Peng Qi1,2#, Ya-ling Zeng1,2#,Xu Ye3#,Ya-mei Li1,2,Feng-jiao Wang8,Wang-yang Pu5,Rong Zhang5,Min Li4, Li Xiao5, Gang Huang6, Sirois Pierre7, Jia Zhang1,2, Duan-fang Liao1,2*, Hongyan Wen1,2*, Kai Li1,2*
  1. Division of Stem Cell Regulation and Application, Key Laboratory for Quality Evaluation of Bulk Herbs of Hunan Province, Hunan University of Chinese Medicine, Changsha 410208, Hunan, China.
  2. National Engineering Research Center of Personalized Diagnostic and Therapeutic Technology, Hunan University of Chinese Medicine, Changsha 410208, Hunan, China.
  3. Department of Radiotherapy,Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine Central South University 283 Tongzipo Road
Changsha 410013, Hunan, China.
  1. Department of Nuclear Medicine, Hunan Provincial People’s Hospital, First Affiliated Hospital of Hunan Normal University, Changsha, 410005, Hunan, China.
  2. The Second Affiliated Hospital of Soochow University, Suzhou 215004, China.
  3. Higentec Co., Ltd, 618 Heping Road, Furong District, Changsha, Hunan 410125, China.
  4. CHUL Research Center, Laval University, Quebec, G1V 4G2, Canada.
  5. Department of Pharmacy,Children’s Hospital of Soochow University,Suzhou, 215000.
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*Correspondence: Duan-fang Liao(dfliao@hnucm.edu.cn), Hongyan Wen(340681499@qq.com) or kai Li (kaili34@yahoo.com) .
Keywords self-ligation,adapters, methylation sequencing
Abstract In our recent effort of developing methylation-dependent endonuclease mediated methylation sequencing technology, efficiency and reproducibility were restrained by adapters’ self-ligation. In the present study, three strategies to prevent self-ligation of adapters used in enzyme mediated methylation sequencing have been developed. Our data have demonstrated that these strategies can either inhibit or eliminate the adapters’ self-ligation. These strategies are crucial in enzyme-mediated methylation sequencing and may be useful in some other genomic sequencing technologies.
Introduction
More than ten types of methylation assays have been developed for epigenetic study[1-5]. Predominantly, sodium bisulfite is used in genomic methylation sequencing[6, 7]. In either regular next generation sequencing or sodium bisulfite-mediated methylation sequencing, adapters with a 3’ extruded T were widely used in library preparation with A-tailing target inserts. These adapters need to be removed before sequencing, which is easily accomplished based on size difference between adapters with and without inserts.
To eliminate the destruction of target DNA by sodium bisulfite [8], methylation-dependent endonuclease was chosen in our effort of developing sulfite-free methylation sequencing technology. We noticed that the efficiency and reproducibility of methylation-dependent endonuclease mediated methylation sequencing were restrained by self-ligation of adapters[9, 10]. Although adapters with 3’ extruded T are still usable as the adapters ligated with inserts containing methylated CpG site, all other methylation site-independent adapters to be used can be self-ligated. Furthermore, methylation site-dependent adapters also can be self-ligated. These self-ligation of adapters has two potential shortages: to decrease the efficiency and reproducibility of methylation sequencing, and to substantially increase the cost[11]. When applied to single cell methylation sequencing in which the ratio of adapters to inserts is usually as high as 200, sequencing data will probably exclusively be consisted of the adapters from the self-ligation products. The latter is due to the sequencing chip competitively occupied by self-ligated adapters without insert. Different from conventional next generation sequencing, it is nearly impossible to physically separate self-ligated adapters and adapter-insert products as their similar sizes.
In addition to adapters with 3’ extruded T, two more methylation site-independent adapters and one methylation site-dependent adapter are designed to the new methylation sequencing technology. These three types of adapters all can be self-ligated. The present study tested strategies to prevent their self-ligation and our data documented that their self-ligation can be either decreased or eliminated.
Materials and methods
2.1 Chemically synthesized templates and adapters
Nine oligonucleotides of the designed templates and adapters were synthesized from Sangon Biotech (Shanghai, China) as listed in Table 1. Oligonucleotides 1 and 2 are the artificially synthesized templates, which were based on the SDC2 gene with addition of two restriction cutting sites of HpaII (5’-CCGG-3’) (New England Biolabs (Beijing) LTD)and MseI (5’-TTAA-3’)(New England Biolabs (Beijing) LTD). The rest are the five double chain adapters to be tested. There are two methylation site-independent adapters with end of CGat (No.3) and TAat (No.4) respectively and their complementary oligonucleotides. The three methylation site-dependent adapters were with YNNY, RNNR, and NNNN in their 5’ termini.
Each of oligonucleotides No. 3 and 4 is complementary paired with oligonucleotides No. 5 to form a double-chain with 2 bases protruding at their 5’ ends (CGat and TAat). These two adapters were named as CGat adapter and TAat adapter. The oligos of No.6, 7 and 8 are respectively complimentary paired with the oligos No.9 to form a double-stranded adapters with 4 base protrusions at their 5’ end with YNNY, RNNR and NNNN, named as YNNY, RNNR and NNNN adapters. Oligos were added to 1X annealing buffer (Beijing Solarbio Science & Technology Co., Ltd.) to gradually anneal the double-stranded templates and adapters with the following procedures: 95 °C 10 min, 85 °C 10 min, 75 °C 10 min, 65 °C 10 min, 55 °C 10 min, 45 °C 10 min, 35 °C 10 min, 25 °C 10 min, 15 °C 10 min ,and stored at 4 °C for use.