Highly sensitive targeted methylome sequencing by post-bisulfite adaptor tagging

DNA Research, 2015, 22(1), 13–18 doi: 10.1093/dnares/dsu034 Advance Access Publication Date: 16 October 2014 Full Paper Full Paper Highly sensitive targeted methylome sequencing by post-bisulfite adaptor tagging 1 Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 1130033, Japan, 2Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Higashi-ku, Fukuoka 812-8582, Japan, and 3Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Higashi-ku, Fukuoka 812-8582, Japan *To whom correspondence should be addressed. Tel. +81 92-642-6095. Fax. +81 92-642-2103. Email: Edited by Dr Katsumi Isono Received 29 August 2014; Accepted 24 September 2014 Abstract The current gold standard method for methylome analysis is whole-genome bisulfite sequencing (WGBS), but its cost is substantial, especially for the purpose of multi-sample comparison of large methylomes. Shotgun bisulfite sequencing of target-enriched DNA, or targeted methylome sequencing (TMS), can be a flexible, cost-effective alternative to WGBS. However, the current TMS protocol requires a considerable amount of input DNA and hence is hardly applicable to samples of limited quantity. Here we report a method to overcome this limitation by using post-bisulfite adaptor tagging (PBAT), in which adaptor tagging is conducted after bisulfite treatment to circumvent bisulfite-induced loss of intact sequencing templates, thereby enabling TMS of a 100-fold smaller amount of input DNA with far fewer cycles of polymerase chain reaction than in the current protocol. We thus expect that the PBAT-mediated TMS will serve as an invaluable method in epigenomics. Key words: DNA methylation, target enrichment, massively parallel sequencing 1. Introduction Methylation occurring at the position 5 of cytosine residues in DNA is an epigenetic modification critically involved in the regulation of eukaryotic genomes. Accordingly, the genome-wide distribution of 5-methylcytosine residues, or the methylome, has been attracting intense attention from a wide audience in a variety of research disciplines. The recent advent of next-generation sequencing has revolutionized the way of interrogating the methylome: it has realized whole-genome bisulfite sequencing (WGBS) or genome-wide methylation analysis at single-base resolution.1,2 The power of WGBS has been well demonstrated by many findings that would never have been achieved with other technologies. Although WGBS represents the gold standard for methylome analysis and is rapidly becoming the method of choice, its cost has remained substantial, thereby preventing it from being widely used for multi-sample comparison of large methylomes including those of mammals. The most popular alternative to WGBS is reduced-representation bisulfite sequencing (RRBS), which efficiently enriches CpG-rich regions through restriction enzyme digestion to reduce the cost of sequencing, while maintaining deep coverage of a subset of CpG sites.3 Notably, RRBS can be applied to a minute amount of input DNA.4 However, it cannot be used to examine particular regions of interest unless they are adequately flanked by the restriction enzyme sites. In this context, the shotgun bisulfite sequencing of subgenomic regions enriched using solution hybridization capture technology, referred to hereafter as targeted methylome sequencing (TMS), is ideal, because it can in principle target any unique genomic region.5–7 However, all of the TMS protocols reported thus far require not only a considerable amount of input DNA (i.e. 3 µg or more), but also a large number of polymerase chain reaction (PCR) cycles (i.e. 10–20 cycles), making it difficult to apply TMS to samples of limited quantity. We recently developed a highly efficient protocol for WGBS library construction termed post-bisulfite adaptor tagging (PBAT).8 Although it is well known that bisulfite treatment destroys DNA, © The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the 13 original work is properly cited. Fumihito Miura1,2,3 and Takashi Ito1,2,3,* 14 Highly sensitive targeted bisulfite sequencing 2. Materials and methods 2.1. Preparation of DNA Both human and mouse genomic DNAs used in the model experiments were purchased from Promega. Genomic DNA from IMR90 primary human lung fibroblasts was a generous gift from Yae Kanai. The indicated amount of genomic DNA was dissolved in 130 µl of 10 mM Tris–HCl ( pH 8.0) and sheared with Covaris S220 to the indicated size. We used AMPure XP to purify the fragmented DNA as follows. First, the shared DNA (130 µl) was mixed with 1.8× volume (234 µl) of the AMPure XP reagent and stood for 15 min at room temperature. Next, the beads were collected using a magnet stand, and the supernatant was removed. The pelleted beads were then rinsed with 70% ethanol and dried by standing at 37°C for 5 min. Finally, DNA was eluted from the beads to 20 µl of RNase-free water. The eluted DNA solution was dried in a vacuum concentrator and dissolved in 7 µl of RNase-free water. 2.2. Target enrichment Enrichment of targets with liquid-phase hybridization capture was performed using the reagents in SureSelect Human or Mouse MethylSeq kit (Agilent). Genomic DNA (7 µl) fragmented and purified as above was supplemented with 3 µl of formamide (Wako, Biochemistry grade) and overlaid with 80 µl of mineral oil (Sigma). The DNA was completely denatured by incubating the tube at 99°C for 10 min, cooled down to 65°C and kept at 65°C for at least 5 min before adding the following reagents. Hybridization buffer was prepared by mixing 7.5, 0.3, 3.0 and 3.0 µl of Hyb#1, #2, #3 and #4, respectively. Capture probe mix was prepared by mixing 5.0, 0.5 and 1.0 µl of capture probe solution, RNase Inhibitor and RNase-free water, respectively. The hybridization buffer and the capture probe mix were individually overlaid with 80 µl of mineral oil and incubated at 65°C for 10 min. These two solutions were then combined and mixed thoroughly by pipetting. The combined solution was transferred to the tube containing the denatured input DNA kept at 65°C as above and mixed thoroughly with the DNA solution by pipetting. The tube was incubated at 65°C for at least 24 h to allow hybridization between the probes and the targets. Figure 1. Two strategies for TMS. (A) Conventional procedures comprise adaptor tagging of fragmented genomic DNAs (Steps 1 and 2), target enrichment by hybridization (Steps 3 and 4) and bisulfite treatment of enriched library DNAs (Step 5) followed by PCR amplification (Step 6). The bisulfite treatment (Step 5) induces DNA breaks, inevitably leading to sever (...truncated)