WO2023202030A1 - 一种小分子rna的高通量测序文库构建方法 - Google Patents
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- the invention relates to the field of high-throughput sequencing library construction, and in particular to a method for constructing a high-throughput sequencing library for small molecule RNA.
- Small RNA small molecule RNA
- rsRNAs small molecule RNA
- ysRNA tsRNAs
- miRNAs small RNA
- siRNAs small RNA
- piRNAs small RNA
- Small RNA can regulate physiological processes such as growth and development, metabolism, and disease occurrence of organisms through mRNA degradation, translation inhibition, heterochromatin formation, and DNA removal.
- small RNA maps at the whole genome level of the species can be obtained, and scientific applications such as identification of differential small RNA between samples and RNA clustering can be realized.
- RNA in plasma can be used as a potential biomarker for disease diagnosis and treatment. treat.
- small RNA maps at the whole genome level of the species can be obtained, enabling the identification of specific small RNA markers in the plasma of patients with different diseases.
- kits for small RNA sequencing such as NEBNext Multiplex Small RNA Library Prep Set.
- the materials targeted by these kits are mainly tissue RNA or cellular RNA, and they can only perform library construction and sequencing analysis on RNA with a phosphorylated modification at the 5' end.
- RNA with non-phosphorylated modifications at the 5' end there are no mature kits that can be used to draw small RNA maps for trace amounts of RNA in plasma.
- the purpose of the present invention is to provide a method for constructing a high-throughput sequencing library of small molecule RNA, aiming to solve the problem that the existing technology is unable to carry out a small amount of small molecule RNA with non-phosphorylation modification at the 5' end. Sequencing issues.
- a method for constructing a high-throughput sequencing library for small molecule RNA which includes the following steps:
- the first strand of the cDNA is denatured and then connected to the double-stranded DNA linker protruding from the end to obtain a ligation product;
- the ligation product is treated with USER enzyme to obtain processed cDNA;
- the processed cDNA was subjected to PCR amplification to obtain a high-throughput sequencing library.
- the method for constructing a high-throughput sequencing library of small molecule RNA wherein polyA tailing enzyme and MMLV reverse transcriptase are used, combined with a reverse transcription primer with polyT, to tail and reverse transcribe the RNA to obtain a reaction system product
- the steps include:
- reaction system product included the synthesized first strand of cDNA.
- the method for constructing a high-throughput sequencing library of small molecule RNA wherein the step of using exonuclease I to remove the remaining reverse transcription primers in the reaction system product includes:
- the method for constructing a high-throughput sequencing library of small molecule RNA, wherein the first strand of the cDNA is denatured and then connected to a double-stranded DNA adapter protruding from the end to obtain the ligation product which includes:
- the first strand of cDNA was denatured by incubating it at 95°C for 5 minutes to obtain the first strand of denatured cDNA;
- the high-throughput sequencing library construction method of small molecule RNA, wherein the step of treating the ligation product with USER enzyme to obtain the processed cDNA includes:
- the ligation product was treated with USER enzyme at 37°C for 15 minutes to obtain the processed cDNA.
- the present invention simultaneously uses the test protocol of polyA tailing enzyme and MMLV reverse transcriptase at the 3' end of RNA to construct a library.
- the use of this technical means does not require a ligation reaction at the 3' end of the RNA, thus greatly enhancing the detection
- the sensitivity of the invention enables library construction of extremely small amounts of small RNA in plasma; in addition, the present invention does not rely on the phosphorylation modification of the 5' end of RNA, so it can construct libraries of more different types of RNA.
- Figure 1 is a flow chart of a method for constructing a high-throughput sequencing library of small molecule RNA in the present invention.
- Figure 2 is a schematic diagram of high-throughput sequencing library construction of small RNA.
- Figure 3 shows the agarose gel electrophoresis results of the high-throughput sequencing library of small RNA in the plasma of four lung cancer patients in Example 1.
- the present invention provides a method for constructing a high-throughput sequencing library of small molecule RNA.
- the present invention is further described in detail below. It should be understood that the specific embodiments described here are only used to explain the present invention and are not intended to limit the present invention.
- Figure 1 is a flow chart of a high-throughput sequencing library construction method for small molecule RNA provided by the present invention. As shown in the figure, it includes the steps:
- kits can be used to extract RNA from different samples to be tested.
