WO2018001258A1 - 一种用于核酸富集捕获的探针及设计方法 - Google Patents

一种用于核酸富集捕获的探针及设计方法 Download PDF

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WO2018001258A1
WO2018001258A1 PCT/CN2017/090446 CN2017090446W WO2018001258A1 WO 2018001258 A1 WO2018001258 A1 WO 2018001258A1 CN 2017090446 W CN2017090446 W CN 2017090446W WO 2018001258 A1 WO2018001258 A1 WO 2018001258A1
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probe
sequence
capture
length
dna
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French (fr)
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施伟杰
林清华
纪斌峰
唐郑华
李旭超
阮力
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厦门艾德生物医药科技股份有限公司
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Priority to JP2018548125A priority Critical patent/JP6931356B2/ja
Priority to EP17819255.5A priority patent/EP3480310A4/en
Publication of WO2018001258A1 publication Critical patent/WO2018001258A1/zh

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • 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
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
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    • 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

Definitions

  • the invention relates to the field of gene sequencing, in particular to a probe and a design method for nucleic acid enrichment capture.
  • PCR enrichment mainly includes multiplex PCR and digital PCR; PCR has limitations such as amplification bias, primer interaction in multiplex amplification, and inability to detect unknown sequences (such as genetic recombination).
  • Hybridization and enrichment mainly include chip hybridization and solution hybridization; hybridization is not suitable for small-area enrichment, and the amount of samples required is large.
  • Chip hybridization is an early technique that promotes the hybridization reaction by increasing the amount of the sample; solution hybridization promotes the hybridization reaction by increasing the amount of the probe, thereby reducing the amount of the sample.
  • Hybridization method the corresponding specific probe is designed for the target gene, and the target gene is captured by the probe and the genomic hybridization; the target gene obtained after enrichment can be subjected to high-throughput sequencing to analyze the variation of the target gene.
  • the company that captures probe design and synthesis is mainly Hoffman-La Roche Limited (Roche Sequencing), Agilent Technologies and IDT (Integrated DNA Technologies, Inc).
  • the probe design is designed for single strands of the target region and is designed to be of different lengths (29-39 bp as described in US Pat. No.
  • the unidirectional design of the probe prevents capture of the complementary strand nucleic acid library during capture, resulting in loss of capture copy number; in addition, the probe is designed based on normal genomic sequences, preferentially capturing normal genomic sequences rather than mutant sequences, resulting in mutations in sequencing results Frequency deviation.
  • the present invention provides a probe for nucleic acid enrichment capture
  • the probe is a bidirectional probe comprising a sense strand probe and an antisense strand probe, wherein the sense strand probe and the antisense strand probe are probe-free design and are 30-89 bases in length.
  • the 3' or 5' of the probe has a biotin label that binds to the avidin on the magnetic beads.
  • sequence design method of the bidirectional probe is as follows:
  • the length of the exon is selected
  • the intron and exon lengths at the fusion breakpoint are selected;
  • the antisense strand template Taking the reverse complement of the reference sequence, ie, the antisense strand template, designing antisense strands of equal length without overlapping probes; the antisense strands without overlapping probes have the same length as the sense strand without overlapping probes, and the sense strand No overlapping probe misalignment: the antisense strand has no overlapping probe length n, if n is odd, the optimal misalignment is (n+1)/2; if n is even, the optimal misalignment is n/2;
  • the probe containing the repeat sequence is eliminated by software analysis;
  • the software analysis includes probe sequence analysis and target sequence analysis, and the software analysis can be performed by using repeated sequence recognition software;
  • the captured non-specific fragment is compared with all the probes, and the probe having the matching result with the non-specific fragment is excluded;
  • a specific probe is added, and the specific probe sequence is: a matching sequence upstream of the mutation site, an abasic spacer group, and a downstream matching sequence of the mutation site; Probe sequence The length is n. If n is an odd number, the length of the upstream matching sequence or the downstream matching sequence is (n+1)/2; if n is even, the length of the upstream matching sequence or the downstream matching sequence is n/2.
  • the specific probe is designed to replace the mutant base of the mutation point with an abasic group.
  • two-way probes designed sense strand probes and antisense strand probes, respectively
  • the sense strand probe has no overlap design for the probe
  • the antisense strand probe also has no overlap design for the probe.
  • the sense strand and the corresponding antisense strand probe are designed to be misaligned, that is, each sense strand and the corresponding antisense strand probe have half of the probe length complementary, The capture of the template duplex is ensured to minimize the interaction between the probes.
  • the probe is 30-89 bases in length, and the 3' or 5' of the probe has a biotin label that binds to avidin on the magnetic beads for capture of the target sequence in a subsequent magnetic environment.
  • a specific probe is added to the two-way probe, and the mutation site in the specific probe is replaced by an abasic spacer group (dSpacer, Spacer9 or Spacer18, etc.).
  • the abasic spacer modification group is flanked by mutant and wild type consensus sequences.
  • the reverse complement of the reference sequence was designed to design an antisense strand of equal length without overlapping probes; the antisense strand probe was the same length as the sense strand probe and was misaligned with the sense strand probe.
  • Use software such as Array Designer or manual probe design.
  • the probe length be n, the probability of misalignment is 1 ⁇ (n-1), if n is odd, the optimal misplacement is (n+1)/2; if n is even, the optimal misalignment is n/2. This misalignment will minimize interaction between the sense strand non-overlapping probe and the antisense strand non-overlapping probe.
  • Probes containing repetitive sequences are eliminated by software analysis or (and) test results.
  • Software analysis includes probe sequence analysis and target sequence analysis.
  • Software analysis can be performed using repeated sequence recognition software, such as online software RepeatMasker, local software Blast and BWA.
  • the results of the test based on the high-throughput sequencing results of the probe-trapped sample DNA, compare the captured non-specific fragments with all the probes (such as background software), and eliminate the matching results with the non-specific fragments. needle.
