非交联染色质免疫共沉淀及其二代测序技术建库方法的优化
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摘要
目的优化非交联染色质免疫共沉淀及其二代测序(Native ChIP-seq)技术,简化操作步骤,得到高质量ChIP-seq数据。方法裂解细胞,MNase切割DNA释放核小体,用组蛋白修饰的特异性抗体将组蛋白与DNA的复合物进行免疫共沉淀,蛋白酶K消化后进行DNA纯化,染色质免疫共沉淀(ChIP)产物用Tn5转座酶建库与传统建库两种方法进行文库构建并测序。结果与传统建库方法相比,Tn5转座酶建库经过Tn5片段化后直接进行目的DNA的扩增,操作简单,更加省时,效率更高;IGV可视化信号分布峰图显示两种建库方式获得的富集峰基本相同;两种建库方法获得的测序数据中,Tn5转座酶建库比传统建库获得更多的富集峰;Tn5转座酶建库后结果显示重复性良好,信噪比达到50%以上。结论 Tn5转座酶建库能提高建库效率并得到更好的数据质量,适用于组蛋白修饰的检测,为表观遗传研究提供更好的技术选择。
Objective To optimize DNA library construction in non-crosslinked chromatin immunoprecipitation coupled with next-generation sequencing(Native ChIP-seq) to obtain high-quality Native ChIP-seq data. Methods Human nasopharyngeal carcinoma HONE1 cell lysate was digested with MNase for release of the nucleosomes, and the histone-DNA complexes were immunoprecipitated with specific antibodies. The protein component in the precipitate was digested with proteinase K followed by DNA purification; the DNA library was constructed for sequence analysis. Results Compared with the conventional DNA library construction, Tn5 transposase method allowed direct enrichment of the target DNA after Tn5 fragmentation, which was simple, time-saving and more efficient. The IGV visualized map showed that the information obtained by the two library construction methods was consistent. The sequencing data obtained by the two methods revealed more signal enrichment with Tn5 transposase library construction than with the conventional approach. H3 K4 me3 ChIP results showed a good reproducibility after Tn5 transposase library construction with a signal-to-noise ratio above 50%.Conclusion Tn5 transposase method improves the efficiency of DNA library construction and the results of subsequent sequence analysis, and is especially suitable for detecting histone modification in the DNA to provide a better technical option for epigenetic studies.
Objective To optimize DNA library construction in non-crosslinked chromatin immunoprecipitation coupled with next-generation sequencing(Native ChIP-seq) to obtain high-quality Native ChIP-seq data. Methods Human nasopharyngeal carcinoma HONE1 cell lysate was digested with MNase for release of the nucleosomes, and the histone-DNA complexes were immunoprecipitated with specific antibodies. The protein component in the precipitate was digested with proteinase K followed by DNA purification; the DNA library was constructed for sequence analysis. Results Compared with the conventional DNA library construction, Tn5 transposase method allowed direct enrichment of the target DNA after Tn5 fragmentation, which was simple, time-saving and more efficient. The IGV visualized map showed that the information obtained by the two library construction methods was consistent. The sequencing data obtained by the two methods revealed more signal enrichment with Tn5 transposase library construction than with the conventional approach. H3 K4 me3 ChIP results showed a good reproducibility after Tn5 transposase library construction with a signal-to-noise ratio above 50%.Conclusion Tn5 transposase method improves the efficiency of DNA library construction and the results of subsequent sequence analysis, and is especially suitable for detecting histone modification in the DNA to provide a better technical option for epigenetic studies.
引文
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[14]Medina-Gali R, Bello-Perez M, Martinez-Lopez A, et al. Chromatin immunoprecipitation and high throughput sequencing of SVCVinfected zebrafish reveals novel epigenetic histone methylation patterns involved in antiviral Immune response[J]. Fish Shellfish Immunol, 2018, 82(4):514-21.
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[16]Seitan VC, Hao BT, Tachibana-Konwalski KA, et al. A role for cohesin in T-cell-receptor rearrangement and thymocyte differentiation[J]. Nature, 2011, 476(7361):467-U126.
[17]Northrup DL, Zhao K. Application of ChIP-Seq and related techniques to the study of immune function[J]. Immunity, 2011, 34(6):830-42.
[18]Gilfillan GD, Hughes T, Sheng Y, et al. Limitations and possibilities of low cell number ChIP-seq[J]. BMC Genomics, 2012, 13:645.
