WO2019223502A1 - 一种基于cfDNA高通量测序检测病原体的方法 - Google Patents

一种基于cfDNA高通量测序检测病原体的方法 Download PDF

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WO2019223502A1
WO2019223502A1 PCT/CN2019/085073 CN2019085073W WO2019223502A1 WO 2019223502 A1 WO2019223502 A1 WO 2019223502A1 CN 2019085073 W CN2019085073 W CN 2019085073W WO 2019223502 A1 WO2019223502 A1 WO 2019223502A1
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pathogen
cfdna
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throughput sequencing
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姜君
朱成林
陈卓
王华贵
邹弈君
张华宇
贾冰清
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江苏宏微特斯医药科技有限公司
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  • the invention relates to the field of pathogen detection, in particular to a method for detecting pathogens based on cfDNA high-throughput sequencing.
  • the existing detection methods such as smear and microscopy have low detection rates; the culture method is time-consuming and cumbersome to operate, and it is not easy to identify cultures for some anaerobic bacteria and viruses; serology, Biochemical and PCR molecular biology tests can only target a limited number of pathogens and cannot determine other infections. Especially for chronic pathogen infections, the sensitivity is difficult to meet the detection needs, which makes it difficult to achieve effective detection in the clinic.
  • tuberculosis and syphilis pathogen infections which are particularly high-frequency and representative, have extremely low pathogenic content, which leads to great difficulties from the early diagnosis to the evaluation of cure.
  • An object of the present invention is a method for detecting pathogens based on cfDNA high-throughput sequencing.
  • the method for detecting pathogens includes the following steps: Step 1. Constructing a characteristic database of pathogens to be detected through a public biological database, and characteristics of the pathogens to be detected The database is a database obtained by removing the redundancy of the whole genome data of the pathogen to be detected and shielding similar homologous sequences; step two, preparing a cfDNA sample from the biological sample to be tested, and constructing a cfDNA high-throughput sequencing library; The cfDNA high-throughput sequencing library performs high-throughput sequencing to obtain cfDNA sequencing data; step four, performing bioinformatics analysis on the cfDNA sequencing data, including: a.
  • Adapter adapter sequence
  • Masking similar homologous sequences refers to shielding sequences that have a certain degree of similarity with other pathogens, which can effectively avoid false positives; the database of characteristic pathogens can be updated from time to time according to the sequencing of the pathogen's genome.
  • the preparation of a cfDNA sample from a biological sample to be tested and the construction of a cfDNA high-throughput sequencing library are aimed at the biological sample to be tested.
  • the supernatant can be purified by secondary centrifugation, the nucleic acid is extracted using a cfDNA extraction kit, and then high-throughput
  • the sequencing library preparation kit prepares the Index sequencing library and performs corresponding quality control.
  • cfDNA high-throughput sequencing library for high-throughput sequencing refers to mixing a certain proportion of the to-be-sequenced index libraries and sequencing on the machine according to the data volume requirements for pathogen detection, and finally obtaining high-throughput sequencing data
  • the pathogen data is standardized using the following calculation formula to determine the pathogen detection value T i
  • the standardized pathogen method can effectively evaluate the relative content between pathogen data and the host, and does not depend on other factors such as the amount of sequencing and the number of pathogens to be detected.
  • the biological sample to be tested is selected from the group consisting of peripheral blood, cerebrospinal fluid, or diseased tissue. .
  • the host genome data is whole genome data; the valid data is compared with the host genome data by bowtie or bwa software.
  • the high-throughput sequencing is performed using an Illumina Nextseq 500 sequencer.
  • the pathogen to be detected is a parasite, a fungus, a bacterium, an actinomycete, a virus, a mycoplasma, a chlamydia, a rickettsia, or a spirochaete.
  • the pathogen to be detected is Treponema pallidum or Mycobacterium tuberculosis.
  • the public biological database is an NCBI database.
  • the second step includes the isolation and purification of the biological sample to be tested, the extraction of pathogenic cfDNA, the construction of a high-throughput sequencing library, and the quality control of the library.
  • the detection object of the method of the present invention is cfDNA. After the pathogen infects a person, its self-degraded DNA fragments will enter the human peripheral blood. Therefore, the peripheral blood cfDNA is deeply sequenced and combined with an efficient and sensitive bioinformatics analysis method Can achieve the purpose of pathogen detection. And the purpose of this method is to solve the detection and identification of specific chronic pathogen infections. Unlike the detection of unknown pathogens in the metagenomics, the data analysis process is clear, efficient and accurate. It can be applied to the early diagnosis, diagnosis and cure evaluation of tuberculosis and syphilis.
  • pathogenic infection samples such as parasites, bacteria, viruses (DNA), mycoplasma, chlamydia, rickettsial body and spirochaete. And identification, to achieve rapid, accurate and high-sensitivity quantifiable pathogen detection.
  • FIG. 1 is a schematic flowchart of detecting a chronic pathogenic infection using the method of the present invention.
  • the non-redundant feature database of the pathogen will be finally constructed and processed by BWA database for comparison software use.
  • Treponema pallidum infection was taken as an example.
  • the clinical diagnosis is secondary syphilis. 10 mL of peripheral blood of the patient is taken and tested according to the method of the present invention. The detailed process is as follows:
  • the processed data was compared with the human genome HG19 using BWA software. After statistical comparison, the data of the host was about 137.79M reads, accounting for 96.55% of the total data;
  • the filtered data was compared with the virus Treponema pallidum database constructed in Example 1 using BWA software, and 57 Treponema pallidum reads were detected;
  • test results are processed using the standardized method of the present invention, and the detection value of Treponema pallidum is 1.09E-3.
  • Example 3 Testing in a large number of syphilis-infected samples

