Construction method and application of high-throughput sequencing library of lncrna

Abstract

The application provides a method for constructing a high-throughput sequencing library of lncRNA and application. The method for constructing the high-throughput sequencing library of lncRNA comprises the following steps: S1, removing ribosomal RNA in total RNA of a sample to be detected to obtain total RNA without ribosomal RNA; S2, reverse transcribing the total RNA without ribosomal RNA to form double-stranded cDNA; S3, connecting sequencing adapters to the 5' and 3' ends of the double-stranded cDNA to obtain an adapter connection product; S4, PCR amplification and enrichment of the adapter connection product, and recovery to obtain a high-throughput sequencing library of lncRNA of the sample to be detected; the sample to be detected comprises an exosome sample or a low-quality FFPR sample. The method can solve the problem that unknown lncRNA is difficult to be subjected to high-throughput sequencing in the prior art and is suitable for the field of life science technology.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of life science technology, in particular, to a method for constructing a high-throughput sequencing library of lncRNA and application thereof. BACKGROUND

[0002] Exosomes are a kind of outer vesicular structure produced in cytoplasm, released through cell membrane, with a diameter of 30-100 nm, carrying a variety of proteins, lipids and nucleic acids and other substances of mother cells. The sources are diverse, and widely distributed in body fluids such as blood, saliva, urine, cerebrospinal fluid, milk, etc. In recent years, exosomes have gradually become one of the research trends in the medical field, due to their unique components and structures different from ordinary samples, and the characteristics of containing rich markers almost associated with any disease. With the excavation and in-depth research of exosomes, its importance in human life activities is increasingly prominent.

[0003] The ncRNA (non-coding RNA) carried in the exosomes includes small RNA, lncRNA (long non-coding RNA) and circRNA (circular RNA), which plays an important role in cell information transmission and gene expression regulation. Research on miRNA (micro RNA), lncRNA and circRNA in exosomes can help to understand the internal mechanism and functional regulation mechanism of diseases or different development stages, and screening of exosome-specific biomarkers also has very high clinical application value. At present, in scientific research, high-throughput sequencing is generally carried out by using chip, but only known lncRNA can be detected, which has obvious data defects.

[0004] FFPE (formalin fixation and paraffin embedding) is a formalin-fixed and paraffin-embedded sample, which is a common biological material in the medical field. However, low-quality FFPE samples (RIN value less than 4.0) generally have serious RNA degradation, which often hinders scientific research. The earliest FFPE samples were mainly used for pathological morphological observation. In recent years, with the penetration and development of precision medicine, FFPE samples have also been widely used in clinical pathological examination and tumor gene detection. Researchers extract nucleic acids from FFPE samples to construct a library, and then use high-throughput sequencing technology to sequence and analyze the nucleic acids of FFPE samples, which can obtain a lot of important information related to pathology and tumor.

[0005] However, because there are various factors affecting the quality of nucleic acids in low-quality FFPE samples: for example, formalin fixation crosslinks nucleic acids with proteins, affecting the quality of nucleic acid extraction; the high-temperature infiltration process of paraffin accelerates the hydrolysis of phosphodiester bonds, leading to nucleic acid degradation; formalin fixation and suboptimal storage conditions cause cytosine deamination to introduce C→T, G→A mutations; fixation and embedding increase the brittleness of nucleic acid molecules, causing high fragmentation of nucleic acids; and sampling site causes microbial contamination, etc., the quality of nucleic acids in low-quality FFPE samples is often poor, and the use of ordinary lncRNA library construction methods to construct libraries for sequencing from low-quality FFPE samples faces many challenges: low yield, poor library quality, low exon proportion, low mutation detection rate, etc. For the above problems, it is generally recommended to use the method of capturing the target region for library construction and data analysis. However, this method has certain defects, such as the inability to obtain the expression of unknown genes, and the inconsistency of the number of genes detected with other methods. In the library construction and sequencing steps of the second-generation sequencing technology, a certain number of PCR amplifications are usually required to meet the library quantity required for sequencing. However, due to the preference and uncertainty of the amplification multiple (during PCR amplification, not all library fragments are amplified at the same rate and in the same amount. The amplification rate is affected by many factors such as fragment length, GC content, fragment concentration, etc. Fragments that are easily amplified are greatly enriched, and some low-content fragments or base-biased fragments are even completely lost, ultimately affecting the accuracy of the sequencing results), the multiple of each fragment being amplified is not necessarily the same. This duplication caused by PCR amplification is called Duplication. Duplication exists, which will cause the expression of each gene in the subsequent analysis to be inconsistent with the true situation, reducing the reliability of the results. The UMI labeling technology is a technology that can accurately identify and remove the Duplication caused by PCR. Before library amplification, a unique identity tag (Unique Identifier, UMI or UID) is added to each reverse-transcribed cDNA fragment, also known as a digital tag. The digital tag will accompany the whole process of fragment amplification, sequencing, and analysis. The products amplified by PCR from the same fragment all have the same digital tag. After sequencing, the source of each fragment can be traced using UMI, and fragments with the same source (having the same sequence and UMI) can be combined to accurately remove PCR amplification duplicates, and the original state before sample amplification can be accurately restored. In this process, PCR amplification and sequencing errors can also be corrected: amplification and sequencing errors will cause the same UMI tag to correspond to multiple different sequences, and then the similarity of these sequences can be compared to correct these errors. SUMMARY

[0006] The main purpose of the present application is to provide a method for constructing a high-throughput sequencing library of lncRNA and an application, so as to solve the problem that unknown lncRNA cannot be subjected to high-throughput sequencing in the prior art.

[0007] In order to achieve the above-mentioned purpose, according to the first method of the present application, a method for constructing a high-throughput sequencing library of lncRNA is provided, which comprises: S1, removing ribosomal RNA in total RNA of a sample to be detected to obtain total RNA without ribosomal RNA; S2, reverse transcribing the total RNA without ribosomal RNA to form double-stranded cDNA; S3, connecting sequencing adapters to the 5' and 3' ends of the double-stranded cDNA to obtain an adapter connection product; S4, PCR amplification and enrichment of the adapter connection product, and recovery to obtain a high-throughput sequencing library of lncRNA of the sample to be detected; the sample to be detected comprises an exosome sample or a low-quality FFPE sample.

[0008] Further, the ribosomal RNA comprises eukaryotic ribosomal RNA and / or prokaryotic ribosomal RNA; preferably, in S1, ribosomal RNA in total RNA of a sample to be detected is removed by using a ribosome removal reagent or magnetic beads to obtain total RNA without ribosomal RNA; preferably, the ribosome removal reagent comprises a eukaryotic ribosome removal reagent and / or a prokaryotic ribosome removal reagent; preferably, the eukaryotic ribosome removal reagent comprises QIAseq FastSelect-rRNA HMR; preferably, the prokaryotic ribosome removal reagent comprises FastSelect 5S / 16S / 23S; preferably, the reaction system for ribosome removal comprises: FastSelect 5S / 16S / 23S 1 μL and / or FastSelect rRNA HMR 1 μL, sample total RNA 12.5 μL and FastSelect FH Buffer 1.5 μL; preferably, the reaction program of the reaction system comprises: 75℃ reaction for 2 min, 70℃ reaction for 2 min, 65℃ reaction for 2 min, 60℃ reaction for 2 min, 55℃ reaction for 2 min, 37℃ reaction for 2 min; 25℃ reaction for 2 min, 4℃ keeping.

