WO2019014936A1 - Procédé d'enrichissement ciblé et kit de détection de mutation à basse fréquence - Google Patents

Procédé d'enrichissement ciblé et kit de détection de mutation à basse fréquence Download PDF

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WO2019014936A1
WO2019014936A1 PCT/CN2017/093914 CN2017093914W WO2019014936A1 WO 2019014936 A1 WO2019014936 A1 WO 2019014936A1 CN 2017093914 W CN2017093914 W CN 2017093914W WO 2019014936 A1 WO2019014936 A1 WO 2019014936A1
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primer
sequencing
pcr amplification
universal
universal primer
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PCT/CN2017/093914
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Chinese (zh)
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杨林
高雅
蒲丹丹
张海萍
程云阳
陈芳
蒋慧
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深圳华大基因研究院
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Priority to PCT/CN2017/093914 priority Critical patent/WO2019014936A1/fr
Priority to CN201780091041.8A priority patent/CN110651050A/zh
Publication of WO2019014936A1 publication Critical patent/WO2019014936A1/fr

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    • C—CHEMISTRY; METALLURGY
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    • 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

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  • the invention relates to the technical field of molecular biology, in particular to a targeted enrichment method and a kit for detecting low frequency mutations.
  • NGS second-generation sequencing
  • target region targeted enrichment sequencing technology is a technique for enriching the target gene of interest and combining with the second generation sequencing technology to obtain the base information of the target region, which has achieved the purpose of detecting the disease, and is compared with the whole genome sequencing.
  • Targeted enrichment sequencing technology can reduce the cost of sequencing, simplify the information analysis process, improve the sequencing depth of the target area, and improve the sensitivity and accuracy of detection.
  • probe-based capture enrichment technology using the principle of complementary hybridization of nucleic acid molecules, designing a reverse-complementary oligonucleotide probe according to the target region, then interrupting the genomic DNA, plus the linker for sequencing The probe is hybridized, the unhybridized DNA is eluted, the target DNA fragment is recovered, and the library preparation is performed for DNA sequencing.
  • This technique requires a high amount of starting DNA of the sample (generally required to reach micrograms), long experimental operation time, and cumbersome experiment, which is not conducive to automated database construction, low data utilization rate, and high cost.
  • the multiplex PCR-based enrichment technique is based on designing primers according to the target region, and then enriching the target region by multiplex PCR, and then performing PCR library sequencing on the PCR product.
  • this technique is short in experiment time, it requires complicated primer design work in the early stage, and a lot of cumbersome primer optimization work is needed in the later stage.
  • the above two techniques have strict requirements on the amount and integrity of the template, and are incapable of testing samples such as cell free DNA, highly degraded DNA, and paraffin-embedded formaldehyde. Therefore, it is particularly important to develop a simple targeted enrichment method for efficient enrichment of short fragment DNA.
  • the invention provides a targeted enrichment method and a kit for detecting low frequency mutations, which can effectively enrich short fragments, single strands, double strands and lost DNA, and can be detected in DNA by combining second generation sequencing technology. Low frequency mutation.
  • an embodiment provides a targeted enrichment method for detecting low frequency mutations, comprising:
  • First PCR amplification is performed by using an upstream specific primer and a first universal primer for the region where the mutation target site is located, wherein the upstream specific primer is anchored with a target sequence complementary thereto, and the first universal primer is as described above.
  • the single nucleotide tail is an anchor anchor
  • the first universal primer includes a contiguous single base complementary to the above-described single nucleotide tail at the 1' and 3' ends of the sequencing primer sequence at the 5' end;
  • the first PCR-specific primer and the second universal primer perform a second PCR amplification on the first PCR-amplified product, wherein the downstream specific primer is located downstream of the upstream specific primer and has a sequencing primer at the 5' end Sequence 2, the second universal primer described above includes the sequencing primer sequence 1 of the 5' end of the first universal primer described above.
