seq), both one day after and over three months post-radiation (>100 d). The results show that the dose rate contributes to radiation-induced epigenomic changes in the liver at both sampling timepoints. Interestingly, chronic low dose rate exposure to a high total dose (3 Gy) did not inflict long-term changes to the epigenome. In contrast to the acute high dose rate given to the same total dose, reduced accessibility at transcriptional start sites (TSS) was identified in genes relevant for the DNA damage response and transcriptional activity. Our findings link dose rate to essential biological mechanisms that could be relevant for understanding long-term changes after ionizing radiation exposure. However, future studies are needed to comprehend the biological consequence of these findings.

Deletion of the s2m RNA Structure in the Avian Coronavirus Infectious Bronchitis Virus and Human Astrovirus Results in Sequence Insertions.

Coronaviruses infect a wide variety of host species, resulting in a range of diseases in both humans and animals. The coronavirus genome consists of a large positive-sense single-stranded molecule of RNA containing many RNA structures. One structure, denoted s2m and consisting of 41 nucleotides, is located within the 3' untranslated region (3' UTR) and is shared between some coronavirus species, including infectious bronchitis virus (IBV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2, as well as other pathogens, including human astrovirus. Using a reverse genetic system to generate recombinant viruses, we investigated the requirement of the s2m structure in the replication of IBV, a globally distributed economically important Gammacoronavirus that infects poultry causing respiratory disease. Deletion of three nucleotides predicted to destabilize the canonical structure of the s2m or the deletion of the nucleotides corresponding to s2m impacted viral replication in vitro. In vitro passaging of the recombinant IBV with the s2m sequence deleted resulted in a 36-nucleotide insertion in place of the deletion, which was identified to be composed of a duplication of flanking sequences. A similar result was observed following serial passage of human astrovirus with a deleted s2m sequence. RNA modeling indicated that deletion of the nucleotides corresponding to the s2m impacted other RNA structures present in the IBV 3' UTR. Our results indicated for both IBV and human astrovirus a preference for nucleotide occupation in the genome location corresponding to the s2m, which is independent of the specific s2m sequence. IMPORTANCE Coronaviruses infect many species, including humans and animals, with substantial effects on livestock, particularly with respect to poultry. The coronavirus RNA genome consists of structural elements involved in viral replication whose roles are poorly understood. We investigated the requirement of the RNA structural element s2m in the replication of the Gammacoronavirus infectious bronchitis virus, an economically important viral pathogen of poultry. Using reverse genetics to generate recombinant IBVs with either a disrupted or deleted s2m, we showed that the s2m is not required for viral replication in cell culture; however, replication is decreased in tracheal tissue, suggesting a role for the s2m in the natural host. Passaging of these viruses as well as human astrovirus lacking the s2m sequence demonstrated a preference for nucleotide occupation, independent of the s2m sequence. RNA modeling suggested deletion of the s2m may negatively impact other essential RNA structures.

Transcriptional profiling of defense responses to Botrytis cinerea infection in leaves of Fragaria vesca plants soil-drenched with ß-aminobutyric acid.

Grey mold caused by the necrotrophic fungal pathogen Botrytis cinerea can affect leaves, flowers, and berries of strawberry, causing severe pre- and postharvest damage. The defense elicitor ß-aminobutyric acid (BABA) is reported to induce resistance against B. cinerea and many other pathogens in several crop plants. Surprisingly, BABA soil drench of woodland strawberry (Fragaria vesca) plants two days before B. cinerea inoculation caused increased infection in leaf tissues, suggesting that BABA induce systemic susceptibility in F. vesca. To understand the molecular mechanisms involved in B. cinerea susceptibility in leaves of F. vesca plants soil drenched with BABA, we used RNA sequencing to characterize the transcriptional reprogramming 24 h post-inoculation. The number of differentially expressed genes (DEGs) in infected vs. uninfected leaf tissue in BABA-treated plants was 5205 (2237 upregulated and 2968 downregulated). Upregulated genes were involved in pathogen recognition, defense response signaling, and biosynthesis of secondary metabolites (terpenoid and phenylpropanoid pathways), while downregulated genes were involved in photosynthesis and response to auxin. In control plants not treated with BABA, we found a total of 5300 DEGs (2461 upregulated and 2839 downregulated) after infection. Most of these corresponded to those in infected leaves of BABA-treated plants but a small subset of DEGs, including genes involved in 'response to biologic stimulus', 'photosynthesis' and 'chlorophyll biosynthesis and metabolism', differed significantly between treatments and could play a role in the induced susceptibility of BABA-treated plants.

