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。The present invention belongs to the field of biotechnology, and more specifically relates to a placeholder primer used in sequencing and a using method and application thereof.
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。In the existing DNA sequencing process, placeholder primers are often needed to prevent the chain from extending from a specific position, or to prevent the template sequence that is still needed from being covered due to the long sequencing length. For example, in the small RNA library sequencing of DNBSEQ technology and the sequencing of SE400, in order to prevent the detection of adapters during sequencing of certain libraries with insert fragments shorter than the sequencing read length, which affects the sequencing of the subsequent barcode sequence, a placeholder primer with a blocked 3' end will be used at the beginning of sequencing to cover the adapter sequence of the barcode part. After the sequencing of the insert fragment is completed, the placeholder primer is removed by some physical or chemical methods, and then the barcode sequence is sequenced with a normal primer.
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。In the existing technology, there are several methods to remove placeholder primers or avoid using placeholder primers: 1) sequence the barcode sequence first and then sequence the insert, but this method will reduce the sequencing quality of the insert; 2) use a high temperature exceeding the primer Tm value to dissociate the placeholder primer from the template, but the disadvantage of this method is that the high temperature may cause some damage to the sequencing template, and it is not applicable to placeholder primers with higher Tm values; 3) use chemical reagents that can denature the double-stranded DNA to dissociate the placeholder primer from the template, such as strong acids, strong bases, formamide, urea, etc., but these chemical reagents will damage the template and affect sequencing; 4) the 3' end of the placeholder primer is modified using a modification method that can restore it to a 3' end that can be extended. When sequencing needs to start from the position of the placeholder primer, the hydroxyl group at the 3' end is restored by a chemical reaction, for example, the 3' end of the placeholder primer is phosphorylated and the 3' end hydroxyl group can be restored by a dephosphorylase, but this method requires additional enzymes or other reagents, which will increase cost, time and operation steps.
发明内容Summary of the invention
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。The purpose of the present invention is to provide a new method for simply and effectively removing placeholder primers when placeholder primers are used in the sequencing process without affecting the sequencing quality, and without requiring special enzymes or other chemical reagents, thereby reducing costs.
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。Therefore, in a first aspect, the present invention provides a placeholder primer for use in sequencing, wherein the placeholder primer includes a blocking group at the 3' end, and the 3' end sequence of the placeholder primer is complementary to a partial sequence of a sequencing template, and the 5' end sequence does not pair with the sequencing template.
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。In one embodiment, 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.
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。In one embodiment, the length of the placeholder primer is 10-300 nt, preferably 30-70 nt, more preferably 40-50 nt.
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。In one embodiment, the length of the non-matching region at the 5' end of the placeholder primer is 3-200 nt, preferably 5-50 nt, and more preferably 10-35 nt.
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。In one embodiment, the length of the 3' end matching region of the placeholder primer is 10-200nt, preferably 20-60nt, and more preferably 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。In a second aspect, the present invention provides a placeholder primer and a removal primer for use in a polymerization reaction, wherein the placeholder primer is the placeholder primer described in the first aspect of the present invention, and the removal primer is a reverse primer that is completely complementary to the placeholder primer.
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，In a third aspect, the present invention provides a method for introducing and removing placeholder primers during polymerization.
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；The introducing of the placeholder primer comprises: introducing a placeholder primer into the sequencing template, wherein the placeholder primer comprises a blocking group at the 3' end, and the 3' end sequence of the placeholder primer is complementary to a partial sequence of the sequencing template, and the 5' end sequence does not pair with the sequencing template;
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。The removal of the placeholder primer includes: introducing a reverse primer that is completely complementary to the placeholder primer, allowing the reverse primer to first bind to the 5' end of the placeholder primer, and then displacing the placeholder primer from the sequencing template.
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。In one embodiment, 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.
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。In one embodiment, the length of the placeholder primer is 10-300 nt, preferably 30-70 nt, more preferably 40-50 nt.
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。In one embodiment, the length of the non-matching region at the 5' end of the placeholder primer is 3-200 nt, preferably 5-50 nt, and more preferably 10-35 nt.
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。In one embodiment, the length of the 3' end matching region of the placeholder primer is 10-200nt, preferably 20-60nt, and more preferably 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。In one embodiment, the method of introducing placeholder primers and removing primers is used for nucleic acid sequence determination.
在第四方面，本发明提供了一种测序方法，所述方法包括：In a fourth aspect, the present invention provides a sequencing method, comprising:
1)如本发明第三方面所述引入占位引物；1) introducing a placeholder primer as described in the third aspect of the present invention;
2)利用测序引物对所述待测核酸进行测序；2) sequencing the nucleic acid to be tested using sequencing primers;
3)如本发明第三方面所述去除所述占位引物。3) Removing the placeholder primer as described in the third aspect of the present invention.
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。In one embodiment, in 2), when the library insert is smaller than the sequencing read length, after the insert is sequenced, it cannot be extended further due to the presence of the placeholder primer.
