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Genomics Core FacilityPublications

Accounting for technical noise in single-cell RNA-seq experiments.
Brennecke, P., Anders, S., Kim, J.K., Kolodziejczyk, A.A., Zhang, X., Proserpio, V., Baying, B., Benes, V., Teichmann, S.A., Marioni, J.C. & Heisler, M.G.
Nat Methods. 2013 Nov;10(11):1093-5. doi: 10.1038/nmeth.2645. Epub 2013 Sep 22.
Single-cell RNA-seq can yield valuable insights about the variability within a population of seemingly homogeneous cells. We developed a quantitative statistical method to distinguish true biological variability from the high levels of technical noise in single-cell experiments. Our approach quantifies the statistical significance of observed cell-to-cell variability in expression strength on a gene-by-gene basis. We validate our approach using two independent data sets from Arabidopsis thaliana and Mus musculus.
Europe PMC

The need for transparency and good practices in the qPCR literature.
Bustin, S.A., Benes, V., Garson, J., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G., Wittwer, C.T., Schjerling, P., Day, P.J., Abreu, M., Aguado, B., Beaulieu, J.F., Beckers, A., Bogaert, S., Browne, J.A., Carrasco-Ramiro, F., Ceelen, L., Ciborowski, K., Cornillie, P., Coulon, S., Cuypers, A., De Brouwer, S., De Ceuninck, L., De Craene, J., De Naeyer, H., De Spiegelaere, W., Deckers, K., Dheedene, A., Durinck, K., Ferreira-Teixeira, M., Fieuw, A., Gallup, J.M., Gonzalo-Flores, S., Goossens, K., Heindryckx, F., Herring, E., Hoenicka, H., Icardi, L., Jaggi, R., Javad, F., Karampelias, M., Kibenge, F., Kibenge, M., Kumps, C., Lambertz, I., Lammens, T., Markey, A., Messiaen, P., Mets, E., Morais, S., Mudarra-Rubio, A., Nakiwala, J., Nelis, H., Olsvik, P.A., Perez-Novo, C., Plusquin, M., Remans, T., Rihani, A., Rodrigues-Santos, P., Rondou, P., Sanders, R., Schmidt-Bleek, K., Skovgaard, K., Smeets, K., Tabera, L., Toegel, S., Van Acker, T., Van den Broeck, W., Van der Meulen, J., Van Gele, M., Van Peer, G., Van Poucke, M., Van Roy, N., Vergult, S., Wauman, J., Tshuikina-Wiklander, M., Willems, E., Zaccara, S., Zeka, F. & Vandesompele, J.
Nat Methods. 2013 Oct 30;10(11):1063-7. doi: 10.1038/nmeth.2697.
Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.
Europe PMC

Multiple epigenetic mechanisms and the piRNA pathway enforce LINE1 silencing during adult spermatogenesis.
Di Giacomo, M., Comazzetto, S., Saini, H., De Fazio, S., Carrieri, C., Morgan, M., Vasiliauskaite, L., Benes, V., Enright, A.J. & O'Carroll, D.
Mol Cell. 2013 May 23;50(4):601-8. doi: 10.1016/j.molcel.2013.04.026.
Transposons present an acute challenge to the germline, and mechanisms that repress their activity are essential for transgenerational genomic integrity. LINE1 (L1) is the most successful retrotransposon and is epigenetically repressed by CpG DNA methylation. Here, we identify two additional important mechanisms by which L1 is repressed during spermatogenesis. We demonstrate that the Piwi protein Mili and the piRNA pathway are required to posttranscriptionally silence L1 in meiotic pachytene cells even in the presence of normal L1 DNA methylation. Strikingly, in the absence of both a functional piRNA pathway and DNA methylation, L1 elements are normally repressed in mitotic stages of spermatogenesis. Accordingly, we find that the euchromatic repressive histone H3 dimethylated lysine 9 modification cosuppresses L1 expression therein. We demonstrate the existence of multiple epigenetic mechanisms that in conjunction with the piRNA pathway sequentially enforce L1 silencing and genomic stability during mitotic and meiotic stages of adult spermatogenesis.
Europe PMC

An efficient method for genome-wide polyadenylation site mapping and RNA quantification.
Wilkening, S., Pelechano, V., Jarvelin, A.I., Tekkedil, M.M., Anders, S., Benes, V. & Steinmetz, L.M.
Nucleic Acids Res. 2013 Mar 1;41(5):e65. doi: 10.1093/nar/gks1249. Epub 2013 Jan7.
The use of alternative poly(A) sites is common and affects the post-transcriptional fate of mRNA, including its stability, subcellular localization and translation. Here, we present a method to identify poly(A) sites in a genome-wide and strand-specific manner. This method, termed 3'T-fill, initially fills in the poly(A) stretch with unlabeled dTTPs, allowing sequencing to start directly after the poly(A) tail into the 3'-untranslated regions (UTR). Our comparative analysis demonstrates that it outperforms existing protocols in quality and throughput and accurately quantifies RNA levels as only one read is produced from each transcript. We use this method to characterize the diversity of polyadenylation in Saccharomyces cerevisiae, showing that alternative RNA molecules are present even in a genetically identical cell population. Finally, we observe that overlap of convergent 3'-UTRs is frequent but sharply limited by coding regions, suggesting factors that restrict compression of the yeast genome.
Europe PMC