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WO2019010775A1 - 分子标签、接头及确定含有低频突变核酸序列的方法 - Google Patents

分子标签、接头及确定含有低频突变核酸序列的方法 Download PDF

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Publication number
WO2019010775A1
WO2019010775A1 PCT/CN2017/100421 CN2017100421W WO2019010775A1 WO 2019010775 A1 WO2019010775 A1 WO 2019010775A1 CN 2017100421 W CN2017100421 W CN 2017100421W WO 2019010775 A1 WO2019010775 A1 WO 2019010775A1
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Prior art keywords
nucleic acid
molecular tag
acid sequence
molecular
linker
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French (fr)
Inventor
曾晓静
高晓峘
韩颖鑫
张印新
何哲
王佳伟
夏伟成
李胜
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Guangzhou Jingke Dx Co Ltd
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Guangzhou Jingke Dx Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

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  • the present invention relates to the field of nucleic acid sequencing technology. Specifically, the present invention relates to a molecular tag and a composition thereof, a molecular tag-containing linker and a composition thereof, and a method for determining a target region of a sample to be tested containing a low frequency mutant nucleic acid sequence.
  • High-throughput sequencing is currently the most widely used sequencing technology.
  • the incidence is 0.1%-0.2% or higher, and the DNA polymerase used in the PCR process is also wrong.
  • the rate and error rate are 10-7 to 10-5, especially as the number of PCR cycles increases, the error rate also increases.
  • Each position of the molecular tag can be one of four bases A, T, C, and G.
  • the length of the molecular tag is selected according to actual experimental needs. According to the length of the molecular tag and the change of four bases, the molecular tag There can be 4 n-th power types. If the molecular tags of the original template are completely randomly distributed, the diversity of the molecular tags ensures that each original template is unique after the molecular tag is attached to the original library.
  • each original template will act as The initial template forms a cluster of "molecular clusters". If there are no sequencing errors and PCR errors, the molecular sequences in each cluster are the error-free "replication strands" of the original template positive and negative strands.
  • the base sequences at each position of the molecular tag are completely randomly distributed.
  • the same amount of A, T, The four bases of C and G because the energy or synthesis efficiency required for the synthesis of these four bases is different, the frequency of occurrence of the four bases A, T, C, and G at each position is not completely equal.
  • the object of the present invention is to provide a molecular tag which has a completely random distribution of bases by optimizing the design of the molecular tag, and a molecular tag composition having a ratio of 0.95-1.05:1 for each molecular tag, and using the molecular tag and The linker synthesized by the composition was constructed and sequenced to effectively distinguish between sequencing errors and low frequency mutations.
  • the invention provides a molecular tag having up to two consecutive identical bases.
  • Another aspect of the present invention also provides a molecular tag composition comprising the above molecular tag, and the ratio of each molecular tag is from 0.95 to 1.05:1.
  • Another aspect of the present invention also provides a linker comprising the above molecular tag, And the molecular tag is located at any position other than the overhang "T" of the linker and the 20 bp base of the non-overhanging end.
  • Another aspect of the present invention also provides a linker composition comprising the above-described linker, and the ratio of each linker is from 0.95 to 1.05:1.
  • Another aspect of the present invention also provides a method for determining a target region of a sample to be tested containing a low frequency mutant nucleic acid sequence, comprising the steps of:
  • the molecular tag provided by the present invention does not have a plurality of identical bases in succession, thereby avoiding a situation in which the sequencing quality is poor due to the appearance of a plurality of consecutive bases. And various kinds of molecular tags inside The proportion of the labels is the same, avoiding the situation of the dominant labels, and maximizing the performance of the molecular labels.
  • FIG. 1 is a schematic view showing the structure of a molecular tag in a fully complementary double link head according to an embodiment of the present invention.
  • FIG. 2 is a schematic view showing the structure of a molecular tag in a Y-type connector with one end complementary to one end at the complementary end in the embodiment of the present invention.
  • FIG. 3 is a schematic view showing the structure of a molecular tag in a Y-type connector with one end complementary to one end in an open end.
  • Figure 4 is a schematic illustration of a Y-type structure in which a molecular tag is not located on a linker, but a linker can be introduced by PCR, in accordance with an embodiment of the present invention.
  • FIG. 5 is a flow chart of a method for determining a target region of a sample to be tested containing a low frequency mutant nucleic acid sequence according to an embodiment of the present invention.
