Please cite this article in press as: C.-P. Soong, et al., Development of a novel method to create double-strand break repair fingerprints using next-generation sequencing, DNA Repair (2014), http://dx.doi.org/10.1016/j.dnarep.2014.12.002 ARTICLE IN PRESS G Model DNAREP-2029; No. of Pages 10 DNA Repair xxx (2014) xxx–xxx Contents lists available at ScienceDirect DNA Repair j ourna l ho me pa ge: www.elsevier.com/locate/dnarepair Development of a novel method to create double-strand break repair fingerprints using next-generation sequencing Chen-Pang Soong a,1 , Gregory A. Breuer b,1 , Ryan A. Hannon b , Savina D. Kim b , Ahmed F. Salem b , Guilin Wang c , Ruoxi Yu b , Nicholas J. Carriero d , Robert Bjornson d , Ranjini K. Sundaram b , Ranjit S. Bindra b,∗ a Department of Internal Medicine, University of Connecticut, Farmington, CT 06030, United States b Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, United States c Yale Center for Genomic Analysis (YCGA), Orange, CT 06477, United States d Department of Computer Science, Yale University, New Haven, CT 06511, United States a r t i c l e i n f o Article history: Received 1 August 2014 Received in revised form 5 November 2014 Accepted 9 December 2014 Available online xxx Keywords: NHEJ HR I-SceI DSB repair a b s t r a c t Efficient DNA double-strand break (DSB) repair is a critical determinant of cell survival in response to DNA damaging agents, and it plays a key role in the maintenance of genomic integrity. Homologous recombi- nation (HR) and non-homologous end-joining (NHEJ) represent the two major pathways by which DSBs are repaired in mammalian cells. We now understand that HR and NHEJ repair are composed of multi- ple sub-pathways, some of which still remain poorly understood. As such, there is great interest in the development of novel assays to interrogate these key pathways, which could lead to the development of novel therapeutics, and a better understanding of how DSBs are repaired. Furthermore, assays which can measure repair specifically at endogenous chromosomal loci are of particular interest, because of an emerging understanding that chromatin interactions heavily influence DSB repair pathway choice. Here, we present the design and validation of a novel, next-generation sequencing-based approach to study DSB repair at chromosomal loci in cells. We demonstrate that NHEJ repair “fingerprints” can be identified using our assay, which are dependent on the status of key DSB repair proteins. In addition, we have validated that our system can be used to detect dynamic shifts in DSB repair activity in response to specific perturbations. This approach represents a unique alternative to many currently available DSB repair assays, which typical rely on the expression of reporter genes as an indirect read-out for repair. As such, we believe this tool will be useful for DNA repair researchers to study NHEJ repair in a high- throughput and sensitive manner, with the capacity to detect subtle changes in DSB repair patterns that was not possible previously. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Homologous recombination (HR) and non-homologous end- joining (NHEJ) represent the two major DSB repair pathways in cells [1–4]. While HR utilizes homologous DNA sequences as a template for repair, NHEJ processes and re-ligates the ends of the breaks [5]. The NHEJ pathway is considered more error prone than HR and occurs more frequently in cells. NHEJ is the predominant pathway in the G0/G1-phases of the cell cycle, while HR increases during S/G2, when a sister chromatid becomes available as a template for ∗ Corresponding author at: Department of Therapeutic Radiology, 333 Cedar Street, New Haven, CT 06520, United States. Tel.: +1 (203) 200 2100. E-mail address: ranjit.bindra@yale.edu (R.S. Bindra). 1 These authors contributed equally to this work. repair. Emerging evidence indicates that many sub-pathways exist within NHEJ and HR repair. In particular, NHEJ repair mainly is comprised of canonical NHEJ and non-canonical NHEJ repair. The latter process is been given many names, including back-up NHEJ (bNHEJ), alternative NHEJ (aNHEJ), and microhomology-mediated NHEJ (MMEJ; [6]). The former pathway results in minimal process- ing of the DSB ends [7], while the latter process typically results in deletions with local sequence microhomology [8–11]. Canonical NHEJ proteins include DNA-PKcs, XRCC4, and Ligase IV [7]. Alterna- tive NHEJ is a poorly defined pathway but appears to require MRE11 [12], and PARP-1 [13]. Ligase III and XRCC1 have also been impli- cated in alternative NHEJ [14,15], although more recent studies have questioned the requirement of these proteins in this pathway [16–18]. Collectively, the non-canonical NHEJ repair processes share a common theme of higher rates of insertions, deletions, http://dx.doi.org/10.1016/j.dnarep.2014.12.002 1568-7864/© 2014 Elsevier B.V. All rights reserved.