Classifying next-generation sequencing data using a zero-inflated Poisson model.

High-quality RNA is crucial for obtaining reliable RNA sequencing (RNA-seq) data, with metrics like RNA Integrity Number (RIN). However, these metrics, while effective for evaluating RNA integrity, do not always correlate with RNA-seq data quality, especially at the transcript level. This gap is particularly evident in total RNA-seq, where existing measures such as the coefficient of variation (CV) for read coverage fail to fully capture data quality and are influenced by confounding factors like read coverage depth. To address this, we developed a novel method to assess RNA-seq data quality by quantifying nonuniformity in read coverage while minimizing the influence of read coverage depth. In this study we invented new matric called windowCV (wCV) and applied to a diverse range of RNA-seq datasets, including fresh frozen (FF) and FFPE total RNA-seq data, as well as poly(A)-enriched mRNA-seq data. In mRNA-seq, our method captured 3' read coverage bias, a hallmark of RNA degradation, particularly in longer transcripts. For total RNA-seq data, we identified noisy coverage patterns associated with poor data quality, even in samples with high RIN values. By fitting regression lines between wCV and mean coverage depth (MCD) and calculating the area under the curve (wCVAUC), we refined the assessment to account for RNA quality variability. Using the TCGA pilot study and our own datasets, we demonstrated that wCVAUC reliably identified low quality RNA-seq data and highlighted its impact on downstream analyses, including gene expression quantification and clustering. Importantly, we observed that low RIN values do not always predict poor RNA-seq data quality, as some samples with RIN values below 7 exhibited high-quality RNA-seq data based on wCVAUC. It means that our analyses showed that wCVAUC effectively distinguished high-quality from low-quality samples, including cases where traditional metrics like RIN and CV were insufficient. Additionally, our investigation into the relationships between nonuniformity of read coverage, exon GC content, and RNA localization revealed that the transcript-level RNA-seq data quality of lncRNA genes in FFPE samples is influenced by low exon GC content and nuclear localization. In conclusion, our method provides robust, transcript-level metrics for assessing RNA-seq data quality across platforms, enabling more accurate identification of low-quality data and minimizing biases in downstream analyses. This approach offers a new standard for integrating RNA-seq data quality with sample variability, particularly for challenging datasets such as FFPE and total RNA-seq. Citation Format: Wonyoung Choi, Miyeon Yeon, Jay Lee, Hyo Young Choi, David Neil Hayes. The new approach for measuring nonuniformity of read coverages reveals the quality of RNA-seq data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2489.

Transcriptome deep sequencing (RNA-seq) data analysis is often affected by reference bias introduced by the use of a single linear reference (SLR) genome. Graph-based pangenomes can mitigate this bias by integrating the SLR genome with complex genetic variations within a species; however, their application remains limited owing to a lack of dedicated analytical tools. Here, we present PanGraphRNA, an integrated bioinformatics platform for RNA-seq data analysis using a graph pangenome as reference. Built on the Galaxy web-based framework, PanGraphRNA provides functional modules for constructing, evaluating, and applying graph pangenomes across different population scales, thus enabling accessibility, traceability, and reproducibility throughout the analysis. Applied to both real and simulated RNA-seq data sets from Arabidopsis (Arabidopsis thaliana), PanGraphRNA outperformed the SLR approach, achieving higher read alignment accuracy and more precise gene expression quantification. PanGraphRNA enabled the identification of drought stress-induced genes and flowering time-related quantitative trait loci that were previously missed with the conventional SLR approach. Furthermore, we successfully applied PanGraphRNA to process RNA-seq data sets from rice (Oryza sativa) and maize (Zea mays). By providing standardized, containerized workflows, PanGraphRNA will facilitate transcriptomic research in key plant species, including Arabidopsis, rice, and maize.