- the sample to be tested is tissue or cells
- the RNAiso Plus kit can be used to extract the sample RNA
- the sample to be tested is When using plasma
- alle MiniMax High Efficiency cfRNA Isolation kit can be used to extract sample RNA.
- the extracted RNA is finally dissolved in RNase-free water for later use.
- the present invention uses a test protocol of polyA tailing enzyme and MMLV reverse transcriptase at the 3' end of RNA to construct a library.
- the use of this technical means can greatly reduce the amount of RNA input and can achieve trace amounts in plasma.
- RNA library construction this technical method has not been reported yet.
- the present invention greatly enhances the sensitivity of detection and realizes the construction of libraries for extremely small amounts of small RNA in plasma; the present invention does not rely on the phosphorylation modification of the 5' end of RNA, so it can carry out library construction on more different types of RNA. Construct.
- polyA tailing enzyme and MMLV reverse transcriptase are used, combined with a reverse transcription primer containing polyT, to tail and reverse transcribe the RNA.
- the steps of obtaining the product of the reaction system include: adding polyA tailing enzyme, MMLV Reverse transcriptase, reverse transcription primers with polyT and reverse transcription buffer were mixed together to obtain a reverse transcription mixture; the RNA was treated at 70°C for 2 minutes to open the secondary structure of the RNA. When the temperature was lowered to 25 At 37°C, the reverse transcription mixture and the RNA are mixed together to obtain a reaction system; after incubating the above reaction system for 30 minutes at 37°C, a reaction system product is obtained, and the reaction system product includes the synthesized first strand of cDNA.
- different reverse transcription primers are used for different samples, and 8 additional marker bases are added to the reverse transcription primers to distinguish different samples during sequencing.
- the step of using exonuclease I to remove the remaining reverse transcription primers in the reaction system product includes: adding exonuclease I to the reaction system product, incubating at 37°C for 30 min, and then incubating at 80°C for 20 min, Remove reverse transcription primers from the reaction system products.
- this embodiment uses exonuclease I to process the reaction system products, thereby removing primers that have not undergone reverse transcription reaction, so that the proportion of self-ligated primers in the sequencing data is reduced to less than 25% ( The normal proportion is 60%-70%).
- the first strand of cDNA is denatured and then connected to a double-stranded DNA adapter with a protruding end.
- the step of obtaining the ligation product includes: incubating the first strand of cDNA at 95°C for 5 minutes to denature it. , to obtain the first strand of denatured cDNA; use double-stranded DNA adapters with protruding ends to incubate the first strand of denatured cDNA at 20°C for 1 hour, and then ligate at 65°C for 10 minutes to obtain the ligation product.
- the ligation product is treated with USER enzyme at 37°C for 15 minutes to obtain processed cDNA.
- USER enzyme to process the ligation product, the number of cycles required in the PCR process can be reduced and redundancy in the library can be reduced.
- this example specifically constructed a high-throughput sequencing library for small RNA in 200 ⁇ L plasma of 4 lung cancer patients.
- the reverse transcription primer for library construction is the sequence shown in SEQ ID NO.1, where "nnnnnnn” represents the 8 bp sample identification base, which is the sequence used to distinguish different samples when multiple samples are mixed and sequenced.
- 4 reverse transcription primers were used for small molecule RNA from 4 lung cancer patients.
- the sequences are SEQ ID NO.2 to SEQ ID NO.5, where v represents a, c, and g; n represents a, t, c, g.
- the cDNA obtained after reverse transcription of small molecule RNA in the plasma of 4 lung cancer patients was connected to the same double-stranded DNA linker, which was formed by the annealing of two DNA molecules with the sequences SEQ ID NO.6 and SEQ ID NO.7.
- the specific reaction system and conditions for constructing a high-throughput sequencing library of small molecule RNA in the plasma of 4 lung cancer patients are as follows:
- reaction solution with a total volume of 2.25 ⁇ L, including 0.625 ⁇ L of 4 ⁇ PolyA tailing buffer, 0.625 ⁇ L of 4 ⁇ reverse transcription buffer, 0.5 ⁇ L of PolyA tailing enzyme, and 0.5 ⁇ L of reverse transcriptase.
