ATAC-seq technology development and related technologies
Reveling in the Revealed this article, for DNase-seq and ATAC-seq There MNase-seq related technical principles, advantages and disadvantages are summarized. details as follows:
First, we have learned to, CHIP-seq i.e. chromatin immunoprecipitation mass spectrometry, it is mainly through known protein, a transcription factor TF purpose usually immunoprecipitation, gripping the DNA fragment that binds to the TF, then further amplification, find potential target genes of TF. This method is usually composed of very limited:
- This method is smart to find a potential target gene transcription factors limited. We can not find all the regions of the protein to be protected within the scope of the genome.
And DNase-seq mentioned in the article is mainly the use of DNase enzymes cut genomic having one pair of DNase-sensitive position, i.e., by enzymatic digestion, digestion fragments were then amplified, and finally out of the sequenced data analysis peak found with protected area is often a protein transcription factor binding and nucleosome positions.
The advantages and disadvantages of this method is
advantage
- The method is simple, easy to set up experimental system, it has been used in a variety of cells, and a better understanding of its cut error;
- By sequencing result, indirect estimation nucleosome binding site, and transcription factors.
Disadvantages:
- This method is difficult to control the optimum digestion conditions, at the same time, the number of cells need to address the requirements of large, time-consuming and laborious, the number of cells normally required to reach millions;
- Due to its sequence number of cells required more required, so the small sample size of some cells, this method is not applicable;
- 1 DNase enzyme for cleavage of DNA has a sequence dependent, and therefore, this method will lead to larger error sequencing. Can not guarantee the result of the cut, it is a region of the protein is completely covered.
在这张图里，作者提到，由于在测序的时候，染色质的状态是一个动态的过程，因此上述的三种方法，MNase-seq以及ATAC-seq和DNase-seq并不能很好的放映染色质的开放状态ATAC-seq和DNase-seq的结果，并不能很好的与MNase-seq的结果互补。
ATAC-seq 这是一种利用超活跃的转座酶来分析染色质的开放性的技术。主要的原理，利用Tn5转座酶复合体，将带有标记的标签，与DNA进行孵育，裸露的DNA片段，将在转座酶的作用下，被置换出来，然后通过特异的标签引物进行PCR扩增，进行后续的测序工作。
这种技术的优缺点:
优点
- 技术相对来说较为简单；
- 所需要的细胞量大大减少，目前差不多只需要5个细胞左右。
缺点
- Tn5转座酶的价格较为昂贵；
- 对于ATAC-seq测序所得结果；记录还不是特别完全，无法充分的去解释这种酶切割的序列偏好问题；
- 每个细胞都是不同的，所以你需要根据你的特殊情况调整细胞数量和裂解条件；
- 理想情况下，需要温和地裂解细胞，使转座酶进入，并且不破坏染色质状态；
- 使用太多的细胞会导致插入的测序适配器减少，从而导致较大的DNA片段；使用太少的细胞会导致较短的DNA片段；
- 细胞的最佳数量可能会有所不同，这取决于细胞起源的组织或机体器官。
单细胞测序中的ATAC-seq存在的问题，单细胞测序的结果较为稀疏，在基因组的任何位置都存在零星的1-2个可接近位点，但是这些数据不准确，难以置信。
MNase-seq数据的原理
研究人员使用金黄色葡萄球菌的微球菌核酸酶（mnase）消化和研究染色质至少已有40年。2010年，他们开始将其与高通量测序配对。
Works
mnase operates by cutting the genomic fragment was digested exposed; associated with nucleosomes dna is recovered and sequenced. This makes mnase-seq least conceptually atac-seq dnase-seq and is complementary to, but to achieve this condition, needs to be some sequence within the same cell, simultaneous sequencing process.
advantage
- Wide range of applications;
Shortcoming
- The number of cells need more, about 10-20 million;
- Chromatin accessibility most enzymes used in the analysis of sequence-specific preferences;
- Preference mnase cleavage enzyme gene group AT-rich region;
- Certain regions of the genome digested more sensitive than other areas of the nuclease, but the exact mechanism remains unclear.
For the following questions, and MNase-seq data analysis sequencing DNase-seq sequencing data are not complementary, Lieb in his view, the process of cell sequencing, there are some sites at the same time is DNase hypersensitive, it may be protected nucleosomes, specific to this issue, we will describe how to further the exact chromosomal most real state?
The method supplement for MNase-seq is called NOMe-seq, the method is carried out using a pull proposed in 2012. This method produces nucleosome positioning and dna methylation status information regarding the range of the whole genome, whereby position information of the nucleosomes and methylation state together. For this NOMe-seq technology, I will be analyzed in the next article.