[0009] Further, in S2, the reverse transcription comprises first-strand synthesis reaction and second-strand synthesis reaction to obtain double-stranded cDNA; preferably, after obtaining the double-stranded cDNA, the double-stranded cDNA is not subjected to breaking operation; preferably, S3 comprises: sequentially performing first purification, end repair and A addition on the double-stranded cDNA to obtain repair DNA; adding sequencing adapters to the 5' and 3' ends of the repair DNA to obtain an adapter connection product; preferably, the first purification comprises first magnetic bead purification.

[0010] Further, the reaction system of the first strand synthesis reaction comprises: total RNA without ribosomal RNA 10 μL, reverse transcription reagent 8 μL, and single strand synthesis enzyme complex 2 μL; preferably, the reaction procedure of the first strand synthesis reaction comprises: reaction at 25°C for 10 min, reaction at 42°C for 15 min, reaction at 70°C for 15 min, and keeping at 4°C.

[0011] Further, the reaction system of the second strand synthesis reaction comprises: the reaction system of the first strand synthesis reaction 20 μL, second strand synthesis reaction buffer 8 μL, second strand synthesis enzyme mixture 4 μL, and nuclease-free water 48 μL; preferably, the reaction procedure of the second strand synthesis reaction comprises: reaction at 16°C for 1 h.

[0012] Further, the reaction solution of the end repair comprises: the first purified solution 42 μL, end system repair solution 1 6.8 μL, and end repair enzyme 1.2 μL; preferably, the reaction procedure of the end repair comprises: reaction at 37°C for 30 min, reaction at 72°C for 30 min, and keeping at 4°C.

[0013] Further, in S3, the sequencing adapter is a sequencing adapter containing UMI; preferably, after the sequencing adapter is connected, second purification is performed; preferably, the second purification comprises second magnetic bead purification; preferably, the reaction solution for connecting the sequencing adapter comprises: the reaction solution containing double-stranded cDNA 50 μL, enzyme reaction buffer 2-1 8.4 μL, enzyme reaction buffer 2-2 15 μL, fast ligase 1.6 μL, UMI-containing sequencing adapter 1 μL, nuclease-free water 3 μL, and Enzyme 2 1 μL; preferably, the reaction procedure for connecting the sequencing adapter comprises: reaction at 20°C for 30 min, and keeping at 4°C.

[0014] Further, in S4, the U-containing adapter ligation product is digested before PCR amplification; preferably, the U-containing adapter ligation product is digested by UDG enzyme; preferably, after the PCR amplification, third purification is performed to obtain a high-throughput sequencing library of the sample lncRNA; preferably, the third purification comprises third magnetic bead purification; preferably, the total RNA without ribosomal RNA comprises one or more of mRNA, lncRNA, or circRNA; preferably, the reaction solution for digestion and PCR amplification comprises: the reaction solution containing the adapter ligation product 22 μL, USER enzyme 1 μL, PCR buffer (2X) 25 μL, Index (X) primer / i7 primer 25 μM 1 μL, P5 PCR primer 25 μM 1 μL; preferably, the reaction procedure for digestion and PCR amplification comprises: reaction at 37°C for 10 min, pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 65°C for 30 s, extension at 72°C for 30 s, 10-15 cycles; extension at 72°C for 5 min; and keeping at 4°C.

[0015] In order to achieve the above-mentioned purpose, according to the second method of the present application, a high-throughput sequencing library of lncRNA is provided, which is constructed by the above-mentioned construction method.

[0016] In order to achieve the above-mentioned purpose, according to the third method of the present application, the above-mentioned construction method is applied in the construction of a high-throughput sequencing library of lncRNA.

[0017] In order to achieve the above-mentioned purpose, according to the fourth method of the present application, a lncRNA sequencing method is provided, which comprises sequencing the above-mentioned high-throughput sequencing library of lncRNA.

[0018] In order to achieve the above-mentioned purpose, according to the fifth method of the present application, the above-mentioned construction method or high-throughput sequencing library of lncRNA is applied in the preparation of a product for early screening, prognosis evaluation, scientific basic research and / or clinical efficacy monitoring of diseases.

[0019] By using the technical solution of the present application, different sources of total RNA samples are used as starting samples, ribosomes are removed, cDNA is synthesized by reverse transcription and sequencing adapters are connected, and a high-throughput sequencing library is obtained by enrichment and recovery. The PCR duplication generated in the library construction process and the on-machine sequencing process can be accurately removed. Compared with conventional sample lncRNA libraries, the present application has the advantages of more accurate quantification, more accurate sequence sequencing, and more accurate quantification of low copy sequences, ensuring that the quantification results of the contained ncRNA and mRNA are unbiased, and unknown lncRNA can be sequenced at high throughput, providing reliable data results for the study of sample RNA. BRIEF DESCRIPTION OF DRAWINGS

[0020] The drawings accompanying the specification of this application are used to provide a further understanding of the present application, and the illustrative embodiments of the present application and their descriptions are used to explain the present application, and do not constitute an improper limitation of the present application. In the drawings:

[0021] Figure 1 A flow chart of a construction method of a sample lncRNA high-throughput sequencing library according to embodiment 1 of the present application is shown.

[0022] Figure 2 A quality control chart obtained by using Agilent 2100 Bioanalyzer for an exosome lncRNA high-throughput sequencing library according to embodiment 1 of the present application is shown.

[0023] Figure 3 A quality control chart obtained by using Agilent 2100 Bioanalyzer for an exosome lncRNA high-throughput sequencing library according to embodiment 2 of the present application is shown.

[0024] Figure 4 The quality control chart obtained by using Agilent 2100 Bioanalyzer for the high-throughput sequencing library of exosome lncRNA according to the comparative example 2 of the present application is shown.

[0025] Figure 5 The quality control chart obtained by using Agilent 2100 Bioanalyzer for the high-throughput sequencing library of exosome lncRNA according to the comparative example 1 of the present application is shown.

[0026] Figure 6 The quality control chart obtained by using Agilent 2100 Bioanalyzer for the high-throughput sequencing library of low-quality FFPE sample lncRNA according to the comparative example 3 of the present application is shown.

[0027] Figure 7 The quality control chart obtained by using Agilent 2100 Bioanalyzer for the high-throughput sequencing library of low-quality FFPE sample lncRNA according to the comparative example 4 of the present application is shown.

[0028] Figure 8 The RNA electrophoresis chart of the low-quality FFPE sample according to the example 2 of the present application is shown.

[0029] Figure 9 The detection chart of the exosome total sample according to the example 1 of the present application is shown. DETAILED DESCRIPTION

[0030] It should be noted that the examples in the present application and the features in the examples can be combined with each other without conflict. The present application will be described in detail below with reference to the examples.

