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*
- 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.
- National Engineering Research Center of Personalized Diagnostic and
Therapeutic Technology, Hunan University of Chinese Medicine, Changsha
410208, Hunan, China.
- 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.
- Department of Nuclear Medicine, Hunan Provincial People’s Hospital,
First Affiliated Hospital of Hunan Normal University, Changsha,
410005, Hunan, China.
- The Second Affiliated Hospital of Soochow University, Suzhou 215004,
China.
- Higentec Co., Ltd, 618 Heping Road, Furong District, Changsha, Hunan
410125, China.
- CHUL Research Center, Laval University, Quebec, G1V 4G2, Canada.
- Department of Pharmacy,Children’s Hospital of Soochow
University,Suzhou, 215000.
____________
*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.