BackgroundEpistasis plays an essential rule in understanding the regulation mechanisms and is an essential component of the genetic architecture of the gene expressions. However, interaction analysis of gene expressions remains fundamentally unexplored due to great computational challenges and data availability. Due to variation in splicing, transcription start sites, polyadenylation sites, post-transcriptional RNA editing across the entire gene, and transcription rates of the cells, RNA-seq measurements generate large expression variability and collectively create the observed position level read count curves. A single number for measuring gene expression which is widely used for microarray measured gene expression analysis is highly unlikely to sufficiently account for large expression variation across the gene. Simultaneously analyzing epistatic architecture using the RNA-seq and whole genome sequencing (WGS) data poses enormous challenges.MethodsWe develop a nonlinear functional regression model (FRGM) with functional responses where the position-level read counts within a gene are taken as a function of genomic position, and functional predictors where genotype profiles are viewed as a function of genomic position, for epistasis analysis with RNA-seq data. Instead of testing the interaction of all possible pair-wises SNPs, the FRGM takes a gene as a basic unit for epistasis analysis, which tests for the interaction of all possible pairs of genes and use all the information that can be accessed to collectively test interaction between all possible pairs of SNPs within two genome regions.ResultsBy large-scale simulations, we demonstrate that the proposed FRGM for epistasis analysis can achieve the correct type 1 error and has higher power to detect the interactions between genes than the existing methods. The proposed methods are applied to the RNA-seq and WGS data from the 1000 Genome Project. The numbers of pairs of significantly interacting genes after Bonferroni correction identified using FRGM, RPKM and DESeq were 16,2361, 260 and 51, respectively, from the 350 European samples.ConclusionsThe proposed FRGM for epistasis analysis of RNA-seq can capture isoform and position-level information and will have a broad application. Both simulations and real data analysis highlight the potential for the FRGM to be a good choice of the epistatic analysis with sequencing data.

Gene fusions are important biomarkers for cancer diagnosis, subtype classification and therapeutic decision-making. While fusion detection using RNA-seq data has become a standard practice, existing computational methods primarily focus on identifying canonical exon-to-exon fusions. However, more complex events such as multi-partner fusions, truncations, enhancer hijacking and internal tandem duplications (ITD) can also lead to abnormal function or aberrant transcription of cancer driver genes. To aid discovery of complex and diverse driver fusions, we developed CICERO (CICERO Is Clipping Extended for RNA Optimization), a local assembly-based algorithm that integrates RNA-seq reads bearing aberrant mapping signatures with extensive annotation for ranking candidate fusions. Our benchmark data set, designed to support the main application of RNA-seq fusion analysis, consists of 184 driver fusions from 170 pediatric leukemia, solid tumor and brain tumor detected by paired tumor-normal WGS and orthogonally validated by capture sequencing, RT-PCR and/or FISH. CICERO detected 95% of these fusions with an average ranking of 1.9, whereas ChimeraScan, deFuse, FusionCatcher and STAR-Fusion detected only 63%, 66%, 77% and 63% with an average ranking of 37.0, 9.0, 18.1 and 4.4, respectively. Notably, events such as ITD and rearrangements involving the highly repetitive IGH locus were detected almost exclusively by CICERO. Our re-analysis of 167 RNA-seq data from the TCGA Glioblastoma Multiforme (GBM) cohort unveiled 158 fusions of cancer genes that were not reported previously. These include kinase fusions (KLHL7-BRAF), ITD of EGFR kinase domain and a 13% prevalence of EGFR C-terminal truncation compared to the 6% reported by the TCGA Network. CICERO has greatly improved our ability to discover non-canonical fusions which are overlooked by existing fusion detection methods, and has been used to analyze >2,000 RNA-seq samples generated by the two largest pediatric cancer genomics initiatives: the St. Jude/Washington University Pediatric Cancer Genome Project (PCGP) and the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) project. We anticipate that CICERO will also improve fusion analysis for adult cancer RNA-seq data, as demonstrated through our re-analysis of TCGA-GBM and our recent discovery of MAP3K8 C-terminal truncation fusion in 2% of TCGA melanoma samples. CICERO is accessible via standard (https://github.com/stjude/Cicero) or cloud-based (https://platform.stjude.cloud/tools/rapid_rna-seq) implementation. To further improve accuracy, fusions predicted by CICERO can be curated by FusionEditor (https://proteinpaint.stjude.org/FusionEditor/), an interactive viewer allowing inspection of protein domains involved in the fusion and the gene expression status of fusion-positive samples. Citation Format: Liqing Tian, Yongjin Li, Michael N. Edmonson, Xin Zhou, Scott Newman, Clay McLeod, Yu Liu, Bo Tang, Michael C. Rusch, John Easton, Jing Ma, Austyn Trull, J. Robert Michael, Andrew Thrasher, Charles Mullighan, Suzanne J. Baker, James R. Downing, David W. Ellison, Jinghui Zhang. CICERO: An accurate method for detecting complex and diverse driver fusions using cancer transcriptome sequencing (RNA-seq) data [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5478.