[0031] Explanation of terms:

[0032] lncRNA: long non-coding RNA, is a non-coding RNA with a length of more than 200 nucleotides, which does not encode proteins in the organism.

[0033] As mentioned in the background, in the prior art, for exosome lncRNA, low-quality FFPE and other samples, high-throughput sequencing is difficult to obtain high-quality library due to the use of conventional library construction method, so the chip method is often used for high-throughput sequencing of exosome lncRNA. However, only known lncRNA can be detected by using the chip, and there are obvious data defects in the obtained sequencing data, and unknown lncRNA cannot be subjected to high-throughput sequencing.

[0034] Because of the characteristics of the exosome sample itself, all cells in the single body environment can secrete exosomes. After exosome extraction, especially human-derived bacterial and fungal infection leading to exogenous pollution, and mycoplasma, bacteria and other pollution of eukaryotic cells during culture, will directly lead to low mapping of data after library construction using second-generation sequencing. Chip technology is a high-throughput qPCR for known lncRNA. Due to the principle limitation of the technology, unknown lncRNA cannot be mined and researched. According to the actual operation and analysis, the inventors found that the sample source for exosome lncRNA library construction is mostly clinical or cell culture, so there is easy to exist part of exogenous microorganism interference in the sample, which may be the reason affecting the quality of lncRNA library. And because the RNA segment contained therein is small, mainly distributed in the range of 25-200 nt, using conventional ncRNA or trace amplification to construct a library often leads to poor data quality.

[0035] There is also serious RNA degradation in low-quality FFPE samples, and multiple factors in low-quality FFPE affect nucleic acid quality. Therefore, the nucleic acid quality in low-quality FFPE samples is often poor, and sequencing with low-quality FFPE samples faces many challenges: low yield, poor library quality, low mutation detection rate, etc. The high-throughput data obtained by ordinary library construction method is of poor quality, and cannot be effectively and accurately analyzed and applied. Therefore, in the present application, the inventors try to explore the construction method of the high-throughput sequencing library of the sample lncRNA, and obtain an effective construction method of the high-throughput sequencing library of the sample lncRNA by optimizing the ribosomal RNA removal experiment before reverse transcription, and thus a series of protection schemes of the present application are proposed.

[0036] In the first typical embodiment of the present application, a construction method of a high-throughput sequencing library of lncRNA is provided, which comprises: S1, removing ribosomal RNA in total RNA of a sample to be tested to obtain total RNA without ribosomal RNA; S2, reverse transcribing the total RNA without ribosomal RNA to form double-stranded cDNA; S3, connecting sequencing adapters to the 5' and 3' ends of the double-stranded cDNA to obtain adapter connection products; S4, PCR amplification and enrichment of the adapter connection products to recover a high-throughput sequencing library of lncRNA of the sample to be tested; and the sample to be tested comprises an exosome sample or a low-quality FFPE sample. The construction method flow chart is shown in Figure 1 .

[0037] The sample containing exosome lncRNA can be derived from exosomes in blood, body fluid, tissue or cell culture supernatant of animals, and total RNA extracted from exosomes secreted by plant tissues. The applicant found that in the above samples and actual operations, there is a risk of contamination by prokaryotic or eukaryotic organisms, so in the above construction method, ribosomal RNA is removed from the total RNA of the exosome to obtain total RNA without ribosomal RNA.

[0038] The RNA in low-quality FFPE is affected by various factors such as sample preparation, preservation, etc., and is in a state of microbial contamination and high degradation of nucleic acids, so the above construction method can reduce the influence of exogenous nucleic acids, impurities, etc. in low-quality FFPE, thereby obtaining a high-quality lncRNA high-throughput sequencing library of low-quality FFPE.

[0039] In the prior art, the library construction and sequencing steps in the second-generation sequencing technology usually require a certain number of PCR amplifications to meet the library quantity required for sequencing. However, due to the preference of amplification and the uncertainty of amplification fold, the fold of each fragment amplified is not necessarily the same, which will lead to inconsistent gene expression and real situation in subsequent analysis, reducing the reliability of the results. In the present application, by reverse transcribing the total RNA without ribosomal RNA into double-stranded cDNA and then connecting the sequencing adapter, the number of molecules of the starting lncRNA is accurately quantified, and the errors generated by sequencing and library preparation and the unevenness caused by PCR amplification are reduced, effectively distinguishing false positive mutations introduced in the library construction process, thereby more effectively detecting ultra-low abundance mutations. It can overcome the interference of exogenous nucleic acids and solve the problems of less effective data caused by sample exogenous contamination and quantitative bias caused by library PCR duplication.

[0040] For small RNAs in the sample, such as small ncRNAs (including miRNAs), they are removed in the library adapter connection and library out-of-library piece selection steps. For circular RNA, no removal is made, so in the above high-throughput sequencing library construction method, circRNA can also be analyzed. Therefore, using the above construction method to construct a library, one library can analyze three types of RNA (mRNA, lncRNA, circRNA), solving the problem that chip sequencing is difficult to achieve in the prior art.

[0041] In a preferred embodiment, the ribosomal RNA comprises eukaryotic ribosomal RNA and / or prokaryotic ribosomal RNA; preferably, the ribosomal RNA in the total RNA of the sample to be detected is removed by using a ribosomal removal reagent or magnetic beads in S1, to obtain total RNA without ribosomal RNA; preferably, the ribosomal removal reagent comprises eukaryotic ribosomal removal reagent and / or prokaryotic ribosomal removal reagent; preferably, the eukaryotic ribosomal removal reagent comprises QIAseq FastSelect-rRNA HMR; preferably, the prokaryotic ribosomal removal reagent comprises FastSelect 5S / 16S / 23S; preferably, the ribosomal removal reaction system comprises: FastSelect 5S / 16S / 23S 1 μL and / or FastSelect rRNA HMR 1 μL, sample total RNA 12.5 μL and FastSelect FH Buffer 1.5 μL; preferably, the reaction procedure of the reaction system comprises: 75°C reaction for 2 min, 70°C reaction for 2 min, 65°C reaction for 2 min, 60°C reaction for 2 min, 55°C reaction for 2 min, 37°C reaction for 2 min; 25°C reaction for 2 min, 4°C holding.

[0042] Since the sample containing lncRNA is easy to be contaminated and interfered by prokaryotic, eukaryotic or other impurities, the ribosomal removal reagent or magnetic beads is used to remove the eukaryotic ribosomal RNA and prokaryotic ribosomal RNA at the same time, so that the best removal effect can be obtained, and the residual ribosomal RNA can be prevented from interfering with the sequencing results and data quality after library construction. In the present application, the commercially available eukaryotic ribosomal removal reagent QIAseq FastSelect-rRNA HMR and the prokaryotic ribosomal removal reagent FastSelect 5S / 16S / 23S are used at the same time, and the two reagents play a role at the same time in the ribosomal removal reaction system, so that a better ribosomal RNA removal effect can be obtained. Other ribosomal removal methods in the prior art can also be flexibly selected to achieve efficient removal of prokaryotic and eukaryotic ribosomal RNA. For the eukaryotic ribosomal removal reagent QIAseq FastSelect-rRNA HMR and the prokaryotic ribosomal removal reagent FastSelect 5S / 16S / 23S, the above ribosomal removal reaction system and reaction procedure can be used to obtain the best removal effect, the ribosomal RNA removal rate is high, the reaction is simple, and the content, activity and subsequent test of the lncRNA are not affected.

