RNA Sequencing Reads Research Articles

Slinker: Visualising novel splicing events in RNA-Seq data.

Visualisation of the transcriptome relative to a reference genome is fraught with sparsity. This is due to RNA sequencing (RNA-Seq) reads being predominantly mapped to exons that account for just under 3% of the human genome. Recently, we have used exon-only references, superTranscripts, to improve visualisation of aligned RNA-Seq data through the omission of supposedly unexpressed regions such as introns. However, variation within these regions can lead to novel splicing events that may drive a pathogenic phenotype. In these cases, the loss of information in only retaining annotated exons presents significant drawbacks. Here we present Slinker, a bioinformatics pipeline written in Python and Bpipe that uses a data-driven approach to assemble sample-specific superTranscripts. At its core, Slinker uses Stringtie2 to assemble transcripts with any sequence across any gene. This assembly is merged with reference transcripts, converted to a superTranscript, of which rich visualisations are made through Plotly with associated annotation and coverage information. Slinker was validated on five novel splicing events of rare disease samples from a cohort of primary muscular disorders. In addition, Slinker was shown to be effective in visualising deletion events within transcriptomes of tumour samples in the important leukemia gene, IKZF1. Slinker offers a succinct visualisation of RNA-Seq alignments across typically sparse regions and is freely available on Github.

Larger, more connected societies of ants have a higher prevalence of viruses.

The benefits of cooperative living for foraging, nesting, defence and buffering environmental challenges lead animals with the most highly social lifestyles to dominate many ecosystems. However, living in larger, more highly connected groups should also increase the risks of pathogen exposure and transmission. While over long timescales selective responses could buffer the impacts of potential higher pathogen prevalence, similar processes are unlikely over short timescales. The red fire ant Solenopsis invicta is ideal for measuring the effects of group size on pathogen prevalence because two types of society coexist in this species: smaller single-nest single-queen colonies that are highly aggressive to their neighbours and larger multiple-queen colonies that exchange resources with neighbouring nests. We compare the presence of viruses between these two colony types using metagenomic sequence classification of RNA-sequencing reads. We find that queens from multiple-queen colonies have 8.3-times higher viral load and 1.5-times higher viral diversity than queens from single-queen colonies. This finding characterizes a rarely considered cost of transitions to more highly social living. Furthermore, our results show that highly social invertebrates can harbour many viruses.

Discovery of clinically relevant fusions in pediatric cancer

BackgroundPediatric cancers typically have a distinct genomic landscape when compared to adult cancers and frequently carry somatic gene fusion events that alter gene expression and drive tumorigenesis. Sensitive and specific detection of gene fusions through the analysis of next-generation-based RNA sequencing (RNA-Seq) data is computationally challenging and may be confounded by low tumor cellularity or underlying genomic complexity. Furthermore, numerous computational tools are available to identify fusions from supporting RNA-Seq reads, yet each algorithm demonstrates unique variability in sensitivity and precision, and no clearly superior approach currently exists. To overcome these challenges, we have developed an ensemble fusion calling approach to increase the accuracy of identifying fusions.ResultsOur Ensemble Fusion (EnFusion) approach utilizes seven fusion calling algorithms: Arriba, CICERO, FusionMap, FusionCatcher, JAFFA, MapSplice, and STAR-Fusion, which are packaged as a fully automated pipeline using Docker and Amazon Web Services (AWS) serverless technology. This method uses paired end RNA-Seq sequence reads as input, and the output from each algorithm is examined to identify fusions detected by a consensus of at least three algorithms. These consensus fusion results are filtered by comparison to an internal database to remove likely artifactual fusions occurring at high frequencies in our internal cohort, while a “known fusion list” prevents failure to report known pathogenic events. We have employed the EnFusion pipeline on RNA-Seq data from 229 patients with pediatric cancer or blood disorders studied under an IRB-approved protocol. The samples consist of 138 central nervous system tumors, 73 solid tumors, and 18 hematologic malignancies or disorders. The combination of an ensemble fusion-calling pipeline and a knowledge-based filtering strategy identified 67 clinically relevant fusions among our cohort (diagnostic yield of 29.3%), including RBPMS-MET, BCAN-NTRK1, and TRIM22-BRAF fusions. Following clinical confirmation and reporting in the patient’s medical record, both known and novel fusions provided medically meaningful information.ConclusionsThe EnFusion pipeline offers a streamlined approach to discover fusions in cancer, at higher levels of sensitivity and accuracy than single algorithm methods. Furthermore, this method accurately identifies driver fusions in pediatric cancer, providing clinical impact by contributing evidence to diagnosis and, when appropriate, indicating targeted therapies.

