WO2019010775A1 - Molecular tag, joint and method for determining nucleotide sequence containing low-frequency mutation - Google Patents

Abstract

The present invention provides a molecular tag and a composition thereof, a joint containing the molecular tag and a composition thereof, as well as a method for determining nucleotide sequence containing low-frequency mutation in a target region of a sample to be detected. The molecular tag at most contains two continuous and same bases.

Description

Molecular tags, linkers, and methods for determining sequences containing low frequency mutant nucleic acids Technical field

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.

Background technique

High-throughput sequencing is currently the most widely used sequencing technology. However, there are still some sequencing errors in the sequencing, 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.

In order to detect base mutations (low frequency mutations) or sequencing errors of less than 0.1%, scholars have invented a method of molecular tagging by adding a special sequence to one or both ends of each sequencing template prior to PCR. 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. In the subsequent PCR process, 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.

In theory, the base sequences at each position of the molecular tag are completely randomly distributed. However, in the synthesis of primers, when a certain base is synthesized, 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. This will cause some of the bases to be in a dominant position, resulting in the fact that not every position in the molecular tag follows the probability of random distribution of four bases A, T, C, and G, and there will be a dominant molecular sequence, and even more The same consecutive bases, for example, 8 A, 8 G, etc., result in the number of random molecular tag types actually obtained is not as theoretical.

Multiple consecutive identical bases not only increase the likelihood of sequencing errors, but also increase the proportion of dominant molecular sequences. Because the ratio is not random, some or even more molecules are linked to the same tag sequence. When these molecules linked to the same tag sequence are highly homologous or the sequences are very similar, the skilled person cannot distinguish between molecules belonging to sequencing errors and low frequency mutations. Further, when the low-frequency mutation and the normal abundance sequence are linked to the same molecular cloning, the low-frequency mutation is caused to be a sequencing error or a PCR error to be missed. Therefore, the non-randomness of the molecular label will reduce its utility and even limit its application.

Summary of the invention

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.

In one aspect, 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:

S1, using the linker as described above, performing a linker reaction on the nucleic acid of the target region of the sample to be tested, and performing PCR amplification on the nucleic acid of the target region of the sample to be tested after the addition of the linker to obtain an amplification product, and the amplification product constitutes the Measuring a target region nucleic acid sequencing library of the sample;

S2, sequencing the target region nucleic acid sequencing library of the sample to be tested, and obtaining the sequenced nucleic acid sequence;

S3, classifying the sequenced nucleic acid sequence according to the molecular tag contained in the linker, and classifying the sequenced nucleic acid sequence carrying the same molecular tag into the same nucleic acid sequence set;

S4, comparing the sequenced nucleic acid sequences in the nucleic acid sequence set with each other, and counting the base types and frequencies of each base position in the nucleic acid sequence set;

S5, according to the base type and frequency of each base position in the nucleic acid sequence set, by data analysis, obtaining a nucleic acid sequence in which the nucleic acid sequence contains a correct base arrangement position;

S6: Comparing the nucleic acid sequence containing the correct base arrangement position with the nucleic acid sequence of the remaining nucleic acid sequence or the parallel nucleic acid sequence set in the nucleic acid sequence to obtain a nucleic acid sequence containing a low frequency mutation.

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.

DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments herein

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.

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.

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.

Detailed ways

Embodiments of the present invention are described in detail below. The embodiments described below with reference to the accompanying drawings are intended to be illustrative of the invention and are not to be construed as limiting.

It should be noted that in the description of the present invention, the meaning of "a plurality" is two or more unless otherwise specified.

The present invention provides a molecular tag having up to two consecutive identical bases on the molecular tag.

According to an embodiment of the invention, the molecular tag is a single strand or a reverse complementary double strand.

According to an embodiment of the invention, the number of bases of the molecular tag is 6-24 bp.

The present invention also provides 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.

According to an embodiment of the present invention, the ratio includes at least one of a molar ratio, a molecular mass ratio, and a molecular ratio.

According to an embodiment of the invention, the number of species of the molecular tag comprises 4n and n is equal to 6-24. For example, according to experimental needs, 4096, 16384, 65536, 262144, 16777216, 268435456, and even more types can be designed.

According to an embodiment of the present invention, 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. Preferably, 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.

When the molecular tag is a double-stranded structure, 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. Preferably, when the molecular tag is a double-stranded structure, 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.

According to an embodiment of the present invention, as shown in FIG. 1, when the linker is a fully complementary double-stranded structure, 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.

