Optimized Repli-seq: improved DNA replication timing analysis by next-generation sequencing.
Optimized Repli-seq: improved DNA replication timing analysis by next-generation sequencing.
复制标题
DOI：
10.1007/s10577-022-09703-7
复制
发表时间：
2022-12
影响因子：
2.6
通讯作者：
Martinez-Cifuentes, Santiago
中科院分区：
文献类型：
--
作者：
Rivera-Mulia, Juan Carlos;Trevilla-Garcia, Claudia;Martinez-Cifuentes, Santiago
人类基因组被分成几个功能单元，在S阶段的特定时间进行复制。这种时间程序被称为复制计时(RT)，与基因组的空间组织和转录活动相协调。RT也是细胞类型特异性的，在发育过程中动态调节，在多种疾病中观察到RT的变化。因此，准确的RT测量对于理解RT在基因功能调节中的作用至关重要。检测RT程序有不同的方法；然而，传统方法需要数千个细胞作为输入，禁止其适用于细胞数量有限的样本，如来自疾病患者或早期发育胚胎的样本。虽然已经开发了单细胞RT分析，但这些方法吞吐量低，需要生成大量文库，增加了测序成本，产生的数据分辨率低。在这里，我们开发了一种改进的方法来测量全基因组RT，从而能够对低输入样本进行高分辨率分析。该方法将直接细胞分选与裂解缓冲液相结合，并在单个试管中进行DNA碎裂和文库准备，从而以较低的成本获得更高的产量、更高的质量和重复性。我们还进行了系统的数据处理分析，为RT测量提供标准化的参数。这种优化的方法便于RT分析，并将使其能够应用于研究RT在基因表达、核结构和疾病中的作用的广泛研究。
The human genome is divided into functional units that replicate at specific times during S-phase. This temporal program is known as replication timing (RT) and is coordinated with the spatial organization of the genome and transcriptional activity. RT is also cell type-specific, dynamically regulated during development, and alterations in RT are observed in multiple diseases. Thus, precise measure of RT is critical to understand the role of RT in gene function regulation. Distinct methods for assaying the RT program exist; however, conventional methods require thousands of cells as input, prohibiting its applicability to samples with limited cell numbers such as those from disease patients or from early developing embryos. Although single-cell RT analyses have been developed, these methods are low throughput, require generation of numerous libraries, increased sequencing costs and produce low resolution data. Here, we developed an improved method to measure RT genome-wide that enables high resolution analysis of low input samples. This method incorporates direct cell sorting into lysis buffer, as well as DNA fragmentation and library preparation in a single tube, resulting in higher yields, increased quality, and reproducibility with decreased costs. We also performed a systematic data processing analysis to provide standardized parameters for RT measurement. This optimized method facilitates RT analysis and will enable its application to a broad range of studies investigating the role of RT in gene expression, nuclear architecture, and disease.
登录
查看更多内容
DOI：
10.1093/bioinformatics/btq033
发表时间：
2010-03-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Quinlan AR;Hall IM
通讯作者：
Hall IM
影响因子：
64.8
作者：
Pope, Benjamin D.;Ryba, Tyrone;Dileep, Vishnu;Yue, Feng;Wu, Weisheng;Denas, Olgert;Vera, Daniel L.;Wang, Yanli;Hansen, R. Scott;Canfield, Theresa K.;Thurman, Robert E.;Cheng, Yong;Guelsoy, Guenhan;Dennis, Jonathan H.;Snyder, Michael P.;Stamatoyannopoulos, John A.;Taylor, James;Hardison, Ross C.;Kahveci, Tamer;Ren, Bing;Gilbert, David M.
通讯作者：
Gilbert, David M.
影响因子：
3.5
作者：
Massey, Dashiell J.;Kim, Dongsung;Koren, Amnon
通讯作者：
Koren, Amnon
影响因子：
16.6
作者：
Dileep V;Gilbert DM
通讯作者：
Gilbert DM
影响因子：
16.6
作者：
通讯作者：
--