Sequencing of RNAs (RNA-Seq) has revolutionized the field of transcriptomics, but the reads obtained often contain errors. Read error correction can have a large impact on our ability to accurately assemble transcripts. This is especially true for de novo transcriptome analysis, where a reference genome is not available. Current read error correction methods, developed for DNA sequence data, cannot handle the overlapping effects of non-uniform abundance, polymorphisms and alternative splicing. Here we present SEquencing Error CorrEction in Rna-seq data (SEECER), a hidden Markov Model (HMM)–based method, which is the first to successfully address these problems. SEECER efficiently learns hundreds of thousands of HMMs and uses these to correct sequencing errors. Using human RNA-Seq data, we show that SEECER greatly improves on previous methods in terms of quality of read alignment to the genome and assembly accuracy. To illustrate the usefulness of SEECER for de novo transcriptome studies, we generated new RNA-Seq data to study the development of the sea cucumber Parastichopus parvimensis. Our corrected assembled transcripts shed new light on two important stages in sea cucumber development. Comparison of the assembled transcripts to known transcripts in other species has also revealed novel transcripts that are unique to sea cucumber, some of which we have experimentally validated.Supporting website: http://sb.cs.cmu.edu/seecer/.

BackgroundDisease progression in the absence of therapy varies significantly in HIV-1 infected individuals. Both viral and host cellular molecules are implicated; however, the exact role of these factors and/or the mechanism involved remains elusive. To understand how microRNAs (miRNAs), which are regulators of transcription and translation, influence host cellular gene expression (mRNA) during HIV-1 infection, we performed a comparative miRNA and mRNA microarray analysis using PBMCs obtained from infected individuals with distinct viral load and CD4 counts.MethodsRNA isolated from PBMCs obtained from HIV-1 seronegative and HIV-1 positive individuals with distinct viral load and CD4 counts were assessed for miRNA and mRNA profile. Selected miRNA and mRNA transcripts were validated using in vivo and in vitro infection model.ResultsOur results indicate that HIV-1 positive individuals with high viral load (HVL) showed a dysregulation of 191 miRNAs and 309 mRNA transcripts compared to the uninfected age and sex matched controls. The miRNAs miR-19b, 146a, 615-3p, 382, 34a, 144 and 155, that are known to target innate and inflammatory factors, were significantly upregulated in PBMCs with high viral load, as were the inflammatory molecules CXCL5, CCL2, IL6 and IL8, whereas defensin, CD4, ALDH1, and Neurogranin (NRGN) were significantly downregulated. Using the transcriptome profile and predicted target genes, we constructed the regulatory networks of miRNA-mRNA pairs that were differentially expressed between control, LVL and HVL subjects. The regulatory network revealed an inverse correlation of several miRNA-mRNA pair expression patterns, suggesting HIV-1 mediated transcriptional regulation is in part likely through miRNA regulation.ConclusionsResults from our studies indicate that gene expression is significantly altered in PBMCs in response to virus replication. It is interesting to note that the infected individuals with low or undetectable viral load exhibit a gene expression profile very similar to control or uninfected subjects. Importantly, we identified several new mRNA targets (Defensin, Neurogranin, AIF) as well as the miRNAs that could be involved in regulating their expression through the miRNA-mRNA interaction.

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Host cells respond to exogenous infectious agents such as viruses, including HIV-1. Studies have evaluated the changes associated with virus infection at the transcriptional and translational levels of the cellular genes involved in specific pathways. While this approach is useful, in our view it provides only a partial view of genome-wide changes. Recently, technological advances in the expression profiling at the microRNA (miRNA) and mRNA levels have made it possible to evaluate the changes in the components of multiple pathways. To understand the role of miRNA and its interplay with host cellular gene expression (mRNA) during HIV-1 infection, we performed a comparative global miRNA and mRNA microarray using human PBMCs infected with HIV-1. The PBMCs were derived from multiple donors and were infected with virus generated from the molecular clone pNL4-3. The results showed that HIV-1 infection led to altered regulation of 21 miRNAs and 444 mRNA more than 2-fold, with a statistical significance of p<0.05. Furthermore, the differentially regulated miRNA and mRNA were shown to be associated with host cellular pathways involved in cell cycle/proliferation, apoptosis, T-cell signaling, and immune activation. We also observed a number of inverse correlations of miRNA and mRNA expression in infected PBMCs, further confirming the interrelationship between miRNA and mRNA regulation during HIV-1 infection. These results for the first time provide evidence that the miRNA profile could be an early indicator of host cellular dysfunction induced by HIV-1.