According to an embodiment of the present invention, as shown in FIGS. 2 and 3, when the joint is a Y-shaped structure in which one end is complementary to one end, 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.

According to an embodiment of the present invention, as shown in FIG. 4, the molecular tag may not be located on the linker, but may be introduced into the Y-type structure of the linker by PCR.

Further, according to an embodiment of the present invention, the molecular tag may also be located at two or more positions of the joint.

According to an embodiment of the invention, the linker further comprises a library tag linked to the 3' or 5' end of the molecular tag.

As can be understood by those skilled in the art, 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.

According to an embodiment of the invention, 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.

According to some specific examples of the invention, the types of the joints include:

As shown in Figure 1, a fully complementary double-linker containing a molecular tag;

As shown in Figures 2 and 3, the one end of the molecular tag is complementary to the Y-type open end;

And as shown in Figure 4, the molecular tag is not located on the linker, but can be introduced into the Y-type structure of the linker by PCR.

According to an embodiment of the invention, a joint having a molecular tag at two or more positions is also included.

According to an embodiment of the present invention, 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:

S1, using the linker as described above, performing a linker reaction on the nucleic acid of the target region of the sample to be tested, and performing PCR amplification on the nucleic acid of the target region of the sample to be tested after the addition of the linker to obtain an amplification product, and the amplification product constitutes the Measuring a target region nucleic acid sequencing library of the sample;

S2, sequencing the target region nucleic acid sequencing library of the sample to be tested, and obtaining the sequenced nucleic acid sequence;

S3, classifying the sequenced nucleic acid sequence according to the molecular tag contained in the linker, and classifying the sequenced nucleic acid sequence carrying the same molecular tag into the same nucleic acid sequence set;

S4, comparing the sequenced nucleic acid sequences in the nucleic acid sequence set with each other, and counting the base types and frequencies of each base position in the nucleic acid sequence set;

S5, according to the base type and frequency of each base position in the nucleic acid sequence set, by data analysis, obtaining a nucleic acid sequence in which the nucleic acid sequence contains a correct base arrangement position;

S6: Comparing the nucleic acid sequence containing the correct base arrangement position with the nucleic acid sequence of the remaining nucleic acid sequence or the parallel nucleic acid sequence set in the nucleic acid sequence to obtain a nucleic acid sequence containing a low frequency mutation.

According to an embodiment of the invention, 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.

The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will appreciate that the following examples are merely illustrative of the invention and are not to be construed as limiting. Unless otherwise stated, the reagents, sequences (linkers, tags and primers), software and instruments not specifically addressed in the following examples are conventionally commercially available or open source.

In the description of the present specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means a specific feature described in connection with the embodiment or example. A structure, material or feature is included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

Example 1 Detection of low frequency mutant gene

1. Design molecular tags and connectors containing the molecular tags

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:

Table 1

SEQ ID NO:	Molecular label	SEQ ID NO:	Molecular label
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

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. As shown in Fig. 1, Fig. 2, Fig. 3, and Fig. 4, NNN...NNN represents a molecular tag, and 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. As shown in Table 2, 16 kinds of linkers containing different molecular tags:

Table 2

When the joint is as shown in Fig. 1 and Fig. 2 and the like, it is necessary to simultaneously design a structure containing the reverse complement of the molecular label. If it is necessary to simultaneously design the F-direction sequence and the R-direction sequence in Table 2 Columns, Figures 3, 4 and similar structures only need to design single-stranded molecular tags, such as the F-directed sequences in Table 2, without the need to design molecular tag reverse complementary sequences.

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.

2. Synthesis of joints containing molecular tags

According to the designed adaptor sequence, 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. As will be appreciated by those skilled in the art, synthetic methods can be employed in methods well known in the art or can be commissioned by a primer synthesis company.

3. Mix the obtained joints in proportion to obtain a set of joint compositions

The synthesized molecular tag-containing linkers were mixed at a molar ratio of 1:1 for different types.

For example, when the types of joints of the structures of Fig. 1, Fig. 2, Fig. 3, and the like are used, each type of joint is mixed in a ratio of 1:1.

When the molecular label is directly mixed with the Y-type joint in a molar ratio of 1:1 as shown in Fig. 5 and the like, a set of the joint composition is obtained.

4, extract sample DNA

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.

5, DNA end repair

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.

5.1 Prepare in a 1.5 ml EP tube as follows:

Reagent	Volume /ul
DNA	85
10×PNK buffer	10
dNTP solution (10mM)	2
T4 DNA polymerase	1
T4PNK	1
KLENOW fragment (dilution 10	1
Total volume / ul	100

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.