  • an embodiment provides a targeted enrichment kit for detecting low frequency mutations, comprising:
  • terminal transferase and a single base nucleotide for the addition of a single base single nucleotide to the 3' end of each strand of single-stranded DNA and/or double-stranded DNA under the action of a terminal transferase tail;
  • the nucleotide tail is an anchor site
  • the first universal primer comprises a single single base complementary to the above-described single nucleotide tail of the sequencing primer sequence 1 and 3' of the 5' end;
  • the downstream specific primer is located downstream of the upstream specific primer and has a 5' end Primer primer sequence 2
  • the second universal primer described above includes the sequencing primer sequence 1 of the 5' end of the first universal primer described above.
  • the method of the invention can effectively enrich short-segment single-stranded DNA, double-stranded DNA and nicked DNA with high stencil utilization; has high detection sensitivity and can detect low-frequency mutations as low as 0.1%; Efficient enrichment of multiple targeted regions and good specificity, uniformity and stability.
  • FIG. 1 is a schematic diagram showing the principle of a target enrichment method for detecting low frequency mutations according to an embodiment of the present invention
  • FIG. 2 is a schematic diagram showing the principle of a target enrichment method for detecting low frequency mutations according to another embodiment of the present invention
  • 3 is a schematic diagram of molecular label correction in an embodiment of the present invention.
  • Example 4 is a diagram showing the results of Agilent 2100 quality inspection of a target-enriched library in Example 1 of the present invention
  • Figure 5 is a graph showing the results of uniformity of each amplicon of HBB in Example 1 of the present invention.
  • Example 6 is a diagram showing the results of Agilent 2100 quality inspection of a targeted enrichment library in Example 2 of the present invention
  • Example 8 is a depth distribution diagram of sequencing data of 10 amplicon regions after molecular label correction in Example 2 of the present invention.
  • Figure 9 is a graph showing the results of the consistency of mutation detection in Example 2 of the present invention.
  • FIG. 1 shows the principle of a targeted enrichment method for detecting low frequency mutations according to an embodiment of the present invention, the method comprising:
  • Step I A single base single nucleotide tail is added to the 3' end of each strand of single-stranded DNA and/or double-stranded DNA under the action of a terminal transferase.
  • a double-stranded DNA molecule (dsDNA) is subjected to denaturing and melting to obtain single-stranded DNA (ssDNA), and 15-30 of the single-stranded DNA is added to the 3' end of the single-stranded DNA by a terminal transferase.
  • Single base C or any of A, T, G
  • the composition consists of a 15-30 bp length of a single nucleotide tail. It should be noted that the double-stranded DNA molecule can be directly added to the 3' end of each strand by the terminal transferase without denaturation.
  • Step II performing the first PCR amplification with the upstream specific primer and the first universal primer for the region where the mutation target site is located, wherein the upstream specific primer is anchored with the target sequence complementary thereto, and the first universal primer is The single nucleotide tail is an anchor site, and the first universal primer includes a 5' end of the sequencing primer sequence 1 and a 3' end of a contiguous single base complementary to the single nucleotide tail.
  • upstream specific primers were designed 25-150 bp before the mutation target site.
  • One end uses a single nucleotide tail as an anchor site, and the other end uses an upstream specific primer (USP) complementary target sequence as an anchor site for 10-20 cycles of amplification.
  • a primer sequence with a single nucleotide tail as an anchor site is referred to as a "first universal primer” (ie, universal primer 1 in Figure 1) including: a 5'-end sequencing primer sequence 1 (eg, BGISEQ-500, Illumina, or Proton) Such as the sequencing primer sequence of the sequencing platform), the continuous base G in the middle (or any one of T, A, C), The number of consecutive bases C or G is 11-15, and the Tm value ranges from 54 to 70 ° C. Too many numbers are not conducive to PCR amplification; the number of consecutive bases A or T is 25-35, Tm The range of values is 54-62 ° C, too many is not conducive to PCR amplification.