[0043] The use of ribosome removal reagent in the present application can improve the efficiency of ribosome removal, and the data quality is obviously improved. On the other hand, this removal method has low requirements for sample quality and initial amount for library construction. It can realize a sample with less than 100 ng and RIN value less than 4. Using sequencing adapters in library construction can improve the accurate quantification of degraded samples, and has obvious application value for improving sequencing quality and data analysis. The existing method uses ribosome probes to hybridize ribosomes in the sample, and then uses magnetic beads adsorption or RNase digestion to remove ribosome contamination in the sample. This method is relatively complex in operation, and cannot meet the initial amount requirements of existing ribosome removal kits for FFPE RNA which generally has a low extraction amount. The construction method of the present application can greatly ignore the initial amount requirements of the sample, and can meet the requirements of ribosome removal efficiency. In a preferred embodiment, in S2, the reverse transcription includes a first strand synthesis reaction and a second strand synthesis reaction to obtain double-stranded cDNA; preferably, after obtaining the double-stranded cDNA, the double-stranded cDNA is not subjected to breaking operation; preferably, S3 includes: sequentially performing first purification, end repair and A addition on the double-stranded cDNA to obtain repaired DNA; adding sequencing adapters to the 5' end and 3' end of the repaired DNA to obtain adapter-ligated products; preferably, the first purification includes first magnetic bead purification.

[0044] The above first strand synthesis reaction is to use reverse transcriptase to reverse transcribe lncRNA into corresponding single-stranded reverse cDNA. Then, the second strand synthesis reaction is performed to obtain single-stranded positive cDNA with the same direction as lncRNA using the obtained single-stranded reverse cDNA as a template. The single-stranded positive cDNA and the single-stranded reverse cDNA can be complementary to each other to form double-stranded cDNA. Since the double-stranded cDNA obtained from lncRNA is short in length, unlike the existing high-throughput sequencing library method, after obtaining the double-stranded cDNA, no breaking operation is performed to prevent the double-stranded cDNA from being damaged and generating too short DNA fragments, which affects the subsequent sequencing quality.

[0045] In the S3 step, after purification, end repair and A addition are performed on the above obtained double-stranded cDNA, protruding A bases are generated at the ends of the double-stranded cDNA, which facilitates the connection with the sequencing adapter using T-A cloning method to obtain adapter-ligated products. Purifying the double-stranded cDNA before end repair can remove lncRNA and other impurities such as small RNA, circular RNA, etc.

[0046] In a preferred embodiment, the reaction system of the first strand synthesis reaction comprises: total RNA without ribosomal RNA 10 μL, reverse transcription reagent (RT Reagent) 8 μL, and first strand synthesis enzyme mix (containing dUTP) 2 μL; preferably, the reaction procedure of the first strand synthesis reaction comprises: reaction at 25°C for 10 min, reaction at 42°C for 15 min, reaction at 70°C for 15 min, and keeping at 4°C.

[0047] In the first strand synthesis reaction, all the reagents used are commercially available reagents, and other brands or functional reagents can also be selected according to actual needs. In order to construct a strand-specific lncRNA library, in the first strand synthesis reaction, the dNTP raw material is replaced by a dUTP raw material, so that there is no T base in the single-stranded reverse sequence cDNA obtained by reverse transcription, and the positions where T bases should exist are actually all U bases. Thus, in subsequent processing, the cDNA single strands of lncRNA sequences or reverse sequences are distinguished, only the sequences are sequenced, and it is ensured that the sequencing results can directly reflect the direction of RNA.

[0048] In a preferred embodiment, the reaction system of the second strand synthesis reaction comprises: the reaction system of the first strand synthesis reaction 20 μL, second strand synthesis reaction buffer 8 μL, second strand synthesis enzyme mix 4 μL, and nuclease-free water 48 μL; preferably, the reaction procedure of the second strand synthesis reaction comprises: reaction at 16°C for 1 h.

[0049] In the second strand synthesis reaction, all the reagents used in the present application are commercially available reagents, and other brands or functional reagents can also be selected according to actual needs. The second strand synthesis reaction uses the single-stranded reverse sequence cDNA obtained above as a template, and through the principle of base complementary pairing, a single-stranded sequence cDNA complementary to the single-stranded reverse sequence cDNA is obtained, and the two cDNA single strands are complementary to each other, forming double-stranded cDNA. The single-stranded sequence cDNA, i.e., the base sequence from 5' to 3', is the same as the base sequence from 5' to 3' of the lncRNA, so the sequencing of the single-stranded sequence cDNA can directly reflect the correct direction of the lncRNA.

[0050] In a preferred embodiment, the reaction solution of the end repair comprises: the first purified solution 42 μL, end system repair solution (Buffer 1) 6.8 μL, and end repair enzyme (Enzyme 1) 1.2 μL; preferably, the reaction procedure of the end repair comprises: reaction at 37°C for 30 min, reaction at 72°C for 30 min, and keeping at 4°C.

[0051] Using the above reagents and reaction procedures, the purified double-stranded cDNA can be end-repaired, so as to perform subsequent end-A.

[0052] In a preferred embodiment, in S3, the sequencing adapter is a UMI-containing sequencing adapter; preferably, after the sequencing adapter is ligated, a second purification is performed; preferably, the second purification comprises a second magnetic bead purification; preferably, the reaction solution for ligating the sequencing adapter comprises: a reaction solution containing double-stranded cDNA, 50 μL; enzyme reaction buffer 2-1 (Buffer 2-1) 8.4 μL; enzyme reaction buffer 2-2 (Buffer 2-2) 15 μL; enhancer 1.6 μL; UMI-containing sequencing adapter 1 μL; nuclease-free water 3 μL; Enzyme 2 1 μL; preferably, the reaction procedure for ligating the sequencing adapter comprises: 20°C reaction for 30 min, 4°C holding.

[0053] By using the UMI-containing sequencing adapter, a unique sequence tag is added to the repaired DNA, which can distinguish different fragments in the same sample or different samples. The UMI will accompany the whole process of fragment amplification, sequencing and analysis. The products amplified by PCR from the same fragment all have the same UMI. After sequencing, the source of each fragment is traced using the UMI. The fragments with the same source (having the same sequence and UMI) are combined, so as to accurately remove the PCR amplification repeats, and accurately restore the original state of the sample before amplification. In this process, PCR amplification and sequencing errors can also be corrected: the errors in amplification and sequencing will cause the same UMI tag to correspond to multiple different sequences, then by comparing the similarity of these sequences, the errors can be corrected. Thus, the quantification of lncRNA, especially low-copy sequences, is more accurate.