Reproducibility is a fundamental expectation in science and enables investigators to have confidence in their research findings and the ability to compare data from disparate sources, but evaluating reproducibility can be elusive. For example, generating RNA sequencing (RNA-seq) data includes multiple steps where variance can be introduced. Thus, it is unclear if RNA-seq data from different sources can be validly compared. While most studies on RNA-seq reproducibility focus on eukaryotes, we evaluate bias in bacteria using Pseudomonas aeruginosa gene expression data from five laboratory models of cystic fibrosis. We leverage a large data set that includes samples prepared in three different laboratories and paired data sets where the same sample was sequenced using at least two different sequencing pipelines. We report here that expression data are highly reproducible across laboratories. In addition, while samples sequenced with different sequencing pipelines showed significantly more variance in expression profiles than between labs, gene expression was still highly reproducible between sequencing pipelines. Further investigation of expression differences between two sequencing pipelines revealed that library preparation methods were the largest source of error, though analyses to identify the source of this variance were inconclusive. Consistent with the reproducibility of expression between sequencing pipelines, we found that different pipelines detected over 80% of the same differentially expressed genes with large expression differences between conditions. Thus, bacterial RNA-seq data from different sources can be validly compared, facilitating the ability to advance understanding of bacterial behavior and physiology using the wide array of publicly available RNA-seq data sets.IMPORTANCERNA sequencing (RNA-seq) has revolutionized biology, but many steps in RNA-seq workflows can introduce variance, potentially compromising reproducibility. While reproducibility in RNA-seq has been thoroughly investigated in eukaryotes, less is known about pipelines and workflows that introduce variance and biases in bacterial RNA-seq data. By leveraging Pseudomonas aeruginosa transcriptomes in cystic fibrosis models from different laboratories and sequenced with different sequencing pipelines, we directly assess sources of bacterial RNA-seq variance. RNA-seq data were highly reproducible, with the largest variance due to sequencing pipelines, specifically library preparation. Different sequencing pipelines detected overlapping differentially expressed genes, especially those with large expression differences between conditions. This study confirms that different approaches to preparing and sequencing bacterial RNA libraries capture comparable transcriptional profiles, supporting investigators' ability to leverage diverse RNA-seq data sets to advance their science.