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Abstract

本发明提供一种基于cfDNA高通量测序检测病原体的方法，所述病原体检测方法包括，通过公共的生物学数据库构建待检测病原体的特征数据库；从待测生物样品制备cfDNA样品，构建cfDNA高通量测序文库；利用所述cfDNA高通量测序文库进行高通量测序，获取cfDNA测序数据；和对所述cfDNA测序数据进行生物信息学分析。本发明方法解决了病毒的临床确诊检测及治愈评价，实现高灵敏度、可量化的病原检测及鉴定。

Description

一种基于cfDNA高通量测序检测病原体的方法 技术领域
本发明涉及病原体检测领域，特别涉及一种基于cfDNA高通量测序检测病原体的方法。
背景技术
感染性疾病的实验室诊断中，现有检测方法如涂片、镜检，检出率较低；培养法耗时且操作繁琐，对一些厌氧菌及病毒则不易进行培养鉴定；血清学、生物化学及PCR分子生物学检测，仅能针对有限的病原种类，无法对其他感染做出判定；尤其对于慢性病原感染，存在灵敏度难以达到检测需求，导致临床难以达到有效检测的目的。
在本发明所涉及的慢性病原感染中，尤其高发且具有代表性的结核及梅毒病原感染，因为其病原含量极低，导致从前期的诊断到治愈的评价等均存在较大困难。鉴于上述检测技术方法的局限性，发明新的快速、准确、高灵敏度的病原体检测技术势在必行。
发明内容
本发明的目的一种基于cfDNA高通量测序检测病原体的方法，所述病原体检测方法包括以下步骤：步骤一，通过公共的生物学数据库构建待检测病原体的特征数据库，所述待检测病原体的特征数据库是所述待检测病原体的全基因组数据去冗余、屏蔽相似同源序列所得的数据库；步骤二，从待测生物样品制备cfDNA样品，构建cfDNA高通量测序文库；步骤三，利用所述cfDNA高通量测序文库进行高通量测序，获取cfDNA测序数据；步骤四，对所述cfDNA测序数据进行生物信息学分析，其包括：a.从所述cfDNA测序数据去除接头序列(Adapter)、复制的序列(Duplication)、连续单碱基重复(Poly)结构以及过滤质量值低于Q20的reads，去除长度小于50bp的reads， 获得有效数据；b.将所述有效数据与宿主基因组数据进行比对，将与之比对上的reads进行去除，统计宿主数据量，保留非宿主数据，获得比对至宿主上的reads数量N h和宿主基因组长度L h；c.将所述非宿主数据与待检测病原体的特征数据库进行比对，获得代表比对至待检测病原体i上的reads数量N i、待检测病原体i的基因组长度L i，筛选比对结果，确定所述待测生物样品是否含待检测病原体。
屏蔽相似同源序列是指屏蔽与其他病原体具有一定相似度的序列，可有效避免假阳性；特征病原体数据库可以根据病原体基因组测序情况进行不定时更新。
所述从待测生物样品制备cfDNA样品，构建cfDNA高通量测序文库是针对待测生物样品，可以采用二次离心分离上清进行纯化，使用cfDNA提取试剂盒进行核酸提取，然后使用高通量测序文库制备试剂盒制备Index测序文库，并进行相应的质量控制。
利用所述cfDNA高通量测序文库进行高通量测序是指根据病原体检测的数据量需求，对待测序Index文库进行一定比例混合后上机测序，最终获得高通量测序数据
在一种实施方式中，如果所述生物样品含待检测病原体，将所述病原数据使用以下计算公式进行标准化，确定该病原体检测值T i
Figure PCTCN2019085073-appb-000001
。所述病原体标准化方法可以有效评价病原数据与宿主间的相对含量，不依赖于测序量、待检测病原体数量等其它因素。
在一种实施方式中，所述待测生物样品选自外周血、脑脊液或病灶组织。。
在一种实施方式中，所述宿主基因组数据为全基因组数据；所述有效数据与宿主基因组数据通过bowtie或者bwa软件进行比对。
在一种实施方式中，所述高通量测序使用illumina Nextseq 500测序仪进行测序。
在一种实施方式中，所述待检测病原体是寄生虫、真菌、细菌、放线菌、病毒、支原体、衣原体、立克次体或螺旋体。
在一种实施方式中，所述待检测病原体是梅毒螺旋体或结核分枝杆菌。
在一种实施方式中，所述公共的生物学数据库是NCBI数据库。
在一种实施方式中，所述步骤二包括待测生物样品的分离纯化、涵盖病原cfDNA提取、高通量测序文库构建以及文库质量控制。