- reaction solution with a total volume of 9 ⁇ L: 10 ⁇ T4DNAligase buffer 1 ⁇ L, 10mM ATP 2 ⁇ L, 50% PEG40002 ⁇ L, adapter 1 ⁇ L, T4DNAligase 1 ⁇ L, nuclease-free water 2 ⁇ L, mix well and place on ice for later use.
- step 2 add the prepared reaction solution to the cDNA in step 2, mix well, and incubate at 20°C for 60 minutes. Incubate at 65°C for 15 minutes to complete the connection of the adapters. Place the product on ice for later use.
- step 4 Add 1 ul USER enzyme to the product in step 4, mix well and incubate at 37°C for 15 minutes to complete the digestion of the adapter. Keep the product on ice for later use.
- the library was quality checked using 3% agarose gel electrophoresis, and the results are shown in Figure 3. Using the method of the present invention, a library with an average length of approximately 180 bp was obtained, which was consistent with the expected results.
- the sample corresponding to each read can be obtained by judging the first 8 bases of R2reads in the sequencing raw data.
- the amount of sequencing data obtained for each sample is greater than 25M reads.
- the adapter sequences were first removed, and then the self-ligation ratio of the adapters was determined by judging the size of the inserted fragments.
- 15 bp is used as the screening condition, that is, the inserted fragment less than 15 bp is the adapter self-ligation product, and the inserted fragment greater than 15 bp is the small molecule RNA reverse transcription product.
- Table 1 after data analysis, it was found that the self-ligation ratios of linkers in the libraries of 4 lung cancer samples were: 22.9%, 25.2%, 22.4% and 24.5% respectively.
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Abstract
提供了一种小分子RNA的高通量测序文库构建方法,其包括步骤:提取待测样品中的RNA;使用polyA加尾酶和MMLV逆转录酶,结合带有polyT的逆转录引物对所述RNA进行加尾和逆转录,得到反应体系产物,所述反应体系产物包括合成的cDNA第一链;利用外切酶I清除反应体系产物中剩余的逆转录引物;对所述cDNA第一链进行变性后与末端突出的双链DNA接头进行连接,得到连接产物;对所述连接产物用USER酶处理,得到处理后cDNA;对处理后cDNA进行PCR扩增,得到高通量测序文库。实现了对血浆中极其微量的small RNA的文库构建,不依赖于RNA5'端为磷酸化修饰,因而可以对更多不同类型的RNA进行文库构建。
Description
本发明涉及高通量测序文库构建领域,特别涉及一种小分子RNA的高通量测序文库构建方法。