No species-level losses of s2m suggests critical role in replication of SARS-related coronaviruses.

The genetic element s2m has been acquired through horizontal transfer by many distantly related viruses, including the SARS-related coronaviruses. Here we show that s2m is evolutionarily conserved in these viruses. Though several lineages of severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) devoid of the element can be found, these variants seem to have been short lived, indicating that they were less evolutionary fit than their s2m-containing counterparts. On a species-level, however, there do not appear to be any losses and this pattern strongly suggests that the s2m element is essential to virus replication in SARS-CoV-2 and related viruses. Further experiments are needed to elucidate the function of s2m.

Perturbed transcriptional profiles after chronic low dose rate radiation in mice.

Adverse health outcomes of ionizing radiation given chronically at low dose rates are highly debated, a controversy also relevant for other stressors. Increased knowledge is needed for a more comprehensive understanding of the damaging potential of ionizing radiation from all dose rates and doses. There is a lack of relevant low dose rate data that is partly ascribed to the rarity of exposure facilities allowing chronic low dose rate exposures. Using the FIGARO facility, we assessed early (one day post-radiation) and late (recovery time of 100-200 days) hepatic genome-wide transcriptional profiles in male mice of two strains (CBA/CaOlaHsd and C57BL/6NHsd) exposed chronically to a low dose rate (2.5 mGy/h; 1200h, LDR), a mid-dose rate (10 mGy/h; 300h, MDR) and acutely to a high dose rate (100 mGy/h; 30h, HDR) of gamma irradiation, given to an equivalent total dose of 3 Gy. Dose-rate and strain-specific transcriptional responses were identified. Differently modulated transcriptional responses across all dose rate exposure groups were evident by the representation of functional biological pathways. Evidence of changed epigenetic regulation (global DNA methylation) was not detected. A period of recovery markedly reduced the number of differentially expressed genes. Using enrichment analysis to identify the functional significance of the modulated genes, perturbed signaling pathways associated with both cancer and non-cancer effects were observed, such as lipid metabolism and inflammation. These pathways were seen after chronic low dose rate and were not restricted to the acute high dose rate exposure. The transcriptional response induced by chronic low dose rate ionizing radiation suggests contribution to conditions such as cardiovascular diseases. We contribute with novel genome wide transcriptional data highlighting dose-rate-specific radiation responses and emphasize the importance of considering both dose rate, duration of exposure, and variability in susceptibility when assessing risks from ionizing radiation.

A genetic element in the SARS-CoV-2 genome is shared with multiple insect species.

SARS-CoV-2 is a member of the subgenus Sarbecovirus and thus contains the genetic element s2m. We have extensively mined nucleotide data in GenBank in order to obtain a comprehensive list of s2m sequences both in the four virus families where s2m has previously been described and in other groups of organisms. Surprisingly, there seems to be a xenologue of s2m in a large number of insect species. The function of s2m is unknown, but our data show a very high degree of sequence conservation both in insects and in viruses and that the version of s2m found in SARS-CoV-2 has unique features, not seen in any other virus or insect strains.

Bartonella spp. detection in ticks, Culicoides biting midges and wild cervids from Norway.

