WO2023216030A1 - 一种占位引物和去除方法 - Google Patents

一种占位引物和去除方法 Download PDF

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WO2023216030A1
WO2023216030A1 PCT/CN2022/091488 CN2022091488W WO2023216030A1 WO 2023216030 A1 WO2023216030 A1 WO 2023216030A1 CN 2022091488 W CN2022091488 W CN 2022091488W WO 2023216030 A1 WO2023216030 A1 WO 2023216030A1
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primer
placeholder
sequencing
sequence
occupying
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PCT/CN2022/091488
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English (en)
French (fr)
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李长英
李计广
徐崇钧
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深圳华大智造科技股份有限公司
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Application filed by 深圳华大智造科技股份有限公司 filed Critical 深圳华大智造科技股份有限公司
Priority to PCT/CN2022/091488 priority Critical patent/WO2023216030A1/zh
Priority to CN202280083891.4A priority patent/CN118401679A/zh
Publication of WO2023216030A1 publication Critical patent/WO2023216030A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the invention belongs to the field of biotechnology, and more specifically relates to a placeholder primer used in sequencing and its use method and purpose.
  • placeholder primers In the existing DNA sequencing process, it is often necessary to use placeholder primers to prevent the chain from extending from a specific position, or to prevent the longer sequencing length from covering the template sequence that still needs to be used.
  • the sequence will be used at the beginning of sequencing.
  • a placeholder primer with a blocked 3' end covers the linker sequence of the barcode part. After the insertion fragment is sequenced, some physical or chemical methods are used to remove the placeholder primer, and then normal primers are used to sequence the barcode. sequence.
  • the object of the present invention is to provide a new method when space-occupying primers are used in the sequencing process, so that the space-occupying primers can be removed simply and effectively without affecting the quality of sequencing, and without the need for special enzymes and other chemical reagents. , can reduce costs.
  • the present invention provides a placeholder primer used in sequencing, the placeholder primer includes a blocking group at the 3' end, and the 3' end sequence of the placeholder primer is consistent with the sequence of the sequencing template. Part of the sequence is complementary, and the 5' end sequence does not pair with the sequencing template.
  • the blocking group is a phosphorylation blocking group, a steric blocking group, a dideoxynucleotide blocking group or other groups that can prevent 3' end extension.
  • the length of the space-occupying primer is 10-300nt, preferably 30-70nt, more preferably 40-50nt.
  • the length of the non-matching region at the 5' end of the placeholder primer is 3-200nt, preferably 5-50nt, more preferably 10-35nt.
  • the length of the matching region at the 3' end of the placeholder primer is 10-200nt, preferably 20-60nt, more preferably 30-50nt.
  • the present invention provides a occupying primer and a removal primer used in a polymerization reaction.
  • the occupying primer is the occupying primer described in the first aspect of the present invention, and the removing primer is the same as the occupying primer.
  • the reverse primer is completely complementary to the primer.
  • the present invention provides a method for introducing a placeholder primer and removing a placeholder primer during a polymerization process
  • the introduction of a placeholder primer includes: introducing a placeholder primer into the sequencing template, the placeholder primer includes a blocking group at the 3' end, and the 3' end sequence of the placeholder primer is consistent with the partial sequence of the sequencing template. Complementary, the 5' end sequence does not pair with the sequencing template;
  • the removal of the spacer primer includes: introducing a reverse primer that is completely complementary to the spacer primer, so that the reverse primer first binds to the 5' end of the spacer primer, and then removes the spacer primer from the spacer primer.
  • the above sequencing template is replaced.
  • the blocking group is a phosphorylation blocking group, a steric blocking group, a dideoxynucleotide blocking group or other groups that can prevent 3' end extension.
  • the length of the space-occupying primer is 10-300nt, preferably 30-70nt, more preferably 40-50nt.
  • the length of the non-matching region at the 5' end of the placeholder primer is 3-200nt, preferably 5-50nt, more preferably 10-35nt.