  • the present invention provides a molecular tag having up to two consecutive identical bases on the molecular tag.
  • the molecular tag is a single strand or a reverse complementary double strand.
  • the number of bases of the molecular tag is 6-24 bp.
  • the present invention also provides a molecular tag composition
  • a molecular tag composition comprising the molecular tag as described above, and the ratio of each molecular tag is from 0.95 to 1.05:1.
  • the ratio includes at least one of a molar ratio, a molecular mass ratio, and a molecular ratio.
  • the number of species of the molecular tag comprises 4n and n is equal to 6-24.
  • n is equal to 6-24.
  • 4096, 16384, 65536, 262144, 16777216, 268435456, and even more types can be designed.
  • the molecular tag when the molecular tag is a single-stranded structure, the molecular tag is in a ratio of a molar number of 0.95-1.05:1, or a molecular mass of 0.95-1.05:1, or a molecular number of 0.95-1.05:1.
  • the ratio is mixed.
  • the molecular tag when the molecular tag is a single-stranded structure, the molecular tag is mixed in a ratio of 1:1 molar ratio, or a molecular mass ratio of 1:1, or a molecular number of 1:1.
  • the single-stranded molecular tag is firstly matched according to the ratio of the molar number of 0.95-1.05:1, or the molecular mass of 0.95-1.05:1, or the ratio of the number of molecules of 0.95-1.05:1.
  • the reverse complementary sequences are annealed to form a molecular tag of a double-stranded structure, and these double-stranded molecular tags are then mixed in a ratio of 0.95-1.05:1.
  • the single-stranded molecular tag is firstly reversed according to a molar ratio of 1:1, or a molecular mass of 1:1, or a molecular number of 1:1.
  • the complementary sequences are annealed to form a molecular tag of the double-stranded structure, and the double-stranded molecular tags are then mixed in a ratio of 1:1.
  • the invention also provides for the use of the molecular tag composition for correcting sequencing errors and PCR errors, detecting low frequency mutations, de-redundancy, and calculating the number of specific molecules or cells carrying a particular molecule.
  • Another aspect of the invention provides a linker comprising a molecule as described above A tag, and the molecular tag is located at any position other than the overhang "T" of the linker and the 20 bp base of the non-overhang end.
  • the molecular tag "NNN...NNN” may be located at the 3' end, 5' of the fully complementary duplex of the linker. End or middle, anywhere except the overhang "T” and non-overhanging end blocks, which are 20 bp base length.
  • the molecular label "NNN...NNN” may be located at one end of the joint Y-shaped structure, open One end or the middle, except for the protruding end "T” and any position other than the 20 bp base of the non-overhanging end.
  • the molecular tag may not be located on the linker, but may be introduced into the Y-type structure of the linker by PCR.
  • the molecular tag may also be located at two or more positions of the joint.
  • the linker further comprises a library tag linked to the 3' or 5' end of the molecular tag.
  • the library tag is used to distinguish different sample libraries, and after PCR amplification, PCR products of multiple samples are mixed and sequenced, and samples of each sequence are further based on library tags. Sources are distinguished.
  • the linker further comprises an identifying characteristic sequence, the identifying characteristic sequence being 4 non-repeating bases, for example: "ATCG” or “TGAC”, the identifying feature sequence and the The 3' or 5' end of the molecular tag is linked.
  • Another aspect of the present invention also provides a linker composition comprising a linker as described above, and the ratio of each linker is from 0.95 to 1.05:1.
  • the types of the joints include:
  • the one end of the molecular tag is complementary to the Y-type open end
  • the molecular tag is not located on the linker, but can be introduced into the Y-type structure of the linker by PCR.
  • a joint having a molecular tag at two or more positions is also included.
  • the ratio of the joint is at least one of a molar ratio, a molecular mass ratio, and a molecular ratio of each type of joint.
  • a further aspect of the present invention provides a method for determining a target region of a sample to be tested containing a low frequency mutant nucleic acid sequence, as shown in FIG. 5, comprising the steps of:
  • said step S6 further comprises filtering sequencing errors brought in by PCR and sequencing.
  • the data analysis can be performed using statistical analysis methods well known to those skilled in the art, such as Z test, T test, run test, and the like.