Bartonella spp. are fastidious, gram-negative, aerobic, facultative intracellular bacteria that infect humans, and domestic and wild animals. In Norway, Bartonella spp. have been detected in cervids, mainly within the distribution area of the arthropod vector deer ked (Lipoptena cervi). We used PCR to survey the prevalence of Bartonella spp. in blood samples from 141 cervids living outside the deer ked distribution area (moose [Alces alces, n = 65], red deer [Cervus elaphus, n = 41] and reindeer [Rangifer tarandus, n = 35]), in 44 pool samples of sheep tick (Ixodes ricinus, 27 pools collected from 74 red deer and 17 from 45 moose) and in biting midges of the genus Culicoides (Diptera: Ceratopogonidae, 120 pools of 6,710 specimens). Bartonella DNA was amplified in moose (75.4%, 49/65) and in red deer (4.9%, 2/41) blood samples. All reindeer were negative. There were significant differences in Bartonella prevalence among the cervid species. Additionally, Bartonella was amplified in two of 17 tick pools collected from moose and in 3 of 120 biting midge pool samples. The Bartonella sequences amplified in moose, red deer and ticks were highly similar to B. bovis, previously identified in cervids. The sequence obtained from biting midges was only 81.7% similar to the closest Bartonella spp. We demonstrate that Bartonella is present in moose across Norway and present the first data on northern Norway specimens. The high prevalence of Bartonella infection suggests that moose could be the reservoir for this bacterium. This is the first report of bacteria from the Bartonella genus in ticks from Fennoscandia and in Culicoides biting midges worldwide.

Genotyping of Salmon Gill Poxvirus Reveals One Main Predominant Lineage in Europe, Featuring Fjord- and Fish Farm-Specific Sub-Lineages.

Salmon gill poxvirus (SGPV) can cause serious gill disease in Atlantic salmon (Salmo salar L.) and represents a significant problem to aquaculture industries in Northern Europe. Here, a single-tube multi-locus variable-number tandem-repeat (VNTR) analysis (MLVA) genotyping assay, targeting eight VNTR loci, was developed for studying the epizootiology of SGPV. Through MLVA typing of SGPV positive samples from 180 farmed and wild Atlantic salmon in Northern Europe, the first molecular population study of this virus was undertaken. Comparison of resulting MLVA profiles by cluster analysis revealed considerable micro-diversity, while only a limited degree of specific clustering by country of origin could be observed, and no clustering relating to the severity of disease outbreaks. Phylogenetic analysis, based on genomic data from six SGPV specimens (three Norwegian, one Scottish, one Faroese and one Canadian), complemented and corroborated MLVA by pointing to a marked transatlantic divide in the species, with one main, relatively conserved, SGPV lineage as predominant in Europe. Within certain fjord systems and individual freshwater salmon smolt farms in Norway, however, discrete MLVA clustering patterns that prevailed over time were observed, likely reflecting local predominance of specific SGPV sub-lineages. MLVA typing was also used to refute two suspected instances of vertical SGPV transmission from salmon broodstock to offspring, and to confirm a failed disinfection attempt in one farm. These novel insights into the previously undocumented population structure of SGPV provide important clues, e.g., regarding the mechanisms underlying spread and recurrence of the virus amongst wild and farmed salmon populations, but so far no indications of more or less virulent SGPV sub-lineages have been found. The MLVA scheme represents a highly sensitive genotyping tool particularly well suited for illuminating SGPV infection routes, and adds to the relatively low number of MLVA protocols that have so far been published for viral species. Typing is reasonably inexpensive, with a moderate technological requirement, and may be completed within a single working day. Resulting MLVA profiles can be readily shared and compared across laboratories, facilitating rapid placement of samples in an international ezpizootiological context.

Molecular underpinnings of methyl jasmonate-induced resistance in Norway spruce.

In response to various stimuli, plants acquire resistance against pests and/or pathogens. Such acquired or induced resistance allows plants to rapidly adapt to their environment. Spraying the bark of mature Norway spruce (Picea abies) trees with the phytohormone methyl jasmonate (MeJA) enhances resistance to tree-killing bark beetles and their associated phytopathogenic fungi. Analysis of spruce chemical defenses and beetle colonization success suggests that MeJA treatment both directly induces immune responses and primes inducible defenses for a faster and stronger response to subsequent beetle attack. We used metabolite and transcriptome profiling to explore the mechanisms underlying MeJA-induced resistance in Norway spruce. We demonstrated that MeJA treatment caused substantial changes in the bark transcriptional response to a triggering stress (mechanical wounding). Profiling of mRNA expression showed a suite of spruce inducible defenses are primed following MeJA treatment. Although monoterpenes and diterpene resin acids increased more rapidly after wounding in MeJA-treated than control bark, expression of their biosynthesis genes did not. We suggest that priming of inducible defenses is part of a complex mixture of defense responses that underpins the increased resistance against bark beetle colonization observed in Norway spruce. This study provides the most detailed insights yet into the mechanisms underlying induced resistance in a long-lived gymnosperm.