[19]杜玲,刘刚,陆健,等.高通量测序技术的发展及其在生命科学中的应用[J].中国畜牧兽医, 2014, 41(12):109-16.
[20]Chaitankar V, Karakulah G, Ratnapriya RA, et al. Next Generation sequencing technology and genomewide data analysis:perspectives for retinal research[J]. Prog Retin Eye Res, 2016(6), 55:1-31.
[21]Nakato R, Shirahige K. Recent advances in Ch IP-seq analysis:from quality management to whole-genome annotation[J]. Brief Bioinform,2017, 18(2):279-90.
[22]Bailey T, Krajewski P, Ladunga I, et al. Practical guidelines for the comprehensive analysis of ChIP-seq data[J]. PLoS Comput Biol,2013, 9(11):1003326.
[23]Landt SG, Marinov GK, Kundaje AA, et al. ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia[J]. Genome Res, 2012, 22(9, SI):1813-31.
[24]Mendoza-Parra M. Gronemeyer H, assessing quality standards for Ch IP-seq and related massive parallel sequencing-generated datasets:when rating goes beyond avoiding the crisis[J]. Genom Data, 2014,10(2):268-73.
[25]Mendoza-Parra M, Saravaki V, Cholley PE, et al. Antibody performance in ChIP-sequencing assays:from quality scores of public data sets to quantitative certification[J]. F1000Res, 2016, 12(5):54.
[26]Mukhopadhyay A, Deplancke B, Walhout AJ, et al. Chromatin immol/Lunoprecipitation(Ch IP)coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in caenorhabditis elegans[J]. Nat Protoc, 2008, 3(4):698-709.
[27]Hashimoto M, He Y, Yeung ES. On-line integration of PCR and cycle sequencing in capillaries:from human genomic DNA directly to called bases[J]. Nucleic Acids Res, 2003, 31(8):e41.
[28]Chen S, Huang T, Zhou Y, et al. AfterQC:automatic filtering,trimmol/Ling, error removing and quality control for fastq data[J]. BMC Bioinformatics, 2017, 18(Suppl 3):80.
[29]Simonsen AT, Hansen MC, Kjeldsen E, et al. Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing[J]. BMC Genomics, 2018, 19(1):681.
[30]Sundaram AY, Hughes T, Biondi S, et al. A comparative study of Ch IP-seq sequencing library preparation methods[J]. BMC Genomics,2016, 17(1):816.
[31]Schmidl C, Rendeiro AF, Sheffield NC, et al. ChIPmentation:fast,robust, low-input ChIP-seq for histones and transcription factors[J].Nat Methods, 2015, 12(10):963-5.
[32]Gustafsson C, De Paepe A, Schmidl CA. High-throughput Ch IPmentation:freely scalable, single day ChIPseq data generation from very low cell-numbers[J]. BMC Genomics, 2019, 20(1):59.
[2] Dupont C, Armant DR, Brenner CA. Epigenetics:definition,mechanisms and clinical perspective[J]. Semin Reprod Med, 2009,27(5):351-7.
[3]Novak K. Epigenetics changes in cancer cells[J]. MedGenMed, 2004,6(4):17.
[4]Kagohara LT, Stein-O'brien GL, Kelley D, et al. Epigenetic regulation of gene expression in cancer:techniques, resources and analysis[J].Brief Funct Genomics, 2018, 17(1):49-63.
[5] Jordan-Pla A, Visa N. Considerations on experimental design and data analysis of chromatin immol/lunoprecipitation experiments[M].Methods Mol Biol, 2018, 1689:9-28.
[6] Hao BT, Naik AK, Watanabe A, et al. An anti-silencer-and SATB1-dependent chromatin hub regulates Rag1 and Rag2 gene expression during thymocyte development[J]. J Exp Med, 2015, 212(5):809-24.
[7] Milne TA, Zhao K, Hess JL. Chromatin immol/Lunoprecipitation(Ch IP)for analysis of histone modifications and chromatin-associated proteins[M]. Methods Mol Biol, 2009, 538:409-23.
[8] Schmidt D, Wilson MD, Spyrou CA, et al. ChIP-seq:using highthroughput sequencing to discover protein-DNA interactions[J].Methods, 2009, 48(3):240-8.
[9]田李,张颖,赵云峰.新一代测序技术的发展和应用[J].生物技术通报, 2015, 31(11):1-8.
[10]Lightbody G, Haberland V, Browne F, et al. Review of applications of high-throughput sequencing in personalized medicine:barriers and facilitators of future progress in research and clinical application[J]. Brief Bioinform, 2018.
