Biology subjects

Dowell, R. D.

Publications and source records attributed to Dowell, R. D..

At least 19 recordsLinked to original sources

bioRxiv2026

Cross-assay RNA modeling reveals cancer biomarkers

The clinical heterogeneity of cancer poses a major challenge for precision medicine. Limited cohort sizes across evolving assay platforms impede reliable biomarker discovery. Here, we systematically evaluate how to integrate data from four transcriptomics platforms: bulk and single-cell (sc) RNA sequencing (RNA-seq), NanoString, and microarray for predictive modeling in cancer. We use high-grade serous carcinoma (HGSC) of tube-ovarian origin as a model system, as it is highly heterogeneous in both biology and assay data. We find that using fold-change of gene expression in patients with matched pre- and post-neoadjuvant chemotherapy samples reduces inter-patient and inter-assay variability but is insufficient to overcome platform-specific biases. Microarray and scRNA-seq data exhibit systematic biases, while RNA-seq and NanoString show the most promise for combination into a single training cohort. To mitigate inter-assay limitations, we generate a new data set of HGSC tumor samples profiled with both RNA-seq and NanoString, and use it to identify the limits of detection and optimal harmonization strategies. Our approaches enable integration of cohorts for separate and combined RNA-seq and NanoString predictive models of disease recurrence (test-set AUROCs > 0.8), validated in external microarray cohorts. We leverage single-cell and bulk RNA-seq network-based analyses to provide mechanistic context for genes in the predictive models. Our models indicate that GBP4 expression is a key predictor of recurrence and marks immune remodeling towards cytotoxicity. We provide an interactive web portal to facilitate exploration of data and results. These findings guide cross-assay harmonization of transcriptomic data and enable improved predictive modeling in heterogeneous cancers. Statement of SignificanceWe present a framework for integrating RNA-seq, NanoString, microarray, and single-cell transcriptomic data for predictive modeling, enabling robust biomarker discovery in heterogeneous cancers and identifying GBP4 as a marker of immune remodeling.

bioinformatics↗

bioRxiv2025

Base Composition Influences the Position andPrecision of RNA Polymerase II Disassociation inBasal and Perturbed Conditions.

RNA Polymerase II (Pol II) transcribes all protein-coding and many non-protein coding genes in the genome. Pol II transcription termination is crucial for mRNA maturation and, when disrupted, can lead to altered mRNA processing and mRNA export. Termination involves two intertwined processes: pre-mRNA cleavage and Pol II release from the DNA (disassociation). Despite its importance, the exact mechanisms underlying Pol II disassociation from the DNA remain poorly understood. Moreover, under certain cellular stress conditions, there is a partial failure of cleavage, leading to a shift of the position of disassociation further downstream, a phenomenon known as run-on transcription. We performed the first-ever systematic analysis of Pol II termination across cell types and species and provide novel insights into the mechanism of disassociation. Using a probabilistic mixture model to quantify Poll II dynamics across an entire gene body from nascent RNA sequencing data, we discovered that genes have two types of conserved regions near the disassociation site: one characterized by a T-rich region upstream of disassociation, and another characterized by a GC-rich region surrounding disassociation. Strikingly, the GC-rich disassociation regions have more accessible chromatin and higher levels of phospho-threonine 4 on the CTD of Pol II. Additionally, we find that upstream T-rich genes are preferentially affected by perturbations that alter disassociation, including heat-shock, viral infection, kinase inhibition, and arsenic treatment. Thus, our work has determined there are two types of Pol II disassociation regions, which are differentially affected by perturbation of cellular homeostasis.

genomics↗

bioRxiv2025

Fragile nucleosomes are essential for RNA Polymerase II to transcribe in eukaryotes

Nucleosomes are barriers to RNA Polymerase II (Pol II) transcribing along gene bodies in eukaryotes. We found that a fragile "tailless nucleosome" could be generated to resolve the Pol II pausing caused by the +1 nucleosome in metazoans. We hypothesized that "pan-acetylated nucleosomes" along gene bodies are fragile and are generated to resolve the resistance of nucleosomes to the elongation of Pol II. To confirm this hypothesis, we systematically analyzed pan-acetylation and pan-phosphorylation of the entire human genome in HEK293T cells. Herein, we show that both pan-acetylation and pan-phosphorylation of histones in nucleosomes along gene bodies are coupled with the activities of Pol II. Our results show that there are four major types of fragile nucleosomes required for Pol II to transcribe along gene bodies: nucleosomes without tails, nucleosomes with dominant acetylation, nucleosomes with dominant phosphorylation, and nucleosomes with both acetylation and phosphorylation.