Mycoplasma conjunctivae-Associated Keratoconjunctivitis in Norwegian Muskox (Ovibos moschatus).

In late summer 2014, an outbreak of ocular disease occurred in the Norwegian muskox (Ovibos moschatus) population. Animals showed rings of pus around their eyes and one euthanized animal was diagnosed with acute keratoconjunctivitis. The DNA sequence analysis of eye-swab samples from this animal revealed a high abundance of Mycoplasma conjunctivae.

Evolution of the Piscine orthoreovirus Genome Linked to Emergence of Heart and Skeletal Muscle Inflammation in Farmed Atlantic Salmon (Salmo salar).

Heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar) was first diagnosed in Norway in 1999. The disease is caused by Piscine orthoreovirus-1 (PRV-1). The virus is prevalent in farmed Atlantic salmon, but not always associated with disease. Phylogeny and sequence analyses of 31 PRV-1 genomes collected over a 30-year period from fish with or without HSMI, grouped the viral sequences into two main monophylogenetic clusters, one associated with HSMI and the other with low virulent PRV-1 isolates. A PRV-1 strain from Norway sampled in 1988, a decade before the emergence of HSMI, grouped with the low virulent HSMI cluster. The two distinct monophylogenetic clusters were particularly evident for segments S1 and M2. Only a limited number of amino acids were unique to the association with HSMI, and they all located to S1 and M2 encoded proteins. The observed co-evolution of the S1-M2 pair coincided in time with the emergence of HSMI in Norway, and may have evolved through accumulation of mutations and/or segment reassortment. Sequences of S1-M2 suggest selection of the HSMI associated pair, and that this segment pair has remained almost unchanged in Norwegian salmon aquaculture since 1997. PRV-1 strains from the North American Pacific Coast and Faroe Islands have not undergone this evolution, and are more closely related to the PRV-1 precursor strains not associated with clinical HSMI.

Molecular and Antigenic Characterization of Piscine orthoreovirus (PRV) from Rainbow Trout (Oncorhynchus mykiss).

Piscine orthoreovirus (PRV-1) causes heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). Recently, a novel PRV (formerly PRV-Om, here called PRV-3), was found in rainbow trout (Oncorhynchus mykiss) with HSMI-like disease. PRV is considered to be an emerging pathogen in farmed salmonids. In this study, molecular and antigenic characterization of PRV-3 was performed. Erythrocytes are the main target cells for PRV, and blood samples that were collected from experimentally challenged fish were used as source of virus. Virus particles were purified by gradient ultracentrifugation and the complete coding sequences of PRV-3 were obtained by Illumina sequencing. When compared to PRV-1, the nucleotide identity of the coding regions was 80.1%, and the amino acid identities of the predicted PRV-3 proteins varied from 96.7% (λ1) to 79.1% (σ3). Phylogenetic analysis showed that PRV-3 belongs to a separate cluster. The region encoding σ3 were sequenced from PRV-3 isolates collected from rainbow trout in Europe. These sequences clustered together, but were distant from PRV-3 that was isolated from rainbow trout in Norway. Bioinformatic analyses of PRV-3 proteins revealed that predicted secondary structures and functional domains were conserved between PRV-3 and PRV-1. Rabbit antisera raised against purified virus or various recombinant virus proteins from PRV-1 all cross-reacted with PRV-3. Our findings indicate that despite different species preferences of the PRV subtypes, several genetic, antigenic, and structural properties are conserved between PRV-1 and-3.

Infection with purified Piscine orthoreovirus demonstrates a causal relationship with heart and skeletal muscle inflammation in Atlantic salmon.