  • a 5'-end sequencing primer sequence 1 eg, BGISEQ-500, Illumina, or Proton
  • the continuous base G in the middle or any one of T, A, C
  • the number of consecutive bases C or G is 11-15, and the Tm value ranges from 54 to 70
  • universal primer 1 is 13 consecutive bases G in the middle, and, preferably, a degenerate base H is added last (in other cases it may be V, B or D) ), the degenerate base at this end is used to fix the length of the 3' end product, and the template with C bases of different lengths is subjected to PCR amplification by primers with degenerate bases, and the 3' end is fixed.
  • a product of base length C (which in other cases may be A, T or G).
  • the product is from the 5' end to the 3' end in turn, the target upstream specific primer sequence, the target region sequence, and the continuous single nucleotide C (in other cases, A, T) Or G), and the final sequencing primer sequence.
  • the method of the embodiments of the present invention is applicable to targeted enrichment of various mutation types, including single nucleotide polymorphism (SNP), insertion and deletion (INDEL), and copy number variation (CNV).
  • Step III performing a second PCR amplification of the first PCR amplified product with a downstream specific primer and a second universal primer, wherein the downstream specific primer is located downstream of the upstream specific primer and has a sequencing primer at the 5' end Sequence 2, the second universal primer comprises the sequencing primer sequence 1 of the 5' end of the first universal primer.
  • downstream specific primers are designed downstream of the upstream specific primer (USP) (eg, 0-10 bp downstream of USP), and downstream specific primers ( The 5' end of DSP) plus sequencing primer sequence 2 (eg, sequencing primer sequences for sequencing platforms such as BGISEQ-500, Illumina or Proton), using downstream specific primers (DSP) with sequencing primer sequence 2 and universal primer 2 ( Also known as "second universal primers") amplification is performed for 15-30 cycles.
  • the second universal primer comprises a sequencing primer sequence 1 at the 5' end of the first universal primer.
  • a sequencing tag primer can also be introduced, and the 3' end of the sequencing tag primer includes the sequencing primer sequence 2 at the 5' end of the downstream specific primer.
  • the downstream specific primer (DSP) and universal primer 2 were amplified, and since the downstream specific primer (DSP) also had the sequencing primer sequence 2 at the 5' end, the product was obtained at both ends.
  • Sequencing the primer sequence starting from the second cycle, using the sequencing primer sequence as the anchor site, two universal primers (ie, universal primer 2 and sequencing tag primer (BC)) are simultaneously amplified with downstream specific primers to obtain a target. Sequencing library.
  • the advantages of introducing sequencing tag primers are: (1) reducing the specific amplification step, increasing the universal amplification step, facilitating the homogeneity of different amplicon regions; (2) reducing the specific primer-introduced linker sequences, and more The linker sequence was introduced by sequencing tag primers.
  • Both single-stranded and double-stranded DNA can be efficiently enriched and have a very high template utilization rate.
  • the terminal transferase can add bases to free single-stranded, double-stranded and lost DNA, and the efficiency of adding the template can reach 99% or more, that is, more than 99% of the templates can be added at the 3' end.
  • the single nucleotide tail Enrichment of the targeted region by specific primers and a single nucleotide tail requires efficient enrichment by only having a specific primer binding site on the template.
  • multiplex PCR amplification is performed using specific primers and anchor primers, and the specific primers may be primers for different targeting regions, and the anchor primers are fixed primer sequences, by mixing specific primers and immobilized targets. Enrichment of multiple regions was performed on the primers, and two-round nested PCR was used to increase the specificity of enrichment, and a multi-region targeted sequencing library was obtained.
  • FIG. 2 illustrates the principle of a targeted enrichment method for detecting low frequency mutations in accordance with another embodiment of the present invention, the method comprising:
  • the double-stranded DNA molecule is subjected to denaturing and melting to obtain single-stranded DNA, and 5-10 random bases are randomly added to the 3' end of the single-stranded DNA template by a terminal transferase as a molecular tag for labeling the original template.
