Peng Wu1,2,3, Yan Gao4,5,6,7, Weilong Guo8, Ping Zhu1,2,3. 1. State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China. 2. Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Tianjin, China. 3. Department of Stem Cell & Regenerative Medicine, Peking Union Medical College, Tianjin, China. 4. Center for Breast Cancer Prevention and TherapyTianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, China. 5. Key Laboratory of Cancer Prevention and Therapy, Tianjin, China. 6. Tianjin's Clinical Research Center for Cancer, Tianjin, China. 7. Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin, China. 8. State Key Laboratory for Agrobiotechnology, College of Agronomy and Biotechnology, China Agricultural University, Beijing, China.
Abstract
MOTIVATION: Single-cell bisulfite sequencing (BS-seq) techniques have been developed for DNA methylation heterogeneity detection and studies with limited materials. However, the data deficiency such as low read mapping ratio is still a critical issue. RESULTS: We comprehensively characterize single-cell BS-seq data and reveal chimerical molecules to be the major source of alignment failures. These chimerical molecules are produced by recombination of genomic proximal sequences with microhomology regions (MR) after bisulfite conversion. In addition, we find DNA methylation within MR is highly variable, suggesting the necessity of removing these regions to accurately estimate DNA methylation levels. We further develop scBS-map to perform quality control and local alignment of bisulfite sequencing data, chimerical molecule determination and MR removal. Using scBS-map, we show remarkable increases in uniquely mapped reads, genomic coverage and number of CpG sites, and recover more functional elements with precise DNA methylation estimation. AVAILABILITY AND IMPLEMENTATION: The scBS-map software is freely available at https://github.com/wupengomics/scBS-map. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
MOTIVATION: Single-cell bisulfite sequencing (BS-seq) techniques have been developed for DNA methylation heterogeneity detection and studies with limited materials. However, the data deficiency such as low read mapping ratio is still a critical issue. RESULTS: We comprehensively characterize single-cell BS-seq data and reveal chimerical molecules to be the major source of alignment failures. These chimerical molecules are produced by recombination of genomic proximal sequences with microhomology regions (MR) after bisulfite conversion. In addition, we find DNA methylation within MR is highly variable, suggesting the necessity of removing these regions to accurately estimate DNA methylation levels. We further develop scBS-map to perform quality control and local alignment of bisulfite sequencing data, chimerical molecule determination and MR removal. Using scBS-map, we show remarkable increases in uniquely mapped reads, genomic coverage and number of CpG sites, and recover more functional elements with precise DNA methylation estimation. AVAILABILITY AND IMPLEMENTATION: The scBS-map software is freely available at https://github.com/wupengomics/scBS-map. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.