Small RNA(小分子RNA)包括rsRNAs、ysRNA、tsRNAs、miRNAs、siRNAs和piRNAs等,是一大类调控分子,几乎存在于所有生物体内。Small RNA可以通过mRNA降解、翻译抑制、异染色质形成以及DNA去除来调控生物体的生长发育、代谢和疾病发生等生理学过程。通过对small RNA进行高通量的测序分析,可以获得物种全基因组水平的small RNA图谱,实现样品间差异small RNA的鉴定、RNA聚类等科学应用。此外,最近研究发现血浆中存在丰富的small RNA,并且在肿瘤、高血压和糖尿病等多种疾病中呈特异性表达,提示血浆中的small RNA可以作为潜在的生物标记物用于疾病的诊断和治疗。通过对small RNA大规模测序分析,可以从中获得物种全基因组水平的small RNA图谱,实现对不同疾病患者血浆中特异的small RNA标志物鉴定。
目前针对small RNA进行测序的方法已有多款成熟的试剂盒,如NEBNext Multiplex Small RNA Library Prep Set。但这些试剂盒针对的材料主要为组织RNA或细胞RNA,并且只能对5’端为磷酸化修饰的RNA进行文库构建与测序分析。目前,针对5’端为非磷酸化修饰的RNA,并没有成熟的试剂盒,此外对于血浆中的微量RNA,亦没有成熟的试剂盒可以用于small RNA图谱的绘制。
因此,现有技术还有待于改进和发展。
发明内容
鉴于上述现有技术的不足,本发明的目的在于提供一种小分子RNA的高通量测序文库构建方法,旨在解决现有技术无法对微量5’端为非磷酸化修饰的小分子RNA进行测序的问题。
本发明的技术方案如下:
一种小分子RNA的高通量测序文库构建方法,其中,包括步骤:
提取待测样品中的RNA,备用;
使用polyA加尾酶和MMLV逆转录酶,结合带有polyT的逆转录引物对所述RNA进行加尾和逆转录,得到反应体系产物,所述反应体系产物包括合成的cDNA第一链;
利用外切酶I清除反应体系产物中剩余的逆转录引物;
对所述cDNA第一链进行变性后与末端突出的双链DNA接头进行连接,得到连接产物;
对所述连接产物用USER酶处理,得到处理后cDNA;
对处理后cDNA进行PCR扩增,得到高通量测序文库。
所述小分子RNA的高通量测序文库构建方法,其中,使用polyA加尾酶和MMLV逆转录酶,结合带有polyT的逆转录引物对所述RNA进行加尾和逆转录,得到反应体系产物的步骤包括:
将polyA加尾酶、MMLV逆转录酶、带有polyT的逆转录引物以及逆转录缓冲液混合在一起,得到逆转录混合物;
在70℃条件下处理所述RNA2min使所述RNA的二级结构打开,当温度降低至25℃时,将所述逆转录混合物和所述RNA混合在一起,得到反应体系;
在37℃将上述反应体系孵育30min后得到反应体系产物,所述反应体系产物包括合成的cDNA第一链。
所述小分子RNA的高通量测序文库构建方法,其中,利用外切酶I清除反应体系产物中剩余的逆转录引物的步骤包括:
将外切酶I加入到反应体系产物中,在37℃保温30min,接着以80℃保温20min,清除反应体系产物中的逆转录引物。
所述小分子RNA的高通量测序文库构建方法,其中,对所述cDNA第一链进行变性后与末端突出的双链DNA接头进行连接,得到连接产物的步骤包括:
对所述cDNA第一链进行95℃,5min孵育使其变性,得到变性cDNA第一链;
使用末端突出的双链DNA接头与变性cDNA第一链在20℃保温1h,接着在65℃保温10min的条件下进行连接,得到连接产物。
所述小分子RNA的高通量测序文库构建方法,其中,对所述连接产物用USER酶处理,得到处理后cDNA的步骤包括:
利用USER酶对所述连接产物进行37℃15min处理,得到处理后cDNA。
有益效果:本发明在RNA3’端同时使用polyA加尾酶和MMLV逆转录酶的试验方案进行文库构建,这一技术手段的使用不需要进行RNA 3’端的连接反应,因而极大的增强了检测的敏感性,实现了对血浆中极其微量的small RNA的文库构建;此外,本发明不依赖于RNA5’端为磷酸化修饰,因而可以对更多不同类型的RNA进行文库构建。
图1为本发明一种小分子RNA的高通量测序文库构建方法流程图。
图2为小分子RNA的高通量测序文库构建的原理图。
图3为实施例1中4例肺癌患者血浆中小分子RNA的高通量测序文库的琼脂糖凝胶电泳结果图。
本发明提供一种小分子RNA的高通量测序文库构建方法,为使本发明的目的、技术方案及效果更加清楚、明确,以下对本发明进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
请参阅图1,图1为本发明提供的一种小分子RNA的高通量测序文库构建方法流程图,如图所示,其包括步骤:
S10、提取待测样品中的RNA,备用;
S20、使用polyA加尾酶和MMLV逆转录酶,结合带有polyT的逆转录引物对所述RNA进行加尾和逆转录,得到反应体系产物,所述反应体系产物包括合成的cDNA第一链;