Association of Tumor Site With the Prognosis and Immunogenomic Landscape of Human Papillomavirus–Related Head and Neck and Cervical Cancers

Human papillomavirus (HPV)-positive status in patients with oropharyngeal squamous cell carcinoma (OPSCC) is associated with improved survival compared with HPV-negative status. However, it remains controversial whether HPV is associated with improved survival among patients with nonoropharyngeal and cervical squamous cell tumors. To investigate differences in the immunogenomic landscapes of HPV-associated tumors across anatomical sites (the head and neck and the cervix) and their association with survival. This cohort study used genomic and transcriptomic data from the Cancer Genome Atlas (TCGA) for 79 patients with OPSCC, 435 with nonoropharyngeal head and neck squamous cell carcinoma (non-OP HNSCC), and 254 with cervical squamous cell carcinoma and/or endocervical adenocarcinoma (CESC) along with matched clinical data from TCGA. The data were analyzed from November 2020 to March 2021. Positivity for HPV was classified by RNA-sequencing reads aligned with the HPV reference genome. Gene expression profiles, immune cell phenotypes, cytolytic activity scores, and overall survival were compared by HPV tumor status across multiple anatomical sites. The study comprised 768 patients, including 514 (66.9%) with HNSCC (380 male [73.9%]; mean [SD] age, 59.5 [10.8] years) and 254 (33.1%) with CESC (mean [SD] age, 48.7 [14.1] years). Human papillomavirus positivity was associated with a statistically significant improvement in overall survival for patients with OPSCC (adjusted hazard ratio [aHR], 0.06; 95% CI, 0.02-0.17; P < .001) but not for those with non-OP HNSCC (aHR, 0.64; 95% CI, 0.31-1.27; P = .20) or CESC (aHR, 0.50; 95% CI, 0.15-1.67; P = .30). The HPV-positive OPSCCs had increased tumor immune infiltration and immunomodulatory receptor expression compared with HPV-negative OPSCCs. Compared with HPV-positive non-OP HNSCCs, HPV-positive OPSCCs showed greater expression of immune-related metrics including B cells, T cells, CD8+ T cells, T-cell receptor diversity, B-cell receptor diversity, and cytolytic activity scores, independent of tumor variant burden. The immune-related metrics were similar when comparing HPV-positive non-OP HNSCCs and HPV-positive CESCs with their HPV-negative counterparts. The 2-year overall survival rate was significantly higher for patients with HPV-positive OPSCC compared with patients with HPV-negative OPSCC (92.0% [95% CI, 84.8%-99.9%] vs 45.8% [95% CI, 28.3%-74.1%]; HR, 0.10 [95% CI, 0.03-0.30]; P = .009). In this cohort study, tumor site was associated with the immune landscape and survival among patients with HPV-related tumors despite presumed similar biologic characteristics. These tumor site-related findings provide insight on possible outcomes of HPV positivity for tumors in oropharyngeal and nonoropharyngeal sites and a rationale for the stratification of HPV-associated tumors by site and the subsequent development of strategies targeting immune exclusion in HPV-positive nonoropharyngeal cancer.

MADS-box transcription factors determine the duration of temporary winter dormancy in closely related evergreen and deciduous Iris spp.

Winter dormancy (WD) is a crucial strategy for plants coping with potentially deadly environments. In recent decades, this process has been extensively studied in economically important perennial eudicots due to changing climate. However, in evergreen monocots with no chilling requirements, dormancy processes are so far a mystery. In this study, we compared the WD process in closely related evergreen (Iris japonica) and deciduous (I. tectorum) iris species across crucial developmental time points. Both iris species exhibit a 'temporary' WD process with distinct durations, and could easily resume growth under warm conditions. To decipher transcriptional changes, full-length sequencing for evergreen iris and short read RNA sequencing for deciduous iris were applied to generate respective reference transcriptomes. Combining results from a multipronged approach, SHORT VEGETATIVE PHASE and FRUITFULL (FUL) from MADS-box was associated with a dormancy- and a growth-related module, respectively. They were co-expressed with genes involved in phytohormone signaling, carbohydrate metabolism, and environmental adaptation. Also, gene expression patterns and physiological changes in the above pathways highlighted potential abscisic acid and jasmonic acid antagonism in coordinating growth and stress responses, whereas differences in carbohydrate metabolism and reactive oxygen species scavenging might lead to species-specific WD durations. Moreover, a detailed analysis of MIKCCMADS-box in irises revealed common features described in eudicots as well as possible new roles for monocots during temporary WD, such as FLOWERING LOCUS C and FUL. In essence, our results not only provide a portrait of temporary WD in perennial monocots but also offer new insights into the regulatory mechanism underlying WD in plants.