  • the length of the matching region at the 3' end of the placeholder primer is 10-200nt, preferably 20-60nt, more preferably 30-50nt.
  • the method of introducing placeholder primers and removing primers is used for nucleic acid sequence determination.
  • the present invention provides a sequencing method, which method includes:
  • the sequencing method further includes 4) sequencing the nucleic acid to be tested using sequencing primers, and the nucleic acid to be tested is located downstream of the placeholder primer binding site.
  • the sequencing primer is a barcode primer
  • the barcode primer is used to sequence the nucleic acid to be tested.
  • the nucleic acid to be tested is a barcode sequence and is located downstream of the barcode primer binding site.
  • the method further includes sequencing the barcode sequence.
  • the present invention introduces a sequence that does not pair with the template at the 5' end of the occupying primer, and uses the branch migration of DNA to displace the occupying primer from the template, so that the occupying primer can be easily removed.
  • Figure 1 is a schematic diagram showing simultaneous hybridization of a placeholder primer and a sequencing primer to a template.
  • the placeholder primer also has a specific sequence connected to its 5' end that is not complementary to the adapter.
  • the schematic diagram in Figure 2 shows that when the insert fragment of the library is smaller than the sequencing read length, after the insert fragment is measured, the placeholder primer prevents the sequencing fragment from continuing to extend along the template.
  • Figure 3 is a schematic diagram illustrating the replacement of a reverse primer with a spacer primer.
  • Figure 4 shows the split rates of the test group and the control group.
  • Figure 5 shows the accuracy of different barcodes.
  • the 3' end sequence of the placeholder primer is complementary to the partial sequence of the template, and the 5' end is a specific sequence that is not complementary to the library or adapter sequence.
  • the length of the placeholder primer is 10-300nt, preferably 30-70nt, more preferably 40-50nt, and the length of its 5'-end non-matching region is 3-200nt, preferably 5-50nt, more preferably 10-35nt, and its 3'-end
  • the length of the matching region is 10-200nt, preferably 20-60nt, more preferably 30-50nt.
  • the partial sequence to be blocked can be any sequence on the template, preferably the linker sequence of the barcode portion of the template.
  • the 3' end of the placeholder primer is a blocking group, which can block the sequencing fragment from continuing to extend along the template.
  • the blocking group can be a phosphorylation blocking group, a steric blocking group, a dideoxynucleotide blocking group or other groups that can prevent 3’ end extension.
  • the 3' end of the occupying primer is complementary to the portion to be blocked on the template, such as the linker sequence of the barcode portion, to achieve the purpose of occupying space.
  • the 5' end of the placeholder primer is not complementary to any sequence and can exist as a single-stranded "tail".
  • the relationship between placeholder primers and templates is shown in Figure 1.
  • the placeholder primer with the blocked 3' end modified and the normal sequencing primer hybridize to the template at the same time.
  • the chain is extended from the 3' end of the normal sequencing primer, and the template sequence is read sequentially.
  • the placeholder primer cannot be extended due to the blocking modification at the 3' end. This prevents the library from being completely tested because the inserted fragment is smaller than the sequencing read length, and the sequencing chain will cover the linker at this position, affecting the subsequent barcode sequencing. effect.
  • the library sequence whose inserted fragment is smaller than the sequencing read length will detect the position of the placeholder primer, and the placeholder primer prevents the sequencing fragment from continuing to extend along the template.
  • the sequencing sequence will use the “tail” at the 5’ end of the placeholder primer as a template and continue to extend forward, as shown in Figure 2.
  • Figure 2 shows that after the library insert is tested, it continues to be extended using the 5’ end of the placeholder primer as a template.
  • a reverse primer that is completely complementary to the occupying primer is introduced and given an appropriate annealing temperature.
  • the annealing temperature depends on the length of the complementary sequence of the placeholder primer and the reverse primer. Generally, the longer the length of the complementary sequence, the higher the annealing temperature required.
