@article {32, title = {Normalization of RNA-sequencing data from samples with varying mRNA levels.}, journal = {PLoS One}, volume = {9}, year = {2014}, month = {2014}, pages = {e89158}, abstract = {

Methods for normalization of RNA-sequencing gene expression data commonly assume equal total expression between compared samples. In contrast, scenarios of global gene expression shifts are many and increasing. Here we compare the performance of three normalization methods when polyA(+) RNA content fluctuates significantly during zebrafish early developmental stages. As a benchmark we have used reverse transcription-quantitative PCR. The results show that reads per kilobase per million (RPKM) and trimmed mean of M-values (TMM) normalization systematically leads to biased gene expression estimates. Biological scaling normalization (BSN), designed to handle differences in total expression, showed improved accuracy compared to the two other methods in estimating transcript level dynamics. The results have implications for past and future studies using RNA-sequencing on samples with different levels of total or polyA(+) RNA.

}, keywords = {Animals, Base Sequence, Gene Expression, Gene Expression Profiling, Polymerase Chain Reaction, RNA, Messenger, Sequence Analysis, RNA, Zebrafish}, issn = {1932-6203}, doi = {10.1371/journal.pone.0089158}, author = {Aanes, H{\r a}vard and Winata, Cecilia L and Moen, Lars F and {\O}strup, Olga and Mathavan, Sinnakaruppan and Collas, Philippe and Rognes, Torbj{\o}rn and Alestr{\"o}m, Peter} } @article {36, title = {Zebrafish mRNA sequencing deciphers novelties in transcriptome dynamics during maternal to zygotic transition.}, journal = {Genome Res}, volume = {21}, year = {2011}, month = {2011 Aug}, pages = {1328-38}, abstract = {

Maternally deposited mRNAs direct early development before the initiation of zygotic transcription during mid-blastula transition (MBT). To study mechanisms regulating this developmental event in zebrafish, we applied mRNA deep sequencing technology and generated comprehensive information and valuable resources on transcriptome dynamics during early embryonic (egg to early gastrulation) stages. Genome-wide transcriptome analysis documented at least 8000 maternal genes and identified the earliest cohort of zygotic transcripts. We determined expression levels of maternal and zygotic transcripts with the highest resolution possible using mRNA-seq and clustered them based on their expression pattern. We unravel delayed polyadenylation in a large cohort of maternal transcripts prior to the MBT for the first time in zebrafish. Blocking polyadenylation of these transcripts confirms their role in regulating development from the MBT onward. Our study also identified a large number of novel transcribed regions in annotated and unannotated regions of the genome, which will facilitate reannotation of the zebrafish genome. We also identified splice variants with an estimated frequency of 50\%-60\%. Taken together, our data constitute a useful genomic information and valuable transcriptome resource for gene discovery and for understanding the mechanisms of early embryogenesis in zebrafish.

}, keywords = {Animals, Base Sequence, Genome, RNA, Messenger, RNA, Messenger, Stored, Sequence Analysis, RNA, Transcriptome, Zebrafish, Zebrafish Proteins, Zygote}, issn = {1549-5469}, doi = {10.1101/gr.116012.110}, author = {Aanes, H{\r a}vard and Winata, Cecilia L and Lin, Chi Ho and Chen, Jieqi P and Srinivasan, Kandhadayar G and Lee, Serene G P and Lim, Adrian Y M and Hajan, Hajira Shreen and Collas, Philippe and Bourque, Guillaume and Gong, Zhiyuan and Korzh, Vladimir and Alestr{\"o}m, Peter and Mathavan, Sinnakaruppan} }