  • the residual dNTPs were removed by magnetic bead purification.
  • terminal transferase may also be A, T, Any of G
  • a single nucleotide tail is obtained, and then the remaining dCTP (or dATP, dTTP, dGTP) is removed by magnetic bead purification.
  • all the free single-stranded, double-stranded and damaged DNA templates have a 5-10 bp molecular tag consisting of four bases and a single base consisting of 15-30 bp in length.
  • Polynucleotide tail it should be noted that the double-stranded DNA molecule can be directly added to the 3' end of each strand with a molecular tag and a single nucleotide tail without denaturing.
  • Upstream specific primers were designed 25-150 bp before the mutation target site. One end uses a single nucleotide tail as an anchor site, and the other end uses an upstream specific primer (USP) complementary target sequence as an anchor site for 10-20 cycles of amplification.
  • a primer sequence with a polynucleotide tail as an anchor site is referred to as a "first universal primer” (ie, universal primer 1 in Figure 2) including: a 5'-end sequencing primer sequence 1 (eg, BGISEQ-500, Illumina, or Proton) Such as the sequencing primer sequence of the sequencing platform), the continuous base G in the middle (or any one of T, A, C), the number of consecutive bases C or G is 11-15, and the Tm value ranges from 54- At 70 ° C, too many numbers are not conducive to PCR amplification; the number of consecutive bases A or T is 25-35, and the Tm value ranges from 54-62 ° C. Too many numbers are not conducive to PCR amplification.
  • a 5'-end sequencing primer sequence 1 eg, BGISEQ-500, Illumina, or Proton
  • universal primer 1 is 13 consecutive bases G in the middle, and, preferably, a degenerate base H is added last (in other cases it may be V, B or D) ), the degenerate base at this end is used to fix the length of the 3' end product, and the template with C bases of different lengths is subjected to PCR amplification by primers with degenerate bases, and the 3' end is fixed.
  • a product of base length C (which in other cases may be A, T or G).
  • the product is, from the 5' end to the 3' end, a target upstream specific primer sequence, a target region sequence, a molecular tag consisting of 5-10 random bases, and a continuous mononucleoside.
  • Acid C which in other cases can be A, T or G
  • the method of the embodiments of the present invention is applicable to targeted enrichment of various mutation types, including single nucleotide polymorphism (SNP), insertion and deletion (INDEL), and copy number variation (CNV). .
  • SNP single nucleotide polymorphism
  • INDEL insertion and deletion
  • CNV copy number variation
  • Downstream specific primers are designed downstream of upstream specific primers (USP) and sequencing primers 2 are added to the 5' end of downstream specific primers (DSP) (eg sequencing platforms such as BGISEQ-500, Illumina or Proton)
  • DSP downstream specific primers
  • the sequencing primer sequence was subjected to 15-30 cycles of amplification using a downstream specific primer (DSP) with sequencing primer sequence 2 and universal primer 2 (also referred to as "second universal primer”).
  • the second universal primer comprises a sequencing primer sequence 1 at the 5' end of the first universal primer.
  • a sequencing tag primer can also be introduced, and the 3' end of the sequencing tag primer includes the sequencing primer sequence 2 at the 5' end of the downstream specific primer.
  • the downstream specific primer (DSP) and universal primer 2 were amplified, and since the downstream specific primer (DSP) also had the sequencing primer sequence 2 at the 5' end, the product was obtained at both ends.
  • Sequencing the primer sequence starting from the second cycle, using the sequencing primer sequence as the anchor site, two universal primers (ie, universal primer 2 and sequencing tag primer (BC)) are simultaneously amplified with downstream specific primers to obtain a target. Sequencing library.