  • the annealing temperature of the complementary sequence can be detected by conventional methods or calculated by a model. method to obtain the annealing temperature of the complementary sequence.
  • the reverse primer sequence After adding the reverse primer sequence, the reverse primer sequence first binds to the "tail" at the 5' end of the placeholder primer, and then under appropriate conditions, the sequence on the placeholder primer that binds to the template adapter is replaced, as shown in Figure 3 Show.
  • Figure 3 shows the reverse primer displacing the spacer primer from the template.
  • the spacer primer and the reverse primer combine, and the double-stranded DNA formed is detached from the template, and the double-stranded spacer primer and reverse primer can be washed away by the buffer.
  • MGISEQ-2000 sequencer MGISEQ-2000 sequencing reagent slide, mini loader, PCR machine, PCR eight-tube strip, a set of Eppendorf pipettes, Effendorf high-speed centrifuge, etc.
  • DNBSEQ-G400RS high-throughput sequencing kit MGI New space-occupying primers bioengineering 5 ⁇ SSC buffer MGI reverse primer bioengineering formamide bioengineering E. coli library MGI
  • Control spacer primer AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAA (blocking modification at 3’ end) (SEQ ID NO.3);
  • Reagent name volume 100 ⁇ m control spacer primer stock solution 100 microliters 5 ⁇ SSC buffer 9.9ml total 10ml
  • Reagent name volume 100 ⁇ M test placeholder primer stock solution 100 microliters 5 ⁇ SSC buffer 9.9ml total 10ml
  • Reagent name volume 100 ⁇ M reverse primer stock solution 100 microliters 5 ⁇ SSC buffer 9.9ml total 10ml
  • the library is a single-stranded circular DNA, and the sequence of its adapter is: AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAAXXXXXXXXXCAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT (X represents the barcode sequence) (SEQ ID NO.4).
  • the inserted fragments of the library are concentrated around 450 bp, with fragments less than 400 bp accounting for approximately 37% of the total.
  • the library contains 8 sub-libraries with barcode numbers 97-104. Prepare two identical chips and load the DNA nanospheres onto the two DNBSEQ-G400RS sequencing reagent slides. Load two lanes per chip.
  • kits that can run 400 cycles of single-end sequencing; in one of the kits, select two free wells and add 3 ml of 1 ⁇ M control spacer primer working solution and 3 ml of formamide as a control group. . In another set of kits, select two free wells and add 3 ml of 1 ⁇ M test placeholder primer working solution and 3 ml of 1 ⁇ M reverse primer working solution respectively to serve as the test group. The two lanes of the control group and the test group serve as two replicates.
  • the control script is set to:
  • hybridization sequencing primers sequence the insert portion for 400 cycles
  • the script settings for the test group are:
  • hybridization sequencing primers sequence the insert portion for 400 cycles
  • Sequence the test group and control group according to the set script. After the sequencing is completed, software analysis is used to obtain the proportion of reads that can be separated by barcode sequences to the total reads (split rate). During splitting, some barcode sequences containing errors can also be accurately split through error tolerance. Calculate the total number of reads (excluding error-tolerant reads) in the eight barcode sub-libraries that are completely and accurately split and the total number of the barcode sub-libraries. The proportion of reads serves as barcode accuracy.
  • Figure 5 shows the splitting rates of the test group and the control group
  • Figure 6 shows the accuracy of different barcodes.
  • the splitting rate and barcode accuracy were significantly improved by using the method of testing placeholder primers and reverse primers.