genetics↗

bioRxiv2025

Improving confidence of differential transcription calls in enhancers

MotivationMost disease-associated genetic variants reside within transcribed regulatory elements (tREs). Patterns of differential transcription at tREs can be leveraged to identify upstream regulators and link enhancers to their target genes. But the low transcription levels and high variability in tREs makes identifying high confidence differentially transcribed elements challenging. ResultsWe present Mu Counts and TFEA-LE, two algorithms for robust identification of differentially transcribed tREs. The first step in accurate identification of differentially transcribed tREs is to obtain accurate RNA lengths and therefore counts over these regions. To this end we developed a method of accurate length inference (LIET-EMG) as wll as a rapid method for counting reads over tREs (Mu Counts). Armed with newly identified and quantified tREs, TFEA-LE then integrates motif information to simultaneously identify responsive tREs and their likely upstream regulators. We show improved precision and recall over general-purpose tools (e.g. DESeq2) in detecting p53-responsive tREs. We then clarify TF-specific responses within multi-TF perturbations in lung cells. Finally we show that the TFEA-LE approach improves TF activity inference, including in complex perturbations where many TFs respond. TFEALE is especially effective in technically challenging datasets, whether due to highly specific or broad responses, outliers, or high GC content. Ultimately, these 1 methods advance the systematic characterization of individual tREs, enabling their integration with regulators, target genes, and disease-associated variants for translational research. Availability and ImplementationTFEA-LE: https://github.com/Dowell-Lab/TFEA/tree/Lead_edge. Nextflow pipeline to run Mu Counts: https://github.com/Dowell-Lab/Bidir_Counting_Analysis. LIET (including modifications for tREs): https://github.com/Dowell-Lab/LIET/tree/LIET_EMGtoo. Source code for this work: https://github.com/Dowell-Lab/Improving_tRE_Analysis_Paper Contactrobin.dowell@colorado.edu

bioinformatics↗

bioRxiv2025

Histone Deacetylase Inhibitor Largazole Deactivates A Subset of Superenchancers and Causes Mitotic Chromosome Mis-alignment by Suppressing SP1 and BRD4

Histone deacetylase inhibitors have been investigated as potential therapeutic agents for cancer and other diseases. HDIs are known to promote histone acetylation, resulting in an open chromatin conformation and generally increased gene expression. In previous work, we reported that a subset of genes, particularly those regulated by superenhancers, can be suppressed by the HDAC inhibitor largazole. To elucidate the molecular mechanisms underlying gene repression by largazole, we conducted transposase-accessible chromatin sequencing, ChIP-seq, and RNA-seq studies. Our findings revealed that while largazole treatment generally enhances chromatin accessibility, it selectively decreases the accessibility of a subset of superenhancer regions. These genomic regions, showing the most significant changes in the presence of largazole, were enriched with transcription factor binding motifs for SP1, BRD4, CTCF, and YY1. ChIP-seq analysis confirmed reduced binding of BRD4 and SP1 at their respective sites on chromatin, particularly at superenhancers regulating genes such as ID1, c-Myc and MCMs. Largazole exerts its effects by inhibiting DNA replication, RNA processing, and cell cycle progression, partially through the suppression of SP1 expression. Depletion of SP1 by shRNA mimics several key biological effects of largazole and increases cellular sensitivity to the drug. Specific to cell cycle regulation, we demonstrated that largazole disrupts G/M transition by interfering with chromosome alignment during metaphase, a phenotype also observed with SP1 depletion. Our results suggest that largazole exerts its growth-inhibitory effect by suppressing BRD4 and SP1 at super-enhancers, leading to cytostatic responses and mitotic dysfunction.

cancer biology↗

bioRxiv2025

Multi-omics and biochemical reconstitution reveal CDK7-dependent mechanisms controlling RNA polymerase II function at gene 5'- and 3'-ends