  • wild-type antisense strand probes can hybridize to capture wild-type sense strand templates
  • wild-type sense strand probes can hybridize to capture wild-type antisense strand templates
  • wild-type antisense strand probes and unbiased antisense The strand probe can hybridize to capture the sense strand wild type template
  • the sense strand probe and the unbiased sense strand probe can hybridize to capture the antisense strand mutant template.
  • Bidirectional probes increase the specificity of capture (ie, reduce capture of genomic DNA to non-target locations) and increase the original copy number of the captured sample nucleic acid.
  • Bidirectional probes of the invention and unbiased groups containing abasic spacer groups The probe also significantly increases capture of the sample mutant DNA.
  • Figure 1 is a schematic diagram of the design of the probe of the present invention.
  • Figure 2 is a schematic diagram of DNA hybridization capture of a sample using the probe of the present invention.
  • Example 1 Effect of bidirectional probe on on Target rate of different GC content fragments
  • Probe design one-way probe 3 times overlapping coverage, or two-way probe misalignment coverage; probe length is 59 bp, and the probe 3' end is Biotin modified.
  • More specific probe sequences with Homo Sapiens, Release 19 (GRCh37.p13) chr2:29446476-chr2:29446775 segment as an example, enumerating 5 unidirectional probes, SEQ ID NO: 1-5; enumerate 5 pairs of bidirectional probes, which are SEQ ID NO: 6-10.
  • 30 ng of leukocyte DNA was taken and constructed by KAPA kit.
  • Hybridization reagent was used for 2h and 24h hybridization capture. The hybrid capture was performed by Roche SeqCap.
  • the EZ Hybridization and Wash Kit kit is operated according to the kit instructions. The sequencing results are shown in Table 1.
  • onTarget is the ratio of the target sequence sequencing results to the total sequencing results (ie, the sequencing results of the target sequence and the non-target sequence); coverage refers to the proportion of the target sequence obtained by sequencing to the entire target sequence; Uniformity refers to each of the target sequences after capture The homogeneity of the base, with 20% of the average depth as the threshold, the base of the target sequence reaching 20% of the average depth accounts for the total base ratio of the target sequence; Depth refers to the average depth of each base in the target sequence in the sequencing result. UID refers to the original template copy calculated by molecular labeling.
  • the yield of the bidirectional probe misalignment coverage (the yield of the amplified library after capture) is significantly higher than that of the one-way overlapping coverage, and the onTarget rate of hybridization capture is also significantly improved, and the effect of the 24h hybridization group is better than that of the 2h group. More obvious; the coverage of each group is consistent; the Uniformity of the two-way probe is lower; due to the limited amount of sequencing data, the UID of the bidirectional probe misalignment increases as the onTarget rate increases.
  • the bidirectional probe is lower than the one-way overlapping probe, mainly because the different sequences of each probe determine the deviation of the capture efficiency, so increasing the frequency of probe coverage can effectively reduce the base of the probe to different positions.
  • the difference in capture ability of the base makes the uniformity better.
  • the unidirectional design of the probe failed to capture the double-stranded stranded nucleic acid library, resulting in a loss of capture copy number, while the two-way probe filled the corresponding defect, resulting in a significant increase in both Depth and UID.
  • the amount of sequencing data is sometimes higher than a single item, sometimes lower than a single item, because high-throughput sequencing, sample loading quality is converted according to the expected amount of data, multiple samples are mixed and then sequenced, so sampling and mixing process There may be deviations that cannot be guaranteed to be absolutely accurate.
  • the sample needs to undergo a PCR amplification process on the sequencing instrument, and there is no guarantee that the amplification efficiency of each sample is completely uniform.
  • probe design was performed as follows: one-way probe without overlap coverage and bidirectional misalignment probe coverage.
  • the length of the probe is 35-80 bp and the probe Tm is 72-78 ° C, and the 3' end of the probe is Biotin modified.
  • More specific probe sequences with Homo Sapiens, Release 19 (GRCh37.p13) chr2:29446201-29448364 section for example, enumerate 5 unidirectional probes, which are SEQ ID NO: 11-15; enumerate 5 pairs of bidirectional probes, which are SEQ ID NO: 16-20, see Table 2.
  • 30 ng of leukocyte DNA was taken and constructed by KAPA kit.
  • Hybridization reagent was used for 2 h of hybridization capture. The hybrid capture was performed by Roche SeqCap.
  • the EZ Hybridization and Wash Kit kit is operated according to the kit instructions. The sequencing results are shown in Table 2.
  • Embodiment 3 Effect of two-way probe on UID
  • More specific probe sequences taking the exon 19 of the EGFR gene as an example, Homo Sapiens, Release 19 (GRCh37.p13) chr7:55242415-55242513, enumerate 5 unidirectional probes that are finally suitable for the reference sequence, SEQ ID NO: 21-25, enumerating the four-way probes that ultimately apply to the reference sequence, SEQ ID NO:26-29; taking exon 31, intron 31 and exon 32 of ROS1 gene as an example, Homo sapiens, Release 19 (GRCh37.p13) Section chr6: 117650492-117658503, listing 5 unidirectional probes finally applied to the reference sequence, SEQ ID NOS: 30-34, listed Obtain 5 pairs of bidirectional probes that are finally suitable for the reference sequence, SEQ ID NOs: 35-39, listing software-cleared two-way probes containing repeat sequences, SEQ ID NO: 40-44, 5 cross-linking probes containing repeated sequences, SEQ ID NO:
  • the variant type of exon 19 deletion NM_005228: exon19:c.2235_2249del:p.745_750del Designs an unbiased probe containing an abasic spacer group to increase capture of the mutant template.