[0054] In a preferred embodiment, in S4, the U-base containing adaptor ligation product is digested before PCR amplification; preferably, the U-base containing adaptor ligation product is digested by a UDG enzyme; preferably, a third purification is performed after PCR amplification to obtain a high-throughput sequencing library of the sample lncRNA; preferably, the third purification comprises a third magnetic bead purification; preferably, the total RNA excluding ribosomal RNA comprises one or more of mRNA, lncRNA or circRNA; preferably, the reaction solution for digestion and PCR amplification comprises: reaction solution containing adaptor ligation product, 22 μL; USER enzyme (UDG enzyme) 1 μL; PCR buffer (PCR Mix, 2X) 25 μL; Index (X) primer / i7 primer 25 μM 1 μL; P5 PCR primer 25 μM 1 μL; preferably, the reaction program for digestion and PCR amplification comprises: 37°C reaction for 10 min, 98°C pre-denaturation for 30 s; 98°C denaturation for 10 s, 65°C annealing for 30 s, 72°C extension for 30 s, 10-15 cycles; 72°C extension for 5 min; 4°C holding.

[0055] In the first strand and second strand synthesis reactions, using raw materials containing dUTP instead of dNTP, the constructed U-base containing double-stranded cDNA, after the formation of adaptor ligation product by connecting sequencing adapters, the U-base containing single strand is digested and treated, which can remove the single-stranded cDNA (single-stranded reverse sequence cDNA) opposite to the sequence of lncRNA, and only keep the cDNA single strand with the same sequence as lncRNA, so that in the subsequent sequencing, the most true and the same direction as lncRNA transcription information is obtained. After digestion, PCR is performed to amplify the single-stranded cDNA with the correct direction into double-stranded DNA, and the double-stranded DNA is continued to be enriched by PCR to obtain double-stranded DNA that can meet the sample amount of subsequent high-throughput sequencing. After PCR, the third purification is performed to remove the DNA fragments generated by digestion, reagents used in PCR and other impurities, and improve the purity of the sequencing sample, i.e. to obtain a high-throughput sequencing library of exosome lncRNA. In this application, the digestion and PCR system are mixed, and the commercially available reagents are used to complete the digestion and PCR process in the same reaction. Other existing technologies or reagents can also be used to flexibly adjust the reaction system and program to achieve the same technical effect.

[0056] In the second typical embodiment of the present application, a device for constructing a high-throughput sequencing library of lncRNA is provided, which utilizes the above-mentioned construction method to construct a high-throughput sequencing library of lncRNA; the device comprises: a ribosome removal module for removing ribosomal RNA in total RNA of a sample to obtain total RNA free of ribosomal RNA; a reverse transcription module for reverse transcribing the total RNA free of ribosomal RNA to form double-stranded cDNA; a linker connection module for connecting sequencing adapters to the 5' and / or 3' end of the double-stranded cDNA to obtain a linker connection product; and an enrichment recovery module for PCR amplification to enrich the linker connection product and recover a high-throughput sequencing library of lncRNA of the sample.

[0057] By using the above-mentioned device, a series of process steps from initial sample processing to final obtaining of a high-throughput sequencing library of lncRNA of the sample can be realized in the device, and batch high-throughput sequencing problems can be conveniently and quickly obtained. The device can also be connected and combined with a subsequent required high-throughput sequencing equipment, and the obtained high-throughput sequencing library of lncRNA of the sample can be sent into the high-throughput sequencing equipment, so that a series of automatic processing procedures from raw sample input to sequencing result output can be completed, which is suitable for implementation of a large batch of high-throughput sequencing projects, and the use of the device for processing can also ensure uniformity of sample processing, which is beneficial to comparative analysis between different samples.

[0058] In the third typical embodiment of the present application, a high-throughput sequencing library of lncRNA is provided, which is constructed by using the above-mentioned construction method or device.

[0059] In the fourth typical embodiment of the present application, the above-mentioned construction method or device is provided, and is applied to construction of a high-throughput sequencing library of lncRNA of a sample.

[0060] In the fifth typical embodiment of the present application, a lncRNA sequencing method is provided, which comprises: sequencing the above-mentioned high-throughput sequencing library of lncRNA.

[0061] In the sixth typical embodiment of the present application, the above-mentioned construction method, or device, or high-throughput sequencing library of lncRNA is provided, and is applied to preparation of a product for early screening, prognosis evaluation, scientific basic research and / or clinical efficacy monitoring of a disease.

[0062] The product for constructing the method or device can obtain a high-throughput sequencing library of lncRNA of a corresponding sample. The sample sources include, but are not limited to, human or animal samples, samples derived from plasma, tissues or existing tissue sections, etc. The product is used to complete the detection and analysis of lncRNA in the samples, so as to reflect the state of lncRNA in the sample, thereby playing an important role in the above-mentioned various application scenarios.

[0063] The beneficial effects of the present application will be further explained in detail below in conjunction with specific examples.

[0064] Example 1

[0065] Step S1, taking the total RNA of plasma-derived exosomes as an example, the detection diagram of the exosome RNA sample is as shown in Figure 9 The RNA concentration is 71 ng / μL, and the RIN value (RNA Integrity Number) is 2.6. The example takes the exosome total RNA of the quality shown in Figure 9 , and is not limited thereto.

[0066] Ribosomal RNA is removed using human / mouse ribosome reagent. Specifically, step S1 further comprises:

[0067] S1.1, ribosome removal system configuration:

[0068] Take 2xFrag / Elute buffer 5 μL, total RNA 4 μL, 1 μL QIAseq FastSelect-rRNA HMR 1 μL, FastSelect 5S / 16S / 23S 1 μL, fully mix and centrifuge.

[0069] S1.2, execute the program shown in Table 1 on the PCR instrument:

[0070] Table 1

[0071]

[0072]

[0073] After the S1.3 program is executed, the reaction system is immediately placed on ice;

[0074] Specifically, step S2 further comprises:

[0075] Because the fragments of the extracted exosome lncRNA are short, in order to avoid the influence of the breaking link of normal NGS library construction on the mapping and dup of the library, the breaking link is not performed.

[0076] S2.1, first strand cDNA synthesis:

[0077] S2.1.1, Take the above reaction system, add RT Reagent 8 μL, First Strand synthesis enzyme mix 2 μL, mix well with pipette, and centrifuge;

[0078] S2.1.2, Perform the program shown in Table 2 on the PCR instrument:

[0079] Table 2

[0080] Step Temperature (°C) Time 1 25 10 min 2 42 15 min 3 70 15 min 8 4 ∞

[0081] S2.1.3, Immediately after the program is executed, place the reaction system on ice;

[0082] S2.2, Second strand cDNA synthesis:

[0083] S2.2.1, Take the above reaction system, add Second strand synthesis reaction buffer 8 μL, Second strand synthesis enzyme mix 4 μL, Nuclease-free water 48 μL, mix well with pipette, and centrifuge;

[0084] S2.2.2, Perform the program shown in Table 3 on the PCR instrument:

[0085] Table 3

[0086]

[0087]

[0088] S2.2.3, Immediately after the program is executed, place the reaction system on ice;

[0089] S2.3, cDNA purification:

[0090] S2.3.1, Take 144 μL XP beads in a 1.5 ml EP tube, add the above reaction system to it, mix well. Incubate at room temperature for 5 min, stand on the magnetic stand for 5 min, discard the supernatant.