We created the PDX Network (PDXNet) Portal to provide an intuitive way for researchers to explore and understand the models, sequencing data, and bioinformatics workflows generated by NCI's PDXNet consortium for research access (https://portal.pdxnetwork.org/). The portal also provides metrics for PDXNet's activities, data processing protocols, and training materials for processing PDX data. The PDXNet Portal highlights model and data resources that include 216 new models across 29 cancer types. The most prevalent cancers represented in the PDX model dataset include invasive breast carcinoma (30.6%), melanoma (18.1%), and adenocarcinoma (14.4%). PDXNet teams have provided 2263 sequencing files from 356 samples across 204 patients, comprising whole exome (82.9%) and RNA seq files (17.1%). The most prevalent cancers represented in the PDXNet sequencing data set include Breast Pleural Effusion (27.2%), Breast Poorly Differentiated (12.5%), and Lung Adenocarcinoma (9.6%). The portal also provides access to 9492 sequencing files across 78 disease types that include 2594 samples across 463 patients uploaded from the NCI Patient-Derived Model Repository. The dataset includes both whole exomes (52.8%) and RNA seq (47.2%) data. The PDMR samples include PDX (82.7%), primary tumor (5.7%), normal germline (5.5), organoid culture (3.2), and Mixed Tumor Culture (2.9). The PDMR dataset also has multiple passages: P0 (21.8%), P1(39.5%), P2 (25.6%), and P3 (8.5%). These models and data resources support ten PDXNet Pilot activities, multiple publications, and international collaborations. PDXNet has also developed a set of 13 robust, validated, and standardized workflows for processing PDXNet whole-exome and RNA seq data. Collectively, these workflows allow for the standardized processing of PDX and complementary human tissues (normal and tumor). Our plan is to continuously update the model and data lists on the PDX portal as resources are generated. We expect that the PDXNet generated models, scheduled to grow to 1000 new models by 2022, will support multi-agent treatment studies, determination of mechanisms of sensitivity and resistance, and pre-clinical trials for example through the COMBO-MATCH program. The robust standard workflows currently processing all PDX sequencing data may also facilitate harmonizing data across studies. Lastly, we expect that the generated sequencing data will support computational approaches for studying cancer evolution and the mechanisms underlying cancer treatments. Citation Format: Soner Koc, Mike Lloyd, Steven Neuhauser, Javad Noodbakhsh, Anuj Srivastava, Xing Yi Woo, Ryan Jeon, Jeffrey Grover, Sara Seepo, Christian Frech, Jack DiGiovanna, PDXNet Consortium, Yvonne A. Evard, Tiffany Wallace, Jeffrey Moscow, James H. Doroshow, Nicholas Mitsuade, Salma Kaochar, Chong-xian Pan, Moon S. Chen, Luis Carvarjal-Carmona, Alana Welm, Bryan Welm, Michael T. Lewis, Govindan Ramaswamy, Li Ding, Shunquang Li, Meenherd Herlyn, Mike Davies, Jack Roth, Funda Meric-Bernstam, Peter Robinson, Carol J. Bult, Brandi Davis-Dusenbery, Dennis A. Dean, Jeffrey H. Chuang. Advancing PDX research through model, data, and bioinformatics with the PDXNet Portal [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3017.

BackgroundRNA sequencing (RNA-seq) is a powerful tool for genome-wide expression profiling of biological samples with the advantage of high-throughput and high resolution. There are many existing algorithms nowadays for quantifying expression levels and detecting differential gene expression, but none of them takes the misaligned reads that are mapped to non-exonic regions into account. We developed a novel algorithm, XBSeq, where a statistical model was established based on the assumption that observed signals are the convolution of true expression signals and sequencing noises. The mapped reads in non-exonic regions are considered as sequencing noises, which follows a Poisson distribution. Given measureable observed and noise signals from RNA-seq data, true expression signals, assuming governed by the negative binomial distribution, can be delineated and thus the accurate detection of differential expressed genes.ResultsWe implemented our novel XBSeq algorithm and evaluated it by using a set of simulated expression datasets under different conditions, using a combination of negative binomial and Poisson distributions with parameters derived from real RNA-seq data. We compared the performance of our method with other commonly used differential expression analysis algorithms. We also evaluated the changes in true and false positive rates with variations in biological replicates, differential fold changes, and expression levels in non-exonic regions. We also tested the algorithm on a set of real RNA-seq data where the common and different detection results from different algorithms were reported.ConclusionsIn this paper, we proposed a novel XBSeq, a differential expression analysis algorithm for RNA-seq data that takes non-exonic mapped reads into consideration. When background noise is at baseline level, the performance of XBSeq and DESeq are mostly equivalent. However, our method surpasses DESeq and other algorithms with the increase of non-exonic mapped reads. Only in very low read count condition XBSeq had a slightly higher false discovery rate, which may be improved by adjusting the background noise effect in this situation. Taken together, by considering non-exonic mapped reads, XBSeq can provide accurate expression measurement and thus detect differential expressed genes even in noisy conditions.