S30、利用外切酶I清除反应体系产物中剩余的逆转录引物;
S40、对所述cDNA第一链进行变性后与末端突出的双链DNA接头进行连接,得到连接产物;
S50、对所述连接产物用USER酶处理,得到处理后cDNA;
S60、对处理后cDNA进行PCR扩增,得到高通量测序文库。
在本发明中,不同的待测样品可使用不同的试剂盒来提取RNA,作为举例,若待测样品为组织或细胞时,则可采用RNAiso Plus试剂盒来提取样品RNA;若待测样品为血浆时,则可采用Apostle MiniMax High Efficiency cfRNA Isolation试剂盒来提取样品RNA,提取的RNA最终溶解于无RNA酶的水中,备用。
如图2所示,本发明是在RNA3’端同时使用polyA加尾酶和MMLV逆转录酶的试验方案进行文库构建,这一技术手段的使用可大大降低RNA投入量,能够实现对血浆中微量RNA的文库构建,这一技术手段还未有过报道。本发明极大的增强了检测的敏感性,实现了对血浆中极其微量的small RNA的文库构建;本发明不依赖于RNA5’端为磷酸化修饰,因而可以对更多不同类型的RNA进行文库构建。
在一些实施方式,使用polyA加尾酶和MMLV逆转录酶,结合带有polyT的逆转录引物对所述RNA进行加尾和逆转录,得到反应体系产物的步骤包括:将polyA加尾酶、MMLV逆转录酶、结合带有polyT的逆转录引物以及逆转录缓冲液混合在一起,得到逆转录混合物;在70℃条件下处理所述RNA2min使所述RNA的二级结构打开,当温度降低至25℃时,将所述逆转录混合物和所述RNA混合在一起,得到反应体系;在37℃将上述反应体系孵育30min后得到反应体系产物,所述反应体系产物包括合成的cDNA第一链。在本实施例操作过程中,对不同的样本采用不同的逆转录引物,逆转录引物上额外的加入8个标识碱基,用于不同样本在测序时能够区分开来。
在一些实施方式中,利用外切酶I清除反应体系产物中剩余的逆转录引物的步骤包括:将外切酶I加入到反应体系产物中,在37℃保温30min,接着以80℃保温20min,清除反应体系产物中的逆转录引物。如图2所示,本实施例通过采用外切酶I对反应体系产物进行处理,从而可以去除掉未发生逆转录反应的引物,使得测序数据中引物自连的比列降低到了25%以下(常规比例在60%-70%)。
在一些实施方式中,对所述cDNA第一链进行变性后与末端突出的双链DNA接头进行连接,得到连接产物的步骤包括:对所述cDNA第一链进行95℃,5min孵育使其变性,得到变性cDNA第一链;使用末端突出的双链DNA接头与 变性cDNA第一链在20℃保温1h,接着在65℃保温10min的条件下进行连接,得到连接产物。
具体来讲,传统方法仅能对5’端为磷酸化修饰的RNA进行文库构建和测序,而本实施例如图2所示,对所述cDNA第一链进行变性后,通过T4DNA连接酶将变性cDNA第一链与末端突出的双链DNA接头进行连接,通过这一技术手段,完全避免了RNA5’端的修饰对文库构建的影响。
在一些实施方式中,利用USER酶对所述连接产物进行37℃15min处理,得到处理后cDNA。在本实施例中,通过采用USER酶对所述连接产物进行处理,可以降低PCR过程所需要的循环数,减少文库中的冗余。
下面通过具体实施例对本发明做进一步的解释说明:
实施例1
根据以上small RNA文库构建方法,本例具体对4例肺癌患者200μL血浆中的小分子RNA进行了高通量测序文库构建。先利用Apostle公司的MiniMax TM High Efficiency cfRNA Isolation Kit对血浆中的小分子RNA进行提取,最终体积均为10μL。
文库构建的逆转录引物为SEQ ID NO.1所示序列,其中,“nnnnnnnn”表示8bp的样本标识碱基,即多个样品混合测序时用于区分不同样品的序列。本例针对4例肺癌患者的小分子RNA用到了4条逆转录引物,序列为SEQ ID NO.2至SEQ ID NO.5,其中v表示a,c,g;n表示a,t,c,g。
4例肺癌患者血浆中小分子RNA逆转录后所得cDNA连接相同的双链DNA接头,其由序列为SEQ ID NO.6和SEQ ID NO.7的两条DNA分子退火形成。
4例肺癌患者血浆中小分子RNA逆转录产物cDNA连接接头后,采用相同的PcR引物进行扩增,即SEQ ID NO.8和SEQ ID NO.9所示。
本例中4例肺癌患者血浆中小分子RNA高通量测序文库构建的具体反应体系和条件如下:
1、小分子RNA加PolyA尾与逆转录
首先将血浆中提取的6.75μL无核酸酶的小分子RNA与0.05μM的逆转录引物1μL混合,制备成混合样品后置于70℃孵育2min后立即放置于冰上,备用。
然后配制总体积为2.25μL的反应液,包括4×PolyA加尾缓冲液0.625μL,4×逆转录缓冲液0.625μL,PolyA加尾酶0.5μL,逆转录酶0.5μL。