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Abstract

一种在测序中使用的占位引物和去除方法。所述占位引物包括3'端的阻断基团，且所述占位引物的3'端序列与测序模板的部分序列互补，5'端序列不与所述测序模板配对。一种去除引物和相关的测序方法。通过在占位引物的5'端引入一段不与模板配对的序列，利用DNA的分支迁移作用在需要去除占位引物时，可以容易地将占位引物去除。

Description

一种占位引物和去除方法 技术领域
本发明属于生物技术领域，更具体而言涉及一种在测序中使用的占位引物及其使用方法和用途。
背景技术
在现有的DNA测序过程中，经常需要利用占位引物来阻止链从特定位置开始延伸，或者防止因为测序长度较长而覆盖住仍需用到的模板序列。例如，在DNBSEQ技术的小RNA文库测序和SE400的测序中，为了防止某些插入片段长度小于测序读长的文库在测序时测到接头影响后序条形码序列的测序，会在测序开始时先用一个3’端被阻断掉的占位引物将条形码部分的接头序列覆盖住，在完成插入片段的测序之后，利用一些物理或化学方法将占位引物去除掉，然后再用正常的引物测序条形码序列。
在现有的技术中有以下几种方法来去除占位引物或者避免使用占位引物：1)先测序条形码序列，后测插入片段的序列，但这种方法会导致插入片段的测序质量降低；2)利用超过引物Tm值的高温使占位引物从模板上解离下来，但这种方法的缺点是高温可能会对测序模板造成一些损伤，而且对于Tm值较高的占位引物不适用；3)利用可以变性DNA双链的化学试剂使占位引物与模板解链，例如利用强酸、强碱、甲酰胺、尿素等，但这些化学试剂会对模板造成损害，影响测序；4)占位引物的3’端使用可以恢复成能够延伸的3’端的修饰方法进行修饰，待需要从占位引物位置处开始测序时，通过化学反应将3’端的羟基恢复，例如在占位引物的3’端用磷酸化修饰，用去磷酸化酶可以恢复3’端羟基，但这种方法需要用到额外的酶或者其他试剂，会增加成本、时间和操作步骤。
发明内容
本发明的目的在于当测序过程中使用了占位引物时，提供一种新的方法使得在不影响测序质量的情况下，可以简单、有效地去除占位引物，并且无需特殊的酶等化学试剂，可以降低成本。
因此，在第一方面，本发明提供一种在测序中使用的占位引物，所述占位 引物包括3’端的阻断基团，且所述占位引物的3’端序列与测序模板的部分序列互补，5’端序列不与所述测序模板配对。
在一个实施方案中，所述阻断基团是磷酸化阻断基团、空间阻断基团、双脱氧核苷酸阻断基团或其他可以阻止3’端延伸的基团。
在一个实施方案中，所述占位引物的长度为10-300nt，优选30-70nt，更优选40-50nt。
在一个实施方案中，所述占位引物的5’端非匹配区的长度为3-200nt，优选5-50nt，更优选10-35nt。
在一个实施方案中，所述占位引物的3’端匹配区的长度为10-200nt，优选20-60nt，更优选30-50nt。
在第二方面，本发明提供了一种在聚合反应中使用的占位引物和去除引物，所述占位引物如本发明第一方面所述占位引物，所述去除引物为与所述占位引物完全互补的反向引物。
在第三方面，本发明提供了一种在聚合过程中引入占位引物和去除占位引物的方法，
所述引入占位引物包括：向测序模板中引入占位引物，所述占位引物包括3’端的阻断基团，且所述占位引物的3’端序列与所述测序模板的部分序列互补，5’端序列不与所述测序模板配对；
所述去除占位引物包括：引入与所述占位引物完全互补的反向引物，使所述反向引物先与所述占位引物的5’端结合，然后将所述占位引物从所述测序模板上置换下来。
在一个实施方案中，所述阻断基团是磷酸化阻断基团、空间阻断基团、双脱氧核苷酸阻断基团或其他可以阻止3’端延伸的基团。
在一个实施方案中，所述占位引物的长度为10-300nt，优选30-70nt，更优选40-50nt。