  • Molecular tags are designed according to the possibility of random distribution of 4 bases per position, and the molecular tag contains up to 2 consecutive identical bases. According to the needs of the experiment, different kinds of molecular labels M were designed.
  • the number of species of the molecular tag sequence includes 4n, and n is equal to 6-24. As shown in Table 1, 16 molecular labels:
  • a linker containing any of the above molecular tags is designed, wherein the molecular tag can be located at any position other than the overhang "T" of the linker and the 20 bp base of the non-overhang end.
  • NNN...NNN represents a molecular tag
  • the type of the linker may be a fully complementary double-stranded structure, a Y-type structure in which one end is complementary to one end, or may be PCR-
  • the molecular tag is introduced into the Y-type structure of the linker.
  • the molecular tag may be located at either or both ends of the linker, or may be distributed at two or more positions.
  • the number of N represents the number of bases of the molecular tag, and the number of molecular tags required increases the number of bases at the position.
  • the number for example, the number of bases of 8 bp, 12 bp, 16 bp, 24 bp or more.
  • 16 kinds of linkers containing different molecular tags As shown in Table 2, 16 kinds of linkers containing different molecular tags:
  • Identification signature sequences and/or library tags can also be added at the 3' or 5' end of the molecular tag as desired for the experiment. For example, when sequencing using the illumina platform, an index sequence that identifies different samples can be added to it.
  • the designed molecular tag or its corresponding reverse complement sequence and its sequence at the 3' end and the 5' end are synthesized to obtain a linker containing the molecular tag.
  • synthetic methods can be employed in methods well known in the art or can be commissioned by a primer synthesis company.
  • the synthesized molecular tag-containing linkers were mixed at a molar ratio of 1:1 for different types.
  • each type of joint is mixed in a ratio of 1:1.
  • the patient's peripheral EDTA anticoagulation was taken 10 ml, and the plasma was separated by fresh centrifugation, and plasma DNA was extracted according to a method well known to those skilled in the art.
  • the extracted DNA solution and the end-repaired reagent mixture are mixed, and the reaction is carried out according to a method of terminal repair well known to those skilled in the art, and the reaction is separated and purified.
  • reaction system After mixing at room temperature, after slight centrifugation, the reaction system was placed in a PCR machine, and reacted at 20 ° C for 30 minutes. After the reaction was completed, it was purified using AMpure XP magnetic beads.
  • terminal-repaired DNA solution and the "A"-added reagent mixture are mixed, and the reaction is carried out according to the method of adding "A" at the end well known to those skilled in the art, and the reaction is separated and purified.
  • reaction system After mixing at room temperature, after slight centrifugation, the reaction system was placed in a PCR machine, and reacted at 37 ° C for 30 minutes. After the reaction was completed, it was purified using AMpure XP magnetic beads.
  • Magnetic bead purification was carried out by the method shown in 5.2, except that 75 ul of magnetic beads were added to the 50 ul system reaction product.
  • the DNA solution after the addition of "A” is mixed with the molecular tag-containing linker and the reaction reagent mixture obtained in the step S3, and the reaction is carried out according to a method of adding a linker well known to those skilled in the art, and after the completion of the reaction, separation and purification are carried out.
  • reaction system After mixing at room temperature, after slight centrifugation, the reaction system was placed in a PCR machine, and reacted at 20 ° C for 15 minutes. After the reaction was completed, it was purified using AMpure XP magnetic beads.
  • Magnetic bead purification was carried out using the method shown in 5.2, except that 75 ul of magnetic beads were added to the 50 ul system reaction product.
  • the DNA after the addition of the linker and the PCR reaction reagent mixture are mixed, and the PCR reaction is carried out according to a method well known to those skilled in the art. After the reaction is completed, the separation and purification are carried out. After the completion of the library construction, the library is subjected to QC detection, and the test is waited after passing the test. Sequencing.
  • Reagent Volume /ul DNA 32 10 ⁇ Pfx amplification buffer 5 dNTP solution (10nM) 2 MgSO 4 (50nM) 2 PCR primer PE1.0 (10pmol/ul) 4 index-X (10pmol/ul) 4 Pfx DNA polymerase 1 Total volume / ul 50
  • reaction system After mixing at room temperature, after slight centrifugation, the reaction system was placed in a PCR machine and reacted according to the following conditions:
  • Magnetic bead purification was carried out using the method shown in 5.2, except that 50 ul of magnetic beads were added to the 50 ul system reaction product. The library construction is over.