[0091] S2.3.2, Keep the EP tube on the magnetic stand, add freshly prepared 80% ethanol to the EP tube, stand for 30 s, discard the supernatant.

[0092] S2.3.3, Repeat step S2.2.2, air dry the magnetic beads at room temperature for 3-5 min;

[0093] S2.3.4, take the EP tube from the magnetic stand, add 43 μL of Nuclease-free water to the 260 sample, mix well, place at room temperature for 5 min, place on the magnetic stand for 5 min, take 42 μL of supernatant in a 0.2 ml EP tube, and proceed to the next step.

[0094] S2.4 End repair, A addition:

[0095] S2.4.1 End repair, A addition to the above supernatant: take 42 μL of the above supernatant, Buffer 1 6.8 μL, Enzyme 1 1.2 μL, mix by blowing, and centrifuge;

[0096] S2.4.2 Perform the program shown in Table 4 on the PCR instrument:

[0097] Table 4

[0098] Step Temperature (°C) Time 1 37 30 min 2 72 30 min 3 4 ∞

[0099] S2.4.3 Immediately after the program is executed, place it on ice, and immediately proceed to the next step of adapter ligation reaction.

[0100] Specifically, step S3 further comprises:

[0101] S3.1 Ligation of sequencing adapters containing UMI to the 5' and 3' ends of cDNA:

[0102] S3.1.1 Add Buffer 2-1 8.4 μL, Buffer 2-2 15 μL, Enhancer 1.6 μL

[0103] UMI Adaptor 1 μL, Nuclease-free water 3 μL, Enzyme 2 1 μL, mix by blowing, and centrifuge (the series of reagents uses Novogene NGS DNA Library Prep kit (Novogene / PT004));

[0104] S3.1.2 Perform the program shown in Table 5 on the PCR instrument:

[0105] Table 5

[0106] Step Temperature (°C) Time 1 20 30 min 3 4 ∞

[0107] S3.1.3 Immediately after the program is executed, take it out and place it at room temperature;

[0108] S3.2 Recovery of adapter ligation product:

[0109] S3.2.1 Add 23 μL of Nuclease-free water to the sample, and supplement the system to 100 μL;

[0110] S3.2.2 Add 30 μL XP beads (0.3X), mix well, and then stand at room temperature for 5 min. Place the mixture on a magnetic stand for 5 min, take the supernatant, and discard the magnetic beads.

[0111] S3.2.3 Carefully take out all the supernatant, add 20 μL XP beads to it, mix well, and then stand at room temperature for 5 min. Place the mixture on a magnetic stand for 5 min, retain the magnetic beads, and discard the supernatant.

[0112] S3.2.4 Add 200 μL of freshly prepared 80% ethanol to the magnetic beads, stand for 30 s, and discard the supernatant.

[0113] S3.2.5 Repeat step S2.4.10 once. After discarding the supernatant, centrifuge for a moment, use a 10 μL pipette to suck away all the liquid, and dry the magnetic beads at room temperature for about 3 min.

[0114] S3.2.6 Add 24 μL of NF-W to the magnetic beads, mix well, stand at room temperature for 5 min, place the mixture on a magnetic stand for 5 min, and take 22 μL of the supernatant for PCR library amplification.

[0115] Specifically, the step S4 process is as follows:

[0116] S4.1, digest the DNA strand containing U base, and perform library amplification:

[0117] S4.1.1 Place the PCR tube on ice, and sequentially add the following reagents: UDG enzyme 1 μL, PCR Mix (2x), P5 PCR primer (25 μM) 1 μL, Index primer (25 μM) 1 μL, mix well by blowing, and centrifuge for a moment.

[0118] S4.1.2 Perform the program shown in Table 6 on a PCR instrument:

[0119] Table 6

[0120]

[0121]

[0122] S4.2, library purification:

[0123] S4.2.1 Add 53 μL of NF-W to the above reaction system, and make up to 100 μL of the system.

[0124] S4.2.2 Take 60 μL (0.6x) of magnetic beads, add them to the above system, mix well, stand at room temperature for 5 min, place the mixture on a magnetic stand for 5 min, take the supernatant, and discard the magnetic beads.

[0125] S4.2.3 Take all supernatant into a new EP tube, add 15 μL (0.15x) magnetic beads, mix well, stand at room temperature for 5 min, place on the magnetic stand for 5 min, leave the magnetic beads, discard the supernatant;

[0126] S4.2.4 Repeat step S4.2.3 once, after aspirating all the liquid, centrifuge instantly, then use a 10 μL pipette to completely aspirate the liquid, dry the magnetic beads at room temperature for about 3 min;

[0127] S4.2.6 Add 15 μL EB (room temperature) to the magnetic beads, mix well, stand at room temperature for 5 min, place on the magnetic stand for 5 min, take 14 μL supernatant, which is the UMI exosome lnc library stock solution;

[0128] Library construction, quality inspection: both DNA fragment and concentration detection are qualified, that is, the size of the target fragment is 300-600 bp,

[0129] The concentration is ≥1 ng, the volume is ≥10 μL, and there is no adapter contamination, which can be detected by machine. A total of 2 parallel tests were performed, and the test results are shown in Table 7, Example 1-1, Example 1-2. The quality inspection graph of Example 1-1 is shown in Figure 2 In the quality inspection graph of the present application, LM and UM are markers used for detection, which are components in Agilent DNA12000 kit (part number: 5067-1508). RFU is the relative fluorescence intensity (Relative Fluorescence Units).

[0130] The construction method flow chart is shown in Figure 1 .

[0131] Example 2

[0132] Step S1, using the extracted low-quality FFPE sample RNA as an example, the total RNA used is 100 ng, and the specific nucleic acid mass is shown in Figure 8 ; The total RNA used in this example is of the mass shown in Figure 8 , which is not limited thereto. For low-quality FFPE samples, generally, they are such a gel map, small amount and highly dispersed.

[0133] Step S1 includes:

[0134] S1.1, ribosome removal system configuration:

[0135] Take 2xFrag / Elute buffer 5μL, total RNA (sample extraction nucleic acid system) 4μL, 1μL QIAseq FastSelect-rRNA HMR 1μL, QIAseq FastSelect 5S / 16S / 24S HMR 1μL, fully mix by blowing, instant separation;

[0136] S1.2, execute the program shown in Table 1 on the PCR instrument.

[0137] S1.3 After the program is executed, immediately place the reaction system on ice;

[0138] Specifically, step S2 further comprises:

[0139] S2.1, First strand cDNA synthesis:

[0140] S2.1.1, take the above reaction system, add RT Reagent 8μL, First Strand synthesis enzyme mix 2μL, mix by blowing with a pipette, instant separation;

[0141] S2.1.2, execute the program shown in Table 2 on the PCR instrument.

[0142] S2.1.3, after the program is executed, immediately place the reaction system on ice;

[0143] S2.2, Second strand cDNA synthesis:

[0144] S2.2.1, take the above reaction system, add Second strand synthesis reaction buffer 8μL, Second strand synthesis enzyme mix 4μL, Nuclease-free water 48μL, mix by blowing with a pipette, instant separation;

[0145] S2.2.2, execute the program shown in Table 3 on the PCR instrument.