将2.25μL的反应液与放置于冰上的混合样品混匀后置于37℃孵育30min,即完成了小分子RNA加PolyA尾与逆转录,得到了cDNA。
2、去除残留逆转录引物
在上述逆转录产物中加入核酸外切酶I 1ul后混匀置于37℃孵育30min,80℃孵育20min,95℃孵育5min后迅速置于冰上备用,即完成了cDNA中残留逆转录引物的去除。
3、接头的制备
首先分别将5μL制备接头所用的100μM的两条DNA分子混合后置于冰上,向混合物中加入5μL的10×NEBbuffer2.1并混匀,加入35μL无核酸酶的水后混匀并放置于冰上备用,总反应体系为50μL。
然后混合液置于Thermal cycler中,并运行下述程序:95℃5min,然后进入70个循环:每个循环温度降低1℃并孵育1min,最终于25℃孵育1min后停止,将混合液置于冰上备用,即得到了接头。
4、逆转录产物连接接头
首先配制总体积为9μL的反应液:10×T4DNAligase buffer 1μL、10mM ATP 2μL、50%PEG40002μL、接头1μL、T4DNAligase 1μL、无核酸酶水2μL,混匀后置于冰上备用。
然后将配好的反应液加入到步骤2中的cDNA中,混匀后置于20℃孵育60min,65℃孵育15min即完成了接头的连接,将产物置于冰上备用。
5、USER酶消化接头
将1ul USER酶加入到步骤4的产物中,混匀后置于37℃孵育15min即完成了对接头的消化,将产物置于冰上备用。
6、PCR扩增制备文库
首先取新的PCR管,配制总体积为35μL的反应液:2×Kapa hifi ready mix25μL、10μM的扩增引物分别1μL,无核酸酶的水8μL,混合后置于冰上备用。
吸取15μL步骤5中的产物并加入至上述PCR反应液中,混合后在Thermal cycler上进行如下反应:95℃孵育1min,然后进入16个循环:98℃20s、60℃30s、72℃30s,循环结束后,72℃孵育1min,15℃保持,即得到了扩增产物。
7、利用XPbeads纯化回收PCR产物
首先向PCR产物中加入90μL XPbead后混匀,室温放置15min,期间振荡混匀1次。
然后置于磁力架上3-5min后丢弃上清,用200μL 80%乙醇清洗两次,清洗时将PCR管前后方向反转5次,使beads在乙醇中得到充分洗涤。
打开PCR管盖,将其置于室温下晾干剩余乙醇。加入30μL蒸馏水后振荡混匀,使beads悬浮于溶液中,室温放置15min,期间再振荡混匀1次。
置于磁力架上3-5min后将上清转移至新的EP管中,即为本例中得到的4例肺癌患者血浆中小分子RNA的高通量测序文库。
采用3%的琼脂糖凝胶电泳对文库进行质检,所得结果如图3所示。采用本发明所述方法得到了平均长度约为180bp的文库,与预期结果一致。
采用Qubit
TMdsDNA Assay Kit对文库进行浓度测定后,分别取30ng文库混匀后在Illumina测序平台进行测序。
对本例的小分子RNA文库测序进行分析,具体如下:
1、对于4个不同样本数据的拆分
根据4个不同肺癌样本血浆小分子RNA在逆转录过程中携带的样本标签不同,对测序原始数据中R2reads的前8个碱基进行判断可得每条reads对应的样本。本例中对原始测序数据进行拆分后,所得每个样本的测序数据量均大于25M reads。
2、小分子RNA文库测序结果分析
通过数据拆分得到4例肺癌样本的测序结果后,首先去除接头序列,然后通过判断插入片段的大小判断接头自连比例。本例中以15bp为筛选条件,即插入片段小于15bp为接头自连产物,插入片段大于15bp为小分子RNA逆转录产物。如表1所示,数据经过分析发现4例肺癌样本文库中接头自连比列分别为:22.9%、25.2%、22.4%和24.5%。
表1 4例肺癌样品测序数据分析表
该结果表明本发明在对微量小分子RNA进行文库构建过程中仅产生少量的接头自连。如表1所示,对去除掉接头自连后的序列进行比对分析发现了5种不同来源的小分子RNA,包括rsRNA、ysRNA、tsRNA、miRNA、piRNA。该结果表明利用本发明可以得到肺癌患者血浆中丰富的5’端不含磷酸化修饰的小分子RNA信息,可以为癌症的早期诊断及预后标志物的筛选奠定基础。
应当理解的是,本发明的应用不限于上述的举例,对本领域普通技术人员来说,可以根据上述说明加以改进或变换,所有这些改进和变换都应属于本发明所附权利要求的保护范围。
Claims (5)
- 一种小分子RNA的高通量测序文库构建方法,其特征在于,包括步骤:提取待测样品中的RNA,备用;使用polyA加尾酶和MMLV逆转录酶,结合带有polyT的逆转录引物对所述RNA进行加尾和逆转录,得到反应体系产物,所述反应体系产物包括合成的cDNA第一链;利用外切酶I清除反应体系产物中剩余的逆转录引物;对所述cDNA第一链进行变性后与末端突出的双链DNA接头进行连接,得到连接产物;对所述连接产物用USER酶处理,得到处理后cDNA;对处理后cDNA进行PCR扩增,得到高通量测序文库。
