技术领域technical field
本发明涉及基因测序技术领域，具体而言，涉及一种基于Nanopore测序技术识别辐射损伤中嘧啶二聚体的模型构建方法。The invention relates to the technical field of gene sequencing, in particular to a model construction method for identifying pyrimidine dimers in radiation damage based on Nanopore sequencing technology.
背景技术Background technique
Nanopore测序技术，一种单分子实时测序的新一代测序方法，其以单分子DNA(RNA)通过生物纳米孔的电信号变化，由于不同的碱基带来的电信号变化是不同的，因此，应用通过机器学习得到的电信号识别模型可以推测其碱基组成，进而实现测序。Nanopore sequencing technology, a next-generation sequencing method for single-molecule real-time sequencing, uses single-molecule DNA (RNA) to change the electrical signal through the biological nanopore. Since the electrical signal changes caused by different bases are different, therefore, Applying the electrical signal recognition model obtained through machine learning can infer its base composition, and then realize sequencing.
紫外线可以造成DNA的损伤，将DNA分子中的胸腺嘧啶以环丁基环形成二聚体，称为胸腺嘧啶二聚体(TT dimer)。这种变化在DNA链上相邻近的胸苷酸容易发生。二聚形成后，RNA引物的合成将停止在二聚体处，DNA的合成也受阻。Ultraviolet light can cause DNA damage, and the thymine in the DNA molecule forms a dimer with a cyclobutyl ring, which is called a thymine dimer (TT dimer). This change occurs readily at adjacent thymidylates on the DNA strand. After the dimerization is formed, the synthesis of the RNA primer will stop at the dimer, and the synthesis of DNA will also be hindered.
但目前来说，在Nanopore测序技术中使用的电信号识别模型针对的都是未经修饰的单分子DNA序列，缺乏针对经修饰(例如嘧啶二聚体、组蛋白修饰、甲基化修饰等)的DNA序列发生变化的电信号识别模型。因此，如果针对特定生物学问题如对于DNA辐射损伤中DNA序列改变的问题等展开分析研究，采用Nanopore测序技术进行测序的容易导致测序结果不准确。However, at present, the electrical signal recognition models used in Nanopore sequencing technology are all aimed at unmodified single-molecule DNA sequences, lacking for modified (such as pyrimidine dimers, histone modifications, methylation modifications, etc.) A model for electrical signal recognition of changes in the DNA sequence. Therefore, if analysis and research are carried out on specific biological issues such as DNA sequence changes in DNA radiation damage, the use of Nanopore sequencing technology for sequencing will easily lead to inaccurate sequencing results.
鉴于此，特提出本发明。In view of this, the present invention is proposed.
发明内容Contents of the invention
本发明的目的在于提供一种基于Nanopore测序技术识别辐射损伤中嘧啶二聚体的模型构建方法，通过该构建方法，可以得到针对辐射损伤中嘧啶二聚体的识别模型，该识别模型可用于DNA损伤例如TT二聚体进行预测。The purpose of the present invention is to provide a model construction method for identifying pyrimidine dimers in radiation damage based on Nanopore sequencing technology. Through this construction method, a recognition model for pyrimidine dimers in radiation damage can be obtained, and the recognition model can be used for DNA Damage such as TT dimer is predicted.
本发明是这样实现的：The present invention is achieved like this:
一种基于Nanopore测序技术识别辐射损伤中嘧啶二聚体的模型构建方法，其包括如下步骤：A method for building a model based on Nanopore sequencing technology to identify pyrimidine dimers in radiation damage, comprising the steps of:
步骤(1)：step 1):
提供目标测序序列及其对应的目标测序序列电信号，所述目标测序序列由Nanopore测序技术对UV照射后的酵母细胞进行测序得到；providing the target sequencing sequence and its corresponding target sequencing sequence electrical signal, the target sequencing sequence is obtained by sequencing the yeast cells after UV irradiation by Nanopore sequencing technology;
步骤(2)：将所述目标测序序列与参考序列比对，获得比对准确的具有连续两个TT以上的位点的连续TT集合S，其中，所述参考序列为酵母基因组序列；Step (2): comparing the target sequencing sequence with a reference sequence to obtain a continuous TT set S with more than two consecutive TT sites, wherein the reference sequence is a yeast genome sequence;
需要说明的是，连续两个TT以上的位点包括连续两个TT、连续三个TT的位点、连续四个TT的位点、连续五个TT的位点、连续六个TT的位点、连续七个TT的位点、连续八个TT的位点、连续九个TT的位点、连续十个TT的位点、连续十一个TT等以上的位点，这些位点都均被纳入连续TT集合S。It should be noted that the sites with more than two TTs in a row include the sites with two TTs in a row, the sites with three TTs in a row, the sites with four TTs in a row, the sites with five TTs in a row, and the sites with six TTs in a row , sites with seven consecutive TTs, sites with consecutive eight TTs, sites with consecutive nine TTs, sites with consecutive ten TTs, sites with consecutive eleven TTs, etc., these sites are all Included in the continuous TT set S.