  • the unbiased probe is replaced by the abasic spacer group Spacer18, the sequence is SEQ ID NO: 50-Spacer18- SEQ ID NO: 51-3'-Biotin;
  • TGAGAAAGTTAAAATTCCCGTCGCTATCAA is SEQ ID NO: 50;
  • CGAAAGCCAACAAGGAAATCCTCGATGTGA is SEQ ID NO:51.
  • Probe coverage Sample type Yield Sampling data volume onTarget Coverage Uniformity Depth UID unidirectional White blood cell DNA 608 211.21M 91.37% 100.00% 100.00% 2483.87 4274.50
  • the yield of the bidirectional probe misalignment coverage (the yield of the amplified library after capture) was significantly higher than that of the one-way probe group, and the onTarget rate of hybridization capture was slightly improved; the coverage of each group was the same as that of Uniformity; Under the condition that the data volume is close to onTarget, the bidirectional probe misalignment coverage increases the UID of leukocyte DNA by about 6.8% and the UID of plasma DNA by about 12.9% compared with the one-way probe 3 fold overlap coverage group.
  • the Depth of the bidirectional probe is higher than that of the unidirectional probe. It is indicated that the two-way probe has better hybridization capture ability to the sample DNA than the one-, two- or three-fold coverage of the one-way probe.
  • Bidirectional probes increase the frequency of probe coverage of the target base, so the introduction of contrast is used to illustrate that the bidirectional probe is designed to be superior to the one-way probe.
  • Multiple genomic segments were selected for probe design. Multiple genomic segments include exon 31 with ROS1 gene, intron 31 and exon 32, and exon 19 of EGFR gene.
  • the probe design the bidirectional probe is misaligned, the probe length is 59 bp, and the 3' end of the probe is modified by Biotin. After software analysis and test results, the probe containing the repeat sequence is eliminated.
  • More specific probe sequences taking the exon 19 of the EGFR gene as an example, Homo Sapiens, Release 19 (GRCh37.p13) chr7:55242415-55242513, listing 4 obtained bidirectional probes, SEQ ID NO:26-29; taking exon 31, intron 31 and exon 32 of ROS1 gene as an example, Homo sapiens, Release 19 (GRCh37.p13) Chr6: 117650492-117658503 segment, enumerating 5 obtained bidirectional probes, SEQ ID NOs: 35-39, listing 5 software-removed bidirectional probes containing repeat sequences, SEQ ID NOs: 40-44, listing five cross-linked probes containing repeats, SEQ ID NOs: 45-49, see attached table.
  • the variant type of exon 19 deletion NM_005228: exon19:c.2235_2249del:p.745_750del Designs an unbiased probe containing an abasic spacer group to increase capture of the mutant template.
  • the unbiased probe is replaced by the abasic spacer group Spacer18, the sequence is SEQ ID NO: 50-Spacer18- SEQ ID NO: 51-3'-Biotin;
  • TGAGAAAGTTAAAATTCCCGTCGCTATCAA is SEQ ID NO: 50;
  • CGAAAGCCAACAAGGAAATCCTCGATGTGA is SEQ ID NO:51.
  • H1650 cell line (NM_005228: exon19: c.2235_2249del: P.745_750del, this cell line is commercially available from ATCC) and was constructed using the KAPA kit.
  • Hybridization reagent was used for 24h+24h secondary hybridization capture.
  • the hybrid capture was performed by Roche SeqCap EZ. Hybridization and Wash Kit kit, according to the kit instructions.
