Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing

Philippe Lefrançois,Ghia M Euskirchen,Joel Rozowsky,Mark Gerstein,Theodore Gibson,Michael Snyder,Raymond K Auerbach,Christopher M Yellman

doi:10.1186/1471-2164-10-37

Philippe Lefrançois, Ghia M Euskirchen + Show 6 more

Open Access

https://doi.org/10.1186/1471-2164-10-37

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Journal: BMC Genomics	Publication Date: Jan 21, 2009
Citations: 209	License type: cc-by

Affiliation: Yale University

Abstract

BackgroundShort-read high-throughput DNA sequencing technologies provide new tools to answer biological questions. However, high cost and low throughput limit their widespread use, particularly in organisms with smaller genomes such as S. cerevisiae. Although ChIP-Seq in mammalian cell lines is replacing array-based ChIP-chip as the standard for transcription factor binding studies, ChIP-Seq in yeast is still underutilized compared to ChIP-chip. We developed a multiplex barcoding system that allows simultaneous sequencing and analysis of multiple samples using Illumina's platform. We applied this method to analyze the chromosomal distributions of three yeast DNA binding proteins (Ste12, Cse4 and RNA PolII) and a reference sample (input DNA) in a single experiment and demonstrate its utility for rapid and accurate results at reduced costs.ResultsWe developed a barcoding ChIP-Seq method for the concurrent analysis of transcription factor binding sites in yeast. Our multiplex strategy generated high quality data that was indistinguishable from data obtained with non-barcoded libraries. None of the barcoded adapters induced differences relative to a non-barcoded adapter when applied to the same DNA sample. We used this method to map the binding sites for Cse4, Ste12 and Pol II throughout the yeast genome and we found 148 binding targets for Cse4, 823 targets for Ste12 and 2508 targets for PolII. Cse4 was strongly bound to all yeast centromeres as expected and the remaining non-centromeric targets correspond to highly expressed genes in rich media. The presence of Cse4 non-centromeric binding sites was not reported previously.ConclusionWe designed a multiplex short-read DNA sequencing method to perform efficient ChIP-Seq in yeast and other small genome model organisms. This method produces accurate results with higher throughput and reduced cost. Given constant improvements in high-throughput sequencing technologies, increasing multiplexing will be possible to further decrease costs per sample and to accelerate the completion of large consortium projects such as modENCODE.

Highlights

Short-read high-throughput DNA sequencing technologies provide new tools to answer biological questions
The strategy was applied to analyze the targets of three yeast DNA binding proteins (Cse4, Ste12 and RNA polymerase II) using chromatin immunoprecipitation (ChIP) and was shown to yield accurate and high quality results
Comparison of polymerase II (PolII) biological replicates By comparing two biological replicates of PolII ChIP DNA with the same barcode (ACGT) we found a close correlation between the target lists (Figure 3a and 3c)

Summary

Introduction

Short-read high-throughput DNA sequencing technologies provide new tools to answer biological questions. Novel high-throughput DNA sequencing technologies have allowed the generation of millions of short reads and have empowered a wide variety of studies such as genome-wide analysis of transcriptomes (RNA-Seq) [1,2,3,4], transcription factor binding sites (ChIP-Seq) [5,6] and whole-genome sequencing and analysis [7,8]. These studies have often been limited by a high cost per sample and low throughput. We included a reference sample for ChIP-Seq termed input DNA

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Conclusion