在一个实施方案中，所述占位引物的5’端非匹配区的长度为3-200nt，优选5-50nt，更优选10-35nt。
在一个实施方案中，所述占位引物的3’端匹配区的长度为10-200nt，优选20-60nt，更优选30-50nt。
在一个实施方案中，所述引入占位引物和去除引物的方法用于核酸序列测定。
在第四方面，本发明提供了一种测序方法，所述方法包括：
1)如本发明第三方面所述引入占位引物；
2)利用测序引物对所述待测核酸进行测序；
3)如本发明第三方面所述去除所述占位引物。
在一个实施方案中，在2)中，当文库插入片段小于测序读长时，对插入片段测序完成后，由于占位引物的存在不能继续往前延伸。
在一个实施方案中，所述测序方法还包括4)利用测序引物对待测核酸进行测序，所述待测核酸位于占位引物结合位点下游。
在一个实施方案中，所述测序引物为条形码引物，利用条形码引物对待测核酸进行测序，所述待测核酸为条形码序列，位于条形码引物结合位点下游。
在一个实施方案中，所述方法还包括对条形码序列进行测序。
本发明通过在占位引物的5’端引入一段不与模板配对的序列，利用DNA的分支迁移作用将占位引物从模板上置换下来，可以容易地将占位引物去除。
附图说明
图1的示意图示出了占位引物与测序引物同时杂交到模板上。该占位引物除了有完全与模板序列互补的序列外，其5’端还连接有一条不与接头互补的特异序列。
图2的示意图示出了当文库的插入片段小于测序读长时，插入片段被测完之后，占位引物阻止测序片段沿着模板继续延伸。
图3的示意图示出了反向引物置换占位引物。
图4示出了测试组和对照组的拆分率。
图5示出了不同条形码的准确率。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
在本发明中，占位引物的3’端序列与模板部分序列互补，5’端是一段特异序列，该特异序列不与文库或者接头序列互补。占位引物的长度为10-300nt，优选30-70nt，更优选40-50nt，其5’端非匹配区的长度为3-200nt，优选5-50nt，更优选10-35nt，其3’端匹配区的长度为10-200nt，优选20-60nt，更优选30-50nt。待阻断部分序列可以是模板上的任意序列，优选模板的条形码部分的 接头序列。
在本发明中，占位引物的3’端是阻断基团，阻断基团可以阻断测序片段沿着模板继续延伸。阻断基团可以是磷酸化阻断基团、空间阻断基团、双脱氧核苷酸阻断基团或其他可以阻止3’端延伸的基团。
在测序时，占位引物的3’端与模板上的待阻断部分，例如条形码部分的接头序列互补，可以达到占位的目的。占位引物的5’端不与任何序列互补，可以作为一条单链状态的“尾巴”存在。占位引物和模板的关系如图1所示。在图1中，3’端被阻断修饰的占位引物和正常的测序引物同时杂交到模板上。测序开始之后，链从正常测序引物的3’端延伸，依次读出模板序列。占位引物由于3’端做了阻断修饰，不可以延伸，从而起到防止文库由于插入片段小于测序读长被完全测完之后，测序链将该位置的接头覆盖，影响后序条形码测序的作用。
在本发明中，在测序过程中，插入片段小于测序读长的文库序列会测到占位引物的位置，占位引物阻止测序片段沿着模板继续延伸。在文库插入片段被完全测完时，测序序列会以占位引物的5’端的“尾巴”作为模板，继续往前延伸，如图2所示。图2示出了文库插入片段被测完之后继续以占位引物的5’端为模板延伸。
在本发明中，在需要去除该占位引物时，引入一条与占位引物完全互补的反向引物并给予合适的退火温度。退火温度取决于占位引物和反向引物互补序列的长度，一般而互补序列的长度越长，需要的退火温度越高，可以通过常规的方法检测互补序列的退火温度，也可以通过模型计算的方式得到互补序列的退火温度。在加入反向引物序列后，反向引物序列先与占位引物5’端的“尾巴”结合，然后在合适的条件下会将占位引物上与模板接头结合的序列置换出来，如图3所示。图3示出了反向引物从模板上置换占位引物。占位引物与反向引物结合，形成的双链DNA从而从模板上脱离下来，形成双链的占位引物与反向引物可以被缓冲液冲走。