  • the sequencing results are as follows. The capture controls were made using a custom commercial capture kit (Roche's SeqCap EZ System custom product).
  • the mutation rate of the bidirectional probe misalignment coverage is significantly higher than that of the commercial kit.
  • the H1650 mutation content was about 62%. This data demonstrates that the present invention is better able to hybridize to capture mutant DNA fragments, effectively capture mutant copies, and to make mutation detection rates higher.
  • the present invention can provide a probe for nucleic acid enrichment capture, which can improve the specificity of capture, and increase the original copy number of the captured sample nucleic acid, and can quickly locate and detect base variation.

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Abstract

一种用于核酸富集捕获的探针及设计方法。该探针由正义链探针和反义链探针组成，其中正义链探针和反义链探针均为探针无重叠设计，其长度均为30-89个碱基，探针的3'或5'具有生物素标记，能与磁珠上的亲和素结合。所述的双向探针能够提高捕获的特异性(即减少对非靶位置的基因组DNA的捕获)，显著提高对样本突变型DNA的捕获，并且增加捕获样品核酸的原始拷贝数。

Description

一种用于核酸富集捕获的探针及设计方法 技术领域
本发明涉及基因测序领域，尤其涉及一种用于核酸富集捕获的探针及设计方法 。
背景技术
人类基因组的序列过于庞大，为了研究靶基因中特定位置的碱基变异情况，需要对靶基因进行富集。富集较常见的方法有PCR富集和杂交富集。PCR富集主要包括多重PCR和数字PCR；PCR具有扩增偏向性、多重扩增中引物相互影响和无法检测未知序列（如基因重组）等局限。杂交富集主要包括芯片杂交法和溶液杂交法；杂交不适合小区域富集、所需样品量较多。芯片杂交，属于早期技术，通过增加样品量以促进杂交反应的进行；溶液杂交则通过增加探针量促进杂交反应的进行，减少了样品用量。杂交法，针对靶基因设计相应的特异探针，通过探针与基因组杂交实现靶基因的捕获；富集后所得靶基因可进行高通量测序分析靶基因的变异情况。
捕获探针设计和合成的公司主要有霍夫曼-拉罗奇有限公司（Roche Sequencing）、安捷伦科技有限公司（Agilent Technologies）和IDT（Integrated DNA Technologies, Inc）。探针设计针对靶区域的单链进行设计，设计成不同长度（专利US 7636637中所述的29-39 bp；参考资料所述>60 bp，https://lifescience.roche.com/wcsstore/RASCatalogAssetStore/Articles/BIOCHEMICA_3_09_p13-14.pdf； agilent专利 US20110184161所述>100 bp；文献PMID 19182786 所述的120 bp）不同重叠数的探针（如1x、1.5x、2x或4x tiling，文献PMID 19835619）。探针的单向设计使捕获过程中无法捕获互补链核酸文库，导致捕获拷贝数损失；另外探针是根据正常基因组序列进行设计，优先捕获正常基因组序列而非突变型序列，导致测序结果中突变频率的偏差。
发明内容
本发明的目的在于提供一种用于核酸富集捕获的探针，能够提高捕获的特异性，并且增加捕获样品核酸的原始拷贝数 。
为实现上述目的，本发明提供一种 用于核酸富集捕获的探针 ，该探针为双向探针，包括正义链探针和反义链探针，其中正义链探针和反义链探针为探针无重叠设计，其长度均为30-89个碱基，探针的3'或5'具有生物素标记，能与磁珠上的亲和素结合。
进一步，所述双向探针的序列设计方法如下:
（1）依据基因突变点在参考基因组对应位置， 选取如下长度参考序列：
若为外显子上的点突变、***突变或缺失突变，则选取该外显子长度；
若为融合突变，则选取融合断点处的内含子和外显子长度；
（2）取参考序列即正义链模板，设计相等长度的正义链无重叠探针；
（3）取参考序列的反向互补序列即反义链模板，设计相等长度的反义链无重叠探针；反义链无重叠探针与正义链无重叠探针长度相同，且与正义链无重叠探针错位：反义链无重叠探针长度为n，若n为奇数，最适错位为（n+1）/2；若n为偶数，最适错位为n/2；
(4) 通过软件分析，剔除含有重复序列的探针；所述软件分析包括探针序列分析以及靶序列分析，软件分析可采用重复序列识别软件进行分析；
(5) 根据探针捕获样品DNA的高通量测序结果，将捕获所得的非特异片段与所有探针的进行比对，剔除与非特异片段有比对匹配结果的探针；
(6) 得到最终适用于参考序列的捕获探针。
进一步，为同时捕获野生型序列和突变型序列，增加特异探针，该特异探针序列依次为：突变位点上游匹配序列、无碱基间隔基团和突变位点下游匹配序列；所述特异探针序列 长度为n，若n为奇数，则 上游匹配序列或下游匹配序列的长度 为（n+1）/2；若n为偶数，则 上游匹配序列或下游匹配序列的长度 为n/2 。
进一步， 针对 所述突变型序列的 突变点，特异探针设计时将突变点的变异碱基用无碱基基团替代。
为了更好的捕获双链基因组靶序列，根据所需捕获的基因组靶序列，设计双向探针（分别设计正义链探针和反义链探针）。正义链探针为探针无重叠设计，反义链探针也为探针无重叠设计。为了减少正义链与反义链探针的互补配对，将正义链与对应反义链探针设计成错位的，即每条正义链与对应反义链探针存在一半探针长度的互补，既保证了对模板双链的捕获又最大程度的减少了探针之间的互作。
探针长度为30-89个碱基，探针的3'或5'具有生物素标记，能与磁珠上的亲和素结合，用于后续磁力环境下捕获靶序列。
同时，为了同时捕获野生型序列和突变型序列，在双向探针的基础上补加特异探针，特异探针中的变异位点用无碱基间隔基团（dSpacer、Spacer9或Spacer18等）替代，无碱基间隔修饰基团两侧为突变型和野生型共有序列。
本发明探针设计原理见图1所示。依据基因突变点在人类参考基因组（如Homo sapiens, Release 19 (GRCh37.p13)）对应位置，选取相应长度参考序列进行探针设计，根据基因组的互补双链分别设计正义链无重叠探针以及反义链无重叠探针，每条正义链探针与相应位置的反义链探针存在一半探针长度的互补，每条反义链探针与相应位置的正义链探针存在一半探针长度的互补；并且根据所需检测基因组突变，在相应位置设计无偏向性探针。所设计探针与样品DNA杂交捕获见图2。