Improved nanopore direct RNA sequencing of cardiac myocyte samples by selective mt-RNA depletion

RNA sequencing is a powerful tool to analyze gene expression transcriptome wide. However, RNA sequencing in general and especially the recently developed methods of long read RNA sequencing are still low-throughput and cost-intensive. Here, one important design choice is to concentrate the sequencing capacity on specific parts of the transcriptome. Especially, abundant transcripts as ribosomal RNAs may dominate the available sequencing space, if not removed prior to sequencing. Several methods exist to reduce ribosomal RNA read numbers: either based on enrichment of the relevant fraction (polyA+ RNA) or depletion, respectively degradation of ribosomal RNAs. Furthermore, commercial kits are available to deplete globin transcripts from blood samples. However, so far, no solution exists to deal with other tissue-specific highly abundant transcripts. This is especially of interest in the heart and other muscle derived samples, where reads originating from mitochondrial RNAs make up to 30% of reads in polyA+ selected libraries and around 70% in single cell sequencing experiments. We present a simple method to diminish sequencing of mitochondrial RNAs in Oxford Nanopore direct RNA sequencing libraries by RNase H based clipping of the polyA tail. We show that mt-clipping enables enhanced detection of cytoplasmic mRNAs, among them genes involved in heart development and pathogenesis. Mt-clipping may be applied as well to other sequencing protocols that are based on oligo(dT) priming and can be easily adapted to other tissue-specific high-abundant transcripts.

Germline DDX41 mutations cause ineffective hematopoiesis and myelodysplasia.

DDX41 mutations are the most common germline alterations in adult myelodysplastic syndromes (MDSs). The majority of affected individuals harbor germline monoallelic frameshift DDX41 mutations and subsequently acquire somatic mutations in their other DDX41 allele, typically missense R525H. Hematopoietic progenitor cells (HPCs) with biallelic frameshift and R525H mutations undergo cell cycle arrest and apoptosis, causing bone marrow failure in mice. Mechanistically, DDX41 is essential for small nucleolar RNA (snoRNA) processing, ribosome assembly, and protein synthesis. Although monoallelic DDX41 mutations do not affect hematopoiesis in young mice, a subset of aged mice develops features of MDS. Biallelic mutations in DDX41 are observed at a low frequency in non-dominant hematopoietic stem cell clones in bone marrow (BM) from individuals with MDS. Mice chimeric for monoallelic DDX41 mutant BM cells and a minor population of biallelic mutant BM cells develop hematopoietic defects at a younger age, suggesting that biallelic DDX41 mutant cells are disease modifying in the context of monoallelic DDX41 mutant BM.

Bistable auto-aggregation phenotype in Lactiplantibacillus plantarum emerges after cultivation in in vitro colonic microbiota

BackgroundAuto-aggregation is a desired property for probiotic strains because it is suggested to promote colonization of the human intestine, to prevent pathogen infections and to modulate the colonic mucosa. We recently reported the generation of adapted mutants of Lactiplantibacillus plantarum NZ3400, a derivative of the model strain WCFS1, for colonization under adult colonic conditions of PolyFermS continuous intestinal fermentation models. Here we describe and characterize the emerge of an auto-aggregating phenotype in L. plantarum NZ3400 derivatives recovered from the modelled gut microbiota.ResultsL. plantarum isolates were recovered from reactor effluent of four different adult microbiota and from spontaneously formed reactor biofilms. Auto-aggregation was observed in L. plantarum recovered from all microbiota and at higher percentage when recovered from biofilm than from effluent. Further, auto-aggregation percentage increased over time of cultivation in the microbiota. Starvation of the gut microbiota by interrupting the inflow of nutritive medium enhanced auto-aggregation, suggesting a link to nutrient availability. Auto-aggregation was lost under standard cultivation conditions for lactobacilli in MRS medium. However, it was reestablished during growth on sucrose and maltose and in a medium that simulates the abiotic gut environment. Remarkably, none of these conditions resulted in an auto-aggregation phenotype in the wild type strain NZ3400 nor other non-aggregating L. plantarum, indicating that auto-aggregation depends on the strain history. Whole genome sequencing analysis did not reveal any mutation responsible for the auto-aggregation phenotype. Transcriptome analysis showed highly significant upregulation of LP_RS05225 (msa) at 4.1–4.4 log2-fold-change and LP_RS05230 (marR) at 4.5–5.4 log2-fold-change in all auto-aggregating strains compared to non-aggregating. These co-expressed genes encode a mannose-specific adhesin protein and transcriptional regulator, respectively. Mapping of the RNA-sequence reads to the promoter region of the msa-marR operon reveled a DNA inversion in this region that is predominant in auto-aggregating but not in non-aggregating strains. This strongly suggests a role of this inversion in the auto-aggregation phenotype.ConclusionsL. plantarum NZ3400 adapts to the in vitro colonic environment by developing an auto-aggregation phenotype. Similar aggregation phenotypes may promote gut colonization and efficacy of other probiotics and should be further investigated by using validated continuous models of gut fermentation such as PolyFermS.