步骤(3)：以所述连续TT集合S与预先设置的TT二聚体集合B的交集作为机器学习的阳性样本训练集合P，以所述连续TT集合S与所述预先设置的TT二聚体集合B的的差集作为机器学习的阴性样本训练集合N；Step (3): Use the intersection of the continuous TT set S and the preset TT dimer set B as the positive sample training set P for machine learning, and use the continuous TT set S and the preset TT dimerization The difference set of body set B is used as the negative sample training set N of machine learning;
步骤(4)：分别将阳性样本训练集合P和阴性样本训练集合N中的连续TT位点向其上下游各拓展4bp，得到覆盖TT二聚体位点的10bp碱基序列，根据步骤(1)中的所述目标测序序列电信号获取每个10bp碱基序列相对应的电信号特征；Step (4): Extend the continuous TT sites in the positive sample training set P and the negative sample training set N to their upstream and downstream respectively by 4 bp to obtain a 10 bp base sequence covering the TT dimer site, according to step (1) The electrical signal of the target sequencing sequence in the acquisition of the electrical signal characteristics corresponding to each 10bp base sequence;
以得到与所述阳性样本集合P对应的阳性样本碱基序列集和阳性样本电信号特征集，以及与所述阴性样本集合N对应的阴性样本碱基序列集和阴性样本电信号特征集；To obtain a positive sample base sequence set and a positive sample electrical signal feature set corresponding to the positive sample set P, and a negative sample base sequence set and a negative sample electrical signal feature set corresponding to the negative sample set N;
步骤(5)：将步骤(4)的阳性样本训练集合P和阴性样本训练集合N作为机器学习输入，构建目标训练模型。Step (5): The positive sample training set P and the negative sample training set N in step (4) are used as machine learning input to construct the target training model.
进一步地，在本发明的一些实施方案中，在步骤(3)中：获得所述预先设置的TT二聚体集合B的步骤，包括：Further, in some embodiments of the present invention, in step (3): the step of obtaining the preset TT dimer set B includes:
统计现有文献报道的经过UV照射后的酵母中存在的所有TT二聚体位点，作为真阳性TT二聚体集合A；统计现有文献报道的未经过UV照射的酵母中存在的所有TT二聚体位点，作为假阳性TT二聚体集合A’；Count all TT dimer sites present in yeast after UV irradiation reported in the existing literature, as the true positive TT dimer set A; count all TT dimer sites present in yeast that have not been irradiated by UV Polymer site, as false positive TT dimer set A';
去除真阳性TT二聚体集合A1中同时包含在假阳性TT二聚体集合A’的TT二聚体位点后，得到真阳性TT二聚体集合A2，并在该真阳性TT二聚体集合A2中去除测序深度小于10的所有位点和所有的具有连续10个T的位点，得到所述预先设置的TT二聚体集合B。After removing the TT dimer sites contained in the false positive TT dimer set A' in the true positive TT dimer set A1, the true positive TT dimer set A2 is obtained, and in the true positive TT dimer set A2 In A2, all sites with a sequencing depth less than 10 and all sites with 10 consecutive Ts are removed to obtain the preset TT dimer set B.
进一步地，在本发明的一些实施方案中，步骤(4)中，所述构成目标训练模型的步骤包括：依据SVM算法及RNN算法训练初始模型，以得到目标训练模型。Further, in some embodiments of the present invention, in step (4), the step of forming the target training model includes: training the initial model according to the SVM algorithm and the RNN algorithm to obtain the target training model.