探针设计步骤：
1 、依据基因突变点在人类参考基因组（如Homo sapiens, Release 19 (GRCh37.p13)）对应位置，选取相应长度参考序列，若为外显子上的点突变、***突变或缺失突变，则选取该外显子长度；若为融合突变，则选取融合断点处的内含子和外显子长度。如EGFR基因T790M点突变，则选取EGFR基因的外显子20；EGFR基因E746_A750del缺失，则选取EGFR基因的外显子19；如发生在ALK基因内含子19的基因融合，则选取ALK外显子19、内含子19、外显子20。
2 、取参考序列（正义链模板），设计相等长度的正义链无重叠探针。使用软件（如Array Designer）或手工进行探针设计。
3 、取参考序列的反向互补序列（反义链模板），设计相等长度的反义链无重叠探针；反义链探针与正义链探针长度相同，且与正义链探针错位。使用软件（如Array Designer）或手工进行探针设计。设探针长度为n，错位的可能性为1~(n-1)，若n为奇数，最适错位为（n+1）/2；若n为偶数，最适错位为n/2。这种错位将使正义链无重叠探针和反义链无重叠探针之间的互作降到最低。
4 、通过软件分析或者（和）试验结果，剔除含有重复序列的探针。软件分析包括探针序列分析以及靶序列分析，软件分析可采用重复序列识别软件进行分析，如在线软件RepeatMasker、本地软件Blast和BWA。试验结果，根据探针捕获样品DNA的高通量测序结果，将捕获所得的非特异片段与所有探针的进行比对（如本底软件），剔除与非特异片段有比对匹配结果的探针。
5 、确定最终适用于参考序列的捕获探针。
应用本发明探针杂交捕获样品DNA原理见图2所示。样品DNA中存在野生型正义链模板、野生型反义链模板、突变型正义链模板和突变型反义链模板；探针含有野生型正义链探针、野生型反义链探针、无偏向性正义链探针和无偏向性反义链探针。在特定条件下，野生型反义链探针可杂交捕获野生型正义链模板，野生型正义链探针可杂交捕获野生型反义链模板，野生型反义链探针和无偏向性反义链探针可杂交捕获正义链野生型模板，正义链探针和无偏向性正义链探针可杂交捕获反义链突变型模板。
本发明的 双向探针能够提高捕获的特异性（即减少对非靶位置的基因组DNA的捕获），并且增加捕获样品核酸的原始拷贝数。 本发明的 双向探针及含无碱基间隔基团的 无偏向性 探针，还能显著提高对样本突变型DNA的捕获。
附图说明
图1是本发明探针设计原理图。
图2是应用本发明探针杂交捕获样品DNA原理图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。 实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：双向探针对不同GC含量片段on Target率的影响
取6个基因组区段（Homo sapiens, Release 19 (GRCh37.p13) chr2:29446476-chr2:29446775；chr2:29447226-chr2:29447525；chr2:29448065-chr2:29448364；chr6:117642641-chr6:117642940；chr6:117645323-chr6:117645622；chr6:117641038-chr6:117641337），进行探针设计：单向探针3倍重叠覆盖，或双向探针错位覆盖；探针长度均为59bp，探针3'端为Biotin修饰。具体探针序列较多，以Homo sapiens, Release 19 (GRCh37.p13) chr2:29446476-chr2:29446775区段为例，列举单向探针5条，为SEQ ID NO:1-5；列举双向探针5条，为SEQ ID NO:6-10。取白细胞DNA各30ng，以KAPA试剂盒进行建库后，采用杂交试剂，进行2h和24h的一次杂交捕获，杂交捕获采用的是Roche公司的SeqCap EZ Hybridization and Wash Kit试剂盒，根据试剂盒说明书操作。测序结果如表1。
表1 双向探针对不同GC含量片段on Target率的影响表
探针覆盖 杂交时间 产量 测序数据量 onTarget Coverage Uniformity Depth UID
单向 24h 990 ng 146.96Mbp 5.36% 100.00% 100.00% 3378.06 2103.65
双向 24h 1065 ng 156.92Mbp 9.45% 100.00% 99.11% 6549.81 2854.83
单向 2h 1050 ng 157.91Mbp 4.61% 100.00% 100.00% 3140.51 1902.92
双向 2h 1140 ng 162.28Mbp 5.57% 100.00% 99.83% 4023.36 2088.54
注：onTarget为靶序列测序结果占总测序结果（即靶序列和非靶序列的测序结果）的比例；coverage指测序所得靶序列部分占整个靶序列的比例；Uniformity指的捕获后靶序列中各个碱基的均一性情况，以平均深度的20%作为阈值时，靶序列中达到20%平均深度的碱基占靶序列总碱基比例；Depth指测序结果中靶序列中各碱基的平均深度；UID指的是经过分子标签推算所得的原始模板拷贝。
从表1可以看出，双向探针错位覆盖的产量（捕获后扩增的文库产量）明显高于单向重叠覆盖，并且杂交捕获的onTarget率也显著提高，并且24h杂交组效果比2h组效果更明显；各组Coverage一致；双向探针的Uniformity较低；因测序数据量有限，双向探针错位覆盖的UID随着onTarget率提高而增高。Uniformity数值中，双向探针低于单向重叠探针，主要是因为各条探针的不同序列决定了捕获效能上的偏差，因此增加探针覆盖的频率，能有效降低探针对不同位置碱基的捕获能力差异，使均一性更佳。探针的单向设计无法捕获双链的链核酸文库，导致捕获拷贝数损失，而双向探针则填补相应缺陷，使Depth和UID均有大幅度提高。测序数据量有时比单项高，有时比单项低，原因是高通量测序时，样品上样质量是依据预期数据量进行换算的，多个样品混成后再进行测序，因此取样和混样过程中可能存在偏差，无法保证绝对的精确，另外样品在测序仪器上需经过PCR放大过程，也无法保证每个样品扩增效率完全一致。
实施例2、双向探针对不同染色体on Target率的影响
取5个基因组区段Homo sapiens, Release 19 (GRCh37.p13) chr2:29446201-29448364，chr7:55241635-55241748，chr7:55242396-55242539，chr7:55249029-55249107，chr7:55259462-55259576；进行如下探针设计：单向探针无重叠覆盖和双向错位探针覆盖。探针长度为35-80bp及探针Tm为72-78℃，探针3'端为Biotin修饰。具体探针序列较多，以Homo sapiens, Release 19 (GRCh37.p13) chr2:29446201-29448364区段为例，列举单向探针5条，为SEQ ID NO:11-15；列举双向探针5条，为SEQ ID NO:16-20，见表2。取白细胞DNA各30ng，以KAPA试剂盒进行建库后，采用杂交试剂，进行2h的一次杂交捕获，杂交捕获采用的是Roche公司的SeqCap EZ Hybridization and Wash Kit试剂盒，根据试剂盒说明书操作。测序结果如表2。