本发明的方法检测对象为cfDNA，在病原感染人后，其自身降解的DNA片段会进入到人体外周血中，因此通过对外周血cfDNA进行深度测序，并结合高效、灵敏的生物信息学分析方法可以实现病原检测目的。且该方法目的在于解决特定慢性病原感染的检测及鉴定，不同于宏基因组未知病原检测，数据分析过程明确、高效、准确。可适用于结核及梅毒的早期诊断确诊及治疗后的治愈评价，也可将该方法拓展到寄生虫、细菌、病毒(DNA)、支原体、衣原体、立克次体及螺旋体等病原感染样本的检测及鉴定中，实现快速、精准、高灵敏度的可量化病原检测。
附图说明
为了更清楚地说明本申请实施例中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请中记载的一些实施例，对于本领域普通技术人员来说，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它的附图。
图1是使用本发明的方法检测慢性病原感染的流程示意图。
具体实施方式
为了使本领域技术领域人员更好地理解本申请中的技术方案，下面将结合下面结合实施例对本发明作进一步说明，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例，都应当属于本申请保护的范围。以下实施例中，如无特殊说明，均为常规方法。
实施例1.梅毒螺旋体数据库构建
1.通过NCBI(美国国立生物技术信息中心)下载梅毒螺旋体基因组数据梅毒螺旋体基因组数据收集；
2.将收集的梅毒螺旋体基因组数据间使用blastn软件进行比对，并进行同源性分析，将同源性大于99％的基因组进行过滤，构建非冗余基因组数据集；
3.去除重复序列，将构建好的非冗余数据集与NT库中其它病原体数据进行blastn比对，将与其它病原数据库比对长度大于50bp的区域使用N进行屏蔽，处理结果显示主要区域为核糖体区域；
4.将最终构建该病原体的非冗余特征数据库并进行BWA建库处理，以备比对软件使用。
实施例2.梅毒感染样本检测
我们以1例梅毒螺旋体感染的临床病例为例，临床确诊为二期梅毒，取患者外周血10mL，按照本发明方法进行检测，详细过程如下：
一.cfDNA样本制备
1.采用4000转/秒，低速离心10分钟后取上清；然后对上清采用12000转/秒，高速离心10分钟后再取上清，获得约4ml上清用于后续cfDNA提取；
2.使用TIANamp Micro DNA Kit试剂盒对制备的上清进行DNA提取；
3.使用Library Prep Kit for Illumina V2试剂盒构建cfDNA的Index测序文库；
4.使用Qubit，Agilent2100对文库进行定量质控，最终获得5ul文库，浓度不小于0.2ng/ul，完成样本制备过程。
二.高通量测序
1.采用Illumina NextSeq 500测序仪进行单端75bp文库测序，共获得156M reads；
2.对测序数据进行质控，Q30占91％，测序质量较好。
三.生物信息学分析
1.使用fastp软件对下机数据进行去除接头序列、复制的序列、连续单碱基重复结构，过滤质量值低于Q20的reads，去除长度小于50bp的reads，并进行相关统计及质控，处理后获得142.72M reads，过滤约8.75％的数据；
2.将处理后的数据使用BWA软件与人基因组HG19进行比对，经统计比对到宿主的数据约137.79M reads，占总数据的96.55％；
3.将过滤后的数据使用BWA软件与实施例1构建的病毒螺旋体数据库进行比对，检测得到57条梅毒螺旋体reads；
4.将检测结果使用本发明标准化方法进行处理，获得梅毒螺旋体检测值为1.09E-3。
样品号 N tp L tp(Mb) N h(M) L h(Gb) T tp
TP0001 57 1.14 137.79 3 1.09E-3
实施例3.在大量梅毒感染样本中进行测试
我们以临床收集梅毒螺旋体感染病例样本150份为例，按照治疗过程分为活跃期，血清固定期和治愈期三组各50份血液样本按照本发明方法进行检测，采用实施例2的检测过程，对检测结果进行统计，结果如下表：
表1.梅毒150例样本检测结果统计表
Figure PCTCN2019085073-appb-000002
由此结果显示，在梅毒感染初期确诊可以达到100％，治疗过程中的抵抗组中有16％的部分病例仍需继续治疗。
应该理解到披露的本发明不仅仅限于描述的特定的方法、方案和物质，因为这些均可变化。还应理解这里所用的术语仅仅是为了描述特定的实施方式方案的目的，而不是意欲限制本发明的范围，本发明的范围仅受限于所附的权利要求。
本领域的技术人员还将认识到，或者能够确认使用不超过常规实验，在本文中所述的本发明的具体的实施方案的许多等价物。这些等价物也包含在所附的权利要求中。