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。In one embodiment, the sequencing method further comprises 4) sequencing the nucleic acid to be tested using a sequencing primer, wherein the nucleic acid to be tested is located downstream of the placeholder primer binding site.
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。In one embodiment, the sequencing primer is a barcode primer, and the barcode primer is used to sequence the nucleic acid to be tested. The nucleic acid to be tested is a barcode sequence located downstream of the barcode primer binding site.
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。In one embodiment, the method further comprises sequencing the barcode sequence.
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。The present invention introduces a sequence that does not pair with the template at the 5' end of the placeholder primer, utilizes the branch migration effect of DNA to displace the placeholder primer from the template, and can easily remove the placeholder primer.
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。The schematic diagram of Figure 1 shows that the placeholder primer and the sequencing primer are hybridized to the template at the same time. In addition to having a sequence that is completely complementary to the template sequence, the placeholder primer has a specific sequence that is not complementary to the adapter connected to its 5' end.
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。The schematic diagram of FIG2 shows that when the insert fragment of the library is smaller than the sequencing read length, after the insert fragment is sequenced, the placeholder primer prevents the sequencing fragment from continuing to extend along the template.
图3的示意图示出了反向引物置换占位引物。Figure 3 is a schematic diagram showing the replacement of the placeholder primer by the reverse primer.
图4示出了测试组和对照组的拆分率。FIG4 shows the split ratios of the test group and the control group.
图5示出了不同条形码的准确率。Figure 5 shows the accuracy of different barcodes.
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。The technical scheme in the embodiment of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, not all of the embodiments. Based on the embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
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。In the present invention, 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, the length of the non-matching region at its 5' end is 3-200nt, preferably 5-50nt, more preferably 10-35nt, and the length of the matching region at its 3' end 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 adapter sequence of the barcode portion of the template.
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。In the present invention, 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 the 3' end from extending.
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。During sequencing, the 3' end of the placeholder primer is complementary to the portion to be blocked on the template, such as the adapter sequence of the barcode portion, to achieve the purpose of placeholder. The 5' end of the placeholder primer is not complementary to any sequence and can exist as a "tail" in a single-stranded state. The relationship between the placeholder primer and the template is shown in Figure 1. In Figure 1, the placeholder primer with a blocking modification at the 3' end and the normal sequencing primer are hybridized to the template at the same time. After the sequencing starts, the chain extends from the 3' end of the normal sequencing primer, and the template sequence is read out in sequence. Since the placeholder primer has a blocking modification at the 3' end, it cannot be extended, thereby preventing the sequencing chain from covering the adapter at this position after the library is completely sequenced due to the insertion fragment being shorter than the sequencing read length, affecting the subsequent barcode sequencing.
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。In the present invention, during the sequencing process, the library sequence with an insert fragment 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. When the library insert fragment is completely sequenced, the sequencing sequence will continue to extend forward using the "tail" at the 5' end of the placeholder primer as a template, as shown in Figure 2. Figure 2 shows that after the library insert fragment is sequenced, it continues to extend using the 5' end of the placeholder primer as a template.
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。In the present invention, when it is necessary to remove the placeholder primer, a reverse primer that is completely complementary to the placeholder primer is introduced and given a suitable 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 required annealing temperature. The annealing temperature of the complementary sequence can be detected by conventional methods, or the annealing temperature of the complementary sequence can be obtained by model calculation. After the reverse primer sequence is added, 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 displaced, as shown in Figure 3. Figure 3 shows that the reverse primer displaces the placeholder primer from the template. The placeholder primer binds to the reverse primer, and the double-stranded DNA formed is thereby detached from the template, and the double-stranded placeholder primer and reverse primer can be washed away by the buffer.
实施例1。Example 1.
1.器材：1. Equipment:
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。MGISEQ-2000 sequencer, MGISEQ-2000 sequencing reagent slide, mini loader, PCR instrument, PCR eight-tube strip, a set of Eppendorf pipettes, Effendorf high-speed centrifuge, etc.
2.试剂：2. Reagents:
3.试剂准备：3. Reagent preparation:
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：1) The required primers for dissolution, test placeholder primers, reverse primers, control placeholder primers and adapter sequences are as follows:
测试占位引物：Testing placeholder primers:
agagtgaccgtgcctAAGTCGGAGGCCAAGCGGTCTTAGGAAGACAA(3’端做阻断修饰)(SEQ ID NO.1)；agagtgaccgtgcctAAGTCGGAGGCCAAGCGGTCTTAGGAAGACAA (blocking modification at the 3' end) (SEQ ID NO.1);
反向引物：Reverse Primer:
TTGTCTTCCTAAGACCGCTTGGCCTCCGACTTaggcacggtcactct(SEQ ID NO.2)；TTGTCTTCCTAAGACCGCTTGGCCTCCGACTTaggcacggtcactct(SEQ ID NO.2);
对照占位引物：AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAA(3’端做阻断修饰)(SEQ ID NO.3)；Control placeholder primer: AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAA (blocking modification at the 3' end) (SEQ ID NO. 3);
说明：illustrate:
文库接头序列：Library adapter sequence:
AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAAXXXXXXXXXXCAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT(X表示条形码序列)(SEQ ID NO.4)。AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAAXXXXXXXXXXCAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT (X represents the barcode sequence) (SEQ ID NO. 4).