进一步地，在本发明的一些实施方案中，Further, in some embodiments of the present invention,
步骤(4)中，所述电信号特征包括：电信号强度、标准差和电信号持续时间。In step (4), the electrical signal characteristics include: electrical signal strength, standard deviation and electrical signal duration.
进一步地，在本发明的一些实施方案中，步骤(1)中的照射时间为1小时。Further, in some embodiments of the present invention, the irradiation time in step (1) is 1 hour.
通过本发明提供的构建方法，所得到的识别模型可以用于Nanopore测序技术，对由Nanopore测序技术测定的电信号进行识别，从而对由辐射损伤引起的嘧啶二聚体进行预测，为针对特定生物学问题如对于DNA辐射损伤中DNA序列改变的问题提供的依据；当然，本发明提供的构建方法不仅适用于由辐射损伤引起的嘧啶二聚体进行预测，也同样可以适用于对其他DNA修饰例如组蛋白修饰、甲基化修饰等的识别模型的构建。Through the construction method provided by the present invention, the obtained recognition model can be used in Nanopore sequencing technology to identify the electrical signal measured by Nanopore sequencing technology, so as to predict the pyrimidine dimer caused by radiation damage. Scientific problems such as the basis provided for DNA sequence changes in DNA radiation damage; of course, the construction method provided by the present invention is not only applicable to the prediction of pyrimidine dimers caused by radiation damage, but also applicable to other DNA modifications such as Construction of recognition models for histone modifications, methylation modifications, etc.
附图说明Description of drawings
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus It should be regarded as a limitation on the scope, and those skilled in the art can also obtain other related drawings based on these drawings without creative work.
图1为本发明实施例中UV照射后TT二聚体训练集的构建流程示意图。Figure 1 is a schematic diagram of the construction process of the TT dimer training set after UV irradiation in the embodiment of the present invention.
具体实施方式Detailed ways
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.
以下结合实施例对本发明的特征和性能作进一步的详细描述。The characteristics and performance of the present invention will be described in further detail below in conjunction with the examples.
实施例1Example 1
基于Nanopore测序技术识别辐射损伤中嘧啶二聚体的模型构建方法，步骤如下：A method for building a model based on Nanopore sequencing technology to identify pyrimidine dimers in radiation damage, the steps are as follows:
(1)首先采用实验手段，对酵母细胞进行UV照射，获得照射后1hr的酵母细胞。(1) Firstly, the yeast cells were irradiated with UV by experimental means, and the yeast cells 1 hr after irradiation were obtained.
当然，照射的时间可以根据实际情况确定，其不限于1hr，所照射的时间长短尽量以能够促使其基因组序列发生TT二聚体的时间为宜。Of course, the irradiation time can be determined according to the actual situation, and it is not limited to 1 hr. The irradiation time should be as long as possible to promote TT dimerization of the genome sequence.
且，所用的酵母细胞也并不限于酵母细胞，可以根据实际情况更换处理对象，例如大肠杆菌等。Moreover, the yeast cells used are not limited to yeast cells, and the treatment object can be replaced according to the actual situation, such as Escherichia coli and the like.
(2)采用Nanopore测序技术对UV照射后的酵母细胞进行测序，得到的照射后1hr的酵母细胞的基因组序列及其基于Nanopore测序技术的电信号，将其作为目标测序序列及其对应的目标测序序列电信号。(2) Use Nanopore sequencing technology to sequence the yeast cells after UV irradiation, and obtain the genome sequence of the yeast cells 1hr after irradiation and the electrical signal based on Nanopore sequencing technology, which are used as the target sequencing sequence and its corresponding target sequencing sequence electrical signals.
(3)使用BWA软件将目标测序序列比对到正常的酵母基因组序列，使用perl脚本获得比对准确的具有连续两个TT以上的位点，将这些位点全部作为连续TT集合S。(3) Use the BWA software to align the target sequencing sequence to the normal yeast genome sequence, and use the perl script to obtain sites with more than two consecutive TTs that are accurately aligned, and use all of these sites as a continuous TT set S.
连续TT集合S中的连续两个TT以上的位点中包括有TT二聚体位点；这些TT二聚体位点包括自然存在的即未经UV照射就存在于酵母基因组上的TT二聚体位点，也包括由UV照射后引起的TT二聚体位点。More than two consecutive TT sites in the continuous TT set S include TT dimer sites; these TT dimer sites include naturally occurring TT dimer sites that exist on the yeast genome without UV irradiation , also including TT dimer sites induced after UV irradiation.