5.2 Add 150 ul of magnetic beads to the 100 ul system reaction product, and after performing AMpure XP magnetic bead purification, repeatedly wash twice with 500 ul of 75% ethanol, and discard the supernatant. Dry at 37 ° C until the beads are dry. Add 34.5 ul of water, mix the magnetic beads, and clarify, and draw 34 ul of the supernatant.

6, add "A" at the end

The 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.

6.1 Prepare the reaction solution from the solution obtained in 5 according to the following system:

Reagent	volume
End repair DNA	34
10× blue buffer	5
dATP (1mM)	10
Klenow 3'-5'exo-	1
Total volume / ul	50

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.

6.2 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.

7, add joint reaction

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.

7.1 Prepare the reaction solution from the solution obtained in 6 according to the following 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.

7.2 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.

8. PCR enrichment and construction of sequencing library

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.

8.1 Prepare the reaction solution in a new PCR tube according to the following system:

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

After mixing at room temperature, after slight centrifugation, the reaction system was placed in a PCR machine and reacted according to the following conditions:

After the reaction was completed, it was purified using AMpure XP magnetic beads.

8.2 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.

9, library quality inspection

The library was subjected to QPCR and Agilent 2100 detection, and the quality-qualified library was arranged on the machine.

10. DNA sequencing of the library

The library can be sequenced using a second generation sequencer such as Hiseq2000, Hiseq 2500, Proton, Miseq, NS500.

11, analysis of sequencing results

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. Class 1 DNA formed by PCR, the "replication strand" of the positive and negative strands of the original DNA molecule.

Count the base types at each base position within the "molecular cluster" and the frequency of their occurrence.

Based on data analysis, identify errors and correct them due to PCR and sequencing.

The correct sequence of the original DNA is thus obtained, and the true mutant sequence is found by internal and parallel comparison of the molecular clusters.

The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to be limiting, and the present invention will be described in detail with reference to the preferred embodiments of the present invention. Neither should the spirit and scope of the technical solutions of the present invention be deviated.

Claims (10)

1. A molecular tag characterized in that the molecular tag contains up to two consecutive identical bases.
2. The molecular tag according to claim 1, wherein the molecular tag is a single-stranded or reverse-complementary double-stranded chain.
3. The molecular tag according to claim 1, wherein the molecular tag has a base number of 6 to 24 bp.
4. A molecular tag composition comprising the molecular tag according to any one of claims 1 to 3, wherein the ratio of each molecular tag is from 0.95 to 1.05:1.
5. The molecular tag composition according to claim 4, wherein the ratio comprises at least one of a molar ratio, a molecular mass ratio, and a molecular ratio.
6. A linker comprising the molecular tag according to any one of claims 1 to 3, wherein the molecular tag is located at the terminus "T" of the linker and 20 bp of the non-overhanging end Any location other than.
7. The linker of claim 6 wherein said linker further comprises a library tag linked to the 3' or 5' end of said molecular tag.
8. The linker according to claim 6, wherein said linker further comprises an identifying characteristic sequence, said identifying characteristic sequence being 4 non-repeating bases, said identifying characteristic sequence and said molecular tag The 3' end or the 5' end is connected.
9. A joint composition, characterized in that the joint composition contains the joint of claim 6, and the ratio of each joint is from 0.95 to 1.05:1.
10. 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:

    S1. Using the linker according to claim 6, the target region nucleic acid to be tested Performing a linker reaction, performing PCR amplification on the nucleic acid of the target region of the sample to be tested after the addition of the linker, to obtain an amplification product, the amplification product constituting the target region nucleic acid sequencing library of the sample to be tested;

    S2, sequencing the target region nucleic acid sequencing library of the sample to be tested, and obtaining the sequenced nucleic acid sequence;

    S3, classifying the sequenced nucleic acid sequence according to the molecular tag contained in the linker, and classifying the sequenced nucleic acid sequence carrying the same molecular tag into the same nucleic acid sequence set;

    S4, comparing the sequenced nucleic acid sequences in the nucleic acid sequence set with each other, and counting the base types and frequencies of each base position in the nucleic acid sequence set;

    S5, according to the base type and frequency of each base position in the nucleic acid sequence set, by data analysis, obtaining a nucleic acid sequence in which the nucleic acid sequence contains a correct base arrangement position;

    S6: Comparing the nucleic acid sequence containing the correct base arrangement position with the nucleic acid sequence of the remaining nucleic acid sequence or the parallel nucleic acid sequence set in the nucleic acid sequence to obtain a nucleic acid sequence containing a low frequency mutation.

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