[0146] S2.2.3, after the program is executed, immediately place the reaction system on ice;

[0147] S2.3, cDNA purification:

[0148] S2.3.1, take 144μL XP beads in a 1.5ml EP tube, add the above reaction system to it, mix well. Room temperature for 5min, stand on the magnetic stand for 5min, discard the supernatant.

[0149] S2.3.2, keep the EP tube on the magnetic stand, add freshly prepared 80% ethanol into the EP tube, stand for 30 s, discard the supernatant.

[0150] S2.3.3, repeat step S2.2.2, dry the magnetic beads at room temperature for 3-5 min;

[0151] S2.3.4, take the EP tube off the magnetic stand, add 43 μL of Nuclease-free water into the 260 sample, mix well, stand at room temperature for 5 min, place on the magnetic stand for 5 min, take 42 μL of the supernatant into a 0.2 ml EP tube, and proceed to the next step.

[0152] S2.4 End repair, A addition:

[0153] S2.4.1 End repair, A addition of the above supernatant: take 42 μL of the above supernatant, 6.8 μL of Buffer 1, 1.2 μL of Enzyme 1, mix well by blowing, and centrifuge;

[0154] S2.4.2 Perform the program shown in Table 4 on a PCR instrument.

[0155] S2.4.3 Immediately place on ice after the completion of the program, and immediately proceed to the next step of adapter ligation reaction.

[0156] Specifically, step S3 further comprises:

[0157] S3.1 Ligation of sequencing adapters containing UMI to the 5' end and 3' end of cDNA:

[0158] S3.1.1 Add 8.4 μL of Buffer 2-1, 15 μL of Buffer 2-2, 1.6 μL of Enhancer, 1 μL of UMI Adaptor, and 3 μL of Nuclease-free water to the above reaction system, mix well by blowing, and centrifuge;

[0159] 1 μL of UMI Adaptor, and 3 μL of Nuclease-free water to the above reaction system, mix well by blowing, and centrifuge;

[0160] S3.1.2 Perform the program shown in Table 5 on a PCR instrument.

[0161] S3.1.3 Immediately take out and place at room temperature after the completion of the program.

[0162] S3.2 Recovery of adapter ligation product:

[0163] S3.2.1 Add 23 μL of Nuclease-free water to the sample, and supplement the system to 100 μL;

[0164] S3.2.2 Add 30 μL XP beads (0.3X), mix well, and then stand at room temperature for 5 min, and then place on a magnetic stand for 5 min, take the supernatant, and discard the magnetic beads;

[0165] S3.2.3 Carefully take out all the supernatant, add 20 μL XP beads to it, mix well, and then stand at room temperature for 5 min, and then place on a magnetic stand for 5 min, leave the magnetic beads, and discard the supernatant;

[0166] S3.2.4 Add 200 μL of freshly prepared 80% ethanol to the magnetic beads, stand for 30 s, and discard the supernatant;

[0167] S3.2.5 Repeat step S2.4.10 once, after discarding the supernatant, centrifuge for a moment, and use a 10 μL pipette to suck away all the liquid, and dry the magnetic beads at room temperature for about 3 min;

[0168] S3.2.6 Add 24 μL of NF-W to the magnetic beads, mix well, stand at room temperature for 5 min, and then place on a magnetic stand for 5 min, take 22 μL of the supernatant for PCR library amplification.

[0169] Specifically, the step S4 process is as follows:

[0170] S4.1, digest the DNA strand containing U base, and perform library amplification:

[0171] S4.1.1 Place the PCR tube on ice, and sequentially add the following reagents: UDG enzyme 1 μL, PCR Mix (2x),

[0172] P5 PCR primer (25 μM) 1 μL, Index primer (25 μM) 1 μL, mix well by blowing, and centrifuge for a moment;

[0173] S4.1.2 Perform the procedure shown in Table 6 on a PCR instrument.

[0174] S4.2, library purification:

[0175] S4.2.1 Add 53 μL of NF-W to the above reaction system, and make up to 100 μL system;

[0176] S4.2.2 Take 60 μL (0.6x) magnetic beads, add to the above system, mix well, stand at room temperature for 5 min, and then place on a magnetic stand for 5 min, take the supernatant, and discard the magnetic beads;

[0177] S4.2.3 Take all the supernatant to a new EP tube, add 15 μL (0.15x) magnetic beads to it, mix well, stand at room temperature for 5 min, and then place on a magnetic stand for 5 min, leave the magnetic beads, and discard the supernatant;

[0178] S4.2.4 Repeat step S4.2.3 once, centrifuge immediately after all liquid is aspirated, then completely aspirate the liquid with a 10 μL pipette, dry the magnetic beads at room temperature for about 3 min;

[0179] S4.2.6 Add 15 μL of EB (room temperature) to the magnetic beads, mix well, stand at room temperature for 5 min, place on the magnetic stand for 5 min, and take 14 μL of supernatant, which is the absolute quantification lncRNA library stock solution of the low-quality FFPE sample;

[0180] Library construction and quality control: both the DNA fragment and the concentration detection are qualified, that is, the target fragment size is 300-600 bp,

[0181] The concentration is ≥1 ng, the volume is ≥10 μL, and there is no adapter contamination, which can be detected by machine, such as Figure 3 .

[0182] Comparative Example 1

[0183] The library construction method is the same as that of Example 1, only the ribosome removal system configuration in S1.1 is different: take 2x Frag / Elute buffer 5 μL, total RNA 4 μL, 1 μL QIAseq FastSelect-rRNA HMR 1 μL, mix well by blowing, and centrifuge immediately. The library detection results of Comparative Example 1 are shown in Figure 5 and Table 7.

[0184] Comparative Example 2

[0185] Use the existing kit Ovation SoLo RNA-Seq System to synthesize cDNA from plasma-derived exosome total RNA, perform first-round amplification, then end repair, add adapters, purify, and then perform second-round amplification. The subsequent amplification product is subjected to human / mouse (only supports these two species) ribosome removal, and then subjected to third-round amplification, and the purified library is obtained. In the prior art, the main source of exosomes is currently animal blood or cell supernatant, and the ribosome removal kit in this kit is only designed for human / mouse ribosome sequences. The library detection results of Comparative Example 2 are shown in Figure 4 and Table 7.

[0186] Table 7

[0187]

[0188] Comparative Example 3

[0189] In the comparative example implementation, the only difference with Example 2 is that in step S1, only QIAseq FastSelect-rRNA HMR is used for eukaryotic ribosome removal, and other operations are completely consistent. The detection results are shown in Figure 6 and Table 8.

[0190] Comparative Example 4

[0191] Common library construction method for FFPE samples.

