title: Impact of sequencing depth in ChIP-seq experiments.
abstract: 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.
authors.affiliation: Center for Biomedical Informatics, Harvard Medical School, Boston, MA 02115, USA Division of Genetics, Brigham and Women's Hospital & Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Youngsook L
authors.initials: YL
authors.lastname: Jung
authors.affiliation: Center for Biomedical Informatics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Lovelace J
authors.initials: LJ
authors.lastname: Luquette
authors.affiliation: Center for Biomedical Informatics, Harvard Medical School, Boston, MA 02115, USA Division of Genetics, Brigham and Women's Hospital & Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Joshua W K
authors.initials: JW
authors.lastname: Ho
authors.affiliation: Center for Biomedical Informatics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Francesco
authors.initials: F
authors.lastname: Ferrari
authors.affiliation: Division of Genetics, Brigham and Women's Hospital & Harvard Medical School, Boston, MA 02115, USA Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Tolstorukov
authors.affiliation: Department of Genome Dynamics, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Aki
authors.initials: A
authors.lastname: Minoda
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
authors.forename: Robbyn
authors.initials: R
authors.lastname: Issner
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
authors.forename: Charles B
authors.initials: CB
authors.lastname: Epstein
authors.affiliation: Department of Genome Dynamics, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Gary H
authors.initials: GH
authors.lastname: Karpen
authors.affiliation: Division of Genetics, Brigham and Women's Hospital & Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Mitzi I
authors.initials: MI
authors.lastname: Kuroda
authors.affiliation: Center for Biomedical Informatics, Harvard Medical School, Boston, MA 02115, USA Division of Genetics, Brigham and Women's Hospital & Harvard Medical School, Boston, MA 02115, USA Informatics Program, Children's Hospital, Boston, MA 02115, USA peter_park@harvard.edu.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Park
mesh_terms: D000465:Algorithms; D000818:Animals; D047369:Chromatin Immunoprecipitation; D004331:Drosophila melanogaster / Q000235:genetics; D015894:Genome, Human; D049750:Genome, Insect; D015698:Genomic Library; D059014:High-Throughput Nucleotide Sequencing; D006657:Histones / Q000378:metabolism; D006801:Humans; D008957:Models, Genetic; D011499:Protein Processing, Post-Translational; D017422:Sequence Analysis, DNA
chemical_list: D006657:Histones