BackgroundRNA-Seq has become one of the most widely used applications based on next-generation sequencing technology. However, raw RNA-Seq data may have quality issues, which can significantly distort analytical results and lead to erroneous conclusions. Therefore, the raw data must be subjected to vigorous quality control (QC) procedures before downstream analysis. Currently, an accurate and complete QC of RNA-Seq data requires of a suite of different QC tools used consecutively, which is inefficient in terms of usability, running time, file usage, and interpretability of the results.ResultsWe developed a comprehensive, fast and easy-to-use QC pipeline for RNA-Seq data, RNA-QC-Chain, which involves three steps: (1) sequencing-quality assessment and trimming; (2) internal (ribosomal RNAs) and external (reads from foreign species) contamination filtering; (3) alignment statistics reporting (such as read number, alignment coverage, sequencing depth and pair-end read mapping information). This package was developed based on our previously reported tool for general QC of next-generation sequencing (NGS) data called QC-Chain, with extensions specifically designed for RNA-Seq data. It has several features that are not available yet in other QC tools for RNA-Seq data, such as RNA sequence trimming, automatic rRNA detection and automatic contaminating species identification. The three QC steps can run either sequentially or independently, enabling RNA-QC-Chain as a comprehensive package with high flexibility and usability. Moreover, parallel computing and optimizations are embedded in most of the QC procedures, providing a superior efficiency. The performance of RNA-QC-Chain has been evaluated with different types of datasets, including an in-house sequencing data, a semi-simulated data, and two real datasets downloaded from public database. Comparisons of RNA-QC-Chain with other QC tools have manifested its superiorities in both function versatility and processing speed.ConclusionsWe present here a tool, RNA-QC-Chain, which can be used to comprehensively resolve the quality control processes of RNA-Seq data effectively and efficiently.

The zero-inflated Poisson (ZIP) regression model is often employed in public health research to examine the relationships between exposures of interest and a count outcome exhibiting many zeros, in excess of the amount expected under sampling from a Poisson distribution. The regression coefficients of the ZIP model have latent class interpretations, which correspond to a susceptible subpopulation at risk for the condition with counts generated from a Poisson distribution and a non-susceptible subpopulation that provides the extra or excess zeros. The ZIP model parameters, however, are not well suited for inference targeted at marginal means, specifically, in quantifying the effect of an explanatory variable in the overall mixture population. We develop a marginalized ZIP model approach for independent responses to model the population mean count directly, allowing straightforward inference for overall exposure effects and empirical robust variance estimation for overall log-incidence density ratios. Through simulation studies, the performance of maximum likelihood estimation of the marginalized ZIP model is assessed and compared with other methods of estimating overall exposure effects. The marginalized ZIP model is applied to a recent study of a motivational interviewing-based safer sex counseling intervention, designed to reduce unprotected sexual act counts.