CDK7 regulates RNA polymerase II (RNAPII) initiation, elongation, and termination through incompletely understood mechanisms. Because contaminating kinases precluded CDK7 analysis with nuclear extracts, we completed biochemical assays with purified factors. Reconstitution of RNAPII transcription initiation showed CDK7 inhibition slowed and/or paused RNAPII promoter-proximal transcription, which reduced re-initiation. These CDK7-regulatory functions were Mediator- and TFIID-dependent. Similarly in human cells, CDK7 inhibition reduced transcription by suppressing RNAPII activity at promoters, consistent with reduced initiation and/or re-initiation. Moreover, widespread 3-end readthrough transcription was observed in CDK7-inhibited cells; mechanistically, this occurred through rapid nuclear depletion of RNAPII elongation and termination factors, including high-confidence CDK7 targets. Collectively, these results define how CDK7 governs RNAPII function at gene 5-ends and 3-ends, and reveal that nuclear abundance of elongation and termination factors is kinase-dependent. Because 3-readthrough transcription is commonly induced during stress, our results further suggest regulated suppression of CDK7 activity may enable this RNAPII transcriptional response.

biochemistry↗

bioRxiv2024

LIET Model: Capturing the kinetics of RNA polymerase from loading to termination

Transcription by RNA polymerases is an exquisitely regulated step of the central dogma. Transcription is the primary determinant of cell-state, and most cellular perturbations impact transcription by altering polymerase activity. Thus, detecting changes in polymerase activity yields insight into most cellular processes. Nascent run-on sequencing provides a direct readout of polymerase activity, but no tools exist to model this activity at genes. We focus on RNA polymerase II--responsible for transcribing protein-coding genes. We present the first model to capture the complete process of gene transcription. For individual genes, this model parameterizes each distinct stage of transcription--Loading, Initiation, Elongation, and Termination, hence LIET--in a biologically interpretable Bayesian mixture, which is applied to nascent run-on data. Our improved modeling of Loading /Initiation demonstrates these are characteristically different between sense and antisense strands. Applying LIET to 24 human cell-types, our analysis indicates the position of dissociation (the last step of Termination) appears to be highly consistent, indicative of a highly regulated process. Furthermore, applying LIET to perturbation experiments, we demonstrate its ability to detect specific changes in pausing (5'end), strand-bias, and dissociation location (3'end)--opening the door to differential assessment of transcription at individual stages of individual genes.

bioinformatics↗

bioRxiv2024

Single nucleus RNA-sequencing reveals transcriptional synchrony across different relationships

As relationships mature, partners share common goals, improve their ability to work together, and experience coordinated emotions. However, the neural underpinnings responsible for this unique, pair-specific experience remain largely unexplored. Here, we used single nucleus RNA-sequencing to examine the transcriptional landscape of the nucleus accumbens (NAc) in socially monogamous prairie voles in peer or mating-based relationships. We show that, regardless of pairing type, prairie voles exhibit transcriptional synchrony with a partner. Further, we identify genes expressed in oligodendrocyte progenitor cells that are synchronized between partners, correlated with dyadic behavior, and sensitive to partner separation. Together, our data indicate that the pair-specific social environment profoundly shapes transcription in the NAc. This provides a potential biological mechanism by which shared social experience reinforces and strengthens relationships.

neuroscience↗

bioRxiv2024

A transcription factor (TF) inference method that broadly measures TF activity and identifies mechanistically distinct TF networks

TF profiler is a method of inferring transcription factor regulatory activity, i.e. when a TF is present and actively regulating transcription, directly directly from nascent sequencing assays such as PRO-seq and GRO-seq. Transcription factors orchestrate transcription and play a critical role in cellular maintenance, identity and response to external stimuli. While ChIP assays have measured DNA localization, they fall short of identifying when and where transcription factors are actively regulating transcription. Our method, on the other hand, uses RNA polymerase activity to infer TF activity across hundreds of data sets and transcription factors. Based on these classifications we identify three distinct classes of transcription factors: ubiquitous factors that play roles in cellular homeostasis, driving basal gene programs across tissues and cell types, tissue specific factors that act almost exclusively at enhancers and are themselves regulated at transcription, and stimulus responsive TFs which are regulated post-transcriptionally but act predominantly at enhancers. TF profiler is broadly applicable, providing regulatory insights on any PRO-seq sample for any transcription factor with a known binding motif.

genomics↗

bioRxiv2023

Atlas of nascent RNA transcripts reveals enhancer to gene linkages

Gene transcription is controlled and modulated by regulatory regions, including enhancers and promoters. These regions are abundant in unstable, non-coding bidirectional transcription. Using nascent RNA transcription data across hundreds of human samples, we identified over 800,000 regions containing bidirectional transcription. We then identify highly correlated transcription between bidirectional and gene regions. The identified correlated pairs, a bidirectional region and a gene, are enriched for disease associated SNPs and often supported by independent 3D data. We present these resources as an SQL database which serves as a resource for future studies into gene regulation, enhancer associated RNAs, and transcription factors.