  • the library was subjected to QPCR and Agilent 2100 detection, and the quality-qualified library was arranged on the machine.
  • the library can be sequenced using a second generation sequencer such as Hiseq2000, Hiseq 2500, Proton, Miseq, NS500.
  • a second generation sequencer such as Hiseq2000, Hiseq 2500, Proton, Miseq, NS500.
  • the sequencing results of the DNA obtained after sequencing are analyzed, and the obtained DNA sequences are classified according to molecular tags, and the sequence carrying the same molecular tag is taken as a "molecular cluster" which is the initial one DNA molecule.

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Abstract

本发明提供了分子标签及其组合物、含有分子标签的接头及其组合物以及一种确定待测样本目标区域含有低频突变核酸序列的方法。其中所述分子标签上最多含有2个连续相同的碱基。

Description

分子标签、接头及确定含有低频突变核酸序列的方法 技术领域
本发明涉及核酸测序技术领域，具体的，本发明涉及分子标签及其组合物、含有分子标签的接头及其组合物、确定待测样本目标区域含有低频突变核酸序列的方法。
背景技术
高通量测序是目前应用范围最广的测序技术，然而其在测序中仍不可避免的存在一些测序错误，发生率为0.1％-0.2％或者更高，并且PCR过程使用的DNA聚合酶也有错误率，错误率为10-7～10-5，特别是随着PCR循环数的增加错误率也有所增加。
为了检测低于0.1％的碱基突变(低频突变)或测序错误，学者发明了分子标签的方法，分子标签是在PCR之前给每个测序模板的一端或者两端加入一段特殊的序列。分子标签的每个位置可以是A、T、C、G 4种碱基中的1种，分子标签的长度根据实际的实验需要选择，根据分子标签的长度及4种碱基的变化，分子标签可以有4的n次方种类。如果原始模板的分子标签是完全随机分布的，那分子标签的多样性能够保证每个原始模板在原始文库中连上分子标签后是独一无二的，在之后的PCR过程中，每个原始模板会作为初始模板形成一簇“分子簇”，如果没有测序错误和PCR错误，这各簇中的分子序列都是初始模板正链和负链的无错误“复制链”。
理论上，分子标签的每个位置的碱基序列是完全随机分布的。然而，在引物合成过程中，合成某一碱基时，会加入等量的A、T、 C、G四种碱基，由于这四种碱基合成所需的能量或者合成效率不一样，使得每个位置上A、T、C、G四种碱基的出现频率并不是完全相等的。这样会造成部分的碱基处于优势地位，导致了分子标签中并不是每个位置都遵循A、T、C、G四种碱基随机分布的概率，并且会出现优势分子序列，甚至会出现多个连续一样的碱基，例如8个A、8个G等，从而导致实际上得到的随机分子标签种类并没有理论上那么多。
多个连续一样的碱基不仅会增加测序错误的可能性，也会增加优势分子序列的比例。由于比例不随机，使得某几种甚至更多的分子连上了同一种标签序列。当这些连上同一种标签序列的分子属于同源性高或者序列十分相似的情况下，技术人员无法区别判断属于测序错误和低频突变的分子。更进一步的，当低频突变和正常丰度的序列连上一样的分子克隆时会导致将低频突变当成测序错误或PCR错误从而漏检。因此分子标签的不随机性会降低其效用，甚至限制了其应用。
发明内容
本发明的目的在于，通过优化分子标签的设计，提供一种碱基完全随机分布的分子标签，及每种分子标签的比例均为0.95-1.05:1的分子标签组合物，利用该分子标签及其组合物合成的接头进行文库构建并对其进行测序，从而有效地区分测序错误和低频突变。