ObjectiveTo propose a more realistic model for disease cluster detection, through a modification of the spatial scan statistic to account simultaneously for inflated zeros and overdispersion.IntroductionSpatial Scan Statistics [1] usually assume Poisson or Binomial distributed data, which is not adequate in many disease surveillance scenarios. For example, small areas distant from hospitals may exhibit a smaller number of cases than expected in those simple models. Also, underreporting may occur in underdeveloped regions, due to inefficient data collection or the difficulty to access remote sites. Those factors generate excess zero case counts or overdispersion, inducing a violation of the statistical model and also increasing the type I error (false alarms). Overdispersion occurs when data variance is greater than the predicted by the used model. To accommodate it, an extra parameter must be included; in the Poisson model, one makes the variance equal to the mean.MethodsTools like the Generalized Poisson (GP) and the Double Poisson [2] may be a better option for this kind of problem, modeling separately the mean and variance, which could be easily adjusted by covariates. When excess zeros occur, the Zero Inflated Poisson (ZIP) model is used, although ZIP’s estimated parameters may be severely biased if nonzero counts are too dispersed, compared to the Poisson distribution. In this case the Inflated Zero models for the Generalized Poisson (ZIGP), Double Poisson (ZIDP) and Negative Binomial (ZINB) could be good alternatives to the joint modeling of excess zeros and overdispersion. By one hand, Zero Inflated Poisson (ZIP) models were proposed using the spatial scan statistic to deal with the excess zeros [3]. By the other hand, another spatial scan statistic was based on a Poisson-Gamma mixture model for overdispersion [4]. In this work we present a model which includes inflated zeros and overdispersion simultaneously, based on the ZIDP model. Let the parameter p indicate the zero inflation. As the the remaining parameters of the observed cases map and the parameter p are not independent, the likelihood maximization process is not straightforward; it becomes even more complicated when we include covariates in the analysis. To solve this problem we introduce a vector of latent variables in order to factorize the likelihood, and obtain a facilitator for the maximization process using the E-M (Expectation-Maximization) algorithm. We derive the formulas to maximize iteratively the likelihood, and implement a computer program using the E-M algorithm to estimate the parameters under null and alternative hypothesis. The p-value is obtained via the Fast Double Bootstrap Test [5].ResultsNumerical simulations are conducted to assess the effectiveness of the method. We present results for Hanseniasis surveillance in the Brazilian Amazon in 2010 using this technique. We obtain the most likely spatial clusters for the Poisson, ZIP, Poisson-Gamma mixture and ZIDP models and compare the results.ConclusionsThe Zero Inflated Double Poisson Spatial Scan Statistic for disease cluster detection incorporates the flexibility of previous models, accounting for inflated zeros and overdispersion simultaneously.The Hanseniasis study case map, due to excess of zero cases counts in many municipalities of the Brazilian Amazon and the presence of overdispersion, was a good benchmark to test the ZIDP model. The results obtained are easier to understand compared to each of the previous spatial scan statistic models, the Zero Inflated Poisson (ZIP) model and the Poisson-Gamma mixture model for overdispersion, taken separetely. The E-M algorithm and the Fast Double Bootstrap test are computationally efficient for this type of problem.

BackgroundPost-FDA approval immune therapy for cancer becomes widely used for locally advanced or metastatic urothelial carcinomas. There are patients that may suffer severe side effects from these immune checkpoint blockades...

Herpesviruses employ extensive bidirectional transcription of overlapping genes to overcome length constraints on their gene product repertoire. As a consequence, many lytic transcripts cannot be measured individually by reverse transcription-quantitative PCR (RT-qPCR) or conventional RNA sequencing (RNA-seq) analysis. A. G. Bruce, S. Barcy, T. DiMaio, E. Gan, et al. (Pathogens 6:11, 2017, https://doi.org/10.3390/pathogens6010011) have proposed an approximation method using unique coding sequences (UCDS) to estimate lytic gene abundance from Kaposi's sarcoma-associated herpesvirus (KSHV) RNA-seq data. Although UCDS has been widely employed, its accuracy, to our knowledge, has never been rigorously validated for any herpesvirus. In this study, we use cap analysis of gene expression sequencing (CAGE-seq) as a gold-standard to determine the accuracy of UCDS for estimating Epstein-Barr virus (EBV) lytic gene expression levels from RNA-seq data. We also introduce the Unique TranScript (UTS) method, which, like UCDS, estimates transcript abundance from changes in mean RNA-seq read depth. UTS is distinguished by its use of empirically determined 5' and 3' transcript ends rather than coding sequence annotations. Compared to conventional read assignment, both UCDS and UTS improved the accuracy of quantitation of overlapping genes, with UTS giving the most-accurate results. The UTS method discards fewer reads and may be advantageous for experiments with less sequencing depth. UTS is compatible with any aligner and, unlike isoform-aware alignment methods, can be implemented on a laptop computer. Our findings demonstrate that the accuracy achieved by complex and expensive techniques such as CAGE-seq can be approximated using conventional short-read RNA-seq data when read assignment methods address transcript overlap. Although our study focuses on EBV transcription, the UTS method should be applicable across all herpesviruses as well as to other genomes with extensively overlapping transcriptomes. IMPORTANCE Many viruses employ extensively overlapping transcript structures. This complexity makes it difficult to quantify gene expression by using conventional methods, including RNA-seq. Although high-throughput techniques that overcome these limitations exist, they are complex, expensive, and scarce in the herpesvirus literature relative to short-read RNA-seq. Here, using Epstein-Barr virus (EBV) as a model, we demonstrate that conventional RNA-seq analysis methods fail to accurately quantify the abundances of many overlapping transcripts. We further show that the previously described Unique CoDing Sequence (UCDS) method and our Unique TranScript (UTS) method greatly improve the accuracy of EBV lytic gene measurements obtained from RNA-seq data. The UTS method has the advantages of discarding fewer reads and being implementable on a laptop computer. Although this study focuses on EBV, the UCDS and UTS methods should be applicable across herpesviruses and for other viruses that make extensive use of overlapping transcription.