Viral diseases pose a significant threat to the productivity in aquaculture. Heart- and skeletal muscle inflammation (HSMI) is an emerging disease in Atlantic salmon (Salmo salar) farming. HSMI is associated with Piscine orthoreovirus (PRV) infection, but PRV is ubiquitous in farmed Atlantic salmon and thus present also in apparently healthy individuals. This has brought speculations if additional etiological factors are required, and experiments focusing on the causal relationship between PRV and HSMI are highly warranted. A major bottleneck in PRV research has been the lack of cell lines that allow propagation of the virus. To bypass this, we propagated PRV in salmon, bled the fish at the peak of the infection, and purified virus particles from blood cells. Electron microscopy, western blot and high-throughput sequencing all verified the purity of the viral particles. Purified PRV particles were inoculated into naïve Atlantic salmon. The purified virus replicated in inoculated fish, spread to naïve cohabitants, and induced histopathological changes consistent with HSMI. PRV specific staining was demonstrated in the pathological lesions. A dose-dependent response was observed; a high dose of virus gave earlier peak of the viral load and development of histopathological changes compared to a lower dose, but no difference in the severity of the disease. The experiment demonstrated that PRV can be purified from blood cells, and that PRV is the etiological agent of HSMI in Atlantic salmon.

Multilevel genomics of colorectal cancers with microsatellite instability-clinical impact of JAK1 mutations and consensus molecular subtype 1.

BACKGROUND: Approximately 15% of primary colorectal cancers have DNA mismatch repair deficiency, causing a complex genome with thousands of small mutations-the microsatellite instability (MSI) phenotype. We investigated molecular heterogeneity and tumor immunogenicity in relation to clinical endpoints within this distinct subtype of colorectal cancers. METHODS: A total of 333 primary MSI+ colorectal tumors from multiple cohorts were analyzed by multilevel genomics and computational modeling-including mutation profiling, clonality modeling, and neoantigen prediction in a subset of the tumors, as well as gene expression profiling for consensus molecular subtypes (CMS) and immune cell infiltration. RESULTS: Novel, frequent frameshift mutations in four cancer-critical genes were identified by deep exome sequencing, including in CRTC1, BCL9, JAK1, and PTCH1. JAK1 loss-of-function mutations were validated with an overall frequency of 20% in Norwegian and British patients, and mutated tumors had up-regulation of transcriptional signatures associated with resistance to anti-PD-1 treatment. Clonality analyses revealed a high level of intra-tumor heterogeneity; however, this was not associated with disease progression. Among the MSI+ tumors, the total mutation load correlated with the number of predicted neoantigens (P = 4 × 10-5), but not with immune cell infiltration-this was dependent on the CMS class; MSI+ tumors in CMS1 were highly immunogenic compared to MSI+ tumors in CMS2-4. Both JAK1 mutations and CMS1 were favorable prognostic factors (hazard ratios 0.2 [0.05-0.9] and 0.4 [0.2-0.9], respectively, P = 0.03 and 0.02). CONCLUSIONS: Multilevel genomic analyses of MSI+ colorectal cancer revealed molecular heterogeneity with clinical relevance, including tumor immunogenicity and a favorable patient outcome associated with JAK1 mutations and the transcriptomic subgroup CMS1, emphasizing the potential for prognostic stratification of this clinically important subtype. See related research highlight by Samstein and Chan 10.1186/s13073-017-0438-9.

Issues with RNA-seq analysis in non-model organisms: A salmonid example.

High throughput sequencing (HTS) is useful for many purposes as exemplified by the other topics included in this special issue. The purpose of this paper is to look into the unique challenges of using this technology in non-model organisms where resources such as genomes, functional genome annotations or genome complexity provide obstacles not met in model organisms. To describe these challenges, we narrow our scope to RNA sequencing used to study differential gene expression in response to pathogen challenge. As a demonstration species we chose Atlantic salmon, which has a sequenced genome with poor annotation and an added complexity due to many duplicated genes. We find that our RNA-seq analysis pipeline deciphers between duplicates despite high sequence identity. However, annotation issues provide problems in linking differentially expressed genes to pathways. Also, comparing results between approaches and species are complicated due to lack of standardized annotation.