本发明一方面提供一种分子标签，所述分子标签上最多含有2个连续相同的碱基。
本发明另一方面还提供一种分子标签组合物，含有上述分子标签，且每种分子标签的比例为0.95-1.05:1。
本发明另一方面还提供一种接头，所述接头含有上述分子标签， 且所述分子标签位于所述接头除突出端“T”和非突出端末端20bp碱基以外的任意位置。
本发明另一方面还提供一种接头组合物，含有上述接头，且每种接头的比例为0.95-1.05:1。
本发明另一方面还提供一种确定待测样本目标区域含有低频突变核酸序列的方法，包括如下步骤：
S1、利用如上所述的接头，对待测样本目标区域核酸进行加接头反应，对加接头后的待测样本目标区域核酸进行PCR扩增，获得扩增产物，所述扩增产物构成所述待测样本的目标区域核酸测序文库；
S2、对所述待测样本的目标区域核酸测序文库进行测序，获得测序后核酸序列；
S3、将所述测序后核酸序列按照所述接头中含有的分子标签进行分类，将携带有相同分子标签的所述测序后的核酸序列归类为同一核酸序列集；
S4、将所述核酸序列集内的测序后核酸序列进行相互比较，统计所述核酸序列集中每个碱基位置的碱基种类及其频率；
S5、根据所述核酸序列集中每个碱基位置的碱基种类及其频率，通过数据分析，得到所述核酸序列集中含有正确的碱基排列位置的核酸序列；
S6、将所述含有正确的碱基排列位置的核酸序列与所述核酸序列集中的其余的核酸序列或平行的核酸序列集中的核酸序列进行比较，得到含有低频突变的核酸序列。
本发明所提供的分子标签没有连续多个相同的碱基，避免由于多个连续碱基出现导致测序质量差的情况。并且分子标签内部各种 标签的比例一致，避免出现优势标签的情况，能够最大程度发挥分子标签的效能。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中
图1为本发明实施例中完全互补双链接头中分子标签结构示意图。
图2为本发明实施例中一端互补一端开放的Y型接头中分子标签位于互补端的结构示意图。
图3为本发明实施例中一端互补一端开放的Y型接头中分子标签位于开放端的结构示意图。
图4为本发明实施例中分子标签不位于接头上，但可通过PCR引入接头的Y型结构的示意图。
图5为本发明实施例中确定待测样本目标区域含有低频突变核酸序列的方法流程图。
具体实施方式
下面详细描述本发明的实施例。下面通过参考附图描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
需要说明的是，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
本发明提供一种分子标签，所述分子标签上最多含有2个连续相同的碱基。
根据本发明的实施例，所述分子标签为单链或反向互补的双链。
根据本发明的实施例，所述分子标签的碱基数目为6-24bp。
本发明还提供一种分子标签组合物，含有如上所述的分子标签，且每种分子标签的比例为0.95-1.05:1。
根据本发明的实施例，所述比例包括摩尔比、分子质量比、分子数比的至少之一。
根据本发明的实施例，所述分子标签的种类数包括4n，n等于6-24。例如根据实验需要，可以设计出4096、16384、65536、262144、16777216、268435456种，甚至更多的种类。
根据本发明的实施例，当分子标签是单链的结构，则将分子标签按照摩尔数0.95-1.05:1的比例，或分子质量0.95-1.05:1的比例，或分子数0.95-1.05:1的比例混合。优选的，当分子标签是单链的结构，则将分子标签按照摩尔数1:1的比例，或分子质量1:1的比例，或分子数1:1的比例混合。
当分子标签是双链的结构，先将单链的分子标签按照摩尔数0.95-1.05:1的比例，或分子质量0.95-1.05:1的比例，或分子数0.95-1.05:1的比例与对应的反向互补的序列进行退火互补形成双链结构的分子标签，再将这些双链分子标签按照0.95-1.05:1的比例混合。优选的，当分子标签是双链的结构，先将单链的分子标签按照摩尔数1:1的比例，或分子质量1:1的比例，或分子数1:1的比例与对应的反向互补的序列进行退火互补形成双链结构的分子标签，再将这些双链分子标签按照1:1的比例混合。
本发明还提供所述分子标签组合物，在纠正测序错误和PCR错误、检测低频突变、去冗余以及计算特定分子或携带有特定分子的细胞数量中的应用。