BackgroundComplex designs are common in (observational) clinical studies. Sequencing data for such studies are produced more and more often, implying challenges for the analysis, such as excess of zeros, presence of random effects and multi-parameter inference. Moreover, when sample sizes are small, inference is likely to be too liberal when, in a Bayesian setting, applying a non-appropriate prior or to lack power when not carefully borrowing information across features.ResultsWe show on microRNA sequencing data from a clinical cancer study how our software ShrinkBayes tackles the aforementioned challenges. In addition, we illustrate its comparatively good performance on multi-parameter inference for groups using a data-based simulation. Finally, in the small sample size setting, we demonstrate its high power and improved FDR estimation by use of Gaussian mixture priors that include a point mass.ConclusionShrinkBayes is a versatile software package for the analysis of count-based sequencing data, which is particularly useful for studies with small sample sizes or complex designs.

Most of the existing methodologies for evaluating heterogeneity in zero-inflated Poisson (ZIP) models are often assuming that the Poisson mean is a function of nuisance parameters. However, these nuisance parameters can be misspecified when performing these methodologies, the validity and the power of the test may be affected. In this article, we primarily focus on investigating the impact of misspecification on the performance of score test for homogeneity in ZIP models. Through an intensive simulation study, we find that: 1) under misspecification, the limiting distribution of the score test statistic under the null no longer follows a distribution. A parametric bootstrap methodology is suggested to use to find the true null limiting distribution of the score test statistic; 2) the power of the test decreases as the number of covariates in the Poisson mean increases. The test with a constant Poisson mean has the highest power, even compared to the test with a well-specified mean. At last, simulation results are applied to the Wuhan Inpatient Care Insurance data which contain excess zeros.

A Fusion Transcript (FT) is a RNA molecule product of cis/trans-splicing of two differently annotated elements, either genes or Transposable Elements (TE). It may be coding or non-coding. There are many examples of gene-gene FTs in cancer. Lately, FTs have been also reported in control conditions in different organisms. Moreover, other authors argue that a FT might be a product of artifacts, either experimental or computational. In a recent work, we have developed a pipeline to predict FTs between an exon and a TE. In it, we have found 813 exon-TE FTs from 438 different genes. This prediction was made starting with RNA-seq data from Nucleus Accumbens in mouse (M. musculus) treated with cocaine vs. control. From that group, 20 candidates were taken and all were validated experimentally through PCR. However, the depth of sequencing is low. We can further gather supporting evidence of their expression by filtering the candidates, which could be regulated by small RNAs, the same way piRNAs regulate TEs. We aligned around 5 million mouse piRNAs against the 813 mouse exon-TE Fts, identifying 112 piRNAs that map only across the juction point of exon-TE FTs, in 56 genes. These piRNAs do not map on the reference genome or on the transcriptome sequences. These results suggest that either: 1) piRNAs might regulate the expression of exon-TE FTs, or 2) the exon-TE Fts may be the origin of piRNAs. In either case, they suggest that exon-TE FTs may express in germline. Grant Fondecyt #11140869.