[0192] After RNA detection, rRNA was removed by epicentre Ribo-ZeroTM kit. Then fragmentation reagent was added to break the RNA into short fragments of 250-300 bp, and short fragment RNA was used as a template to synthesize single-stranded cDNA with random hexamers. Then buffer, dNTPs (dUTP, dATP, dGTP and dCTP) and DNA polymerase I were added to synthesize double-stranded cDNA, and then double-stranded cDNA was purified by AMPureXP beads. The purified double-stranded cDNA was then subjected to end repair, A tailing and ligation of sequencing adapters, and then AMPureXP beads were used for fragment size selection. Then USER enzyme was used to degrade the second strand of cDNA containing U, and finally PCR enrichment was performed to obtain strand-specific cDNA library.

[0193] The detection results are shown in Table 7 and Table 8. RFU is Relative Fluorescence Units. Figure 7

[0194] Table 8

[0195]

[0196] From the above description, it can be seen that the above-mentioned embodiments of the present application achieve the following technical effects: the present application directly reverse transcribes cDNA from total RNA after ribosome removal, then connects adapters containing UMI sequences, and further amplifies to obtain an absolute quantification lncRNA library of low-quality FFPE samples, so as to achieve the purpose of accurately quantifying the number of molecules at low initial amount and low-quality FFPE sample lncRNA library and reducing errors generated by sequencing and library preparation and unevenness caused by PCR amplification, effectively distinguishing false positive mutations introduced in the library construction process, thereby more effectively detecting ultra-low abundance mutations and avoiding data bias caused by sample quality differences.

[0197] ​As can be seen from the above description, the above embodiments of the present invention achieve the following technical effects: cDNA is synthesized by direct reverse transcription of total RNA after ribosome removal, and then a adapter containing UMI sequence is ligated to further amplify and obtain UMI sample lncRNA library, thereby achieving the purpose of accurately quantifying the number of molecules starting in low-abundance sample lncRNA library and reducing errors caused by sequencing and library preparation as well as heterogeneity caused by PCR amplification, effectively distinguishing false positive mutations introduced in the library construction process, and thus more effectively detecting ultra-low abundance mutations.

[0198] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing a high-throughput sequencing library of lncRNA, characterized in that, The construction method includes: S1. Use a ribosome removal reagent to remove ribosomal RNA from the total RNA of the sample to be tested, and obtain total RNA without ribosomal RNA. S2, reverse transcribe the total RNA that does not contain ribosomal RNA to form double-stranded cDNA; S3, the double-stranded cDNA is sequentially purified, end-repaired, and A-added to obtain repaired DNA; sequencing adapters are added to the 5' and 3' ends of the repaired DNA to obtain adapter ligation products; the sequencing adapters are sequencing adapters containing UMI. S4, PCR amplification enriches the adapter ligation product, and a high-throughput sequencing library of lncRNA from the sample to be tested is obtained. The samples to be tested include exosome samples or low-quality FFPE samples; The ribosomal RNA includes eukaryotic ribosomal RNA and prokaryotic ribosomal RNA; The ribosome removal reagents include eukaryotic ribosome removal reagents and prokaryotic ribosome removal reagents.

2. The construction method according to claim 1, characterized in that, In S2, the reverse transcription includes a first-strand synthesis reaction and a second-strand synthesis reaction to obtain the double-stranded cDNA.

3. The construction method according to claim 1, characterized in that, After obtaining the double-stranded cDNA, no fragmentation operation is performed on the double-stranded cDNA.

4. The construction method according to claim 1, characterized in that, The first purification includes first magnetic bead purification.

5. The construction method according to claim 2, characterized in that, The reaction system for the first chain synthesis reaction includes: 10 μL of total RNA without ribosomal RNA, 8 μL of reverse transcription reagent, and 2 μL of one-chain synthase complex.

6. The construction method according to claim 2, characterized in that, The reaction procedure for the first chain synthesis reaction includes: reacting at 25°C for 10 min, reacting at 42°C for 15 min, reacting at 70°C for 15 min, and holding at 4°C.

7. The construction method according to claim 2, characterized in that, The reaction system for the second chain synthesis reaction includes: 20 μL of the reaction system for the first chain synthesis reaction, 8 μL of the second chain synthesis reaction buffer, 4 μL of the second chain synthase mixture, and 48 μL of nuclease-free water.

8. The construction method according to claim 2, characterized in that, The reaction procedure for the second chain synthesis reaction includes: reacting at 16°C for 1 h.

9. The construction method according to claim 1, characterized in that, The reaction solution for end repair comprises: 42 μL of the first purified solution; 6.8 μL of end repair solution 1; and 1.2 μL of end repair enzyme.

10. The construction method according to claim 5, characterized in that, The reaction procedure for end repair includes: reaction at 37°C for 30 min, reaction at 72°C for 30 min, and holding at 4°C.

11. The construction method according to claim 1, characterized in that, After connecting the sequencing adapter, a second purification process is performed.

12. The construction method according to claim 11, characterized in that, The second purification includes a second magnetic bead purification.

13. The construction method according to claim 1, characterized in that, The reaction solution for ligating the sequencing adapter comprises: 50 μL of reaction solution containing the double-stranded cDNA; 8.4 μL of enzyme reaction buffer 2-1; 15 μL of enzyme reaction buffer 2-2; 1.6 μL of rapid ligase; 1 μL of sequencing adapter containing UMI; 3 μL of nuclease-free water; and 1 μL of Enzyme 2.

14. The construction method according to claim 1, characterized in that, The reaction procedure for connecting the sequencing adapter includes: reaction at 20°C for 30 min, followed by holding at 4°C.

15. The construction method according to claim 1, characterized in that, In step S4, the adapter ligation product containing U bases is digested before PCR amplification.

16. The construction method according to claim 15, characterized in that, The linker ligation product containing the U base was digested using UDG enzyme.

17. The construction method according to claim 1, characterized in that, After PCR amplification, a third purification was performed to obtain a high-throughput sequencing library of the sample's lncRNA.

18. The construction method according to claim 17, characterized in that, The third purification includes a third magnetic bead purification.

19. The construction method according to claim 1, characterized in that, The total RNA that does not contain ribosomal RNA includes one or more of mRNA, lncRNA, or circRNA.

20. The construction method according to claim 15, characterized in that, The reaction solution for digestion and PCR amplification includes: 22 μL of reaction solution containing the adapter ligation product; 1 μL of USER enzyme; 25 μL of PCR buffer (2X); 1 μL of 25 μM Index(X) primer / i7 primer; and 1 μL of 25 μM P5 PCR primer.

21. The construction method according to claim 15, characterized in that, The digestion and PCR amplification reaction procedure included: 37℃ reaction for 10 min, 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 65℃ annealing for 30 s, 72℃ extension for 30 s, 10-15 cycles; 72℃ extension for 5 min; and 4℃ hold.

22. A high-throughput sequencing library of lncRNA, characterized in that, The high-throughput sequencing library of the lncRNA was constructed using the construction method described in any one of claims 1 to 21.

23. A lncRNA sequencing method for non-disease diagnosis and treatment purposes, characterized in that, The sequencing method includes sequencing the high-throughput sequencing library of lncRNA as described in claim 22.

24. The construction method of any one of claims 1 to 21, or the high-throughput sequencing library of lncRNA as described in claim 22, is used in the preparation of products for early disease screening, prognostic assessment, basic scientific research, and / or clinical efficacy monitoring.

Images