  • the advantages of introducing sequencing tag primers are: (1) reducing the specific amplification step, increasing the universal amplification step, facilitating the homogeneity of different amplicon regions; (2) reducing the specific primer-introduced linker sequences, and more The linker sequence was introduced by sequencing tag primers.
  • the method shown in Fig. 2 has the following advantageous effects in addition to the advantageous effects of the method shown in Fig. 1: a high degree of detection sensitivity, and a low frequency mutation further reduced to 0.1% can be detected. Specifically, a random sequence of 5-10 bp in length consisting of four bases randomly added to the 3' end of the free DNA template by a terminal transferase, the sequence of which can be up to one million species, which can be opposed The initial template is uniquely labeled, and a low frequency mutation further reduced to 0.1% can be detected by molecular marker binding information analysis.
  • the targeted sequencing library obtained in the present embodiment is subjected to double-end sequencing, and the specific primer is used for detecting the targeted enrichment region, and the other end is used for reading the molecular tag information for labeling the template.
  • the detection of very low frequency mutations is achieved by combining PCR errors and sequencing errors with molecular tags in conjunction with specific data analysis algorithms.
  • the plasma source is plasma free DNA of pregnant women at 12 weeks, mother carries CD41/42 ⁇ E (del CTTT) mutation, and father carries CD71/72 (Ins A) mutation.
  • CD41/42 ⁇ E del CTTT
  • father carries CD71/72 (Ins A) mutation.
  • CD71/72 mutation.
  • the cfDNA was first heat denatured at 95 ° C for 5 minutes, then rapidly inserted into the ice, followed by an enzymatic reaction.
  • the oligonucleotide tail was added to the 3' end of the DNA by a terminal transferase (Terminal Transferase, NEB, USA, Cat. No. M0315S).
  • the upstream specific primer pool is shown in Table 3, and the universal primer 1 is shown in Table 4.
  • HBB-USP14 CCTTAAACCTGTCTTGTAACCTTGAT SEQ ID NO: 14 HBB-USP15 CAGTAACGGCAGACTTCTCCTC SEQ ID NO: 15 HBB-USP16 GTTGTGTCAGAAGCAAATGTAAGC SEQ ID NO: 16 HBB-USP17 CTGACTTTTATGCCCAGCC SEQ ID NO: 17 HBB-USP18 CTAGGGTGTGGCTCCACAG SEQ ID NO:18 HBB-USP19 CAGCCGTACCTGTCCTTGG SEQ ID NO: 19
  • the upstream specific primer pool consisted of a mixture of the equimolar numbers of the primers shown in Table 3.
  • the downstream specific primer pool is shown in Table 7, and the universal primer 2 and sequencing primer primers are shown in Table 4.
  • the downstream specific primer pool consisted of a mixture of the equimolar numbers of the primers shown in Table 7.
  • Targeted enriched libraries were detected with Agilent 2100, and the quality results are shown in Figure 4.
  • the BGISEQ-500 sequencing platform was used for sequencing, single-ended 100 bp sequencing, and the obtained data was obtained. After data conversion and quality filtering, the following information analysis is used.
  • the obtained data was de-joined, and single-ended sequencing results were obtained.
  • the genome (reference genome hg19) was compared, and the mutation of the target site was statistically analyzed by data analysis to obtain information of the target site.
  • the results are shown in Table 9-10.
  • the depth of each target area is calculated according to the location of the target area, and the uniformity information of the target area is obtained. The result is shown in FIG. 5, and the uniformity is good from the figure.
  • Design primers for 10 hotspot mutations related to lung cancer construct a targeted sequencing library for plasma free DNA, and combine high-throughput sequencing and specific information analysis to detect lung cancer-related hotspots.
  • the plasma free DNA was Horizon cfDNA standard: 0.1% Multiplex I cfDNA Reference Standard (Cat. No. HD779), and the mutation information was as shown in Table 11, starting at 10 ng, and the experiment was carried out as follows.