genomics↗

bioRxiv2023

Cellular zinc status alters chromatin accessibility and binding of transcription factor TP53 to genomic sites

Zinc (Zn2+) is an essential metal required by approximately 2500 proteins. Nearly half of these proteins act on DNA, including > 850 human transcription factors, polymerases, DNA damage response factors, and proteins involved in chromatin architecture. How these proteins acquire their essential Zn2+ cofactor and whether they are sensitive to changes in the labile Zn2+ pool in cells remain open questions. Here, we examine how changes in the labile Zn2+ pool affect chromatin accessibility and transcription factor binding to DNA. We observed both increases and decreases in accessibility in different chromatin regions via ATAC-seq upon treating MCF10A cells with elevated Zn2+ or the Zn2+-specific chelator tris(2-pyridylmethyl)amine (TPA). Transcription factor enrichment analysis was used to correlate changes in chromatin accessibility with transcription factor motifs, revealing 477 transcription factor motifs that were differentially enriched upon Zn2+ perturbation. 186 of these transcription factor motifs were enriched in Zn2+ and depleted in TPA, and the majority correspond to Zn2+ finger transcription factors. We selected TP53 as a candidate to examine how changes in motif enrichment correlate with changes in transcription factor occupancy by ChIP-qPCR. Using publicly available ChIP-seq and nascent transcription datasets, we narrowed the 50,000+ ATAC-seq peaks to 2164 TP53 targets and subsequently selected 6 high-probability TP53 binding sites for testing. ChIP-qPCR revealed that for 5 of the 6 targets, TP53 binding correlates with the local accessibility determined by ATAC-seq. These results demonstrate that changes in labile zinc directly alter chromatin accessibility and transcription factor binding to DNA.

genomics↗

bioRxiv2023

Transcription dosage compensation does not occur in Down syndrome

BackgroundTrisomy 21, also known as Down syndrome, describes the genetic condition of having an extra copy of chromosome 21. The increase in DNA copy number has led to the "DNA dosage hypothesis", which claims that the level of gene transcription is proportional to the genes DNA copy number. Yet many reports have suggested that a proportion of chromosome 21 genes are dosage compensated back towards typical expression levels (1.0x). In contrast, other reports suggest that dosage compensation is not a common mechanism of gene regulation in Trisomy 21, providing support to the DNA dosage hypothesis. ResultsIn our work, we use both simulated and real data to dissect the elements of differential expression analysis that can lead to the appearance of dosage compensation even when compensation is demonstrably absent. Using lymphoblastoid cell lines derived from a family of an individual with Down syndrome, we demonstrate that dosage compensation is nearly absent at both nascent transcription (GRO-seq) and steady-state RNA (RNA-seq) levels. ConclusionsTranscriptional dosage compensation does not occur in Down syndrome. Simulated data containing no dosage compensation can appear to have dosage compensation when analyzed via standard methods. Moreover, some chromosome 21 genes that appear to be dosage compensated are consistent with allele specific expression.

bioinformatics↗

bioRxiv2023

Characterizing Primary transcriptional responses to short term heat shock in paired fraternal lymphoblastoid lines with and without Down syndrome.

Heat shock stress induces genome wide changes in transcription regulation, activating a coordinated cellular response to enable survival. Using publicly available transcriptomic and proteomic data sets comparing individuals with and without trisomy 21, we noticed many heat shock genes are up-regulated in blood samples from individuals with trisomy 21. Yet no major heat shock response regulating transcription factor is encoded on chromosome 21, leaving it unclear why trisomy 21 itself would cause a heat shock response, or how it would impact the ability of blood cells to subsequently respond when faced with heat shock stress. To explore these issues in a context independent of any trisomy 21 associated co-morbidities or developmental differences, we characterized the response to heat shock of two lymphoblastoid cell lines derived from brothers with and without trisomy 21. To carefully compare the chromatin state and the transcription status of these cell lines, we measured nascent transcription, chromatin accessibility, and single cell transcript levels in the lymphoblastoid cell lines before and after acute heat shock treatment. The trisomy 21 cells displayed a more robust heat shock response after just one hour at 42{degrees}C than the matched disomic cells. We suggest multiple potential mechanisms for this increased heat shock response in lymphoblastoid cells with trisomy 21 including the possibility that cells with trisomy 21 may exist in a hyper-reactive state due to chronic stresses. Whatever the mechanism, abnormal heat shock response in individuals with Down syndrome may hobble immune responses during fever and contribute to health problems in these individuals.

