Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq).
Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq).
复制标题
DOI：
10.1093/nar/gkw760
复制
发表时间：
2016-12-01
影响因子：
14.9
通讯作者：
Krude T
中科院分区：
文献类型：
--
作者：
Langley AR;Gräf S;Smith JC;Krude T
下一代测序使人类DNA复制起源的全基因组鉴定成为可能。然而，绘制复制起源的不同方法，即：(i)对分离的小的新生DNA链进行测序（SNS-seq）；（ii）测序复制泡（bubble-seq）和（iii）测序Okazaki片段（OK-seq），仅显示有限的一致性。为了解决这一争议，我们在这里描述了一种独立的高分辨率起源制图技术，我们称之为起始位点测序（ini-seq）。在这种方法中，在无细胞系统中，新复制的DNA在其起始位点附近直接用地高辛- dutp标记。然后免疫沉淀标记的DNA，基因组位置由DNA测序确定。利用这种技术，我们在人类基因组上以亚千碱基的分辨率确定了bb50 000个离散的起源位点，生物复制之间具有高度的一致性。通过ini-seq鉴定的大多数活化起始点位于转录起始位点，并含有g -四重体（G4）基序。它们倾向于聚集在早期复制区域，提供了早期复制时间和激活起源的局部密度之间的相关性。ini-seq鉴定的位点与SNS-seq鉴定的位点的一致性最高，其次是OK-seq和bubble-seq。此外，与SNS-seq或bubble-seq鉴定的位点相比，通过阳性核苷酸分布斜跳鉴定的种系起源与ini-seq和OK-seq鉴定的起源重叠的频率和特异性更高。
Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins. However, different approaches to mapping replication origins, namely (i) sequencing isolated small nascent DNA strands (SNS-seq); (ii) sequencing replication bubbles (bubble-seq) and (iii) sequencing Okazaki fragments (OK-seq), show only limited concordance. To address this controversy, we describe here an independent high-resolution origin mapping technique that we call initiation site sequencing (ini-seq). In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP near the sites of its initiation in a cell-free system. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. Using this technique we identify >25,000 discrete origin sites at sub-kilobase resolution on the human genome, with high concordance between biological replicates. Most activated origins identified by ini-seq are found at transcriptional start sites and contain G-quadruplex (G4) motifs. They tend to cluster in early-replicating domains, providing a correlation between early replication timing and local density of activated origins. Origins identified by ini-seq show highest concordance with sites identified by SNS-seq, followed by OK-seq and bubble-seq. Furthermore, germline origins identified by positive nucleotide distribution skew jumps overlap with origins identified by ini-seq and OK-seq more frequently and more specifically than do sites identified by either SNS-seq or bubble-seq.
登录
查看更多内容
影响因子：
4.3
作者：
Baker A;Audit B;Chen CL;Moindrot B;Leleu A;Guilbaud G;Rappailles A;Vaillant C;Goldar A;Mongelard F;d'Aubenton-Carafa Y;Hyrien O;Thermes C;Arneodo A
通讯作者：
Arneodo A
影响因子：
4.3
作者：
Guilbaud G;Rappailles A;Baker A;Chen CL;Arneodo A;Goldar A;d'Aubenton-Carafa Y;Thermes C;Audit B;Hyrien O
通讯作者：
Hyrien O
影响因子：
16.6
作者：
Bartholdy B;Mukhopadhyay R;Lajugie J;Aladjem MI;Bouhassira EE
通讯作者：
Bouhassira EE
影响因子：
7
作者：
Cayrou, Christelle;Coulombe, Philippe;Mechali, Marcel
通讯作者：
Mechali, Marcel
影响因子：
16.8
作者：
Besnard, Emilie;Babied, Amelie;Lemaitre, Jean-Marc
通讯作者：
Lemaitre, Jean-Marc