  • the cfDNA was first heat denatured at 95 ° C for 5 minutes, then rapidly inserted into the ice, followed by an enzymatic reaction. 5-10 random bases added to the 3' end of the DNA by terminal transferase (Terminal Transferase, NEB, USA, Cat. No. M0315S).
  • the upstream specific primer pool is shown in Table 15, and the universal primer 1 is shown in Table 4.
  • the upstream specific primer pool consisted of a mixture of the equimolar numbers of the primers described in Table 15.
  • the amplification system is shown in Table 16 below:
  • the downstream specific primer pool is shown in Table 18, and the universal primer 2 and sequencing primer primers are shown in Table 4.
  • the downstream specific primer pool consisted of a mixture of equimolar numbers of primers shown in Table 18.
  • Targeted enriched libraries were detected using the Agilent 2100 and the results are shown in Figure 6.
  • the BGISEQ-500 sequencing platform was used for sequencing, and the double-ended 50 bp sequencing was performed. After the data was converted and mass filtered, the following information was analyzed.
  • the obtained data was deligated to obtain double-end sequencing results.
  • One end of the sequencing results was used to compare the genome (reference genome hg19), and the other end of the result removed the consecutive G bases, and then 10 base sequences were intercepted from the end.
  • Molecular label used to mark the sequence information at the front end; perform basic parameter statistics (Table 20) to compare the proportion of data on the genome, and calculate the depth of each target area according to the target area position, and obtain the target area uniformity information (Figure 7 -8), wherein FIG. 7 shows the depth of sequencing of the original data, and FIG. 8 shows the depth of sequencing after the deduplication of the reads. The results showed good homogeneity.
  • the depth of the target site and the ratio of the four bases were counted, and the molecular tag was used to remove the repetition and sequencing errors, PCR errors, and the mutation information of the target site was obtained by a specific information analysis algorithm.
  • the results are shown in Table 21.
  • Table 22 shows the results of the consistency of the test results.
  • Figure 9 shows the mutation detection identity information indicating that the detected mutation information of the target site is consistent with the expectation.
  • the mutation ratio detected by our method is 0.08%, 0.10%, 0.10%, 0.15%, 0.12%, 0.11%, 0.15%, and the detected value and the true value are within ⁇ 0.02% error range, indicating The method can accurately detect mutations as low as 0.10%. It can be seen that the molecular marker (UID) correction can remove the repetition and sequencing errors, and reduce the sequencing background from 0.60% to 0.00%. (It should be noted that the error value before the V600E correction is the largest, which means that the error rate of the method can be 0.60% dropped to 0.00%).
  • UID molecular marker

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Abstract

La présente invention concerne un procédé d'enrichissement ciblé et un kit de détection de mutation à basse fréquence. Le procédé comprend : l'ajout d'un mononucléotide ou d'une queue polynucléotidique d'une base unique à l'extrémité 3'd'ADN simple brin et/ou de chaque brin d'ADN double brin sous l'action d'une transférase terminale ; la conduite d'une première instance d'amplification par PCR sur une région où un site cible de mutation est situé au moyen d'une amorce spécifique en amont et d'une première amorce universelle, la première amorce universelle comprenant une séquence d'amorce de séquençage 1 à l'extrémité 5'de celle-ci et des bases uniques continues, complémentaires du mononucléotide ou de la queue polynucléotidique, à l'extrémité 3'de celle-ci ; et la conduite, au moyen d'une amorce spécifique en aval et d'une deuxième amorce universelle, d'une deuxième instance d'amplification par PCR sur le produit, qui a subi la première instance d'amplification par PCR, la deuxième amorce universelle comprenant la séquence d'amorce de séquençage 1 de la première amorce universelle.
PCT/CN2017/093914 2017-07-21 2017-07-21 Procédé d'enrichissement ciblé et kit de détection de mutation à basse fréquence WO2019014936A1 (fr)

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