bioinformatics↗

bioRxiv2022

Integrated Genomics Approaches Identify Mediators of Transcriptional and Epigenetic Responses to Afghan Desert Particulate Matter in Human Small Airway Epithelial Cells

Military Deployment to Southwest Asia and Afghanistan and exposure to toxic airborne particulates has been associated with an increased risk of developing respiratory disease, collectively termed deployment-related respiratory diseases (DRRD). Our knowledge about how particulates mediate respiratory disease is limited, precluding the appropriate recognition or management. Central to this limitation is the lack of understanding of how exposures translate into dysregulated cell identity with dysregulated transcriptional programs. The small airway epithelium is involved in both the pathobiology of DRRD and fine particulate matter deposition. To characterize small airway epithelial cell epigenetic and transcriptional responses to Afghan desert particulate matter (APM) and investigate the functional interactions of transcription factors that mediate these responses, we applied two genomics assays, the assay for transposase accessible chromatin with sequencing (ATAC-seq) and Precision Run-on sequencing (PRO-seq). We identified activity changes in a series of transcriptional pathways as candidate regulators of susceptibility to subsequent insults, including signal-dependent pathways, such as loss of cytochrome P450 or P53/P63, and lineage-determining transcription factors, such as GRHL2 loss or TEAD3 activation. We further demonstrated that TEAD3 activation was unique to APM exposure despite similar inflammatory responses when compared with wood smoke particle exposure and that P53/P63 program loss was uniquely positioned at the intersection of signal-dependent and lineage-determining transcriptional programs. Our results establish the utility of an integrated genomics approach in characterizing responses to exposures and identify genomic targets for the advanced investigation of the pathogenesis of DRRD.

genomics↗

bioRxiv2021

Myonuclear maturation dynamics in aged and adult regenerating mouse skeletal muscle

Skeletal muscle cells are multinucleated syncytial cells arising from cell fusion, yet despite sharing a common cytoplasm individual myonuclei express distinct transcriptional programs. Whether individual myonuclei acquire heterogenous transcriptional states via differences in their progenitors, during differentiation, or once their anatomical position is acquired, is not known. We performed transcriptome and pseudotime analysis of single myogenic nuclei from uninjured and post-injury murine skeletal muscle to assess when myonuclear heterogeneity is acquired. Two distinct progenitors contribute to myonuclei, one a non-myogenic fibroblast subtype, and skeletal muscle stem cells the other. Both progenitors enter a single pseudotime trajectory that bifurcates as myonuclei mature into two branches segregated by myosin isoform expression and metabolic profiles, suggesting transcriptional heterogeneity is acquired as myonuclei mature. In aged skeletal muscle myogenic progenitor expansion is perturbed and nuclei from aged muscle display distinct pseudotemporal kinetics compared to nuclei from young mice. In aged mice, the inferred myogenic differentiation trajectory is delayed, altering the distribution of myogenic nuclei in pseudotime, suggesting that altered transcriptional dynamics in nuclei in aged mice may drive age-associated muscle deficits and bias myonuclei towards acquiring oxidative metabolic profiles.

molecular biology↗

bioRxiv2021

Infection of immune competent macrophages expressing functional Slc11a1 alters global gene expression, regulation of metal ions, and infection outcomes

