Project description:Many library preparation methods are available for gene expression quantification. Here, we sequenced and analysed Universal Human Reference RNA (UHRR) prepared using Smart-Seq2, TruSeq (public data) and a protocol using unique molecular identifiers (UMIs) that all include the ERCC spike-in mRNAs to investigate the effects of amplification bias on expression quantification. UHRR 10 and 12 replicates for Smart-seq2 and UMI-seq library preparation methods, respectively.
Project description:Many library preparation methods are available for gene expression quantification. Here, we sequenced and analysed Universal Human Reference RNA (UHRR) prepared using Smart-Seq2, TruSeq (public data) and a protocol using unique molecular identifiers (UMIs) that all include the ERCC spike-in mRNAs to investigate the effects of amplification bias on expression quantification.
Project description:Analysis of RNA samples by massive parallel sequencing holds the promise to assay gene expression in both a quantitative and qualitative fashion and therefore allows for digital gene expression (DGE) profiling. We assessed the effect of different experimental approaches by generating small RNA libraries from a biological sample as well as an equimolar pool of synthetic miRNAs and analyzed the results using capillary dideoxy sequencing and next-generation sequencing platforms (Roche/454, AB/SOLiD and Illumina/Solexa). Whereas different sequencing platforms provided highly similar results, large differences in DGE profiles were observed depending on the library preparation method used. Nevertheless, our results indicate that the preferential nature of the library preparation methods is systematic and highly reproducible and we show that DGE is well suited for the quantification of relative expression differences between samples. Keywords: Transcriptome analysis Examination of three different library preparation methods for small RNAs, two replicates per library method
Project description:Analysis of RNA samples by massive parallel sequencing holds the promise to assay gene expression in both a quantitative and qualitative fashion and therefore allows for digital gene expression (DGE) profiling. We assessed the effect of different experimental approaches by generating small RNA libraries from a biological sample as well as an equimolar pool of synthetic miRNAs and analyzed the results using capillary dideoxy sequencing and next-generation sequencing platforms (Roche/454, AB/SOLiD and Illumina/Solexa). Whereas different sequencing platforms provided highly similar results, large differences in DGE profiles were observed depending on the library preparation method used. Nevertheless, our results indicate that the preferential nature of the library preparation methods is systematic and highly reproducible and we show that DGE is well suited for the quantification of relative expression differences between samples. Keywords: Transcriptome analysis Examination of three different library preparation methods for small RNAs, two replicates per library method
Project description:We performed a parallel analysis of commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se generates pronounced sequencing biases, and subsequent fragmentation and amplification steps lead to several-fold overrepresentation of these artefacts. Standard pre-bisulfite library preparation methods lead to a significantly biased genomic sequence representation and a marked overestimation of methylation levels. We have integrated a bias diagnostic tool in the Bismark package and propose that amplification-free and post-bisulfite procedures should become the gold standard for WGBS library preparation.
Project description:Transcriptome profiling of wild type and cfo1 mutant with fluconazole treatment in Cryptococcus neoformans var. grubii H99 Purpose: The goals of this study are to compare cfo1 mutant transcriptome profiling (RNA-seq) to wild-type with or without fluconazole treatment in Cryptococcus neoformans var. grubii H99. Methods: mRNA profiles of wild-type and cfo1 mutant with or without fluconazole treatment were generated by RNA-Seq, using Illumina GAIIx. The sequence reads that passed quality filters were mapped to reference genome and the normalized RPKM values were calculated by CLC Genomics Workbench. Results: Compared to wild-type, a number of genes were differentially expressed in the cfo1 mutant, especially genes involved in iron homeostasis and transport, ergosterol biosynthesis, mitochondrial function and respiration. Conclusions: Our data suggested reduced expression of the genes in the respiratory chain is the main reason for altered antifungal sensitivity of the cfo1 mutant. The results of our study revealed that iron uptake plays a key role in fluconazole sensitivity of C. neoformans.
Project description:We systematically evaluated alternate strategies for WGBS studies, taking into account opportunites around library preparation methods, sequencing platforms and analysis pipeline optimization. We also assessed the performance and precision of the WGBS method relative to the methylation arrays, in an effort to provide data-driven recommendations for future WGBS studies, in particularly with respect to minimum coverage.
Project description:We systematically evaluated alternate strategies for WGBS studies, taking into account opportunites around library preparation methods, sequencing platforms and analysis pipeline optimization. We also assessed the performance and precision of the WGBS method relative to the methylation arrays, in an effort to provide data-driven recommendations for future WGBS studies, in particularly with respect to minimum coverage.