Identification of Chicken Transglutaminase 1 and In Situ Localization of Transglutaminase Activity in Avian Skin and Esophagus

Transglutaminase 1 (TGM1) is a membrane-anchored enzyme that cross-links proteins during terminal differentiation of epidermal and esophageal keratinocytes in mammals. The current genome assembly of the chicken, which is a major model for avian skin biology, does not include an annotated region corresponding to TGM1. To close this gap of knowledge about the genetic control of avian cornification, we analyzed RNA-sequencing reads from organotypic chicken skin and identified TGM1 mRNA. By RT-PCR, we demonstrated that TGM1 is expressed in the skin and esophagus of chickens. The cysteine-rich sequence motif required for palmitoylation and membrane anchorage is conserved in the chicken TGM1 protein, and differentiated chicken keratinocytes display membrane-associated transglutaminase activity. Expression of TGM1 and prominent transglutaminase activity in the esophageal epithelium was also demonstrated in the zebra finch. Altogether, the results of this study indicate that TGM1 is conserved among birds and suggest that chicken keratinocytes may be a useful model for the study of TGM1 in non-mammalian cornification.

Found in Translation: Novel Insights Into Type 1 Diabetes and β-Cell Biology.

Although essential features in the natural history of type 1 diabetes are generally understood (1), many key questions remain. In particular, despite decades of research, the precise events that cause immune tolerance mechanisms to fail, triggering autoimmunity, remain unclear (2). One theory that has attracted considerable recent attention is that β-cell “stress” can trigger or potentiate autoimmunity by generating neoantigens (3–5), for example, by posttranslational modifications, hybrid peptide formation, or protein splicing (6). In this issue, Thomaidou et al. (7) significantly expand our knowledge of two other potential sources of “neo-antigens,” namely, the products of translation from noncanonical start sites, and alternatively spliced transcripts. Using a combination of ribosomal profiling (8) and long read RNA sequencing, they show that β-cells can use at least 15,014 unique translation start sites within transcripts from the 5,529 genes whose expression they detected. Ribosome profiling was initially developed by Ingolia, Weissman, and colleagues (8) and is based on next-generation sequencing of the ∼30 base pairs fragments of mRNA that are protected from nuclease digestion by bound ribosomes. Combining this approach with pharmacological treatment to accumulate ribosomes at the sites of initiation allows accurate inference of translational reading frames. This powerful technique is now widely used to study many aspects of translation but had not previously been applied to human β-cells. Surprisingly, only 17% of the …

Transcriptome analysis provides genome annotation and expression profiles in the central nervous system of Lymnaea stagnalis at different ages

BackgroundThe pond snail, Lymnaea stagnalis (L. stagnalis), has served as a valuable model organism for neurobiology studies due to its simple and easily accessible central nervous system (CNS). L. stagnalis has been widely used to study neuronal networks and recently gained popularity for study of aging and neurodegenerative diseases. However, previous transcriptome studies of L. stagnalis CNS have been exclusively carried out on adult L. stagnalis only. As part of our ongoing effort studying L. stagnalis neuronal growth and connectivity at various developmental stages, we provide the first age-specific transcriptome analysis and gene annotation of young (3 months), adult (6 months), and old (18 months) L. stagnalis CNS.ResultsUsing the above three age cohorts, our study generated 55–69 millions of 150 bp paired-end RNA sequencing reads using the Illumina NovaSeq 6000 platform. Of these reads, ~ 74% were successfully mapped to the reference genome of L. stagnalis. Our reference-based transcriptome assembly predicted 42,478 gene loci, of which 37,661 genes encode coding sequences (CDS) of at least 100 codons. In addition, we provide gene annotations using Blast2GO and functional annotations using Pfam for ~ 95% of these sequences, contributing to the largest number of annotated genes in L. stagnalis CNS so far. Moreover, among 242 previously cloned L. stagnalis genes, we were able to match ~ 87% of them in our transcriptome assembly, indicating a high percentage of gene coverage. The expressional differences for innexins, FMRFamide, and molluscan insulin peptide genes were validated by real-time qPCR. Lastly, our transcriptomic analyses revealed distinct, age-specific gene clusters, differentially expressed genes, and enriched pathways in young, adult, and old CNS. More specifically, our data show significant changes in expression of critical genes involved in transcription factors, metabolisms (e.g. cytochrome P450), extracellular matrix constituent, and signaling receptor and transduction (e.g. receptors for acetylcholine, N-Methyl-D-aspartic acid, and serotonin), as well as stress- and disease-related genes in young compared to either adult or old snails.ConclusionsTogether, these datasets are the largest and most updated L. stagnalis CNS transcriptomes, which will serve as a resource for future molecular studies and functional annotation of transcripts and genes in L. stagnalis.