- 根据权利要求1所述小分子RNA的高通量测序文库构建方法,其特征在于,使用polyA加尾酶和MMLV逆转录酶,结合带有polyT的逆转录引物对所述RNA进行加尾和逆转录,得到反应体系产物的步骤包括:将polyA加尾酶、MMLV逆转录酶、结合带有polyT的逆转录引物以及逆转录缓冲液混合在一起,得到逆转录混合物;在70℃条件下处理所述RNA2min使所述RNA的二级结构打开,当温度降低至25℃时,将所述逆转录混合物和所述RNA混合在一起,得到反应体系;在37℃将上述反应体系孵育30min后得到反应体系产物,所述反应体系产物包括合成的cDNA第一链。
- 根据权利要求1所述小分子RNA的高通量测序文库构建方法,其特征在于,利用外切酶I清除反应体系产物中剩余的逆转录引物的步骤包括:将外切酶I加入到反应体系产物中,在37℃保温30min,接着以80℃保温20min,清除反应体系产物中的逆转录引物。
- 根据权利要求1所述小分子RNA的高通量测序文库构建方法,其特征在于,对所述cDNA第一链进行变性后与末端突出的双链DNA接头进行连接,得到连接产物的步骤包括:对所述cDNA第一链进行95℃,5min孵育使其变性,得到变性cDNA第一链;使用末端突出的双链DNA接头与变性cDNA第一链在20℃保温1h,接着在65℃保温10min的条件下进行连接,得到连接产物。
- 根据权利要求1所述小分子RNA的高通量测序文库构建方法,其特征在于,对所述连接产物用USER酶处理,得到处理后cDNA的步骤包括:利用USER酶对所述连接产物进行37℃15min处理,得到处理后cDNA。
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105002569A (zh) * | 2015-07-15 | 2015-10-28 | 北京诺禾致源生物信息科技有限公司 | 转录组文库及其构建方法 |
CN107385018A (zh) * | 2017-06-22 | 2017-11-24 | 哈尔滨博泰生物科技有限公司 | 一种优化的rna高通量测序文库构建的方法及其应用 |
US20170355981A1 (en) * | 2014-11-26 | 2017-12-14 | Bgi Shenzhen | Method and reagent for constructing nucleic acid double-linker single-strand cyclical library |
CN113308514A (zh) * | 2021-05-19 | 2021-08-27 | 武汉大学 | 微量m6A的检测文库构建方法和试剂盒、高通量检测方法 |
WO2021184146A1 (zh) * | 2020-03-16 | 2021-09-23 | 深圳华大智造科技有限公司 | 一种待测样本rna的测序文库的构建方法 |
US20210363517A1 (en) * | 2020-05-20 | 2021-11-25 | Paragon Genomics, Inc. | High throughput amplification and detection of short rna fragments |
CN114736951A (zh) * | 2022-04-20 | 2022-07-12 | 深圳大学 | 一种小分子rna的高通量测序文库构建方法 |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102154505B (zh) * | 2011-04-20 | 2013-03-20 | 苟德明 | 一种检测miRNA的方法及引物及其应用 |
CN104372414B (zh) * | 2012-10-25 | 2016-05-04 | 盛司潼 | 一种构建测序文库的方法 |
CN105624270B (zh) * | 2014-10-27 | 2020-03-24 | 深圳华大基因股份有限公司 | 一种用于Small RNA二代测序建库的接头处理方法 |
CN105985945B (zh) * | 2015-01-30 | 2019-04-23 | 深圳华大智造科技有限公司 | mRNA片段化方法及基于其构建测序文库的方法 |
CN105297144A (zh) * | 2015-10-27 | 2016-02-03 | 北京百迈客生物科技有限公司 | 一种原核生物小rna的高通量文库构建方法 |
US11441169B2 (en) * | 2016-06-17 | 2022-09-13 | Ludwig Institute For Cancer Research Ltd | Methods of small-RNA transcriptome sequencing and applications thereof |