Claims (9)

  1. 一种基于cfDNA高通量测序检测病原体的方法，其特征在于，所述病原体检测方法包括以下步骤：
    步骤一，通过公共的生物学数据库构建待检测病原体的特征数据库，所述待检测病原体的特征数据库是所述待检测病原体的全基因组数据去冗余、屏蔽相似同源序列所得的数据库；
    步骤二，从待测生物样品制备cfDNA样品，构建cfDNA高通量测序文库；
    步骤三，利用所述cfDNA高通量测序文库进行高通量测序，获取cfDNA测序数据；
    步骤四，对所述cfDNA测序数据进行生物信息学分析，其包括：
    a.从所述cfDNA测序数据去除接头序列、复制的序列、连续单碱基结构以及过滤质量值低于Q20的reads，去除长度小于50bp的reads，获得有效数据；
    b.将所述有效数据与宿主基因组数据进行比对，将与之比对上的reads进行去除，统计宿主数据量，保留非宿主数据，获得比对至宿主上的reads数量N h和宿主基因组长度L h；
    c.将所述非宿主数据与待检测病原体的特征数据库进行比对，获得代表比对至待检测病原体i上的reads数量N i、待检测病原体i的基因组长度L i，筛选比对结果，确定所述待测生物样品是否含待检测病原体。
  2. 根据权利要求2所述的方法，其特征在于，如果所述生物样品含待检测病原体，将所述病原数据使用以下计算公式进行标准化，确定该病原体检测值T i，
    Figure PCTCN2019085073-appb-100001
  3. 根据权利要求1所述的方法，其特征在于，所述待测生物样品选自外周血、脑脊液或病灶组织。
  4. 根据权利要求1所述的方法，其特征在于，所述宿主基因组数据为全基因组数据；所述有效数据与宿主基因组数据通过bowtie或者bwa软件进行比对。
  5. 根据权利要求1所述的方法，其特征在于，所述高通量测序使用illumina Nextseq 500测序仪进行测序。
  6. 根据权利要求1所述的方法，其特征在于，所述待检测病原体是寄生虫、真菌、细菌、放线菌、病毒、支原体、衣原体、立克次体或螺旋体。
  7. 根据权利要求6所述的方法，其特征在于，所述待检测病原体是梅毒螺旋体或结核分枝杆菌。
  8. 根据权利要求1所述的方法，其特征在于，所述公共的生物学数据库是NCBI数据库。
  9. 根据权利要求1所述的方法，其特征在于，所述步骤二包括待测生物样品的分离纯化、涵盖病原cfDNA提取、高通量测序文库构建以及文库质量控制。
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