Characterization of the virome associated with Haemagogus mosquitoes in Trinidad, West Indies

Currently, there are increasing concerns about the possibility of a new epidemic due to emerging reports of Mayaro virus (MAYV) fever outbreaks in areas of South and Central America. Haemagogus mosquitoes, the primary sylvan vectors of MAYV are poorly characterized and a better understanding of the mosquito’s viral transmission dynamics and interactions with MAYV and other microorganisms would be important in devising effective control strategies. In this study, a metatranscriptomic based approach was utilized to determine the prevalence of RNA viruses in field-caught mosquitoes morphologically identified as Haemagogus janthinomys from twelve (12) forest locations in Trinidad, West Indies. Known insect specific viruses including the Phasi Charoen-like and Humaiata-Tubiacanga virus dominated the virome of the mosquitoes throughout sampling locations while other viruses such as the avian leukosis virus, MAYV and several unclassified viruses had a narrower distribution. Additionally, assembled contigs from the Ecclesville location suggests the presence of a unique uncharacterized picorna-like virus. Mapping of RNA sequencing reads to reference mitochondrial sequences of potential feeding host animals showed hits against avian and rodent sequences, which putatively adds to the growing body of evidence of a potentially wide feeding host-range for the Haemagogus mosquito vector.

Transposable element sequence fragments incorporated into coding and noncoding transcripts modulate the transcriptome of human pluripotent stem cells.

Transposable elements (TEs) occupy nearly 40% of mammalian genomes and, whilst most are fragmentary and no longer capable of transposition, they can nevertheless contribute to cell function. TEs within genes transcribed by RNA polymerase II can be copied as parts of primary transcripts; however, their full contribution to mature transcript sequences remains unresolved. Here, using long and short read (LR and SR) RNA sequencing data, we show that 26% of coding and 65% of noncoding transcripts in human pluripotent stem cells (hPSCs) contain TE-derived sequences. Different TE families are incorporated into RNAs in unique patterns, with consequences to transcript structure and function. The presence of TE sequences within a transcript is correlated with TE-type specific changes in its subcellular distribution, alterations in steady-state levels and half-life, and differential association with RNA Binding Proteins (RBPs). We identify hPSC-specific incorporation of endogenous retroviruses (ERVs) and LINE:L1 into protein-coding mRNAs, which generate TE sequence-derived peptides. Finally, single cell RNA-seq reveals that hPSCs express ERV-containing transcripts, whilst differentiating subpopulations lack ERVs and express SINE and LINE-containing transcripts. Overall, our comprehensive analysis demonstrates that the incorporation of TE sequences into the RNAs of hPSCs is more widespread and has a greater impact than previously appreciated.

Accurate spliced alignment of long RNA sequencing reads.

MotivationLong-read RNA sequencing technologies are establishing themselves as the primary techniques to detect novel isoforms, and many such analyses are dependent on read alignments. However, the error rate and sequencing length of the reads create new challenges for accurately aligning them, particularly around small exons.ResultsWe present an alignment method uLTRA for long RNA sequencing reads based on a novel two-pass collinear chaining algorithm. We show that uLTRA produces higher accuracy over state-of-the-art aligners with substantially higher accuracy for small exons on simulated and synthetic data. On simulated data, uLTRA achieves an accuracy of about 60% for exons of length 10 nucleotides or smaller and close to 90% accuracy for exons of length between 11 and 20 nucleotides. On biological data where true read location is unknown, we show several examples where uLTRA aligns to known and novel isoforms containing small exons that are not detected with other aligners. While uLTRA obtains its accuracy using annotations, it can also be used as a wrapper around minimap2 to align reads outside annotated regions.Availabilityand implementationuLTRA is available at https://github.com/ksahlin/ultra.Supplementary information Supplementary data are available at Bioinformatics online.