Nutritional immunity involves cellular and physiological responses to invading pathogens, such as limiting iron availability, increasing exposure to bactericidal copper, and manipulating zinc to restrict the growth of pathogens. Manipulation of zinc at the host-pathogen interface depends on both the pathogens identity and the nature of the host cell. Here we examine infection of bone marrow-derived macrophages from 129S6/SvEvTac mice by Salmonella Typhimurium. Unlike Balb/c and C57BL/6 mice, 129S6/SvEvTac mice possess a functional Slc11a1 (Nramp-1), a phagosomal transporter of divalent cations. We carried out global RNA sequencing upon treatment with live or heat-killed Salmonella at 2 Hrs and 18 Hrs post-infection and observed widespread changes in metal transport, metal-dependent, and metal homeostasis genes, suggesting significant remodeling of iron, copper, and zinc availability by host cells. Changes in host cell gene expression suggest infection increases cytosolic zinc while simultaneously limiting zinc within the phagosome. Using a genetically encoded sensor, we demonstrate that cytosolic labile zinc increases 36-fold 12 hrs post-infection. Further, manipulation of zinc in the media alters bacterial clearance and replication, with zinc depletion inhibiting both processes. Comparing our results to published data on infection of C57BL/6 macrophages revealed notable differences in metal regulation and the global immune response, with 129S6 macrophages transitioning from M1 to M2 polarization over the course of infection and showing signs of recovery. Our results reveal that functional Slc11a1 profoundly affects the transcriptional landscape upon infection. Further, our results indicate that manipulation of zinc at the host-pathogen interface is more nuanced than that of iron or copper. 129S6 macrophage leverage intricate means of manipulating zinc availability and distribution to limit the pathogens access to zinc while simultaneously ensuring sufficient zinc to support the immune response. Author summaryMetal ions play an important role in influencing how immune cells such as macrophages respond to infection by pathogens. Because metal ions are both essential to survival, as well toxic when present is excessive amounts, the host and the pathogen have evolved diverse strategies to regulate metal acquisition and availability. Here, we show that the metal transporter slc11a1 plays a critical role in defining the host response to Salmonella infection. Infection causes widespread changes in expression of metal regulatory genes to limit the pathogens access to iron, increase its exposure to copper, and remodel zinc to ensure increased zinc in the cytosol and limited zinc for the pathogen. Macrophages expressing functional slc11a1 have a different profile of metal regulation and vastly different outcomes compared to immune compromised macrophage, demonstrating significantly different nutritional immune responses in immune competent versus immune compromised macrophages.

cell biology↗

bioRxiv2021

Nascent RNA sequencing defines dynamic aryl hydrocarbon receptor signaling responses to wood smoke particles

Heterogeneity of respirable particulates and compounds complicates understanding transcriptional responses to air pollution. Here, we address this problem through applying precision nuclear run-on sequencing (PRO-seq) to measure nascent transcription and the assay for transposase-accessible chromatin using sequencing (ATAC-seq) to airway epithelial cells after wood smoke particle (WSP) exposure. We used transcription factor enrichment analysis to agnostically identify temporally distinct roles for the TCF/SRF family, the aryl hydrocarbon receptor (AHR), and NFkB in regulating transcriptional changes induced by WSP. Transcription of canonical targets of the AHR, such as CYP1A1 and AHRR, was robustly increased after just 30 minutes of WSP exposure, and we discovered novel AHR-regulated pathways and targets including the DNA methyltransferase, DNMT3L. Transcription of these genes and associated enhancers rapidly returned to near baseline by 120 minutes. The kinetics of AHR- and NFkB-regulated responses to WSP were distinguishable based on the timing of both transcriptional responses and chromatin remodeling, with induction of several cytokines implicated in maintaining NFkB responses through 120 minutes of exposure. In aggregate, our data establish a direct and primary role for AHR in mediating airway epithelial responses to WSP and identify crosstalk between AHR and NFkB signaling in controlling pro-inflammatory gene expression. This work also defines an integrated genomics-based strategy for deconvoluting multiplexed transcriptional responses to heterogeneous environmental exposures.

genomics↗

bioRxiv2021

Application of a bioinformatic pipeline to RNA-seq data identifies novel virus-like sequence in human blood

Numerous reports have suggested that infectious agents could play a role in neurodegenerative diseases, but specific etiological agents have not been convincingly demonstrated. To search for candidate agents in an unbiased fashion, we have developed a bioinformatic pipeline that identifies microbial sequences in mammalian RNA-seq data, including sequences with no significant nucleotide similarity hits in GenBank. Effectiveness of the pipeline was tested using publicly available RNA-seq data. We then applied this pipeline to a novel RNA-seq dataset generated from a cohort of 120 samples from amyotrophic lateral sclerosis (ALS) patients and controls, and identified sequences corresponding to known bacteria and viruses, as well as novel virus-like sequences. The presence of these novel virus-like sequences, which were identified in subsets of both patients and controls, were confirmed by quantitative RT-PCR. We believe this pipeline will be a useful tool for the identification of potential etiological agents in the many RNA-seq data sets currently being generated.

bioinformatics↗