实施例1。
1.器材：
MGISEQ-2000测序仪、MGISEQ-2000测序试剂载片、迷你装载仪、PCR仪、PCR八连管、Eppendorf移液器一套、Effendorf高速离心机等。
2.试剂：
试剂名称 品牌
DNBSEQ-G400RS高通量测序试剂盒 MGI
新型占位引物 生工生物
5×SSC缓冲液 MGI
反向引物 生工生物
甲酰胺 生工生物
大肠杆菌文库 MGI
3.试剂准备：
1)所需引物的溶解、测试占位引物、反向引物、对照占位引物以及接头序列如下：
测试占位引物：
agagtgaccgtgcctAAGTCGGAGGCCAAGCGGTCTTAGGAAGACAA(3’端做阻断修饰)(SEQ ID NO.1)；
反向引物：
TTGTCTTCCTAAGACCGCTTGGCCTCCGACTTaggcacggtcactct(SEQ ID NO.2)；
对照占位引物：AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAA(3’端做阻断修饰)(SEQ ID NO.3)；
说明：
文库接头序列：
AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAAXXXXXXXXXXCAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT(X表示条形码序列)(SEQ ID NO.4)。
一链测序引物序列：
Figure PCTCN2022091488-appb-000001
条形码引物序列：
Figure PCTCN2022091488-appb-000002
将装有测试占位引物粉末和反向引物粉末的1.5毫升的离心管在Eppendorf高速离心机(5415D)上，最高转速离心5分钟；按照引物标签上的说明，用1×TE缓冲液将引物溶解至100μM的测试占位引物母液；
2)1μM对照占位引物工作液、1μM测试占位引物工作液和1μM反向引物工作液的配制：
试剂名称 体积
100Μm对照占位引物母液 100微升
5×SSC缓冲液 9.9毫升
总计 10毫升
试剂名称 体积
100μM测试占位引物母液 100微升
5×SSC缓冲液 9.9毫升
总计 10毫升
试剂名称 体积
100μM反向引物母液 100微升
5×SSC缓冲液 9.9毫升
总计 10毫升
4.操作步骤：
1)测序模板的制备，参考《DNBSEQ-G400RS高通量测序试剂套装使用说明书》对大肠杆菌文库进行DNA纳米球的制备和定量，该文库为单链环状DNA，其接头的序列为：AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAAXXXXXXXXXXCAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT(X表示条形码序列)(SEQ ID NO.4)。文库的插入片段集中在450bp左右，其中小于400bp的片段含量约占总量的37％。文库中含有条形码编号为97-104的8个子文库。准备两张一样的芯片，将DNA纳米球装载到这两张DNBSEQ-G400RS测序试剂载片上。每张芯片装载两个泳道。
2)准备两套可以跑单末端测序400个循环的试剂盒；其中一套试剂盒选取两个空闲的孔位分别加入3毫升1μM的对照占位引物工作液和3毫升甲酰胺，作为对照组。另一套试剂盒选两个空闲的孔位，分别加入3毫升1μM的测试占位引物工作液和3毫升1μM的反向引物工作液，作为测试组。对照组与测试组的两个泳道作为两个重复。
3)按照《DNBSEQ-G400RS高通量测序试剂套装使用说明书》将测序试剂盒、芯片放在DNBSEQ-G400RS测序仪上。
对照组脚本设置为：
芯片装载好之后先使测序模板杂交对照占位引物，使其将DNB模板上与条形码引物杂交的部分被覆盖住；
