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Ferrari, Francesco

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Ferrari

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Francesco

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Ferrari, Francesco
Author's Publications: search.filters.isAuthorOfPublication.529757e0-0cf4-4539-8904-71f01c38f042

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    Impact of sequencing depth in ChIP-seq experiments
    (Oxford University Press, 2014) Jung, Lucy; Luquette, Joe; Ho, Joshua W.K.; Ferrari, Francesco; Tolstorukov, Michael; Minoda, Aki; Issner, Robbyn; Epstein, Charles B.; Karpen, Gary H.; Kuroda, Mitzi; Park, Peter
    In a chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) experiment, an important consideration in experimental design is the minimum number of sequenced reads required to obtain statistically significant results. We present an extensive evaluation of the impact of sequencing depth on identification of enriched regions for key histone modifications (H3K4me3, H3K36me3, H3K27me3 and H3K9me2/me3) using deep-sequenced datasets in human and fly. We propose to define sufficient sequencing depth as the number of reads at which detected enrichment regions increase <1% for an additional million reads. Although the required depth depends on the nature of the mark and the state of the cell in each experiment, we observe that sufficient depth is often reached at <20 million reads for fly. For human, there are no clear saturation points for the examined datasets, but our analysis suggests 40–50 million reads as a practical minimum for most marks. We also devise a mathematical model to estimate the sufficient depth and total genomic coverage of a mark. Lastly, we find that the five algorithms tested do not agree well for broad enrichment profiles, especially at lower depths. Our findings suggest that sufficient sequencing depth and an appropriate peak-calling algorithm are essential for ensuring robustness of conclusions derived from ChIP-seq data.
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    Rearranging the chromatin for pluripotency
    (Landes Bioscience, 2014) Ferrari, Francesco; Apostolou, Effie; Park, Peter; Hochedlinger, Konrad
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    Ascorbic Acid Prevents Loss of Dlk1-Dio3 Imprinting and Facilitates Generation of All-iPS Cell Mice from Terminally Differentiated B Cells
    (Nature Publishing Group, 2012) Stadtfeld, Matthias; Apostolou, Effie; Chen, Taiping; Oi, Steen; Bestor, Tim; Ferrari, Francesco; Choi, Jiho; Walsh, Ryan M.; Kim, Sang Yong; Shioda, Toshi; Park, Peter; Hochedlinger, Konrad
    The generation of induced pluripotent stem cells (iPSCs) often results in aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster, compromising the ability to generate entirely iPSC-derived adult mice ('all-iPSC mice'). Here, we show that reprogramming in the presence of ascorbic acid attenuates hypermethylation of Dlk1-Dio3 by enabling a chromatin configuration that interferes with binding of the de novo DNA methyltransferase Dnmt3a. This approach allowed us to generate all-iPSC mice from mature B cells, which have until now failed to support the development of exclusively iPSC-derived postnatal animals. Our data show that transcription factor–mediated reprogramming can endow a defined, terminally differentiated cell type with a developmental potential equivalent to that of embryonic stem cells. More generally, these findings indicate that culture conditions during cellular reprogramming can strongly influence the epigenetic and biological properties of the resultant iPSCs.