Insights into Hematopoiesis in GATA2 Deficiency Using Single-Cell Transcriptomics

GATA2 is an essential transcription factor in hematopoiesis. Germline GATA2 mutations have been identified as the cause of familial syndromes with immunodeficiency and predisposition to myeloid malignancies. GATA2 deficiency results in varying clinical outcomes manifesting at either young or older age. Reason for the variation of the disease phenotype preceding hematological malignancy is not known. GATA2 mutations appear to cause loss of function of the mutated allele resulting in haploinsufficiency. Several different types of mutations in GATA2 have been identified; truncating, missense, large deletions, and regulatory region mutations. The most direct GATA2 control pathway is the effect it has on hematopoietic stem cells (HSCs). Through the progeny of the hematopoietic stem cells that function under GATA2 control, GATA2 indirectly has a great effect on immune system, both protection against external infection and internal tumor formation. We aimed at deciphering how do GATA2-mutated cells differ from wild type (wt) and what are the differences between GATA2 wt vs. GATA2-mutated cells' progeny and downstream signaling? One of the genes positively regulated by GATA2 is NFE2 . NFE2 encodes a component of the NF-E2 complex essential for regulating erythroid and megakaryocytic maturation and differentiation. As GATA2-positive cells are scarce in PB we focused in our analyses on NFE2-positive cells.We conducted single-cell transcriptomic analysis using 10x Genomics Chromium Single Cell 3′ Solution (Zheng et al . Nature Communications, 2017) on peripheral blood (PB) samples from five GATA2 mutation carriers and two healthy controls. The studied GATA2 variants were p.Q328X (two cases), p.T354M, p.G237D, and a sample where a GATA2 mutation has not been identified, but only one allele GATA2 is expressed i.e. haploinsufficiency. Erythrocytes were lysed prior to washing, filtering and diluting the white blood cells in suitable concentration (1x106 cells/mL) for the analysis. The gel beads in emulsion (GEM) generation was performed using the Chromium Single Cell 3‘ v2 Reagent Kit (10x Genomics) aiming for a 3000-cell capture per sample. The Illumina ready sequencing libraries were prepared according to the 10x Genomics instructions and the sample libraries were sequenced in Illumina HiSeq 2500 using Rapid mode. The Cell Ranger v1.3 analysis pipelines (10x Genomics) were used to demultiplex and convert Chromium single cell 3‘ RNA-sequencing barcode and read data to FASTQ files and to generate align reads and gene-cell matrices.Significant gene expression differences in NFE2 expressing cells between patients carrying different variants in GATA2 were observed. Intriguingly, significant expression changes were noted e.g. in CD74, CFD, CTSS, CLEC1B, and CMTM5, all linked to cell signaling and immunological responses. Comparison of GATA2 -mutated vs. controls in NFE2 expressing cells revealed significant changes in expression patterns in hemoglobin genes HBA1, HBA2, and HBB, as well as in e.g. other erythrocyte-associated genes ALAS2 and AHSP. Further analyses also revealed significantly distinct expression patterns in PB eosinophils between study patients and controls in e.g. CEBPD, VCAN, and RGS2 . Also, when comparing the eosinophils from individual patients with controls and each other, we identified distinct expression patterns in e.g. CAMP, and NKG7, which play important roles in the immune system.Following germline GATA2 mutation discovery in acute myeloid leukemia / myelodysplastic syndrome / immunodeficiency patients, we conducted whole transcriptome analysis to decipher the GATA germline mutation defects on single-cell level. Single-cell transcriptomics enables analyzing the role of e.g. GATA2 in a selected cell type and its' progeny. Phenotype of GATA2-deficient patients is multi-faced but generally manifested by challenges in immune functions and autoinflammation. Connection between our findings and patients' clinical manifestations is interesting and triggers us to investigate the possible link further. We will furthermore study the bone marrow sample single-cell transcriptomes to deepen the understanding of GATA2 deficiency on stem cell level and validate our transcriptome analysis findings. All this will hopefully increase our understanding on GATA2 deficiency disease mechanisms and improve patient (family) care. DisclosuresNo relevant conflicts of interest to declare.

Comprehensive Identification of Fusion Transcripts in Multiple Myeloma: An Mmrf Commpass Analysis