然后杂交测序引物测序400个循环的插入片段部分；
用阻断试剂阻止插入片段部分的继续延伸；
用甲酰胺室温处理2分钟后用清洗试剂清洗，以去除对照占位引物；
杂交条形码引物，测序条形码部分10个循环。
测试组的脚本设置为：
芯片装载好之后先杂交测试占位引物，使其将DNB模板上与条形码引物杂交的部分被覆盖住；
然后杂交测序引物测序400个循环的插入片段部分；
再用阻断试剂阻止插入片段部分的继续延伸；
加入反向引物设置55℃10分钟，使反向引物与测试占位引物杂交并将其重模板上置换下来；
杂交条形码引物，测序条形码部分10个循环。
按照设置好的脚本对测试组和对照组进行测序，测序完成之后，通过软件分析得出能够通过条形码序列被拆分出来的读段占总读段的比例(拆分率)。在拆分时，部分含有错误的条形码序列也可以通过容错被准确拆分，分别计算8个条形码子文库中被完全准确拆分的读段(不含容错的读段)与该条形码子文库总读段的比例作为条形码准确率。
5.结果：
结果如图5和图6所示。图5示出了测试组和对照组的拆分率，图6示出了不同条形码的准确率。如图所示，用测试占位引物和反向引物的方法相比对照组拆分率与条形码的准确率均有明显提升。

Claims (14)

  1. 一种在聚合反应中使用的占位引物，所述占位引物包括3’端的阻断基团，且所述占位引物的3’端序列与测序模板的部分序列互补，5’端序列不与所述测序模板配对。
  2. 根据权利要求1所述的占位引物，所述阻断基团是磷酸化阻断基团、空间阻断基团、双脱氧核苷酸阻断基团或其他可以阻止3’端延伸的基团。
  3. 根据权利要求1或2所述的占位引物，所述占位引物的长度为10-300nt，优选30-70nt，更优选40-50nt。
  4. 根据权利要求1或2所述的占位引物，所述占位引物的5’端非匹配区的长度为3-200nt，优选5-50nt，更优选10-35nt。
  5. 根据权利要求1或2所述的占位引物，所述占位引物的3’端匹配区的长度为10-200nt，优选20-60nt，更优选30-50nt。
  6. 一种在聚合反应中使用的占位引物和去除引物，所述占位引物如权利要求1-5任意所述占位引物，所述去除引物为与所述占位引物完全互补的反向引物。
  7. 根据权利要求6所述的去除引物，所述去除引物的长度为10-100nt，优选30-70nt，更优选40-50nt。
  8. 一种在聚合过程中引入占位引物和去除占位引物的方法，
    所述占位引物为根据权利要求1-5任一项所述的占位引物，
    所述引入占位引物包括：向测序模板中引入占位引物，所述占位引物包括3’端的阻断基团，且所述占位引物的3’端序列与所述测序模板的部分序列互补，5’端序列不与所述测序模板配对；
    所述去除占位引物包括：引入与所述占位引物完全互补的反向引物，
    使所述反向引物先与所述占位引物的5’端结合，然后将所述占位引物从所述测序模板上置换下来。
  9. 根据权利要求8所述的方法，其中所述引入占位引物和去除引物的方法用于核酸序列测定。
  10. 一种测序方法，所述方法包括：
    1)如权利要求8所述引入占位引物；
    2)利用测序引物对所述待测核酸进行测序；
    3)如权利要求8所述引入去除引物去除所述占位引物。
  11. 根据权利要求10所述的方法，在2)中，当文库插入片段小于测序读 长时，对插入片段测序完成后，由于占位引物的存在不能继续延伸。
  12. 根据权利要求10或11所述的方法，还包括4)利用测序引物对待测核酸进行测序，所述待测核酸位于占位引物结合位点下游。
  13. 根据权利要求12所述的方法，其中所述测序引物为条形码引物，待测核酸为条形码序列，所述条形码序列位于条形码引物结合位点下游。
  14. 根据权利要求13所述的方法，还包括对条形码序列进行测序。
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