表2 双向探针对不同染色体on Target率的影响表
探针覆盖 杂交时间 产量 测序数据量 onTarget Coverage Uniformity Depth UID
单向 24h 798 ng 167.21M 3.95% 100.00% 100.00% 2151.75 1272.26
双向 24h 896 ng 156.20M 10.95% 100.00% 100.00% 5381.34 2690.09
从表2可以看出，双向探针错位覆盖的产量（捕获后扩增的文库产量）明显高于单向无重叠覆盖，并且杂交捕获的onTarget率也显著提高；各组Coverage和Uniformity一样；因测序数据量有限，双向探针错位覆盖的UID随着onTarget率提高而增高。
实施例3、双向探针对UID的影响
取多个基因组区段进行探针设计，多个基因组区段包含区段有ROS1基因的外显子31、内含子31及外显子32和EGFR基因的外显子19等，进行如下方案的探针设计：单向探针3倍重叠覆盖，双向探针错位覆盖，探针长度均为59bp，探针3'端为Biotin修饰，经过软件分析和试验结果，剔除含有重复序列的探针。具体探针序列较多，以EGFR基因的外显子19为例，Homo sapiens, Release 19(GRCh37.p13) chr7:55242415-55242513，列举得到最终适用于参考序列的单向探针5条，SEQ ID NO:21-25，列举得到最终适用于参考序列的双向探针4条，SEQ ID NO:26-29；以ROS1基因的外显子31、内含子31及外显子32为例，Homo sapiens, Release 19 (GRCh37.p13) chr6:117650492-117658503区段，列举 得到最终适用于参考序列的 单向探针5条，SEQ ID NO:30-34，列举 得到最终适用于参考序列的 双向探针5条，SEQ ID NO:35-39，列举软件剔除的含有重复序列的双向探针5条， SEQ ID NO:40-44，列举试验结果剔除的含有重复序列的双向探针5条，SEQ ID NO:45-49，见附表。同时，针对EGFR 外显子19缺失的变异类型NM_005228:exon19:c.2235_2249del:p.745_750del设计含无碱基间隔基团的无偏向性探针，以增加对突变型模板的捕获。无偏向性探针以无碱基间隔基团Spacer18替代，序列为SEQ ID NO:50-Spacer18- SEQ ID NO:51-3'-Biotin所示；
其中TGAGAAAGTTAAAATTCCCGTCGCTATCAA为SEQ ID NO:50；
CGAAAGCCAACAAGGAAATCCTCGATGTGA 为SEQ ID NO:51。
具体为：
TGAGAAAGTTAAAATTCCCGTCGCTATCAA-Spacer18-CGAAAGCCAACAAGGAAATCCTCGATGTGA-3'-Biotin 。取人体白细胞DNA或血浆DNA各30ng，以KAPA试剂盒进行建库后，采用杂交试剂，进行24h+24h的二次杂交捕获，杂交捕获采用的是Roche公司的SeqCap EZ Hybridization and Wash Kit试剂盒，根据试剂盒说明书操作。测序结果如表3。
表3 双向探针对UID的影响表
探针覆盖 样本类型 产量 测序数据量 onTarget Coverage Uniformity Depth UID
单向 白细胞DNA 608 211.21M 91.37% 100.00% 100.00% 2483.87 4274.50
双向 白细胞DNA 1022 212.95M 93.31% 100.00% 100.00% 2582.17 4564.90
单向 血浆DNA 630 233.69M 92.14% 100.00% 100.00% 418.84 1140.32
双向 血浆DNA 865 245.13M 94.50% 100.00% 100.00% 481.23 1287.16
从表3可以看出，双向探针错位覆盖的产量（捕获后扩增的文库产量）明显高于单向探针各组，杂交捕获的onTarget率略有提高；各组Coverage和Uniformity一样；在数据量和onTarget相近的条件下，双向探针错位覆盖与单向探针3倍重叠覆盖组相比，白细胞DNA的UID提高约6.8%，血浆DNA的UID提高约12.9%。双向探针的Depth比单向探针的高。说明与1倍、2倍或3倍覆盖的单向探针相比，双向探针对样品DNA具有更好的杂交捕获能力。
双向探针增加了探针对目的碱基的覆盖频率，因此引入对比是用来说明双向探针从设计上优于单向探针。
实施例4、双向探针对突变检测的影响
取多个基因组区段进行探针设计，多个基因组区段包含区段有ROS1基因的外显子31、内含子31及外显子32和EGFR基因的外显子19等，进行如下方案的探针设计：双向探针错位覆盖，探针长度均为59bp，探针3'端为Biotin修饰，经过软件分析和试验结果，剔除含有重复序列的探针。具体探针序列较多，以EGFR基因的外显子19为例，Homo sapiens, Release 19 (GRCh37.p13) chr7:55242415-55242513，列举4条得到的双向探针，SEQ ID NO:26-29；以ROS1基因的外显子31、内含子31及外显子32为例，Homo sapiens, Release 19 (GRCh37.p13) chr6:117650492-117658503区段，列举5条得到的双向探针，SEQ ID NO:35-39，列举5条软件剔除的含有重复序列的双向探针， SEQ ID NO:40-44，列举5条经试验结果剔除的含有重复序列的双向探针，SEQ ID NO:45-49，见附表。同时，针对EGFR 外显子19缺失的变异类型NM_005228:exon19:c.2235_2249del:p.745_750del设计含无碱基间隔基团的无偏向性探针，以增加对突变型模板的捕获。无偏向性探针以无碱基间隔基团Spacer18替代，序列为SEQ ID NO:50-Spacer18- SEQ ID NO:51-3'-Biotin所示；
其中TGAGAAAGTTAAAATTCCCGTCGCTATCAA为SEQ ID NO:50；
CGAAAGCCAACAAGGAAATCCTCGATGTGA 为SEQ ID NO:51。
具体为：
TGAGAAAGTTAAAATTCCCGTCGCTATCAA-Spacer18-CGAAAGCCAACAAGGAAATCCTCGATGTGA-3'-Biotin 。
取20ng H1650细胞系（NM_005228: exon19: c.2235_2249del: p.745_750del，该细胞系可购自于ATCC），以KAPA试剂盒进行建库。
采用杂交试剂，进行24h+24h的二次杂交捕获，杂交捕获采用的是Roche公司的SeqCap EZ Hybridization and Wash Kit试剂盒，根据试剂盒说明书操作。测序结果如下。捕获对照采用定制的商业化捕获试剂盒（Roche公司的SeqCap EZ System定制产品）。
表4 双向探针对突变检测的影响结果表
探针覆盖 样本类型 Frequency of EGFR c.2235_2249del15
双向探针 细胞系H1650 70.58%
商业试剂 细胞系H1650 38.46%