本发明另一方面提供一种接头，所述接头含有如上所述的分子 标签，且所述分子标签位于所述接头除突出端“T”和非突出端末端20bp碱基以外的任意位置。
根据本发明的实施例，如图1所示，当所述接头为完全互补的双链结构时，所述分子标签“NNN…NNN”可位于接头完全互补双链中的3’端、5’端或中间，除突出端“T”和非突出端末端方框以外的任意位置，所述方框内为20bp碱基长度。
根据本发明的实施例，如图2和3所示，当所述接头为一端互补一端开放的Y型结构时，所述分子标签“NNN…NNN”可位于接头Y型结构互补的一端、开放的一端或中间，除突出端“T”和非突出端末端20bp碱基以外的任意位置。
根据本发明的实施例，如图4所示，所述分子标签还可以不位于接头上，但可通过PCR引入接头的Y型结构中。
进一步的，根据本发明的实施例，所述分子标签还可以位于接头的2个或2个以上的位置。
根据本发明的实施例，所述接头还含有文库标签，所述文库标签与所述分子标签的3’端或5’端相连。
本领域技术人员可以理解的，所述文库标签用于区分不同样品文库，能够在进行PCR扩增后，将多个样本的PCR产物进行混合测序，进而基于文库标签的不同，对各序列的样本来源进行区分。
根据本发明的实施例，所述接头还含有识别性特征序列，所述识别性特征序列为4个不重复的碱基，例如：“ATCG”或“TGAC”，所述识别性特征序列与所述分子标签的3’端或5’端相连。
本发明另一方面还提供一种接头组合物，所述接头组合物含有如上所述的接头，且每种接头的比例为0.95-1.05:1。
根据本发明的一些具体示例，所述接头的种类包括：
如图1所示，含有分子标签的完全互补的双链接头；
如图2和3所示，含有分子标签的一端互补一端开放的Y型接头；
以及如图4所示，分子标签不位于接头上，但可通过PCR引入接头的Y型结构中。
根据本发明的实施例，还包括分子标签位于2个或2个以上的位置的接头。
根据本发明的实施例，所述接头的比例为各个种类的接头的摩尔比、分子质量比、分子数比的至少之一。
本发明再一方面还提供一种确定待测样本目标区域含有低频突变核酸序列的方法，如图5所示，包括如下步骤：
S1、利用如上所述的接头，对待测样本目标区域核酸进行加接头反应，对加接头后的待测样本目标区域核酸进行PCR扩增，获得扩增产物，所述扩增产物构成所述待测样本的目标区域核酸测序文库；
S2、对所述待测样本的目标区域核酸测序文库进行测序，获得测序后核酸序列；
S3、将所述测序后核酸序列按照所述接头中含有的分子标签进行分类，将携带有相同分子标签的所述测序后的核酸序列归类为同一核酸序列集；
S4、将所述核酸序列集内的测序后核酸序列进行相互比较，统计所述核酸序列集中每个碱基位置的碱基种类及其频率；
S5、根据所述核酸序列集中每个碱基位置的碱基种类及其频率，通过数据分析，得到所述核酸序列集中含有正确的碱基排列位置的核酸序列；
S6、将所述含有正确的碱基排列位置的核酸序列与所述核酸序列集中的其余的核酸序列或平行的核酸序列集中的核酸序列进行比较，得到含有低频突变的核酸序列。
根据本发明的实施例，所述步骤S6还包括过滤由PCR和测序带入的测序错误。
所述数据分析可采用本领域技术人员所熟知的统计学分析方法进行分析，例如Z检验、T检验、游程检验等。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面示例仅用于解释本发明，而不能理解为对本发明的限制。除另有交待，以下实施例中涉及的未特别交待的试剂、序列(接头、标签和引物)、软件及仪器，都是常规市售产品或者开源的。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
实施例一 检测低频突变基因
1、设计分子标签及含有该分子标签的接头
按照每个位置4种碱基随机分布的可能性设计分子标签，分子标签上最多含有2个连续相同的碱基。按照实验需要，设计不同种类的分子标签M种。分子标签序列的种类数包括4n，n等于6-24。 如表1所示，16种分子标签：
表1
SEQ ID NO: 分子标签 SEQ ID NO: 分子标签
1 ATCGATGC 9 ACTGCATC
2 ACGTGCAC 10 AGACTAGC
3 TCAGCATC 11 TAGTCAGC
4 TGCATGAC 12 TCGACTAC
5 GCATCATC 13 GTATCGAC
6 GTACTATC 14 GACTGATC