[11]Heather JM, Chain B. The sequence of sequencers:The history of sequencing DNA[J]. Genomics, 2015, 107(1):1-8.
[12]Gu H, Smith ZD, Bock C, et al. Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling[J]. Nat Protoc, 2010, 6(4):468-81.
[13]Glaser R, Zhang HY, Yao KT, et al. Two epithelial tumor cell lines(HNE-1 and HONE-1)latently infected with Epstein-Barr virus that were derived from nasopharyngeal carcinomas[J]. Proc Natl Acad Sci USA, 1989, 86(23):9524-8.
[14]Medina-Gali R, Bello-Perez M, Martinez-Lopez A, et al. Chromatin immunoprecipitation and high throughput sequencing of SVCVinfected zebrafish reveals novel epigenetic histone methylation patterns involved in antiviral Immune response[J]. Fish Shellfish Immunol, 2018, 82(4):514-21.
[15]Karlic R, Chung HR, Lasserre J, et al. Histone modification levels are predictive for gene expression[J]. Proc Natl Acad Sci USA, 2010, 107(7):2926-31.
[16]Seitan VC, Hao BT, Tachibana-Konwalski KA, et al. A role for cohesin in T-cell-receptor rearrangement and thymocyte differentiation[J]. Nature, 2011, 476(7361):467-U126.
[17]Northrup DL, Zhao K. Application of ChIP-Seq and related techniques to the study of immune function[J]. Immunity, 2011, 34(6):830-42.
[18]Gilfillan GD, Hughes T, Sheng Y, et al. Limitations and possibilities of low cell number ChIP-seq[J]. BMC Genomics, 2012, 13:645.
[19]杜玲,刘刚,陆健,等.高通量测序技术的发展及其在生命科学中的应用[J].中国畜牧兽医, 2014, 41(12):109-16.
[20]Chaitankar V, Karakulah G, Ratnapriya RA, et al. Next Generation sequencing technology and genomewide data analysis:perspectives for retinal research[J]. Prog Retin Eye Res, 2016(6), 55:1-31.
[21]Nakato R, Shirahige K. Recent advances in Ch IP-seq analysis:from quality management to whole-genome annotation[J]. Brief Bioinform,2017, 18(2):279-90.
[22]Bailey T, Krajewski P, Ladunga I, et al. Practical guidelines for the comprehensive analysis of ChIP-seq data[J]. PLoS Comput Biol,2013, 9(11):1003326.
[23]Landt SG, Marinov GK, Kundaje AA, et al. ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia[J]. Genome Res, 2012, 22(9, SI):1813-31.
[24]Mendoza-Parra M. Gronemeyer H, assessing quality standards for Ch IP-seq and related massive parallel sequencing-generated datasets:when rating goes beyond avoiding the crisis[J]. Genom Data, 2014,10(2):268-73.
[25]Mendoza-Parra M, Saravaki V, Cholley PE, et al. Antibody performance in ChIP-sequencing assays:from quality scores of public data sets to quantitative certification[J]. F1000Res, 2016, 12(5):54.
[26]Mukhopadhyay A, Deplancke B, Walhout AJ, et al. Chromatin immol/Lunoprecipitation(Ch IP)coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in caenorhabditis elegans[J]. Nat Protoc, 2008, 3(4):698-709.
[27]Hashimoto M, He Y, Yeung ES. On-line integration of PCR and cycle sequencing in capillaries:from human genomic DNA directly to called bases[J]. Nucleic Acids Res, 2003, 31(8):e41.
[28]Chen S, Huang T, Zhou Y, et al. AfterQC:automatic filtering,trimmol/Ling, error removing and quality control for fastq data[J]. BMC Bioinformatics, 2017, 18(Suppl 3):80.
[29]Simonsen AT, Hansen MC, Kjeldsen E, et al. Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing[J]. BMC Genomics, 2018, 19(1):681.
[30]Sundaram AY, Hughes T, Biondi S, et al. A comparative study of Ch IP-seq sequencing library preparation methods[J]. BMC Genomics,2016, 17(1):816.
[31]Schmidl C, Rendeiro AF, Sheffield NC, et al. ChIPmentation:fast,robust, low-input ChIP-seq for histones and transcription factors[J].Nat Methods, 2015, 12(10):963-5.
[32]Gustafsson C, De Paepe A, Schmidl CA. High-throughput Ch IPmentation:freely scalable, single day ChIPseq data generation from very low cell-numbers[J]. BMC Genomics, 2019, 20(1):59.