Shortcut barcoding and early pooling for scalable multiplex single-cell reduced-representation CpG methylation sequencing at single nucleotide resolution

Author:

Mai Liyao12,Wen Zebin2,Zhang Yulong2,Gao Yu2,Lin Guanchuan2,Lian Zhiwei2,Yang Xiang23,Zhou Jingjing2,Lin Xianwei24,Luo Chaochao2,Peng Wanwan2,Chen Caiming2,Peng Jiajia2,Liu Duolian2,Marjani Sadie L5,Tao Qian6,Cui Yongping7,Zhang Junxiao24,Wu Xuedong3,Weissman Sherman M8,Pan Xinghua21379ORCID

Affiliation:

1. Department of Hepatobiliary Surgery II, Zhujiang Hospital, Southern Medical University , Guangzhou  510280 , Guangdong Province, China

2. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Southern Medical University, and Guangdong Provincial Key Laboratory of Single Cell Technology and Application , Guangzhou  510515 , Guangdong Province, China

3. Department of Pediatrics, Nanfang Hospital, Southern Medical University , Guangzhou  510515 , Guangdong Province, China

4. SequMed Institute of Biomedical Sciences , Guangzhou  510530 , Guangdong Province, China

5. Department of Biology, Central Connecticut State University , New Britain , CT  06050 , USA

6. Cancer Epigenetics Laboratory, Department of Clinical Oncology, State Key Laboratory of Oncology in South China, Sir YK Pao Center for Cancer and Li Ka Shing Institute of Health Sciences, Chinese University of Hong Kong , 999077  Hong Kong , China

7. Institute of Cancer Research, Shenzhen Bay Laboratory , Shenzhen  518035 , Guangdong , China

8. Department of Genetics, Yale School of Medicine , New Haven , CT  06520 , USA

9. Key Laboratory of Mental Health of the Ministry of Education, Southern Medical University , Guangzhou  510515 , Guangdong Province, China

Abstract

Abstract DNA methylation is essential for a wide variety of biological processes, yet the development of a highly efficient and robust technology remains a challenge for routine single-cell analysis. We developed a multiplex scalable single-cell reduced representation bisulfite sequencing (msRRBS) technology. It allows cell-specific barcoded DNA fragments of individual cells to be pooled before bisulfite conversion, free of enzymatic modification or physical capture of the DNA ends, and achieves read mapping rates of 62.5 ± 3.9%, covering 60.0 ± 1.4% of CpG islands and 71.6 ± 1.6% of promoters in K562 cells. Its reproducibility is shown in duplicates of bulk cells with close to perfect correlation (R = 0.97–0.99). At a low 1 Mb of clean reads, msRRBS provides highly consistent coverage of CpG islands and promoters, outperforming the conventional methods with orders of magnitude reduction in cost. Here, we use this method to characterize the distinct methylation patterns and cellular heterogeneity of six cell lines, plus leukemia and hepatocellular carcinoma models. Taking 4 h of hands-on time, msRRBS offers a unique, highly efficient approach for dissecting methylation heterogeneity in a variety of multicellular systems.

Funder

National Nature Science Foundation of China

Open Fund Programs of Shenzhen Bar Laboratory

Guangdong Major Basic Cultivation Project

Guangdong Natural Science Foundation Major Projects of Basic and Applied Basic Research

Pearl River Talents Program Local Innovative and Research Teams

Publisher

Oxford University Press (OUP)

Subject

Genetics

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