(4)获得TT二聚体集合B(4) Obtain TT dimer set B
统计现有文献(Chromosomal landscape of UV damage formation and repairat single-nucleotide resolution)报道的经过UV照射后的酵母中存在的所有TT二聚体位点，作为真阳性TT二聚体集合A1；统计上述现有文献报道的不是经过UV照射的酵母中的存在的所有TT二聚体位点，作为假阳性TT二聚体集合A’；Count all the TT dimer sites present in the yeast after UV irradiation reported in the existing literature (Chromosomal landscape of UV damage formation and repairat single-nucleotide resolution), as the true positive TT dimer set A1; Not all TT dimer sites present in UV-irradiated yeast were reported in the literature as false positive TT dimer set A';
去除真阳性TT二聚体集合A1中同时包含在假阳性TT二聚体集合A’的TT二聚体位点后，得到真阳性TT二聚体集合A2，并在该真阳性TT二聚体集合A2中去除测序深度小于10的所有位点和所有的具有连续10个T的位点，得到所述预先设置的TT二聚体集合B。After removing the TT dimer sites contained in the false positive TT dimer set A' in the true positive TT dimer set A1, the true positive TT dimer set A2 is obtained, and in the true positive TT dimer set A2 In A2, all sites with a sequencing depth less than 10 and all sites with 10 consecutive Ts are removed to obtain the preset TT dimer set B.
(5)以所述连续TT集合S与预先设置的TT二聚体集合B的交集作为机器学习的阳性样本训练集合P，共计27793个样本，以所述连续TT集合S与所述预先设置的TT二聚体集合B的的差集作为机器学习的阴性样本训练集合N，共计130315个样本。训练集的构建流程可结合图1参考。(5) Use the intersection of the continuous TT set S and the preset TT dimer set B as the positive sample training set P for machine learning, a total of 27793 samples, and use the continuous TT set S and the preset The difference set of TT dimer set B is used as the negative sample training set N of machine learning, with a total of 130315 samples. The construction process of the training set can be referred to in Figure 1.
(6)分别将阳性样本训练集合P和阴性样本训练集合N中的连续TT位点向其上下游各拓展4bp，得到覆盖TT二聚体位点的10bp碱基序列，根据步骤(1)中的所述目标测序序列电信号获取每个10bp碱基序列相对应的电信号特征：电信号强度、标准差和电信号持续时间；每个10bp碱基序列共30个信号特征。(6) Expand the continuous TT sites in the positive sample training set P and the negative sample training set N to the upstream and downstream respectively by 4 bp, and obtain the 10 bp base sequence covering the TT dimer site, according to the step (1) The electrical signal of the target sequencing sequence obtains electrical signal features corresponding to each 10bp base sequence: electrical signal intensity, standard deviation and electrical signal duration; each 10bp base sequence has a total of 30 signal features.
得到与所述阳性样本集合P对应的阳性样本碱基序列集和阳性样本电信号特征集，以及与所述阴性样本集合N对应的阴性样本碱基序列集和阴性样本电信号特征集。A positive sample base sequence set and a positive sample electrical signal feature set corresponding to the positive sample set P, and a negative sample base sequence set and a negative sample electrical signal feature set corresponding to the negative sample set N are obtained.
(7)将步骤(6)处理后的阳性样本训练集合P和阴性样本训练集合N作为机器学习输入，采用SVM算法、RNN算法构建训练模型，训练完成后即为基于Nanopore测序技术辐射损伤中嘧啶二聚体的识别模型。(7) The positive sample training set P and the negative sample training set N processed in step (6) are used as machine learning input, and the training model is constructed by using the SVM algorithm and the RNN algorithm. After the training is completed, the pyrimidine in the radiation damage based on Nanopore sequencing technology A recognition model for dimers.
该识别模型可用于Nanopore测序技术，对DNA序列发生的变化例如由UV照射引起的TT二聚体进行预测。This recognition model can be used in Nanopore sequencing technology to predict changes in DNA sequences such as TT dimers caused by UV irradiation.
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.
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| CN201810627357.0ACN108804867B (en) | 2018-06-15 | 2018-06-15 | Model construction method for identifying pyrimidine dimer in radiation damage based on Nanopore sequencing technology |
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