CN108624666B (zh) * | 2017-03-16 | 2021-12-24 | 深圳华大基因股份有限公司 | 用于构建测序文库的接头核酸分子 |
US20180362968A1 (en) * | 2017-06-14 | 2018-12-20 | Somagenics, Inc. | Methods of library construction for target polynucleotides |
WO2019191900A1 (en) * | 2018-04-03 | 2019-10-10 | Burning Rock Biotech | Compositions and methods for preparing nucleic acid libraries |
CN110872609B (zh) * | 2018-09-04 | 2024-06-04 | 深圳华大基因科技服务有限公司 | 对小rna分子精准建库和测序的方法以及应用 |
CN109957611B (zh) * | 2019-03-22 | 2020-06-26 | 健生生物技术有限公司 | 一种特异性定量PCR反应混合液、miRNA定量检测试剂盒以及检测方法 |
US11390915B2 (en) * | 2019-04-26 | 2022-07-19 | New England Biolabs, Inc. | Polynucleotide adapter design for reduced bias |
CN112251821A (zh) * | 2020-10-20 | 2021-01-22 | 南京实践医学检验有限公司 | 一种快速高效的构建二代测序文库的试剂盒 |
CN113249437A (zh) * | 2021-04-20 | 2021-08-13 | 成都罗宁生物科技有限公司 | 一种用于sRNA测序的建库方法 |
-
2022
- 2022-04-20 CN CN202210414840.7A patent/CN114736951A/zh active Pending
- 2022-11-01 WO PCT/CN2022/128855 patent/WO2023202030A1/zh unknown
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20170355981A1 (en) * | 2014-11-26 | 2017-12-14 | Bgi Shenzhen | Method and reagent for constructing nucleic acid double-linker single-strand cyclical library |
CN105002569A (zh) * | 2015-07-15 | 2015-10-28 | 北京诺禾致源生物信息科技有限公司 | 转录组文库及其构建方法 |
CN107385018A (zh) * | 2017-06-22 | 2017-11-24 | 哈尔滨博泰生物科技有限公司 | 一种优化的rna高通量测序文库构建的方法及其应用 |
WO2021184146A1 (zh) * | 2020-03-16 | 2021-09-23 | 深圳华大智造科技有限公司 | 一种待测样本rna的测序文库的构建方法 |
US20210363517A1 (en) * | 2020-05-20 | 2021-11-25 | Paragon Genomics, Inc. | High throughput amplification and detection of short rna fragments |
CN113308514A (zh) * | 2021-05-19 | 2021-08-27 | 武汉大学 | 微量m6A的检测文库构建方法和试剂盒、高通量检测方法 |
CN114736951A (zh) * | 2022-04-20 | 2022-07-12 | 深圳大学 | 一种小分子rna的高通量测序文库构建方法 |
Non-Patent Citations (1)
Title |
---|
ANTHEIA KISSOPOULOU, JON JONASSON, TOMAS L. LINDAHL, ABDIMAJID OSMAN: "Next Generation Sequencing Analysis of Human Platelet PolyA+ mRNAs and rRNA-Depleted Total RNA", PLOS ONE, vol. 8, no. 12, pages e81809, XP055437269, DOI: 10.1371/journal.pone.0081809 * |
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