上表可以看出，双向探针错位覆盖的突变率明显高于商业试剂盒。根据数字PCR方法的检测结果显示，H1650突变含量约为62%。这个数据说明本发明能更好地杂交捕获变异DNA片段，有效捕获突变拷贝，使突变检出率更高。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在不脱离本发明的原理和宗旨的情况下在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
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<400> 16
ggccgccctg gtcctggctt tctccggcat catga 35
<210> 17
<211> 36
<212> DNA
<213> 人工合成
<400> 17
gctctgtgca ctcaccaatc atgatgccgg agaaag 36
<210> 18
<211> 35
<212> DNA
<213> 人工合成
<400> 18
ttggtgagtg cacagagccc cagggactcc caagg 35
<210> 19
<211> 35
<212> DNA
<213> 人工合成
<400> 19
agtcctgcct tcctgccccc ttgggagtcc ctggg 35
<210> 20
<211> 40
<212> DNA
<213> 人工合成
<400> 20
gggcaggaag gcaggactga atagtgtctc aggctgtgcc 40
<210> 21
<211> 59
<212> DNA
<213> 人工合成
<400> 21
tccttctctc tctgtcatag ggactctgga tcccagaagg tgagaaagtt aaaattccc 59
<210> 22
<211> 59
<212> DNA
<213> 人工合成
<400> 22
ggactctgga tcccagaagg tgagaaagtt aaaattcccg tcgctatcaa ggaattaag 59
<210> 23
<211> 59
<212> DNA
<213> 人工合成
<400> 23
tgagaaagtt aaaattcccg tcgctatcaa ggaattaaga gaagcaacat ctccgaaag 59
<210> 24
<211> 59
<212> DNA
<213> 人工合成
<400> 24
tcgctatcaa ggaattaaga gaagcaacat ctccgaaagc caacaaggaa atcctcgat 59
<210> 25
<211> 59
<212> DNA
<213> 人工合成
<400> 25
gaagcaacat ctccgaaagc caacaaggaa atcctcgatg tgagtttctg ctttgctgt 59
<210> 26
<211> 59
<212> DNA
<213> 人工合成
<400> 26
tccttctctc tctgtcatag ggactctgga tcccagaagg tgagaaagtt aaaattccc 59
<210> 27
<211> 59
<212> DNA
<213> 人工合成
<400> 27
atgttgcttc tcttaattcc ttgatagcga cgggaatttt aactttctca ccttctggg 59
<210> 28
<211> 59
<212> DNA
<213> 人工合成
<400> 28
tcgctatcaa ggaattaaga gaagcaacat ctccgaaagc caacaaggaa atcctcgat 59
<210> 29
<211> 59
<212> DNA
<213> 人工合成
<400> 29
tggaccccca cacagcaaag cagaaactca catcgaggat ttccttgttg gctttcgga 59
<210> 30
<211> 59
<212> DNA
<213> 人工合成
<400> 30
caagtacttt gcaaacacac ataccttatc tcaaggatat agtatgtaat tctacatcc 59
<210> 31
<211> 59
<212> DNA
<213> 人工合成
<400> 31
ataccttatc tcaaggatat agtatgtaat tctacatcca ttatcttcag ctttctccc 59
<210> 32
<211> 59
<212> DNA
<213> 人工合成
<400> 32
agtatgtaat tctacatcca ttatcttcag ctttctccca ctgtattgaa tttttactc 59
<210> 33
<211> 59
<212> DNA
<213> 人工合成
<400> 33
ttatcttcag ctttctccca ctgtattgaa tttttactcc cttctagtaa tttgggaat 59
<210> 34
<211> 59
<212> DNA
<213> 人工合成
<400> 34
ctgtattgaa tttttactcc cttctagtaa tttgggaatg cctggtttat ttgggactc 59
<210> 35
<211> 59
<212> DNA
<213> 人工合成
<400> 35
caagtacttt gcaaacacac ataccttatc tcaaggatat agtatgtaat tctacatcc 59
<210> 36
<211> 59
<212> DNA
<213> 人工合成
<400> 36
ttcaatacag tgggagaaag ctgaagataa tggatgtaga attacatact atatccttg 59
<210> 37
<211> 59
<212> DNA
<213> 人工合成
<400> 37
ttatcttcag ctttctccca ctgtattgaa tttttactcc cttctagtaa tttgggaat 59
<210> 38
<211> 59
<212> DNA
<213> 人工合成
<400> 38
ccctaaagct ggagtcccaa ataaaccagg cattcccaaa ttactagaag ggagtaaaa 59
<210> 39
<211> 59
<212> DNA
<213> 人工合成
<400> 39
cctggtttat ttgggactcc agctttaggg aaaaaaagaa aatattggtt gatatgttt 59
<210> 40
<211> 59
<212> DNA
<213> 人工合成
<400> 40
gggaaaacac ccttcgggat attatccagg agaacttccc caacctagca agacaggcc 59
<210> 41
<211> 59
<212> DNA
<213> 人工合成
<400> 41
gtgttctctg tgtttgccga atttgcatgt tggcctgtct tgctaggttg gggaagttc 59
<210> 42
<211> 59
<212> DNA
<213> 人工合成
<400> 42
acatgcaaat tcggcaaaca cagagaacac cattaagata ctccacgaga agatcaacc 59
<210> 43
<211> 59
<212> DNA
<213> 人工合成
<400> 43