Multiple myeloma is a hematological malignancy characterized by the clonal expansion of plasma cells in the bone marrow. Unlike many other blood cancers there is a diverse array of somatic alterations with patients being split into two large groups of non-hyperdiploid and hyperdiploid based on recurrent trisomies of the odd number chromosomes in ~58% of patients. Though the remaining non-hyperdiploid patients are frequently characterized by immunoglobulin translocations these rarely create fusion transcripts like the quintessential BCR-ABL fusions seen in CML, as the majority represent promoter or enhancer replacements driving the ectopic expression of a target gene with oncogenic potential. The only well defined fusion transcripts in myeloma are the IGH-MMSET fusions created in the majority of patients with t(4;14) translocations. However, these fusions are simple byproducts of the enhancer replacement event and do not create hybrid poly-peptides. We have used the comprehensive nature of the MMRF CoMMpass dataset with matched tumor RNA sequencing and tumor/normal whole genome sequencing to define the landscape of fusion transcripts in myeloma.In this IA11 analysis of the MMRF CoMMpass dataset, which represents the first release of the complete baseline cohort, we utilized the RNA sequencing data from 806 samples with matching WGS for 704 specimens. Our approach leverages a discovery step using Tophat-Fusion to identify potential fusion candidates. These potential fusions are then validated with three independent processes: Tophat-Fusion post, a guided assembly based approach of the RNA sequencing reads to create the fusion transcript in silico, and genomic validation using the WGS data to confirm a somatic structural event exists that could explain the observed fusion.In this large cohort we identified 45,769 potential fusions from 806 samples, Tophat-Fusion Post filters the list to 36,267, with numerous highly recurrent false positive between highly expressed genes. The guided assembly was able to construct 17,420 fusion transcripts with many being proximal fusions of adjacent genes that likely occur in normal cells. Genomic validation identified somatic structural events occurring proximal and in the correct orientation to create 1192 of the observed fusions. All three processes validate 930 fusion events.This analysis found the expected fusions between IGH-JH segments and WHSC1 as the most common fusion event detected in 100 patients. In addition, we observed several other fusion transcripts associated with common IgH rearrangements; CCND1/MYEOV and MYC plus several novel IgH fusions with TOP1MT and MAP3K14. There are very few recurrent gene pairs outside of novel fusions of KANSL1-ARL17A and TFG-GPR128. However, there are several genes like FCHSC2, TXNDC11, TXNDC5, MAP3K14, NEDD9, and TNFRSF17. Several like FCHSC2 and TNXDC5 are clear promoter replacements putting a strong B-cell promoter in front of myeloma promoting genes like CD40, LTBR, or MYC. Others appear to be the target gene of the fusion such as those involving MAP3K14 that removes the protein degradation region leading constitutive non-canonical NF-kB signaling. Another frequent set of fusions observed were highly random events associated with inactivation of known tumor suppressor genes in myeloma like TRAF3, RB1, FAM46C, and NF1.In conclusion, our comprehensive analysis of the landscape of fusion transcripts across several hundred samples with in-silico validation in RNA and DNA brings forth several fusion genes in myeloma. These genes are informative of the malignant processes occurring in multiple myeloma. Further investigation is warranted to understand the significance of the recurrently observed gene pairs or hub genes to understand how they contribute to the development and pathogenesis of multiple myeloma. DisclosuresNo relevant conflicts of interest to declare.

SARS-CoV-2-Host Chimeric RNA-Sequencing Reads Do Not Necessarily Arise From Virus Integration Into the Host DNA.

The human genome bears evidence of extensive invasion by retroviruses and other retroelements, as well as by diverse RNA and DNA viruses. High frequency of somatic integration of the RNA virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the DNA of infected cells was recently suggested, based on a number of observations. One key observation was the presence of chimeric RNA-sequencing (RNA-seq) reads between SARS-CoV-2 RNA and RNA transcribed from human host DNA. Here, we examined the possible origin specifically of human-SARS-CoV-2 chimeric reads in RNA-seq libraries and provide alternative explanations for their origin. Chimeric reads were frequently detected also between SARS-CoV-2 RNA and RNA transcribed from mitochondrial DNA or episomal adenoviral DNA present in transfected cell lines, which was unlikely the result of SARS-CoV-2 integration. Furthermore, chimeric reads between SARS-CoV-2 RNA and RNA transcribed from nuclear DNA were highly enriched for host exonic, rather than intronic or intergenic sequences and often involved the same, highly expressed host genes. Although these findings do not rule out SARS-CoV-2 somatic integration, they nevertheless suggest that human-SARS-CoV-2 chimeric reads found in RNA-seq data may arise during library preparation and do not necessarily signify SARS-CoV-2 reverse transcription, integration in to host DNA and further transcription.

Predicting MHC I restricted T cell epitopes in mice with NAP-CNB, a novel online tool

Lack of a dedicated integrated pipeline for neoantigen discovery in mice hinders cancer immunotherapy research. Novel sequential approaches through recurrent neural networks can improve the accuracy of T-cell epitope binding affinity predictions in mice, and a simplified variant selection process can reduce operational requirements. We have developed a web server tool (NAP-CNB) for a full and automatic pipeline based on recurrent neural networks, to predict putative neoantigens from tumoral RNA sequencing reads. The developed software can estimate H-2 peptide ligands, with an AUC comparable or superior to state-of-the-art methods, directly from tumor samples. As a proof-of-concept, we used the B16 melanoma model to test the system’s predictive capabilities, and we report its putative neoantigens. NAP-CNB web server is freely available at http://biocomp.cnb.csic.es/NeoantigensApp/ with scripts and datasets accessible through the download section.

MirMachine: A One-Stop Shop for Plant miRNA Annotation.