7 CATGATGC 15 CTAGTGAC
8 CGGTATTC 16 CGTACATC
设计含有上述任意一种分子标签的接头，其中分子标签可位于接头除突出端“T”和非突出端末端20bp碱基以外的任意位置。如图1、图2、图3、图4所示，NNN...NNN代表分子标签，接头的种类可以是，完全互补的双链结构、一端互补一端开放的Y型结构，或者可通过PCR将分子标签引入接头的Y型结构。分子标签可以仅位于接头的任意一端或中间，也可以分布于2个或者2个以上的位置，N的个数代表分子标签的碱基数目，需要的分子标签种类多就增加该位置的碱基个数，比如采用8bp、12bp、16bp、24bp或者更多的碱基个数。如表2所示，16种含有不同分子标签的接头：
表2
Figure PCTCN2017100421-appb-000001
Figure PCTCN2017100421-appb-000002
Figure PCTCN2017100421-appb-000003
当接头如图1和图2及其类似的结构，需要同时设计含有分子标签反向互补的结构，如需要同时设计表2中的F向序列和R向序 列，图3、图4及其类似的结构则只需要设计单链分子标签，如表2中的F向序列而不需要设计分子标签反向互补序列。
根据实验的需要，还可以在分子标签的3’或5’端添加识别性特征序列和/或文库标签。例如，使用illumina平台测序时，可以将识别不同样本的index序列加入其中。
2、合成含有分子标签的接头
根据所设计的接头序列，将设计出来的分子标签或及其对应的反向互补序列及其3'端、5'端的序列进行合成，得到含有分子标签的接头。本领域人员可以理解的，合成方法可采用本领域熟知的方法，也可委托给引物合成公司合成。
3、将得到的接头按比例混合，得到一组接头组合物
将合成的含有分子标签的接头按照不同的种类的摩尔数1:1的比例进行混合。
例如当如图1、图2、图3及其类似的结构的接头种类时，每种种类的接头按摩尔数1:1的比例混合。
当如图5及其类似的结构，将分子标签直接与Y型接头按照摩尔数1:1的比例混合，得到一组接头组合物。
4、提取样本DNA
抽取病人外周EDTA抗凝血10ml，并新鲜离心分离血浆，按照本领域技术人员熟知的方法提取血浆DNA。
5、DNA末端修复
将提取得到的DNA溶液和末端修复的试剂混合液混合，按照本领域技术人员熟知的末端修复的方法进行反应，反应结束后进行分离纯化。
5.1 按如下反应体系在1.5mlEP管中配制：
试剂 体积/ul
DNA 85
10×PNK缓冲液 10
dNTP溶液(10mM) 2
T4DNA聚合酶 1
T4PNK 1
KLENOW片段(稀释10 1
总体积/ul 100
室温混匀，轻微离心后，反应体系置于PCR仪中，20℃反应30分钟，反应结束后，使用AMpure XP磁珠纯化。
5.2 在100ul体系反应产物中加入150ul磁珠，进行AMpure XP磁珠纯化后，反复用500ul 75％乙醇洗涤两次，弃上清液。37℃烘干，至磁珠干燥。加入34.5ul水，混匀磁珠，待澄清，吸取34ul上清液。
6、末端加“A”
将末端修复的DNA溶液和加“A”的试剂混合液混合，按照本领域技术人员熟知的末端加“A”的方法进行反应，反应结束后进行分离纯化。
6.1 将5中得到的溶液按照以下体系配制反应液：
试剂 体积
末端修复DNA 34
10×蓝色缓冲液 5
dATP(1mM) 10
Klenow 3'-5'exo- 1
总体积/ul 50
室温混匀，轻微离心后，反应体系置于PCR仪中，37℃反应30分钟，反应结束后，使用AMpure XP磁珠纯化。
6.2 采用如5.2所示的方法进行磁珠纯化，其区别在于50ul体系反应产物中加入75ul磁珠。
7、加接头反应
将加“A”后的DNA溶液和步骤S3中得到的含有分子标签的接头、连接反应试剂混合液混合，按照本领域技术人员熟知的加接头的方法进行反应，反应结束后进行分离纯化。
7.1 将6中得到的溶液按照以下体系配制反应液：
Figure PCTCN2017100421-appb-000004
室温混匀，轻微离心后，反应体系置于PCR仪中，20℃反应15分钟，反应结束后，使用AMpure XP磁珠纯化。
7.2 采用如5.2所示的方法进行磁珠纯化，其区别在于50ul体系反应产物中加入75ul磁珠。
8、PCR富集，构建测序文库