ccttggagaa tctgatgatt atgtgtcttg gggttgatct tctcgtggag tatcttaat 59
<210> 44
<211> 59
<212> DNA
<213> 人工合成
<400> 44
caagacacat aatcatcaga ttctccaagg ttgaaatcaa gtaaaaactg ttaagggca 59
<210> 45
<211> 59
<212> DNA
<213> 人工合成
<400> 45
agaaatgcag catccatttc tatagctcca ggctgtgctt ttcccctgcg ggagccagc 59
<210> 46
<211> 59
<212> DNA
<213> 人工合成
<400> 46
gacaagtctt gggaccaagc tgtccagcct cgctggctcc cgcaggggaa aagcacagc 59
<210> 47
<211> 59
<212> DNA
<213> 人工合成
<400> 47
aggctggaca gcttggtccc aagacttgtc gccacaaccc aacacaccag ctgtggtag 59
<210> 48
<211> 59
<212> DNA
<213> 人工合成
<400> 48
gttagacctg aagaggcact ctggccgctg actaccacag ctggtgtgtt gggttgtgg 59
<210> 49
<211> 59
<212> DNA
<213> 人工合成
<400> 49
cagcggccag agtgcctctt caggtctaac cctcacccat ccttcctcag tgggtgggg 59
<210> 50
<211> 30
<212> DNA
<213> 人工合成
<400> 50
tgagaaagtt aaaattcccg tcgctatcaa 30
<210> 51
<211> 30
<212> DNA
<213> 人工合成
<400> 51
cgaaagccaa caaggaaatc ctcgatgtga 30
工业实用性
本发明能够提供一种用于核酸富集捕获的探针，能够提高捕获的特异性，并且增加捕获样品核酸的原始拷贝数，能够快速定位并检测碱基变异情况。

Claims (4)

  1. 一种用于核酸富集捕获的探针 ，其特征在于， 该探针为双向探针，包括正义链探针和反义链探针，其中正义链探针和反义链探针为探针无重叠设计，其长度为30-89个碱基，探针的3'或5'具有生物素标记，能与磁珠上的亲和素结合。
  2. 根据权利要求1所述用于核酸 富集 捕获的探针，其特征在于，所述双向探针的序列设计方法如下:
    （1）依据基因突变点在参考基因组对应位置， 选取 如下 长度参考序列：
    若为外显子上的点突变、***突变或缺失突变，选取该外显子长度；
    若为融合突变，选取融合断点处的内含子和外显子长度 ；
    （2）取参考序列即正义链模板，设计相等长度的正义链无重叠探针；
    （3）取参考序列的反向互补序列即反义链模板，设计相等长度的反义链无重叠探针；
    反义链无重叠探针与正义链无重叠探针长度相同，且与正义链无重叠探针错位：反义链无重叠探针长度为n，若n为奇数，错位为（n+1）/2；若n为偶数，错位为n/2；
    (4) 通过软件分析，剔除含有重复序列的探针；所述软件分析包括探针序列分析以及靶序列分析，软件分析可采用重复序列识别软件进行分析；
    (5) 根据探针捕获样品DNA的高通量测序结果，将捕获所得的非特异片段与所有探针的进行比对，剔除与非特异片段有比对匹配结果的探针；
    (6) 得到最终适用于参考序列的捕获探针。
  3. 根据权利要求1或2所述用于 核酸富集捕获的探针 ，其特征在于，该特异探针序列依次为：突变位点上游匹配序列、无碱基间隔基团和突变位点下游匹配序列；所述特异探针序列 长度为n，若n为奇数，则 上游匹配序列或下游匹配序列的长度 为（n+1）/2；若n为偶数，则 上游匹配序列或下游匹配序列的长度 为n/2 。
  4. 根据权利要求3所述用于 核酸富集捕获的探针 ，其特征在于， 针对 所述突变型序列的 突变点，特异探针设计时将突变点的变异碱基用无碱基基团替代。
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CN111500679A (zh) * 2019-06-28 2020-08-07 北京希望组生物科技有限公司 长片段捕获测序探针组的制备方法
CN111696627A (zh) * 2020-03-26 2020-09-22 上海生物芯片有限公司 一种长链rna特异性探针的设计方法
CN112927756A (zh) * 2019-12-06 2021-06-08 深圳华大基因科技服务有限公司 鉴别转录组rRNA污染源的方法、装置和改善rRNA污染的方法
CN113092209A (zh) * 2021-04-02 2021-07-09 复旦大学附属肿瘤医院 一种富集、鉴定待测病样中分子标志物的方法
CN114369650A (zh) * 2022-03-21 2022-04-19 深圳市仙湖植物园(深圳市园林研究中心) 捕获探针的设计方法、捕获探针及其应用
CN115011594A (zh) * 2022-05-16 2022-09-06 纳昂达(南京)生物科技有限公司 一种用于检测hpv的液相杂交捕获探针、应用及其试剂盒

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CN112927756A (zh) * 2019-12-06 2021-06-08 深圳华大基因科技服务有限公司 鉴别转录组rRNA污染源的方法、装置和改善rRNA污染的方法
CN111424115A (zh) * 2020-03-13 2020-07-17 南京农业大学 一种利用血糖仪检测新型冠状病毒的方法
CN111696627A (zh) * 2020-03-26 2020-09-22 上海生物芯片有限公司 一种长链rna特异性探针的设计方法
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CN113092209A (zh) * 2021-04-02 2021-07-09 复旦大学附属肿瘤医院 一种富集、鉴定待测病样中分子标志物的方法
CN113092209B (zh) * 2021-04-02 2023-10-27 复旦大学附属肿瘤医院 一种富集、鉴定待测病样中分子标志物的方法
CN114369650A (zh) * 2022-03-21 2022-04-19 深圳市仙湖植物园(深圳市园林研究中心) 捕获探针的设计方法、捕获探针及其应用
CN115011594A (zh) * 2022-05-16 2022-09-06 纳昂达(南京)生物科技有限公司 一种用于检测hpv的液相杂交捕获探针、应用及其试剂盒
CN116083423A (zh) * 2022-05-16 2023-05-09 纳昂达(南京)生物科技有限公司 一种靶向富集核酸的探针
CN115011594B (zh) * 2022-05-16 2023-10-20 纳昂达(南京)生物科技有限公司 一种用于检测hpv的液相杂交捕获探针、应用及其试剂盒
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