一链测序引物序列：First strand sequencing primer sequence:
条形码引物序列：Barcode primer sequences:
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；Place the 1.5 ml centrifuge tube containing the test placeholder primer powder and the reverse primer powder in an Eppendorf high-speed centrifuge (5415D) at the highest speed for 5 minutes; Dissolve the primers to 100 μM test placeholder primer stock solution with 1×TE buffer according to the instructions on the primer label;
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：2) Preparation of 1 μM control placeholder primer working solution, 1 μM test placeholder primer working solution and 1 μM reverse primer working solution:
4.操作步骤：4. Operation steps:
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。1) Preparation of sequencing templates. Refer to the "DNBSEQ-G400RS High-throughput Sequencing Reagent Set Instructions" to prepare and quantify DNA nanoballs for the E. coli library. The library is a single-stranded circular DNA, and the sequence of its linker is: AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAAXXXXXXXXXXCAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT (X represents the barcode sequence) (SEQ ID NO.4). The insert fragments of the library are concentrated at around 450bp, of which the fragment content of less than 400bp accounts for about 37% of the total. The library contains 8 sub-libraries with barcode numbers 97-104. Prepare two identical chips and load the DNA nanoballs onto the two DNBSEQ-G400RS sequencing reagent slides. Each chip is loaded with two lanes.
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。2) Prepare two kits that can run single-end sequencing for 400 cycles; select two free wells in one kit and add 3 ml of 1 μM control placeholder primer working solution and 3 ml of formamide respectively as the control group. Select two free wells in the other kit and add 3 ml of 1 μM test placeholder primer working solution and 3 ml of 1 μM reverse primer working solution respectively as the test group. The two lanes of the control group and the test group are used as two replicates.
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。3) Place the sequencing kit and chip on the DNBSEQ-G400RS sequencer according to the "DNBSEQ-G400RS High-throughput Sequencing Reagent Set Instructions".
对照组脚本设置为：The control group script settings are:
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；After the chip is loaded, the sequencing template is first hybridized to the control placeholder primer so that the portion of the DNB template that hybridizes to the barcode primer is covered;
然后杂交测序引物测序400个循环的插入片段部分；Then the hybridization sequencing primer was used to sequence the insert fragment portion for 400 cycles;
用阻断试剂阻止插入片段部分的继续延伸；Using a blocking agent to prevent further extension of the inserted fragment;
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；Treat with formamide at room temperature for 2 minutes and then wash with a washing reagent to remove the control placeholder primer;
杂交条形码引物，测序条形码部分10个循环。Hybridize the barcoded primers and sequence the barcoded portion for 10 cycles.
测试组的脚本设置为：The script settings for the test group are:
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；After the chip is loaded, the test placeholder primer is hybridized first so that the portion of the DNB template that hybridizes with the barcode primer is covered;
然后杂交测序引物测序400个循环的插入片段部分；Then the hybridization sequencing primer was used to sequence the insert fragment portion for 400 cycles;
再用阻断试剂阻止插入片段部分的继续延伸；Then use a blocking reagent to prevent the further extension of the inserted fragment;
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；Add the reverse primer and set it at 55°C for 10 minutes to allow the reverse primer to hybridize with the test placeholder primer and displace it from the heavy template;
杂交条形码引物，测序条形码部分10个循环。Hybridize the barcoded primers and sequence the barcoded portion for 10 cycles.
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。The test group and the control group were sequenced according to the set script. After the sequencing was completed, the software analyzed the proportion of the reads that could be split by the barcode sequence to the total reads (splitting rate). During the splitting, some barcode sequences containing errors can also be accurately split through error tolerance. The ratio of the reads that were completely and accurately split in the 8 barcode sub-libraries (without error tolerance) to the total reads of the barcode sub-library was calculated as the barcode accuracy rate.
5.结果：5. Results:
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。The results are shown in Figures 5 and 6. Figure 5 shows the splitting rates of the test group and the control group, and Figure 6 shows the accuracy of different barcodes. As shown in the figures, the splitting rate and barcode accuracy of the control group using the test placeholder primer and reverse primer method were significantly improved.
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| PCT/CN2022/091488WO2023216030A1 (en) | 2022-05-07 | 2022-05-07 | Site-occupying primer and removal method |
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| CN118401679Atrue CN118401679A (en) | 2024-07-26 |
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| CN202280083891.4APendingCN118401679A (en) | 2022-05-07 | 2022-05-07 | Space occupying primer and removing method |
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