Of different types of noncoding RNAs, microRNAs (miRNAs) have arguably been in the spotlight over the last decade. As post-transcriptional regulators of gene expression, miRNAs play key roles in various cellular pathways, including both development and response to a/biotic stress, such as drought and diseases. Having high-quality reference genome sequences enabled identification and annotation of miRNAs in several plant species, where miRNA sequences are highly conserved. As computational miRNA identification and annotation processes are mostly error-prone processes, homology-based predictions increase prediction accuracy. We developed and have improved the miRNA annotation pipeline, SUmir, in the last decade, which has been used for several plant genomes since then. This study presents a fully automated, new miRNA pipeline, mirMachine (miRNA Machine), by (i) adding an additional filtering step on the secondary structure predictions, (ii) making it fully automated, and (iii) introducing new options to predict either known miRNA based on homology or novel miRNAs based on small RNA sequencing reads using the previous pipeline. The new miRNA pipeline, mirMachine, was tested using The Arabidopsis Information Resource, TAIR10, release of the Arabidopsis genome and the International Wheat Genome Sequencing Consortium (IWGSC) wheat reference genome v2.

POS-378 SELECTIVE ETA ANTAGONIST ATRASENTAN, RAPIDLY REDUCES ALBUMINURIA AND DOWNREGULATES INTRA-RENAL PRO-INFLAMMATORY AND PRO-FIBROTIC TRANSCRIPTIONAL NETWORKS IN THE GDDY MOUSE MODEL OF SPONTANEOUS IGA NEPHROPATHY

Endothelin pathway activation has been observed in kidney biopsies from IgA nephropathy (IgAN) patients which may be an important driver of disease progression by promoting proteinuria along with kidney inflammation and fibrosis via ETA receptor activation. Atrasentan is a potent and selective ETA antagonist which has demonstrated rapid and sustained reductions in proteinuria, preservation of kidney function and improved kidney outcomes in diabetic kidney disease patients. However, the effects of atrasentan have not been previously investigated in IgAN. The objective of this study was to evaluate the effect of short-term treatment of varying doses of atrasentan in the gddY mouse model of spontaneous IgAN, with a focus on dynamic changes in the intra-renal transcriptional profile. 6-week-old male gddY mice were administered atrasentan (10, 20 or 30 mg/kg/day, n=3/group or 0 mg/kg/day, n=2/group) in drinking water for 5 days. Urine albumin to creatinine ratio (UACR) was measured at baseline and on Day 4 of treatment. On Day 5, a terminal plasma sample was collected for the measurement of atrasentan plasma concentrations and kidney cortex was flash-frozen for RNA-seq which was analyzed by pairwise differential gene expression using edgeR’s quasi-likelihood F-test (FDR adjusted p-value < 0.05) and gene pathway analysis (FDR adjusted p-value filter of < 0.001). At baseline, gddY mice had substantial albuminuria, with a mean UACR of 293.1 ± 16 mg/g. Atrasentan reduced UACR from baseline by 28 ± 44 % (p=ns), 62 ± 8 % (p=0.0498) and 63 ± 6 % (P=0.029) at 10, 20 and 30 mg/kg/day, respectively. Free terminal atrasentan plasma concentrations were 90 pM, 100 pM and 207 pM at the 10, 20 and 30 mg/kg/day doses respectively, providing free atrasentan concentrations 2.6-fold, 2.9-fold and 6.1-fold above the atrasentan ETA Ki (34 pM), respectively and in the same range as observed clinically at the optimized atrasentan dose of 0.75 mg. High quality RNA sequencing reads revealed dose-dependent effects of atrasentan on differential gene expression compared to untreated control ddY mice, including reductions in known ETA target genes ET-1 and ETA. Ingenuity Pathway Analysis to calculate enrichment scores showed that atrasentan produced prominent, dose-dependent downregulation of inflammatory, proliferative, and fibrotic gene networks. Atrasentan was observed to downregulate signaling pathways previously implicated in IgAN pathogenesis including the PDGF, NF-κB, IL-6 and TNF pathways. In the gddY mouse model of spontaneous IgAN, selectively blocking ETA with atrasentan leads to rapid reductions in albuminuria and results in intra-renal transcriptional downregulation of inflammatory and fibrotic signaling. The dynamic transcriptional changes in the kidney, including downregulation of known direct ETA receptor target genes, following only 5 days of treatment and prior to sustained long-term reductions in albuminuria and blood pressure that could mediate this benefit, is consistent with direct anti-inflammatory and antifibrotic effects of ETA blockade in IgAN. These results support further characterization of the effects of long-term treatment of atrasentan in the gddY mouse model and also support the therapeutic potential of atrasentan in IgAN patients to reduce proteinuria and kidney inflammation and fibrosis, key drivers of IgAN progression.