Abstract
Multiplexing samples in sequencing experiments is a common approach to maximize information yield while minimizing cost. In most cases the number of samples that are multiplexed is determined by financial consideration or experimental convenience, with limited understanding on the effects on the experimental results. Here we set to examine the impact of multiplexing ChIP-seq experiments on the ability to identify a specific epigenetic modification. We performed peak detection analyses to determine the effects of multiplexing. These include false discovery rates, size, position and statistical significance of peak detection, and changes in gene annotation. We found that, for histone marker H3K4me3, one can multiplex up to 8 samples (7 IP + 1 input) at ~21 million single-end reads each and still detect over 90% of all peaks found when using a full lane for sample (~181 million reads). Furthermore, there are no variations introduced by indexing or lane batch effects and importantly there is no significant reduction in the number of genes with neighboring H3K4me3 peaks. We conclude that, for a well characterized antibody and, therefore, model IP condition, multiplexing 8 samples per lane is sufficient to capture most of the biological signal.
Highlights
Knowledge of protein-DNA interactions contributes to the understanding of gene expression regulation, and understanding of biological processes and disease states
The technique of chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) is commonly used for genome-wide identification of protein-DNA interaction sites, and epigenetic modifications [1, 2]
Diffuse large B-cell lymphoma (DLBCL) cell line OCI-Ly7 (ACC688 Deutsche Sammlung von Mikroorganismen und Zellkulturen Braunschweig, Germany) was grown in medium containing 90% Iscove's, 10% fetal calf serum and supplemented with penicillin G and streptomycin
Summary
Knowledge of protein-DNA interactions contributes to the understanding of gene expression regulation, and understanding of biological processes and disease states. The technique of chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) is commonly used for genome-wide identification of protein-DNA interaction sites (e.g. transcription factors), and epigenetic modifications (e.g. histone and DNA modifications) [1, 2]. In contrast to its micro-array predecessor ChIP-ChIP, the ChIP-seq assay provides unbiased genome-wide survey of all protein-DNA interactions and higher genomic resolution of PLOS ONE | DOI:10.1371/journal.pone.0129350. In recent years ChIP-seq has become the primary method for surveying protein-DNA interactions it remains a challenging technique to master in part because of vast differences in efficiency of DNA capture. Despite increasing experience and knowledge about the technique [3] there has been no systematic detailed analysis of the impact of sequencing depth on the results of ChIP-seq experiments
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
