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WO2019010775A1 - Étiquette moléculaire, jonction et procédé de détermination de séquence nucléotidique contenant une mutation à basse fréquence - Google Patents

Étiquette moléculaire, jonction et procédé de détermination de séquence nucléotidique contenant une mutation à basse fréquence 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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WIPO (PCT)
Prior art keywords
nucleic acid
molecular tag
acid sequence
molecular
linker
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Ceased
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PCT/CN2017/100421
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English (en)
Chinese (zh)
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

Definitions

  • 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

La présente invention concerne une étiquette moléculaire et une composition de cette dernière, une jonction contenant l'étiquette moléculaire et une composition de cette dernière, ainsi qu'un procédé de détermination de séquence nucléotidique contenant la mutation à basse fréquence dans une région cible d'un échantillon à détecter. L'étiquette moléculaire contient au plus deux bases continues et identiques.
PCT/CN2017/100421 2017-07-14 2017-09-04 Étiquette moléculaire, jonction et procédé de détermination de séquence nucléotidique contenant une mutation à basse fréquence Ceased WO2019010775A1 (fr)

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CN201710573052.1A CN107385030A (zh) 2017-07-14 2017-07-14 分子标签、接头及确定含有低频突变核酸序列的方法
CN201710573052.1 2017-07-14

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CN109295049A (zh) * 2018-09-26 2019-02-01 刘强 血液微量cfDNA文库的标签特异性接头、引物组和建库方法

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US20160017410A1 (en) * 2014-07-17 2016-01-21 Jay Shendure Highly multiplex single amino acid mutagenesis for massively parallel functional analysis
CN106086162B (zh) * 2015-11-09 2020-02-21 厦门艾德生物医药科技股份有限公司 一种用于检测肿瘤突变的双标签接头序列及检测方法

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WO2009140191A2 (fr) * 2008-05-12 2009-11-19 Biomedomics, Inc. Détection d'une maladie liée à des mutations de gène par une amplification de molécule unique avec des dosages de ligature multiplex
CN106811460A (zh) * 2015-11-30 2017-06-09 安诺优达基因科技(北京)有限公司 用于低频突变检测的二代测序文库的构建方法及试剂盒
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