将加接头后的DNA和PCR反应试剂混合液混均，按照本领域技术人员熟知的方法进行PCR反应，反应结束后进行分离纯化，到此文库构建结束，对文库进行QC检测，检测合格后等待测序。
8.1 在1个新的PCR管中按照以下体系配制反应液：
试剂 体积/ul
DNA 32
10×Pfx扩增缓冲液 5
dNTP溶液(10nM) 2
MgSO4(50nM) 2
PCR引物PE1.0(10pmol/ul) 4
index-X(10pmol/ul) 4
Pfx DNA聚合酶 1
总体积/ul 50
室温混匀，轻微离心后，反应体系置于PCR仪中，按照以下条件进行反应：
Figure PCTCN2017100421-appb-000005
反应结束后，使用AMpure XP磁珠纯化。
8.2 采用如5.2所示的方法进行磁珠纯化，其区别在于50ul体系反应产物中加入50ul磁珠。文库构建结束。
9、文库质检
对文库进行QPCR和Agilent 2100检测，质检合格文库安排上机。
10、对文库进行DNA测序
可使用Hiseq2000、Hiseq2500、Proton、Miseq、NS500等二代测序仪对文库进行测序。
11、分析测序结果
将测序后得到的DNA的测序结果进行分析，按照分子标签将得到的DNA序列进行分类，将携带有相同的分子标签的序列作为1个“分子簇”，这个分子簇是初始1个DNA分子通过PCR形成的1类DNA，即原始DNA分子的正链和负链的“复制链”。
统计“分子簇”内部每个碱基位置的碱基种类及其出现的频数。
根据数据分析，找出由于PCR和测序带入的错误并纠正。
从而得到原始DNA的正确序列，并通过分子簇内部和平行比较，找出真正的突变序列。
以上实施方式仅用以说明本发明的技术方案而非限制，尽管参照以上较佳实施方式对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或等同替换都不应脱离本发明技术方案的精神和范围。

Claims (10)

  1. 一种分子标签，其特征在于，所述分子标签上最多含有2个连续相同的碱基。
  2. 根据权利要求1所述的分子标签，其特征在于，所述分子标签为单链或反向互补的双链。
  3. 根据权利要求1所述的分子标签，其特征在于，所述分子标签的碱基数目为6-24bp。
  4. 一种分子标签组合物，其特征在于，含有如权利要求1-3任一项所述的分子标签，且每种分子标签的比例为0.95-1.05:1。
  5. 根据权利要求4所述的分子标签组合物，其特征在于，所述比例包括摩尔比、分子质量比、分子数比的至少之一。
  6. 一种接头，其特征在于，所述接头含有如权利要求1-3任一项所述的分子标签，且所述分子标签位于所述接头除突出端“T”和非突出端末端20bp碱基以外的任意位置。
  7. 如权利要求6所述的接头，其特征在于，所述接头还含有文库标签，所述文库标签与所述分子标签的3’端或5’端相连。
  8. 如权利要求6所述的接头，其特征在于，所述接头还含有识别性特征序列，所述识别性特征序列为4个不重复的碱基，所述识别性特征序列与所述分子标签的3’端或5’端相连。
  9. 一种接头组合物，其特征在于，所述接头组合物含有如权利要求6所述的接头，且每种接头的比例为0.95-1.05:1。
  10. 一种确定待测样本目标区域含有低频突变核酸序列的方法，包括如下步骤：
    S1、利用如权利要求6所述的接头，对待测样本目标区域核酸 进行加接头反应，对加接头后的待测样本目标区域核酸进行PCR扩增，获得扩增产物，所述扩增产物构成所述待测样本的目标区域核酸测序文库；
    S2、对所述待测样本的目标区域核酸测序文库进行测序，获得测序后核酸序列；
    S3、将所述测序后核酸序列按照所述接头中含有的分子标签进行分类，将携带有相同分子标签的所述测序后的核酸序列归类为同一核酸序列集；
    S4、将所述核酸序列集内的测序后核酸序列进行相互比较，统计所述核酸序列集中每个碱基位置的碱基种类及其频率；
    S5、根据所述核酸序列集中每个碱基位置的碱基种类及其频率，通过数据分析，得到所述核酸序列集中含有正确的碱基排列位置的核酸序列；
    S6、将所述含有正确的碱基排列位置的核酸序列与所述核酸序列集中的其余的核酸序列或平行的核酸序列集中的核酸序列进行比较，得到含有低频突变的核酸序列。
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